The Date Palm Genome, Vol. 1: Phylogeny, Biodiversity and Mapping 9783030737467, 3030737462

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Compendium of Plant Genomes

Jameel M. Al-Khayri S. Mohan Jain Dennis V. Johnson   Editors

The Date Palm Genome, Vol. 1 Phylogeny, Biodiversity and Mapping

Compendium of Plant Genomes Series Editor Chittaranjan Kole, Raja Ramanna Fellow, Government of India, ICAR-National Research Center on Plant Biotechnology, Pusa, New Delhi, India

Whole-genome sequencing is at the cutting edge of life sciences in the new millennium. Since the first genome sequencing of the model plant Arabidopsis thaliana in 2000, whole genomes of about 100 plant species have been sequenced and genome sequences of several other plants are in the pipeline. Research publications on these genome initiatives are scattered on dedicated web sites and in journals with all too brief descriptions. The individual volumes elucidate the background history of the national and international genome initiatives; public and private partners involved; strategies and genomic resources and tools utilized; enumeration on the sequences and their assembly; repetitive sequences; gene annotation and genome duplication. In addition, synteny with other sequences, comparison of gene families and most importantly potential of the genome sequence information for gene pool characterization and genetic improvement of crop plants are described. Interested in editing a volume on a crop or model plant? Please contact Prof. C. Kole, Series Editor, at [email protected]

More information about this series at http://www.springer.com/series/11805

Jameel M. Al-Khayri • S. Mohan Jain Dennis V. Johnson

•

Editors

The Date Palm Genome, Vol. 1 Phylogeny, Biodiversity and Mapping

123

Editors Jameel M. Al-Khayri Department of Agricultural Biotechnology King Faisal University Al-Ahsa, Saudi Arabia

S. Mohan Jain Department of Agricultural Sciences University of Helsinki Helsinki, Finland

Dennis V. Johnson Cincinnati, OH, USA

ISSN 2199-4781 ISSN 2199-479X (electronic) Compendium of Plant Genomes ISBN 978-3-030-73745-0 ISBN 978-3-030-73746-7 (eBook) https://doi.org/10.1007/978-3-030-73746-7 © Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

The Garden of Essai, Algiers, Algeria, 1881, Pierre-Auguste Renoir. Image Source: Wikimedia https://commons. wikimedia.org/wiki/File:Renoir_-_algiers-the-garden-of-essai-1881.jpg!PinterestLarge.jpg

This book series is dedicated to my wife Phullara and our children Sourav and Devleena Chittaranjan Kole

Preface of the Series

Genome sequencing has emerged as the leading discipline in the plant sciences coinciding with the start of the new century. For much of the twentieth century, plant geneticists were only successful in delineating putative chromosomal location, function, and changes in genes indirectly through the use of a number of “markers” physically linked to them. These included visible or morphological, cytological, protein, and molecular or DNA markers. Among them, the first DNA marker, the RFLPs, introduced a revolutionary change in plant genetics and breeding in the mid-1980s, mainly because of their infinite number and thus potential to cover maximum chromosomal regions, phenotypic neutrality, absence of epistasis, and codominant nature. An array of other hybridization-based markers, PCR-based markers, and markers based on both facilitated construction of genetic linkage maps, mapping of genes controlling simply inherited traits, and even gene clusters (QTLs) controlling polygenic traits in a large number of model and crop plants. During this period, a number of new mapping populations beyond F2 were utilized and a number of computer programs were developed for map construction, mapping of genes, and for mapping of polygenic clusters or QTLs. Molecular markers were also used in the studies of evolution and phylogenetic relationship, genetic diversity, DNA fingerprinting, and map-based cloning. Markers tightly linked to the genes were used in crop improvement employing the so-called marker-assisted selection. These strategies of molecular genetic mapping and molecular breeding made a spectacular impact during the last one and a half decades of the twentieth century. But still they remained “indirect” approaches for elucidation and utilization of plant genomes since much of the chromosomes remained unknown and the complete chemical depiction of them was yet to be unraveled. Physical mapping of genomes was the obvious consequence that facilitated the development of the “genomic resources” including BAC and YAC libraries to develop physical maps in some plant genomes. Subsequently, integrated genetic–physical maps were also developed in many plants. This led to the concept of structural genomics. Later on, emphasis was laid on EST and transcriptome analysis to decipher the function of the active gene sequences leading to another concept defined as functional genomics. The advent of techniques of bacteriophage gene and DNA sequencing in the 1970s was extended to facilitate sequencing of these genomic resources in the last decade of the twentieth century. ix

x

As expected, sequencing of chromosomal regions would have led to too much data to store, characterize, and utilize with the-then available computer software could handle. But the development of information technology made the life of biologists easier by leading to a swift and sweet marriage of biology and informatics, and a new subject was born—bioinformatics. Thus, the evolution of the concepts, strategies, and tools of sequencing and bioinformatics reinforced the subject of genomics—structural and functional. Today, genome sequencing has traveled much beyond biology and involves biophysics, biochemistry, and bioinformatics! Thanks to the efforts of both public and private agencies, genome sequencing strategies are evolving very fast, leading to cheaper, quicker, and automated techniques right from clone-by-clone and whole-genome shotgun approaches to a succession of second-generation sequencing methods. The development of software of different generations facilitated this genome sequencing. At the same time, newer concepts and strategies were emerging to handle sequencing of the complex genomes, particularly the polyploids. It became a reality to chemically—and so directly—define plant genomes, popularly called whole-genome sequencing or simply genome sequencing. The history of plant genome sequencing will always cite the sequencing of the genome of the model plant Arabidopsis thaliana in 2000 that was followed by sequencing the genome of the crop and model plant rice in 2002. Since then, the number of sequenced genomes of higher plants has been increasing exponentially, mainly due to the development of cheaper and quicker genomic techniques and, most importantly, the development of collaborative platforms such as national and international consortia involving partners from public and/or private agencies. As I write this preface for the first volume of the new series “Compendium of Plant Genomes,” a net search tells me that complete or nearly complete whole-genome sequencing of 45 crop plants, eight crop and model plants, eight model plants, 15 crop progenitors and relatives, and 3 basal plants is accomplished, the majority of which are in the public domain. This means that we nowadays know many of our model and crop plants chemically, i.e., directly, and we may depict them and utilize them precisely better than ever. Genome sequencing has covered all groups of crop plants. Hence, information on the precise depiction of plant genomes and the scope of their utilization are growing rapidly every day. However, the information is scattered in research articles and review papers in journals and dedicated Web pages of the consortia and databases. There is no compilation of plant genomes and the opportunity of using the information in sequence-assisted breeding or further genomic studies. This is the underlying rationale for starting this book series, with each volume dedicated to a particular plant. Plant genome science has emerged as an important subject in academia, and the present compendium of plant genomes will be highly useful both to students and teaching faculties. Most importantly, research scientists involved in genomics research will have access to systematic deliberations on the plant genomes of their interest. Elucidation of plant genomes is of interest not only for the geneticists and breeders, but also for practitioners of an array of plant science disciplines, such as taxonomy, evolution, cytology,

Preface of the Series

Preface of the Series

xi

physiology, pathology, entomology, nematology, crop production, biochemistry, and obviously bioinformatics. It must be mentioned that information regarding each plant genome is ever-growing. The contents of the volumes of this compendium are, therefore, focusing on the basic aspects of the genomes and their utility. They include information on the academic and/or economic importance of the plants, description of their genomes from a molecular genetic and cytogenetic point of view, and the genomic resources developed. Detailed deliberations focus on the background history of the national and international genome initiatives, public and private partners involved, strategies and genomic resources and tools utilized, enumeration on the sequences and their assembly, repetitive sequences, gene annotation, and genome duplication. In addition, synteny with other sequences, comparison of gene families, and, most importantly, the potential of the genome sequence information for gene pool characterization through genotyping by sequencing (GBS) and genetic improvement of crop plants have been described. As expected, there is a lot of variation of these topics in the volumes based on the information available on the crop, model, or reference plants. I must confess that as the series editor, it has been a daunting task for me to work on such a huge and broad knowledge base that spans so many diverse plant species. However, pioneering scientists with lifetime experience and expertise on the particular crops did excellent jobs editing the respective volumes. I myself have been a small science worker on plant genomes since the mid-1980s and that provided me the opportunity to personally know several stalwarts of plant genomics from all over the globe. Most, if not all, of the volume editors are my longtime friends and colleagues. It has been highly comfortable and enriching for me to work with them on this book series. To be honest, while working on this series I have been and will remain a student first, a science worker second, and a series editor last. And I must express my gratitude to the volume editors and the chapter authors for providing me the opportunity to work with them on this compendium. I also wish to mention here my thanks and gratitude to the Springer staff, Dr. Christina Eckey and Dr. Jutta Lindenborn in particular, for all their constant and cordial support right from the inception of the idea. I always had to set aside additional hours to edit books beside my professional and personal commitments—hours I could and should have given to my wife, Phullara, and our kids, Sourav, and Devleena. I must mention that they not only allowed me the freedom to take away those hours from them but also offered their support in the editing job itself. I am really not sure whether my dedication of this compendium to them will suffice to do justice to their sacrifices for the interest of science and the science community. Kalyani, India

Chittaranjan Kole

Preface

The date palm is unquestionably the keystone tree species of agriculture in semiarid and arid lands of the Near East and North Africa, and it is now successfully being grown commercially in South Asia, Southern Africa, Iberia, the Americas and Australia. This important multipurpose palm is an essential local nutritional resource for humans and animals, providing a source of income to farmers through fruit sales, the leaves and wood offering an array of other useful products and the trees themselves delivering ecological services by creating a favorable microenvironment for habitation, cultivation of other agricultural crops and animal husbandry. In the coming decades, the major challenge for plant breeders is to sustain and improve upon date fruit production, through genetic improvement to counter biotic threats such that presented by the red palm weevil and bayoud disease, as well as abiotic stresses caused by drought, extreme temperatures and high soil or water salinity. Genomic studies are the best way forward to achieve such ends, along with the adoption of best practices for the cultivation of the palms and improved postharvest fruit handling and marketing to minimize diminished quality and marketable quantity. A deeper knowledge of the date palm genome also offers a promising opportunity to explore such topics as biofortification to discover new products. This book is the first comprehensive assemblage of contemporary knowledge and research work relevant to genomics and other omics in date palm. It highlights the recent progress in the development of plant biotechnology, associated molecular tools and their usage in plant breeding. Two volumes of this book are concurrently published. Volume 1 subtitled Phylogeny, Biodiversity and Mapping consists of 11 chapters arranged in 3 parts grouped according to the subject. Part I Biology and Phylogeny with 3 chapters focusing on date palm biology, evolution and origin. Part II Biodiversity and Molecular Identification with 4 chapters covering conformity of in vitro derived plants, molecular markers, barcoding, pollinizer genetics and gender determination. Part III Genome Mapping and Bioinformatics with 3 chapters addressing genome mapping of nuclear, chloroplast and mitochondrial DNA, in addition to a chapter on progress made in date palm bioinformatics. This volume represents the efforts of 30 international scientists from 10 countries and contains 78 figures and 30 tables to illustrate presented concepts.

xiii

xiv

Preface

Volume 2 subtitled Omics and Molecular Breeding consists of 11 chapters arranged in 4 parts grouped according to the subject. Part I Nutritional and Pharmaceuticals Properties with 3 chapters on the utilization of date palm as an ingredient of various food products, a source of bioactive compounds, along with a chapter on the production of nanomaterials derived from the date palm. Part II Omics Technologies consists of a chapter addressing an overview of omics resources followed by 2 chapters on proteomics and metabolomics. Part III Molecular Breeding and Genome Modification with 3 chapters focusing on genetic improvement technologies based on mutagenesis, quantitative traits loci and genome editing. Part IV Genomics of Abiotic and Biotic Stress contains a chapter covering metagenomics of beneficial microbes to enhance tolerance to abiotic stress, and another chapter addressing the various genomics advances as they apply to insect control in date palm. This volume represents the efforts of 34 international scientists from 12 countries and contains 65 figures and 19 tables to illustrate presented concepts. Participating authors were selected based on their reputable scientific expertise in date palm genomic research. Manuscripts were evaluated through a rigorous review process to assure quality presentation and scientific accuracy and edited for language precision. Each chapter begins with an introduction covering related background materials and provides in-depth discussion of the subject matter supported with high-quality color photos, illustrations and relevant tabulated data. The chapter concludes with recommendations for future research directions and a comprehensive list of pertinent references to facilitate further reading. This book is an excellent reference source for scientists engaged in genetics and modern breeding research based on biotechnology and genomic approaches. It is also a valuable source for advanced undergraduate and postgraduate students specializing in biotechnology and molecular breeding as well as for agricultural industries and policymakers especially those concerned with date palm. We are greatly appreciative of the stalwart efforts of all chapter authors for their contributions toward the success and quality of this book. We are also grateful to Prof. Chittaranjan Kole, the Compendium of Plant Genomes Series editor, for his invitation and supervision and to Springer for the opportunity to publish this book. Al-Ahsa, Saudi Arabia Helsinki, Finland Cincinnati, OH, USA

Jameel M. Al-Khayri S. Mohan Jain Dennis V. Johnson

Contents

Part I 1

2

3

Biology and Phylogeny

Date Palm (Phoenix dactylifera L.) Biology and Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Robert R. Krueger Systematics and Evolution of the Genus Phoenix: Towards Understanding Date Palm Origins . . . . . . . . . . . . . . . . . . . . . . Muriel Gros-Balthazard, William J. Baker, Ilia J. Leitch, Jaume Pellicer, Robyn F. Powell, and Sidonie Bellot A Brief History of the Origin of Domesticated Date Palms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Muriel Gros-Balthazard and Jonathan M. Flowers

Part II

3

29

55

Biodiversity and Molecular Identification 77

4

Genome Conformity of In Vitro Cultures of Date Palm . . . . . Sherif F. El-Sharabasy, Ehab M. B. Mahdy, and Hesham S. Ghazzawy

5

Date Palm Genetic Identification and Improvement Utilizing Molecular Markers and DNA Barcoding . . . . . . . . . 101 Ehab M. B. Mahdy and Sherif F. El-Sharabasy

6

DNA Fingerprinting of Date Palm Pollen Sources and Their Relevance to Yield and Fruit Traits . . . . . . . . . . . . 135 Mohamed M. S. Saleh, Esam A. M. Mostafa, Nagah E. Ashour, and Samy A. A. Heiba

7

Gender Determination of Date Palm . . . . . . . . . . . . . . . . . . . . 161 Summar Abbas Naqvi, Waqar Shafqat, Muhammad Salman Haider, Faisal Saeed Awan, Iqrar Ahmad Khan, and Muhammad Jafar Jaskani

xv

xvi

Contents

Part III

Genome Mapping and Bioinformatics

8

Whole-Genome Mapping of Date Palm (Phoenix dactylifera L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 Zafar Iqbal, Muhammad Naeem Sattar, and Jameel M. Al-Khayri

9

Date Palm (Phoenix dactylifera L.) Chloroplast Genome . . . . 201 M. Kamran Azim

10 Comparative Analysis of Date Palm (Phoenix dactylifera L.) Mitochondrial Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Sajjad Asaf, Abdul Latif Khan, Ahmed Al-Harrasi, and Ahmed Al-Rawahi 11 Date Palm Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Vadivel Arunachalam Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

Contributors

Ahmed Al-Harrasi Natural and Medical Science Research Center, University of Nizwa, Nizwa, Oman Jameel M. Al-Khayri Department of Agricultural Biotechnology, College of Agriculture and Food Sciences, King Faisal University, Al-Ahsa, Kingdom of Saudi Arabia Ahmed Al-Rawahi Natural and Medical Science Research Center, University of Nizwa, Nizwa, Oman Vadivel Arunachalam ICAR-Central Coastal Agricultural Research Institute Ela, Goa, India Sajjad Asaf Natural and Medical Science Research Center, University of Nizwa, Nizwa, Oman Nagah E. Ashour Pomology Department, Agricultural and Biological Division, National Research Centre, Cairo, Egypt Faisal Saeed Awan Center for Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad, Pakistan M. Kamran Azim Department of Biosciences, Mohammad Ali Jinnah University, Karachi, Pakistan William J. Baker Royal Botanic Gardens Kew, Richmond, United Kingdom Sidonie Bellot Royal Botanic Gardens Kew, Richmond, United Kingdom Sherif F. El-Sharabasy Central Laboratory of Date Palm Research and Development (CLDPRD), Agricultural Research Centre (ARC), Giza, Egypt Jonathan M. Flowers Center for Genomics and Systems Biology, New Yok University Abu Dhabi, Abu Dhabi, United Arab Emirates Hesham S. Ghazzawy Central Laboratory of Date Palm Research and Development (CLDPRD), Agricultural Research Centre (ARC), Giza, Egypt; Date Palm Research Center of Excellence, King Faisal University, Al-Ahsa, Saudi Arabia

xvii

xviii

Muriel Gros-Balthazard Center for Genomics and Systems Biology, New Yok University Abu Dhabi, Abu Dhabi, United Arab Emirates; Institut de Recherche pour le Développement, UMR DIADE, Montpellier, France Muhammad Salman Haider Key Laboratory of Genetics and Fruit Development, College of Horticulture, Nanjing Agricultural University, Nanjing, China Samy A. A. Heiba Genetics and Cytology Department, Genetic engineering and Biotechnology Division, National Research Centre, Cairo, Egypt Zafar Iqbal Central Laboratories, King Faisal University, Al-Ahsa, Kingdom of Saudi Arabia Muhammad Jafar Jaskani Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan Abdul Latif Khan Natural and Medical Science Research Center, University of Nizwa, Nizwa, Oman Iqrar Ahmad Khan Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan Robert R. Krueger USDA–ARS National Clonal Germplasm Repository for Citrus & Dates, University of California, Riverside, CA, USA Ilia J. Leitch Royal Botanic Gardens Kew, Richmond, United Kingdom Ehab M. B. Mahdy National Gene Bank (NGB), Agricultural Research Centre (ARC), Giza, Egypt Esam A. M. Mostafa Pomology Department, Agricultural and Biological Division, National Research Centre, Cairo, Egypt Summar Abbas Naqvi Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan Jaume Pellicer Royal Botanic Gardens Kew, Richmond, United Kingdom; Institut Botànic de Barcelona (IBB, CSIC-Ajuntament de Barcelona), Barcelona, Spain Robyn F. Powell Royal Botanic Gardens Kew, Richmond, United Kingdom Mohamed M. S. Saleh Pomology Department, Agricultural and Biological Division, National Research Centre, Cairo, Egypt Muhammad Naeem Sattar Central Laboratories, King Faisal University, Al-Ahsa, Kingdom of Saudi Arabia Waqar Shafqat Department of Horticultural Sciences, Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Fort Pierce, FL, USA

Contributors

Abbreviations

ABySS AFLP CDDP COD FISH cpDNA DArT DPMMD DRDB EST FISH GAPIT GBS GWAS HTPP ISH ISSR ITAP KASPTM LD MAS MSAP NCBI NGS PAGE PCR PGSB QTL RAD RAPD RFLP RRL SCAR SCoT sgRNA SKY SNP

Assembly by short sequencing Amplified fragment length polymorphism Conserved DNA-derived polymorphism Concomitant oncoprotein detection Chloroplast DNA Diversity array technology Date palm molecular marker database Date palm genome database Expressed sequence tag Fluorescent in situ hybridization Genome association and prediction integration tool Genotyping by sequencing Genome wide association study High-throughput phenotyping platform In situ hybridization Inter simple sequence repeats Intron-targeted amplified polymorphism Kompetitive allele specific PCR Linkage disequilibrium Marker-assisted selection Methylation-sensitive amplified polymorphism National Center for Biotechnology Information Next generation sequencing Polyacrylamide gel electrophoresis Polymerase chain reaction Plant genome and system biology Quantitative trait loci Restriction site associated DNA sequencing Random amplified polymorphic DNA Restriction fragment length polymorphism Reduced representation library Sequence characterized amplified region Start codon targeted polymorphism Sequence of gene recognition Spectral karyotyping Single nucleotide polymorphism xix

xx

SNVs SSR TASSEL TILLING VNTR WGRS

Abbreviations

Single nucleotide variants Simple sequence repeat Trait analysis by association, evolution and linkage Targeting induced local lesions in genome Variable number of tandem repeats Whole genome resequencing

Part I Biology and Phylogeny

1

Date Palm (Phoenix dactylifera L.) Biology and Utilization Robert R. Krueger

Abstract

The date palm (Phoenix dactylifera L.) is the type species for the genus Phoenix of the family Arecaceae (Palmaceae). Phoenix species are native to tropical and subtropical areas of Asia and Africa. The date palm has been domesticated for several millennia in its centres of origin, diversity and domestication in the Middle East and North Africa. From there, date culture spread to other areas having a suitable climate (hot and arid or semiarid) with sufficient water available either as groundwater or irrigation. The date palm was introduced into the Western Hemisphere much later and has a more limited production area there. The date palm is a unique, arborescent monocotyledonous plant with distinctive anatomical features and environmental adaptations. It grows in arid, sandy environments but requires large volumes of water for growth and fruit production; it tolerates saline conditions but is not a true halophyte. Numerous labour-intensive cultural practices including pruning, pollination and bunch management are necessary for successful date production. Postharvest handling may include dehydration

R. R. Krueger (&) USDA–ARS National Clonal Germplasm Repository for Citrus & Dates, University of California, Riverside, CA 92507–5437, USA e-mail: [email protected]

or hydration, depending upon the fruit moisture content at harvest. Date fruit is also processed into pastes, syrups, butters and other products. Production of dates has increased in recent decades and will likely continue to increase in the near-term future.

1.1

Introduction

The date palm (Phoenix dactylifera L) is the type species of the genus Phoenix. Phoenix species are native to South Asian and African regions with tropical or subtropical climates (Fig. 1.1). Some Phoenix species are short statured with very short trunks, whereas other species may be over 30 m tall, and different species may have a single trunk or clumped multiple trunks. Phoenix is featherleafed palms with a terminal leaflet and all leaflets having a central fold or ridge that causes the leaflets to remain erect. Basal leaflets are modified into spines. Phoenix species are dioecious, with separate staminate and pistillate trees. Inflorescences on both staminate and pistillate trees arise from lateral meristems among the leaves. The three-petalled flowers are small, single, and pale yellow, with the sepals united into a cupule. Female flowers have three carpels; one of which matures into a fruit while the other two dehisce. Male flowers generally have six stamens. The fruits are drupes, with a single grooved seed. While most Phoenix species have limited or no economic value, the date palm is an important

© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2021 J. M. Al-Khayri et al. (eds.), The Date Palm Genome, Vol. 1, Compendium of Plant Genomes, 2021 https://doi.org/10.1007/978-3-030-73746-7_1

3

4

R. R. Krueger

Fig. 1.1 Distribution of Phoenix species throughout the world. Source Munier (1973)

food crop in areas in which it can be successfully cultivated. Its cultivation began in ancient times in its centre of origin and diversity in the Middle East and later spread westward into North Africa and eastward into the Indian subcontinent. Domestication and spread of date culture are discussed in other chapters in this book. The date palm and dates as food items are also culturally important in Arabic countries and Islamic countries with Arabic cultural influence. This chapter describes the basic biology of the date palm, reproductive characteristics including fruit development, adaptation to environmental and climatic conditions. In addition, the basics of date cultivation and packing are presented. This chapter will provide a basis for understanding some of the genomic information presented in the main body of the book.

1.2

Taxonomy

The genus Phoenix is the only member of the tribe Phoeniceae of the subfamily Coryphyoideae of the monocot family Arecaceae (Bailey and Bailey 1976; Moore 1963). Despite being

distinctive plants, Phoenix taxonomy was not firmly established until recently. Different taxonomic treatments were developed with some variation in species names and applications. As stated by Uhl and Dransfield (1987), Despite the economic importance of Phoenix and the great ease of recognition of the genus, the species remain poorly known and in much need of a careful revision. The most useful account remains that of Beccari (1890). Phoenix species also hybridise readily, which led Wrigley (1995) to suggest that the genus Phoenix is monotypic. Interspecific hybridisation of Phoenix species can lead to confusion or doubt with regard to individual plants, particularly seedlings. This is especially true when several species are present, as may occur in ex situ collections where Phoenix species are often mislabelled (Hodel 1995). Twenty-seven species of Phoenix have been named, but most taxonomic treatments have accepted about 12 species as valid (Table 1.1). The taxonomy presented in the monograph of Barrow (1998), summarised in Table 1.2, recognised 13 species and has become widely accepted. Prior to Barrow (1998), the most recent monograph dealing with Phoenix was Beccari

1

Date Palm (Phoenix dactylifera L.) Biology and Utilization

(1890), which recognised 10 species. Three additional species were published after Beccari (1980): P. caespitosa Chiov. from Somalia, P. atlantica A. Chev. from the Cape Verde Islands, and P. theophrasti Greuter from Crete. Moore (1963) recognised 12 species (Table 1.2). Barrow (1998) sunk Moore’s (1963) P. abyssinica into P. reclinata and his P. farinifera into P. pusilla, recognised the recently-published P. caespitosa and P. theophrasti and two varieties of P. loureiri, included a new species P. andamanensis from the Andaman Islands, and considered P. atlantica to be incompletely known. This resulted in 13 species of Phoenix being recognised, with 33 synonyms and 57 nomina nuda (invalid or unpublished names). Characteristics and geographic distributions of Phoenix species other than P. dactylifera are summarised in Table 1.2.

5

1.3

Botanical Description

A generalised diagram of the date palm is presented in Fig. 1.2. This figure shows a generalised female tree with a single trunk and an attached offshoot. More detailed anatomical descriptions of the various organs and tissues are available in Tomlinson (1961).

1.3.1 Root System As a monocot, the date palm has a fibrous root system without a tap root. The genus Phoenix frequently has roots with pneumathodes, swollen areas caused by the development of loose tissue containing large air spaces. These organs are believed to ventilate underground roots and allow the palm to withstand extended periods of

Table 1.1 Species of Phoenix recognised by several investigators Species

Martius (1836– 1850)

Beccari (1890)

Miller et al. (1930)

Chevalier (1952)

Mowry et al. (1952)

+

+

+

+

+

abyssinica Drude acaulis Roxb

Moore (1963)

Munier (1973, 1974)

+ +

Bailey and Bailey (1976)

Barrow (1998)

+ +

+

andamanensis S Barrow

+ +

atlantica A Chev

+

+

?

caespitosa Chiov

+

canariensis Hort ex Chab

+

+ +

dactylifera L

+

+

farinifera Roxb

+

+

+

+

+

+

+

hanceana Naudin

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

humilis Royle

+

+

loueirii Kunth

+

ouseleyana Griff

+

paludosa Roxb

+

+

pusilla Gaertn

+

+

+

reclinata Jacq

+

+

+

pumila Hort

+

+

+

+ +

+

+

+

+

+

+

+

+

+

+

+ +

+

+

+ (continued)

6

R. R. Krueger

Table 1.1 (continued) Species

Martius (1836– 1850)

Beccari (1890)

Miller et al. (1930)

Chevalier (1952)

+

+

+

+

+

+

+

+

roebelinii O’Brien rupicola T Anderson spinosa FC Schum

+

sylvestris (L) Roxb

+

Mowry et al. (1952)

Moore (1963)

Munier (1973, 1974)

Bailey and Bailey (1976)

Barrow (1998)

+

+

+

+

+

+

+

+

+

+

+

+

+

+

theophrasti Greuter zeylanica Trimen

+ +

submersion in water (Tomlinson 1961). This is consistent with the evolution of Phoenix along the rivers and oases in the Middle East. Although the date palm can withstand extended periods of submersion, eventually the roots and fruit production may be negatively affected (Zaid and De Wet 2002a). Although individual roots of the date palm may extend 10 m or more from the trunk and an equal distance in the soil profile, 85% of the roots are found within 2 m of the trunk and in the upper 2 m of the soil profile (Munier 1973). Munier (1973) divided the date palm root system into four zones (Fig. 1.2). Zone I, also called the respiratory zone, is localised at the base of the trunk, within 0.5 m of the trunk and 0.25 m in the soil profile. The mostly primary and secondary roots in this zone play a respiratory role. Zone II, also called the nutritional zone, extends somewhat beyond the canopy and 1.5 m into the soil profile. The majority of the tree’s root mass is found in this zone, the roots being mostly primary and secondary. Zone III, also called the absorbing zone, is a sort of extension of zone II in the 1.5–1.8 m zone of the soil profile with mostly primary roots. Zone IV extends deeper than 1.8 m in the soil profile and consists of most primary and secondary roots. The depth to which

+

+

this zone extends depends upon soil water presence deep in the soil profile. The actual size and development of a date palm’s root system depend on exogenous factors such as soil characteristics, ground water availability and cultural practices. The lack of roots at the top of the soil profile allows the cultivation of other crops (intercropping). The presence of roots deep in the soil profile may convey some drought–resistance to the date palm (Zaid and De Wet 2002a).

1.3.2 Stem The date palm has a vertical, cylindrical, columnar stem with short internodes; the stem having the same diameter as its entire length. Initial increases in stem diameter result from a fascicular cambium that at some point disappears, after which all stem growth is vertical (Zaid and De Wet 2002a). Basal meristems result in the formation of new aerial stems (offshoots or offsets) at the base of the parent palm. This process may proceed indefinitely, resulting in dense clusters of multi-trunked palms (Tomlinson 1961). However, in cultivation, the offshoots are generally removed and used to propagate

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Date Palm (Phoenix dactylifera L.) Biology and Utilization

7

Table 1.2 The genus Phoenix: a summary Species

Common name

Distribution

Notes

Synonyms

P. acaulis

–

N India, Myanmar

Stemless; fruit edible; sometimes confused with P. loureiri; conservation status uncertain; local populations possibly threatened by development

–

P. andamanensis

–

Bay of Bengal

Single trunk; semi-dwarf; species status somewhat questionable; rare, may be considered threatened

–

P. caespitosa

–

Somalia, Arabian peninsula

Stemless; fruit edible; habitat: wadis; species status somewhat questionable; restricted area, may be considered threatened.

P. arabica

P. canariensis

Canary (Island) date palm

Canary Islands

Single trunk; fruit edible; widely cultivated as ornamental; wide range of habitats within distribution; genetic erosion from hybridisation threatens genetic integrity

P. cycadiflora, P. jubae, P. tenuis

P. dactylifera

Date palm

Middle East to W India, N Africa

Habitat: wadis, oases; widely cultivated in suitable climates for fruit; many other plant parts utilised

P. atlantica

P. loureiri

–

India, China, Indochina, Philippines

Dwarf; fruit edible; other plant parts utilised; taxonomy somewhat confused: 2 varieties (loureiri, humilis); development threatens local populations but overall not threatened

P. P. P. P.

P. paludosa

–

Bay of Bengal, Indochina, Malaysia

Semi-dwarf; habitat mangrove swamps and estuaries; not considered threatened as a species but specific populations might be threatened

P. siamensis

P pusilla

–

S India, Sri Lanka

Fruits edible; other plant parts utilised; conservation status unclear

P. farinifera, P. zeylanica

P. reclinata

Senegal date palm

tropical & subtropical Africa, Madagascar, Comoro Islands

Habitat and morphology variable; fruit edible; other plant parts utilised; widely cultivated as ornamental; not considered threatened

P. P. P. P. P. P. P.

P. roebelenii

Pygmy date palm

Laos, Vietnam, S China

Rheophytic; dwarf; widely cultivated as ornamental; conservation status unclear, use as ornamental may result in removal of native populations

–

formosana, hanceana, humilis, ousleyana

abyssinica, baoulensis, comorensis, madagascariensis, senegalensis, spinosa, zanzibarensis, etc.

(continued)

8

R. R. Krueger

Table 1.2 (continued) Species

Common name

Distribution

Notes

Synonyms

P. rupicola

Cliff date palm

N India

Single trunk; semi-dwarf; fruits eaten by animals but not humans; conservation status unclear

–

P. sylvestris

Indian date palm

India & Pakistan

Wide range of habitats; utilised for sugar, fruit; not threatened

–

P. theophrasti

Cretan date palm

Crete, Turkey

Habitat: coastal areas; species status questionable; restricted growing area, threatened by population pressure

–

Source Compiled from Barrow (1998) and Johnson (1996) Fig. 1.2 Schematic of the date palm (Phoenix dactylifera L). Source USDA archival diagram, after Munier (1973)

1

Date Palm (Phoenix dactylifera L.) Biology and Utilization

additional trees at a different location. With sufficient time and removal of offshoots, a date palm stem may reach a height of 20 m or more.

9

1942). These spirals may be either right- or lefthanded, right-handed spirals predominating. Left-handed palms have been reported to yield more than right-handed palms (Reuvani 1986a).

1.3.3 Leaves 1.3.4 Vascular System Date palm leaves are irregularly pinnate with an odd terminal leaflet in most varieties. This is a distinctive characteristic of Phoenix, the only other group having this characteristic being the small group of Caryotoid palms. The leaflets are lanceolate, with sharp tips and induplicate (V-shaped) folding, the basal leaflets being reduced to spines (Tomlinson 1961). The axes (petiole or midrib) of date palm leaves are 3–6 m in length depending upon age and variety. The midrib is broad at its base but rapidly narrows towards the apex of the leaf. The leaflets are 15–100 cm in length and 1– 6 cm in width, depending upon age and variety, and number between 120 and 240 per frond (Zaid and De Wet 2002a). These leaf characteristics, along with others, have been used to differentiate between different varieties (Mason 1915). A mature date palm has 60–180 green leaves, with 10–25 being produced annually. There are three categories of leaves within the date palm’s canopy. The youngest leaves are white and not yet photosynthetically active, accounting for approximately 40% of the tree’s leaves. The next youngest leaves are green and account for approximately 10% of the leaves. The oldest leaves, accounting for approximately 50% of the leaves, are green and photosynthetically active. Normally each leaf can support the production of 1.0–1.5 kg of fruit (Zaid and De Wet 2002a). The photosynthetic capacity of the leaves decreases with age, a 4-year-old leaf having only about 65% of the photosynthetic efficiency as a 1-yearold leaf (Nixon and Wedding 1956). Leaves of the date palm emerge from the apical meristem one at a time, appearing in a spiral pattern between 137–138° around the trunk at a slightly higher level. This results in spirals of leaves of 5, 8 and 13, the numbers indicating the number of times the trunk must be encircled in order to arrive at a leaf directly above another (Fig. 1.3) (Aldrich et al. 1942; Mathez and Bliss

As a monocot, the date palm has a vascular system consisting of vascular bundles of xylem and phloem distributed throughout the roots, stem and leaves.

1.4

Reproductive Biology

1.4.1 Reproductive Organs The date palm is dioecious, having a separate female (pistillate) and male (staminate) trees. However, hermaphroditic trees and male trees developing female characteristics have occasionally been observed (Sudhersan and El-Nil 1999). Inflorescences of female and male trees are morphologically different (Nixon and Carpenter 1978; Swingle 1904). A flat, tapering peduncle or rachis, commonly known as the fruit stalk in female trees bears many unbranched rachillae, known as strands, arranged in spirals. Each rachilla bears hundreds of flowers. Male flowers usually have three sepals, three petals, six stamens, three reduced sterile carpels that may rarely develop parthenocarpically and are usually waxy white. Female flowers are usually yellowish-green with three sepals, three petals, six staminodes and three separate carpels, of which only one normally develops into a mature fruit (Fig. 1.4) (Reuvani 1985). During the early stages of flower development, the inflorescences are enclosed in a spathe, a hard, fibrous covering that protects them from heat and sunlight.

1.4.2 Flowering The flowering of the date palm begins when the shade temperature increases to more than 18 °C, and fruit forms when it is more than 25 °C (Zaid

10

R. R. Krueger

Fig. 1.3 Phyllotaxy of the date palm (Phoenix dactylifera L), showing the spiral of 13. Source USDA archival image

and De Wet 2002b). Just prior to flowering, the inflorescences arise in the axis of the leaves, pushing through the spathes, causing the spathes to crack longitudinally at anthesis. Only the portion of the rachillae that bears flowers is exposed. About 50–60 days after anthesis, the fruit stalk lengthens and pushes out the nonflower-bearing portion of the inflorescence to a length of 60–120 cm. After pollination, the fruit normally develops from one of the 3 carpels

within each pistillate flower, while the other 2 carpels abort. All 3 carpels may develop into tiny parthenocarpic fruit if pollination does not occur. Natural fruit abscission occurs 25–35 days after spathe crack, and some cultivars have a second abscission about 100 days after spathe crack (sometimes referred to in the Northern Hemisphere as June drop) (Reuvani 1986b). Date palms normally flower between February and April in the Northern Hemisphere. However,

1

Date Palm (Phoenix dactylifera L.) Biology and Utilization

11

b

a

c

Fig. 1.4 Male (left) and female (right) flowers of the date palm (Phoenix dactylifera L). (a) Flowers in spathe; (b) Inflorescence; (c) Close–up. Source USDA archival images

in some years, flowering may occur earlier or later than normal. This is related to temperatures during the flowering season but also appears to be related to crop levels the previous season and possibly other factors (Reuvani 1985).

1.4.3 Fruit Development The date palm’s binomial Phoenix dactylifera is derived from its fruit characteristics. In Greek, phoenix means purple or red (the colour of the fruit) and dactylifera (finger) refers to the appearance of the fruit bunch. The fruit of the date palm, usually referred to as a date, is a berry. Dates are usually oblong or ellipsoid with a

terminal stigma, a fleshy pericarp, and a membranous endocarp between the seed and the flesh. Dates range in weight from about 2–60 g, with lengths of 1.8–11.0 cm, and widths of 0.8– 3.2 cm. Different varieties have distinct fruit characteristics within these parameters, and a range of colours and tastes. Dates usually have a single oblong, ventrally grooved seed with a small embryo. Date seeds are 0.5–4.0 g in weight, 2.3–3.6 cm in length and 0.6–1.3 cm in width. As with the fruit, the seed of the date palm has characteristics that vary by variety (Reuvani 1986b; Zaid and De Wet 2002a). The growth rate and development of seeded date fruit follow a sigmoid growth curve early in the season but decrease later in the season as the

12

fruit loses water. Starting with the immature green kimri stage, the fruit increase in size and weight rapidly until reaching the physiologically mature, fully coloured khalal stage (sometimes called bisr). Fruit colour at khalal is a characteristic of the specific cultivar. Khalal stage fruit is turgid and astringent and contain a substantial amount of water-soluble tannins. During khalal, the rate of gain in size and weight decreases slightly and the fruit reach maximum size and weight. During the soft, brown rutab stage, the date’s skin darkens to amber, brown, or nearly black, accompanied by decreasing water content, increasing softness, decreasing astringency, and increasing insoluble tannins. During the hard, raisin-like tamar stage, dates lose much of their water and the sugar-to-water ratio is high enough to prevent fermentation, similar to raisins or dried prunes. Water content is 75–80% in young fruit, decreasing to 40–60% at the beginning of postkhalal ripening, and decreasing rapidly later. Soft date fruit is usually harvested commercially at 18–24% water content. The sugar content is about 20% dry matter during early kimri, increasing steadily to 50% dry matter at the beginning of khalal, and then accumulating at a faster rate until reaching 72–88% of dry matter at maturation, due partly to decreased fruit weight from the dehydration process (Reuveni 1986b). Additional information on date fruit development and composition is presented in another chapter of this book.

1.4.4 Metaxenia Xenia is the effect of different pollen sources can have on the size and shape of the seeds, whereas metaxenia is the effect of pollen on the tissue outside the embryo and endosperm (Nixon 1934, 1936; Reuveni 1986b). Date palm pollen has been documented as metaxenic. For example, pollen from cv. Fard No. 4 consistently produced earlier but smaller fruit and pollen from cv. Mosque consistently produced larger but later fruit than the average of all pollen sources

R. R. Krueger

studied by Nixon (1934). Pollen from different Phoenix species also exhibited metaxenic effects when applied to date palms (Nixon 1936). Similar effects have been reported from other studies, but in some cases, different pollen sources did not produce metaxenia. This has been attributed to metaxenic effects being less pronounced under favourable climatic conditions (i.e. higher heat units) or the specific pollen tested simply not producing metaxenic effects (Reuveni 1986b). In addition to the direct effect of pollen source on date size and seasonality, pollen source has also been shown to affect the chemical composition of cv. Medjool fruit (Salomon-Torres et al. 2018, 2019) and seeds (Salomon-Torres et al. 2019, 2020).

1.5

Climatic Requirements and Responses

The date palm’s centre of origin in the Middle East has an arid, subtropical climate and consequently, the date palm is adapted to high temperatures and low precipitation and relative humidity. The fact that the date palm’s natural habitat is in oases or river flood plains indicates that its water requirements are not necessarily low as it was not dependent upon rainfall in these situations. The date palm endures temperatures of 56 °C for several days when irrigated, and during the winter, temperatures below 0 °C are also endured. Zaid and De Wet (2002b) point out several interesting anomalies regarding the date palm: • The date palm is neither arenaceous nor aquatic. Growing frequently in sandy soil, it has the root pneumathodes mentioned previously. • Although it grows well under saline conditions, it grows better under less saline conditions and so is not a halophyte. • Although having some adaptations to arid conditions (waxy leaf cuticles, reduced leaf surface, leaflet orientation) it requires large amounts of water and is not a xerophyte.

1

Date Palm (Phoenix dactylifera L.) Biology and Utilization

1.5.1 Responses to Temperature Seasonal growth of the date palm begins when a 7 °C average daily temperature is attained (which maintains the temperature of the apical meristem at 9–10 °C), with flowering occurring when average daily temperatures reach 18–22 °C, depending upon the region and variety (Dowson 1982; Mason 1925a,b). Growth of the date palm increases between 7 °C and 32 °C, remaining stable between that temperature and 40 °C, above which growth decreases (average daily temperatures). The fruiting period of the date palm starts at the fruit set and ends at maturation, lasting for 120–200 days depending upon region and variety (Zaid and De Wet 2002b). Calculations based upon the accumulation of heat above a certain threshold between flowering and fruit maturation provide an estimation of the heat units required for specific varieties in different locations. There have been various ways of estimating the heat units required (Zaid and De Wet 2002b), the most commonly cited being those of Swingle (1904) and Munier (1973). Swingle (1904) based his estimate upon the sum of daily temperature maximums starting when that value reached 18 ° C in different date growing regions and arrived at a figure of approximately 3000–4000 heat units for successful date production. Munier (1973) used the same technique as Swingle (1904) but differentiated between varieties based upon days of maturation and arrived at a figure of 1000 heat units as a minimum for successful date production. In more subtropical or tropical environments, heat accumulation may theoretically be sufficient for good date production. However, the warmer winter temperatures, lower seasonal temperature variations and higher relative humidity generally do not support date production as well as more arid, subtropical conditions. Although the centre of origin of the date palm and current date production areas have relatively mild winter temperatures, occasional cold periods can occur, and the date palm is considered one of the most cold-tolerant palm species. When temperatures fall to below 0 °C for several hr, damage to leaf tissue occurs. At –6 °C, pinnae margins become chlorotic and desiccated.

13

Between –9 and –15 °C, leaves in the middle and outside of the canopy will become damaged and desiccated. If these temperatures persist for more than 12 h, all leaves will show damage and there is the possibility of reduced fruit quality the following growing season. Inflorescences can also be damaged by low temperatures and should be protected by paper bags (Zaid and De Wet 2002b).

1.5.2 Responses to Rain Most date production occurs in areas with low rainfall that occurs mainly in the winter months. The actual water requirements are mainly supplied by ground water (in traditional oasis and river flood plain production) or irrigation. Rain during the flowering period can reduce pollination effectiveness or knock flowers off inflorescences. Rain during the early khalal stage does not seriously damage dates, but during the late khalal, rutab and tamar stages rain may cause severe problems with rot or physiological damage. Growing seasons in which dates ripen late (for instance when temperatures are lower than normal) or in which rains arrive early are potentially the most damaging to date production. The conditions in which rain events occur can also affect the amount of damage caused by rain. A light rain followed by a period of high relative humidity can cause more damage than a heavy rain followed by sunshine and windy conditions (Nixon and Carpenter 1978). Different varieties also show differing sensitivities to rain damage to fruit development (Nixon 1950).

1.5.3 Responses to Relative Humidity and Wind Relative humidity can affect date production. High humidity can promote the growth of fungal pathogens while discouraging some arthropod pests; conversely, low humidity can have the opposite effect. Humid conditions can cause very soft, sticky fruits; whereas low humidity, especially coupled with windy conditions, can result

14

in overly dry or even desiccated fruit. High humidity during late ripening can cause physiological responses that result in market defects. Wind is generally not damaging to the date palm but can cause fruit damage from sand or abrasion when it occurs during the fruit development period. Wind, especially drying wind at certain times in the pollination season, can cause problems with pollination. Drying out of the flowers can reduce the receptivity period. A light wind during the pollination period can increase pollination efficiency, whereas a heavier wind may blow a large proportion of the pollen out of the orchard (Zaid and De Wet 2002b).

1.5.4 Responses to Soil Conditions Although dates can be grown on a variety of soils, the date palm does best on a deep, welldrained soil that can hold water to a depth of 2– 3 m (Nixon and Carpenter 1978). In contrast to most other perennial crops, the date palm is considered tolerant of saline conditions. The threshold for yield decline of the date palm is 4.0 mmho cm−1, with a 3.6-fold decrease in yield per unit increase of salinity beyond that (Maas and Hoffman 1977). However, very high levels of salinity can result in decreased growth and yield (Alhammadi and Kurup 2012). The date palm does not appear to accumulate sodium or chloride under saline conditions; however, boron does accumulate when boron levels are high in soil or water and may be toxic (Tripler et al. 2007).

1.5.5 Responses to Climate Change Climate change is modelled in various ways with differing assumptions and conclusions. See, for instance, CCSP (2008) and USGCRP (2017). Most likely scenarios project increases in average and extreme temperatures, but the magnitude of these changes varies from slight to large, depending on the model. Conversely, the effect on precipitation is not as well understood, and

R. R. Krueger

varies depending on the geographic region of the earth. CCSP (2008) concludes that the production of forage and grain crops will be affected less by climate change than will production of many horticultural crops. Annual crops and weeds will likely change their geographic range and lifespans of annual crops may be shorter. CCSP (2008) deals mainly with staple agronomic crops with little said regarding perennial crops other than to note that there will likely be fewer chill hours under most models. As stated above, date palms are adapted to high temperatures and low humidity. Thus, increases in temperature will likely have less effect on date palms than on most other crops. Depending on the scope of temperature increase, areas with suitable climates for date production may change little; more extreme temperature increases could push date production farther north and south. The effects of precipitation changes are not as easy to predict. In traditional oasis production, where date palms grow in standing water, the effect of altered precipitation may be negligible if standing water remains available. The same is true for irrigated industrial production, if water remains available for irrigation. A caveat here is that increased precipitation during fruit ripening can cause decreases in fruit quality or cause crop losses. Shabani et al. (2012) used a climate change model to project that some date-producing areas in North Africa, Iran, Iraq and Saudi Arabia may become unsuited to date production in less than 100 years, while some areas in North and South America will become more suitable. In addition, some areas in Spain not currently suitable for date cultivation were predicted to become suitable (Shabani et al. 2013a). The model, focusing on Iran, was later modified to include additional parameters such as soil moisture and low temperature (Shabani et al. 2014a,b,c). These studies predicted that areas suitable for date palm production in Iran would increase; as some areas become too hot for date production, cooler areas will become suitable. Shabani et al. (2013b) also modelled the threat from Fusarium oxysporum f. sp. albidensis in possible new date-producing areas.

1

Date Palm (Phoenix dactylifera L.) Biology and Utilization

1.6

Cultivation

The economic lifespan of a date garden is generally 40–50 years, but some may last up to 150 years. There are date palms that are probably hundreds of years old in North African and the Middle Eastern oases. Because of its biological characteristics, date palm cultivation is distinct from that of other perennial crops. There are cultural practices requiring access to the crown of the tree, which can be challenging and dangerous in mature trees reaching tens of meters in height. Good yields of high-quality fruit require that the

Fig. 1.5 Traditional oasis agriculture is still practiced in the Middle East and North Africa, as shown by this image of the Khattwa Oasis in Oman. Source RR Krueger

15

crown of the date palm be accessed for pollination, bunch management, harvesting, and pruning. Although access to the crown of the tree by climbing is still found in all date-producing areas, the use of mechanical lifts is common in more advanced or industrialised production areas, particularly with taller trees (Nixon and Carpenter 1978). Traditional oasis cultivation of date palms continues in rural areas of the Middle East and North Africa (Fig. 1.5). This type of cultivation is based upon low inputs with the resulting low yields. Most consumption is local. What may be

16

R. R. Krueger

Fig. 1.6 Industrial cultivation of dates in the Coachella Valley of California. Source RR Krueger

termed industrial production of dates occurs in countries outside the Middle East and North African region; in addition, most newly planted areas of date palms in the Middle East and North Africa are industrially cultivated (Fig. 1.6). In industrial cultivation, inputs are higher, as are yields and fruit quality. In this type of cultivation, production costs are higher; consequently, the sale price must also be higher. Most consumption in industrial cultivation is not local and the market may be in a different country. The cultural practices described below apply to all forms of date cultivation but are more highly developed and intensely practiced in industrial cultivation.

1.6.1 Pollination The date palm is wind pollinated (anemophily) in nature, but insect pollination (entomophily) is possible. When date palms are cultivated, artificial pollination is necessary for good fruit production and has been practiced in North Africa and the Middle East for thousands of years. Adequate fruit set requires pollination of 60– 80% of the female flowers. Cultivars differ greatly in their fruit set percentage, and incompatibility or partial incompatibility between different female and male varieties is known but the cause is not understood. Date producers

1

Date Palm (Phoenix dactylifera L.) Biology and Utilization

generally maintain a few male trees from which they collect pollen for artificial pollination. Pollen may also be stored under refrigerated conditions for use the next growing season (Nixon and Carpenter 1978; Zaid and De Wet 2002c).

1.6.2 Bunch Management Fruit thinning is often done in date cultivation. It is used to decrease alternate bearing, increase fruit size, improve fruit quality, advance fruit ripening and facilitate bunch management. Fruit thinning is generally done about a month after pollination. There are three different methods of fruit thinning used in date production: removal of entire bunches, in order to reduce fruit load on the tree; reduction in the number of strands per bunch, in order to reduce the number of fruit serving as sinks for the adjacent leaves and reduction in the number of fruits per strand, in order to reduce overall fruit number and provide more volume for fruit expansion. This last is most commonly done with cv. ‘Medjool’ and other large-fruited varieties. Cultivar, climate and cultural practices influence the degree of fruit thinning. Approximately 1 month after pollination, the bunches are often tied to the base of an adjacent leaf’s petiole in order to support the weight of the bunch. Bunches of dates are usually covered (bagged) in the United States with brown craft paper, white paper, or cotton or nylon mesh bags, depending upon cultivar. Bagging is done approximately 1 month after tie down in order to protect fruit bunches from high humidity and rain, minimise damage from sunburn, and decrease losses from birds (Nixon and Carpenter 1978; Zaid and De Wet 2002c).

1.6.3 Soil and Water Management in Date Cultivation Date palms can withstand prolonged drought under high temperatures; however, large volumes of water are required for vigorous growth and high yields of high-quality fruit. In California, annual water consumption was estimated at *

17

1.3 m under flood irrigation (Furr and Armstrong 1956). The majority of this was from the upper 2 m of the soil, which coincided with root distribution. Also in California, excessive application of irrigation did not increase the rate of growth of date palms or fruit yield, but withholding water during the summer months decreased the rate of tree growth and decreased fruit moisture content (Reuther and Crawford 1945). Flood irrigation is the oldest form of irrigation and is still used in many areas; however, lower volume, higher precision methods such as micro-sprinklers, fan sprayers and drip irrigation are often used in newer plantations (Abdul-Baki et al. 2002). Recent research suggests that actual water use of date palms, although large, is somewhat less than previously estimated (Krueger and Perring, unpublished data; Montazar et al. 2020). Date palms are usually fertilised, although in many cases only with nitrogen (Nixon and Carpenter 1978). Responses to fertilisation reported in the literature are inconsistent and probably depend upon cultivar, soil characteristics, irrigation and other factors. For instance, in California, nitrogen concentrations were higher in the leaves of fertilised trees of cvs. Deglet Noor and Khadrawy compared with nonfertilised trees, but neither fertilisation nor leaf nitrogen levels showed a consistent correlation with yield. There was an apparent weak (not statistically significant) response from Deglet Noor, but not from Khadrawy (Furr and Cook 1962). Healthy and declining palms showed no significant differences in nutrient concentrations (Labanauskas and Nixon 1962). Leaf nutrient concentrations reported from date palms in various countries are inconsistent but generally have low nitrogen concentrations compared with other perennial crops. Leaf mineral concentrations (not just nitrogen) from date palms grown in California and receiving high levels of nitrogen fertilisation have consistently been lower than those reported for date palms in the Middle East and North Africa (Krueger 2007). There is a lack of studies in the area of date palm mineral nutrition, and additional research in this area is probably warranted. In California, at least, there are often

18

visual indications of micronutrient deficiencies, but since no diagnostic levels for nutrients have been established for date palms it is not clear that a deficiency actually exists (personal observation of the author). Manure has been used in date production for many centuries, and many date farmers consider it superior to inorganic fertilisers even in industrial production, although inorganic fertilisers are also used. Organic production of dates is relatively common, and manure is valuable in this situation. Cover crops are also often utilised in date production (Abdul-Baki et al. 2002). Intercropping of shade-providing date palms with fruits, vegetables and pasture is common in traditional areas of date production.

1.6.4 Propagation Date palms are propagated in three ways: by seed, vegetatively by offshoots and by tissue culture (Nixon and Carpenter 1978; Zaid and De Wet 2002b). The most common traditional method of date palm propagation is by planting offshoots, which ensures the genetic identity of maternal varieties (Fig. 1.2). Offshoots develop from axillary buds on the trunk during the date palm’s juvenile stage; at maturity, these axillary buds develop into inflorescences. As a juvenile characteristic, offshoots generally develop near the base of the trunk, although some varieties characteristically produce offshoots above the soil level. After 3–5 years of attachment to the parental palm, offshoots produce roots and can be removed and planted. This is about the age that the offshoots will begin to flower and, in female lines, fruit. Seedlings resulting from chance or human-performed pollinations are not identical to the maternal tree. They are also not genetically uniform and vary greatly in their production and fruit quality. About 50% of the seedlings are male, but these cannot be identified visually until trees began to flower at about 4–5 years of age. There are DNA-based methods available for sex determination at earlier ages (Torres et al. 2018)

R. R. Krueger

but these techniques are not generally available to date producers. Fruit yield and quality in seedling-derived groves are greatly reduced compared with groves planted from offshoots. Traditional oasis culture utilises seedling plants (khalt) as well as offshoots. Superior trees derived from seedlings may be selected and propagated from offshoots, resulting in the establishment of local varieties that may be further disseminated to nearby oases (Krueger 2011). Date palms are also propagated by tissue culture (TC) (Zaid and De Wet 2002d). Tissue culture propagation of date palms was first developed in the 1970s and 1980s. Organogenesis can be achieved using axillary and apical meristems, whereas embryogenesis can be done through the callus stage from various tissues like shoots, young leaves, stem, rachilla and so forth. Cultivars respond to TC differently, and different conditions are needed for the successful propagation of different cultivar. About 6 years are needed for TC date palms to reach production and 8 years to reach commercial fruit yields. Although TC progenies generally have characteristics similar to those of offshoots of the same variety, one of the main problems with TC propagation is somaclonal variation (off types). Somaclonal variants display several typical phenotypes, including variegation in leaves, variable leaf structure and overall plant growth patterns, lack of inflorescences, abnormal floral development, and production of seedless parthenocarpic fruits. Most somaclonal variants can be detected early in the process; however, some can only be detected several years after planting or after flowering, fruit set and maturation of the trees. Somaclonal variation in TC-derived date palms is sometimes very high, but the causes are unclear (Gurevich et al. 2005). TC date palms are produced under sterile conditions and so may be healthier and under less stress than offshoots. This may account for observations that TCderived date palms grow faster than offshoots after transplanting into the soil (Mauk et al. unpublished data).

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Date Palm (Phoenix dactylifera L.) Biology and Utilization

1.6.5 Cultivation Constraints In addition to pests and diseases (see next section), various other factors may have adverse effects on date production. Problems commonly observed in traditional date cultivation include crowding of trees; retention of old or unproductive trees; mixtures of cultivars; use of seedlings; salt accumulation; poor drainage; insufficient irrigation, fertilisation or tillage; lack of pest control; competition from other crops when interplanted; soil degradation and water scarcity (Carpenter 1981). Drought, high salinity, aged trees, bayoud disease and genetic erosion are considered the major constrains for future date palm production worldwide (Jaradat 2001).

1.7

Pests and Diseases

Although date palms are attacked by different diseases and arthropod pests throughout their cultivation range, few of these represent existential threats to date palms on a worldwide scale. However, palm weevils are a potentially lethal insect pest, and diseases caused by Fusarium spp. fungi and Candidatus Phytoplasma spp. bacteria are also potentially lethal.

1.7.1 Palm Weevil Pests Palm weevils include the genus Rhynchophorus in the subfamily Dryophthorinae of the family Curculionidae. Palm weevils are large insects whose larvae chew or tunnel into various organs of palm species, causing damage or death due to destruction of the apical meristem (Giblin-Davis and Howard 1988). Of the various insects that bore into palms, the palm-associated members of the Dryophthorinae are the most damaging on the global scale (Giblin-Davis 2001). There are approximately nine species of Rhyncophorus distributed throughout the world (Wattanapongsiri 1966). The red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier), considered the most destructive pest of date palm worldwide, also

19

causes economic losses in coconut (Cocos nucifera L.), and affects many other species of palms (Malumphy and Moran 2007). Rhynchophorus ferrugineus is native to Southeastern Asia but beginning in the 1980s began spreading westward into the date producing areas of the Middle East and North Africa, and from thence into the regions of Southern Europe that have climates suitable for cultivation of Phoenix species (Giblin-Davis 2001; Malumphy and Moran 2007; Molet et al. 2011a; USDA-APHIS 2010). Spread of R. ferrugineus is generally believed to be associated with the movement of infested date palm offshoots, and perhaps secondarily through the movement of entire Phoenix sp. trees for landscape purposes (Giblin-Davis 2001). The South American palm weevil (SAPW), also known as the giant palm weevil, Rhynchophorus palmarum L., has historically been established in much of South and Central America, the Caribbean and north into Central Mexico, where it is considered an important pest of palms in general (Molet et al. 2011b; Hodel et al. 2016). Primary palm host species include Phoenix spp. as well as Cocos nucifera; there are a number of other palm hosts as well as many secondary hosts (Molet et al. 2011b). In addition to direct damage and potential lethality to palms, R. palmarum presents a threat to palms by vectoring Bursaphelenchus cocophilus (Cobb) Baujard, the red ring nematode (RRN), which causes red ring disease (RRD) (Molet et al. 2011b). RRD primarily is an economic disease of coconut due to its geographic distribution but can also affect other palms, including Phoenix spp. (Sullivan 2013). RRD is a vascular wilt that may result in death of a palm, particularly when the SAPW is also present and damaging the palm (Sullivan 2013).

1.7.2 Fusarium Diseases Fusarium is a fungal genus with sometimes conflicting taxonomies. Of the over 300 published species, the most important plant pathogens are found in only 4 species complexes: F. fujikori, F. graminearum, F. solani and F.

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oxysporum (Aoki et al. 2014). Fusarium oxysporum Schlecht emend Snyder and Hansen is unique in that it has a long history of mostly sexual reproduction; it colonises root xylem tissue, causing wilt diseases and it displays apparent gene-for-gene relationships with several hosts, resulting in host-specific formae speciales (Michielse and Rep 2009). The formae speciales do not describe formal taxonomic relationships but rather physiological capabilities and hostrelationships and are useful because they identify a subset of isolates that affect crops susceptible to vascular wilts (Gordon and Martyn 1997). There are 3 formae speciales of F. oxysporum that cause vascular wilts of date palms: F. oxysporum f. sp. albidensis (Killian and Maire), Malencon and Gordon; F. oxysporum f. sp. canariensis Mercier and Louvet; and F. oxysporum f. sp. palmarum Elliott. The bayoud disease of date palms in North Africa is caused by Fusarium oxysporum f. sp. albidensis. This disease was first observed in Morocco prior to 1890, and has since spread to Algeria and Mauritania (Abdelmonem and Tasmy 2007; Carpenter and Elmer 1978; Djerbi 1983). Bayoud is a very destructive disease, and has had particularly devastating effects in Morocco, having destroyed almost 70% of plantings and taking most of the elite cultivars. Phoenix canariensis is also susceptible to F. oxysporum f. sp. albidensis (Carpenter and Elmer 1978). Fusarium oxysporum f. sp. canariensis, or Canary Island date palm wilt (CIDPW), was first reported in southern France by Mercier and Louvet (1973) and subsequently in Southern California (Feather et al. 1989) and many other areas around the world where P. canariensis is cultivated (Downer et al. 2009). Date palms and other Phoenix spp. are also susceptible with the exception of P. roebelinii (Downer et al. 2009; Feather et al. 1989). In Florida, a new and novel forma specialis (F. oxysporum f. sp. palmarum) has been reported on P. canariensis (Elliott 2011) and various other ornamental palms. Although F. oxysporum f. sp. palmarum has apparently not been reported in nor assayed in P. dactylifera, its virulence against P. dactylifera should be assumed, especially as F. oxysporum f.

R. R. Krueger

sp. palmarum is apparently closely related to F. oxysporum f. sp. albidensis (Elliott et al. 2010). Fusarium proliferatum (Matush) Nirenberg ex Gerlach and Nirenberg (1976) is a fungus with a worldwide distribution that is pathogenic to various crops and can also cause keratitis and other medical problems in humans (Sun et al. 2018). Fusarium proliferatum has been identified as a wilt pathogen of date palms in various countries and has been associated with bunch rots in others. Fusarium solani (Mart.) Sacch 1881 has been associated with wilt of date palms in Saudi Arabia (although less frequently than F. proliferatum) (Saleh et al. 2017) and other countries.

1.7.3 Phytoplasma Diseases Phytoplasmas, classified as Candidatus Phytoplasma spp., are heretofore uncultured bacteria lacking cell walls and having reduced genomes. Phytoplasma taxonomy has historically been based upon 16S ribosomal gene sequences. Phytoplasmas of various taxonomic designations have been associated with or shown to cause diseases in a wide range of plant species, both cultivated and wild. In recent years, longestablished and newly-reported declines or diseases of date palms have been associated with phytoplasmas (Gurr et al. 2015). For purposes of discussion, these declines may be divided into Old and New World problems. The following discussion is based upon Gurr et al. (2015), which may be consulted for additional information and primary sources. Lethal yellowing (LY) is an often-fatal disease of coconuts that was first reported in Jamaica in 1891 and much later (1972) associated with a phytoplasma in the taxonomic subgroup 16SrIV-A, vectored by the leaf hopper Haplaxius crudus Van Duzee. An epidemic of LY occurred in southern Florida in the 1960s to 1970s. In addition to coconuts, this epidemic also attacked Phoenix spp., with P. dacylifera being the most susceptible. Symptomology of Phoenix spp. was distinct compared to that in C. nucifera. Starting in the late 1970s, Phoenix spp. palms growing the Lower Rio Grande Valley of south

1

Date Palm (Phoenix dactylifera L.) Biology and Utilization

Texas were observed to be declining in a manner similar to Phoenix spp. in south Florida associated with phytoplasmas. These declines were associated with phytoplasmas of the taxonomic subgroup 16SrIV-D and have been designated as Texas Phoenix Decline (TPD). In 2007, declining trees were detected in Florida and found to be associated with the TPD phytoplasma and a closely related phytoplasma in the subgroup 16SrIV-F. Although the vector for TPD is still not definitively known; in Florida Haplaxius crudus has consistently been found in areas with declining palms and associated phytoplasmas; additional potential vectors have also been identified (Halbert et al. 2014). Al Wijam is a minor disease of date palms reported in Saudi Arabia at least as early as the 1950s (Abdelmonem and Tasmy 2007; Carpenter and Elmer 1978; Djerbi 1983). At the time of the referenced reports, the cause of Al Wijam was unknown. Sequences associated with phytoplasmas in the 16SrI (C Phytoplasma asteris) and 16SrII groups were found associated with Al Wijam, the apparent vector being the leafhopper Cicadulina bipunctata (Melichar). Two new declines of date palm associated with phytoplasmas were reported from Sudan in the year 2000: white tip disease and slow decline. In Kuwait, phytoplasmas were found associated with the yellowing disease of date palms.

1.8

Harvest and Postharvest Handling

1.8.1 Harvesting of Dates Dates are harvested at or near maturity, based upon the fruit’s appearance and related to moisture content. Harvest generally begins in August (Northern Hemisphere) for cultivars that are consumed in the khalal stage and runs from September through December when harvested at the rutab and tamar stages. In many cases, entire bunches are harvested at the same time. In some cases, particularly with high-quality elite cultivars, sequential harvests are made so that only the fruit that is at optimal harvest condition is

21

removed. Dates are harvested by hand, with access to the crown of the tree being by climbing or the use of mechanical lifts. Mechanised harvest, for instance by shaker, such as those used in some other perennial crops, is not developed enough for routine commercial use at this time (Glasner et al. 2002).

1.8.2 Packing and Storage of Dates In traditional date production by small farms with limited resources, such as occurs in the area of origin, dates are usually transported directly to open-air markets. Dates can be successfully stored for a considerable time without specialised storage conditions due to their low moisture content. In more industrial date production, specialised equipment and facilities are used to pack and store dates. Packinghouses for dates often use equipment that has been modified from devices used for other crops, although a few large packing houses have custom-built equipment. Various postharvest processes are used to maintain or improve fruit quality in the packing house. Dates are generally harvested between 18 and 24% moisture. If the dates are too moist, it is necessary to dry them. This may be done outdoors exposed to the sun or in plastic tunnels, or inside the packing facility using heated air. Conversely, date harvested too dry need to be hydrated by dipping in hot water or exposing to steam at 60–65 °C and 100% humidity (Kader and Hussein 2008). Fumigation for the elimination of insect pests is commonly practiced in date packing houses (Glasner et al. 2002; Kader and Hussein 2008; Rygg 1975). Methyl bromide has been the major fumigant used; however, it is being phased out due to environmental concerns. Alternatives to methyl bromide treatment include alternative fumigants, controlled atmospheres and physical control methods (Glasner et al. 2002). Organic dates may be treated with 100% CO2, heated air at 50–55 °C, or freezing at –18 °C or lower (Kader and Hussein 2008). Dates are generally stored under refrigerated conditions in industrial production (Glasner et al.

22

2002; Kader and Hussein 2008; Rygg 1975). Dates harvested at khalal are stored at 0 °C at 85– 95%RH. Dates harvested at tamar can be stored at 0 °C and 65–75% RH for 6–12 months and for longer periods at lower temperatures. Dates may be cleaned by air pressure and/or water, after which they are dried and sorted into grades. Sorting and grading of dates are generally performed by hand rather than by electronic devices at this time, although the latter is becoming more common in larger packing facilities. Dates are sometimes coated with wax or other materials to reduce stickiness and/or improve appearance. Fresh dates (which are actually partially dried) are generally packaged in cardboard or plastic for shipment and retail sale. In addition to entire fruits, dates are sometimes pitted, cut into small pieces, or macerated and are sometimes stuffed with nuts or other food products (Glasner et al. 2002; Kader and Hussein 2008; Rygg 1975).

1.8.3 Market Defects of Dates The fruit quality of dates can be decreased by physiological and biotic factors (Rygg 1975). Physiological defects include blacknose, associated with high humidity during the khalal stage; black scald, associated with abnormally high temperatures and puffiness (skin separation) of the dates, associated with high temperature and humidity. Storage environment may also promote fruit defects such as sugar spotting and darkening of the skin. In addition, dates are sometimes attacked by various pathogens including Aspergillus, Alternaria, and Penicillium, and by arthropod pests (Glasner et al. 2002; Kader and Hussein 2008; Rygg 1975).

R. R. Krueger

More commonly, lower quality cultivars, or dates of lower quality and lower sugar concentrations, are used. Most processed products are made from pitted dates. Kimri stage dates may be used for pickles, chutney, or steak sauces; khalal stage dates may be processed into jam or syrup and tamar stage dates processed into paste, syrup, date bars, or pitted, pressed dates. Date processing byproducts and low-quality dates may be used for sugar extraction or production of sugar alcohols, ethanol, vinegar or yeast. Date fruit, being high in sugar, may also be used to ferment alcoholic beverages (Glasner et al. 2002; Kader and Hussein 2008).

1.9

Uses of Dates and Date Palms

1.9.1 Date Fruit Date palms produce many products used by humans. The main use is as food. Dates can be eaten fresh, dried, or in various processed forms. In North Africa and the Middle East, some dates are eaten when at the khalal stage (true horticultural or physiological maturity), when the fruit is still very astringent with a high tannin content. Certain varieties, such as cvs. Barhee and Samany, are lower in astringency and preferred for consumption at khalal. Most dates are harvested during the fully ripened rutab and tamar stages, at which time sugar concentration is high and moisture and tannin concentrations are low. Cultivars of dates are classified as soft, semidry or dry, depending upon the time of harvest and water content. Some cultivars are used in more than one manner (Glasner et al. 2002; Kader and Hussein 2008). Since the composition and development of date fruits are discussed in another chapter in this book, the reader is directed thereto.

1.8.4 Processing of Dates In addition to fresh fruit, dates are sometimes processed into other products. Elite cultivars, such as cv. Medjool is usually not processed unless conditions result in lower quality fruit.

1.9.2 Other Uses of the Date Palm Almost every part of the date palm can be used for some purpose (Barreveld 1993; Dowson and

1

Date Palm (Phoenix dactylifera L.) Biology and Utilization

Aten 1962; Nixon 1951). Timber, wood and fuel are derived from the trunk. The leaf bases and fruit stalks can also be used for fuel. Bags, baskets, camel saddles, cords, crates, fans, food covers, furniture, mats, paper, ropes, trays and twine can be made from fiber from the trunk and leaves. Structural components such as shades, roofs, separating walls and enclosures can be made from dried bundles of leaves called barusti. The leaf midrib can be used to build small fishing boats called a shasha and fish traps. The pith of the palms can be made into date palm flour. Heart of palm (the terminal bud) can be eaten as a salad or as a cooked vegetable. Date seeds can be used as livestock feed and for decorative purpose as beads. Oil from date seeds can be manufactured into soap. Date fruit also has folk medicinal uses, such as an astringent for treating intestinal problems; treatment for sore throat and colds; relief of fever, cystitis, oedema, liver, and abdominal problems; to counteract hangovers; etc. The gum or exudate of dates is used for treating diarrhoea and the roots of the date palm are used to treat toothache in India. Many of these medicinal uses are based upon secondary metabolic products of date fruit, such as anthocyanins, phenolics, sterols, carotenoids and flavonoids (El-Far et al. 2019).

1.9.3 Environmental Uses of Date Palms Groves or oases of date palms play a central role in the desert ecological systems of the Middle East and North Africa, providing environmental niches with water for local fauna. Date palms have been effective for the control of desertification and land reclamation in the Arabian Peninsula, especially in the United Arab Emirates. Recently, increased plantings of date palms in the Sahara Desert in Algeria have been reported to be positively correlated with indicators of decreased desertification (Mihi et al. 2019). In some instances, traditional oases still

23

support small farming villages. More negatively, feral date trees are sometimes considered invasive species in southern Australia (Gotch et al. 2006). Date palms are also used for ornamental and landscape purposes in southern Europe, the United States (Arizona, California, Nevada, Texas, Florida), and other salubrious areas.

1.10

Date Production

On a worldwide basis, date production is concentrated in the Middle East and North Africa between 15 ºN and 35 ºN. This region is the centre of origin and domestication for dates, and date consumption is deeply ingrained in its inhabitants. Date culture has spread into other areas with climates suitable for their production. However, most of the surface area of the Middle Eastern and North African countries has climates suitable for date production, whereas only limited areas in other countries producing dates (e.g. United States, Australia and Mexico) have suitable climates. According to FAO (http://www.fao.org/ faostat/en/#data/QC), the countries with the largest planted areas of dates in 2018 were Iran, Algeria, Iraq, Saudi Arabia, and Pakistan (Table 1.3). However, yields per area in these countries are not high, and the countries with the largest date production in 2017 were Egypt, Saudi Arabia, Iran, Algeria, and Iraq. FAO does not report income from date production in this tool. It should also be noted that not all countries producing dates are shown in the FAO tool (e.g. India, Australia) and these are omitted from Table 1.3. According to FAO, worldwide date production increased from 1,809,091 mt in 1962 to 8,684,512 mt in 2018. Most of this increase has occurred in the Middle Eastern and North African regions; however, almost all of this increase has occurred as industrial production rather than traditional oasis culture. Per capita date consumption is high in the Middle East and North

24

R. R. Krueger

Table 1.3 World date production 2018 Country

Area harvested (ha)

Yield (hg/ha)

Production (mt)

Egypt

49,184

317,615

1,562,171

Saudi Arabia

116,125

112,195

1,302,859

Iran (Islamic Republic of)

171,647

70,153

1,204,158

Algeria

168,855

64,831

1,094,700

Iraq

147,900

41,554

614,584

Pakistan

100,611

46,880

471,670

Sudan

37,225

118,436

440,871

Oman

25,125

146,789

368,808

United Arab Emirates

38,117

90,542

345,119

Tunisia

57,329

42,096

241,333

Libya

32,500

54,225

176,229

China

13,876

114,078

158,294

China, mainland

13,876

114,078

158,294

Morocco

59,127

18,892

111,701

Kuwait

3,353

288,267

96,656

Yemen

13,736

35,024

48,108

Israel

4,733

94,190

44,580

United States of America

5,301

70,251

37,240

Turkey

2,610

136,332

35,577

Qatar

2,417

120,033

29,012

Mauritania

9,058

24,341

22,049

Chad

11,535

18,521

21,364

Jordan

3,146

62,257

19,588

Niger

6,648

29,387

19,537

Somalia

2,666

51,715

13,785

Albania

482

278,485

13,423

Bahrain

3,177

33,620

10,682

Mexico

1,573

56,888

8,946

Palestine

1,428

24,561

3,508

Syrian Arab Republic

370

81,081

3,000

Spain

465

35,226

1,638

Benin

683

21,075

1,439

Kenya

499

22,944

1,144

Mali

57

125,851

717

Cameroon

160

39,592

635

Namibia

141

25,397

357

Eswatini

98

32,007

312

Peru

141

19,433

274

Djibouti

*

*

117

Colombia

8

41,841

33

World

1,105,982

3,120,683

8,684,512

Source FAO (2020), * Not reported

1

Date Palm (Phoenix dactylifera L.) Biology and Utilization

Africa as compared to other regions of the world due to the fact that the date palm is indigenous to this region and the date palm and its fruits have great cultural and traditional significance. As people migrate from the Middle East and North Africa to other countries and the native populace of countries, outside the region, become exposed to dates, it is possible that per capita date consumption will increase outside the Middle Eastern/North African region in the future. This has occurred over the decades with other formerly regional crops, such as avocado.

1.11

Conclusions and Prospects

The date palm has been domesticated for several millennia in its centres of origin, diversity and domestication in the Middle East and North Africa. Its culture has spread to other areas having a suitable climate (hot, arid or semiarid) with sufficient water available either as groundwater or irrigation. The date palm is a unique, monocotyledonous plant with distinctive anatomical features. Intensive and labourintensive inputs are necessary for successful date cultivation. Production of dates has increased in recent decades and will most probably continue to increase for some time. As a nutritious fruit, its consumption can be promoted for its health benefits as well as its flavourful quality. It is possible that the area suitable for date production will change in the future due to climate change. The exact nature of this change will depend on the nature of the climate change. It is possible that the range of date production will expand to the north and south if temperatures rise sufficiently. However, the precipitation and availability of irrigation water will influence the spread of date production and possibly constrain production in areas it is presently possible to grow dates. Excessive moisture during the maturation phase can have a detrimental effect on fruit quality and sometimes result in the loss of entire crops. The need of the date palm for large amounts of water, generally applied as irrigation in non-oasis

25

environments, may also limit date production if less water is available for agriculture. Recently, there has been more awareness of pests and diseases of date palms. The palm weevils and Fusarium and Phytoplasma pathogens discussed above have moved from their areas of origin to many date production areas around the world. Research is needed on management and control of these threats to date production. Research is also needed on technological inputs to date production. As populations shift from rural to urban areas, less workers are available for labour-intensive date palm cultivation. More complete mechanisation, automation, and use of artificial intelligence may be needed to maintain diet production, particularly in firstworld countries. Additional research is needed in the area of date palm’s adaptation to environmental change and the long-term sustainability of date production. Although many challenges lie ahead, dates have a millennia-long history of sustained cultivation, and appropriate research can help extend this for more millennia into the future.

References Abdelmonem AM, Rasmy MR (2007) Major diseases of date palm and their control. Commun Inst Forestalis Bohemicae 23:9–23 Abdul-Baki A, Aslan A, Linderman R et al (2002) Soil, water, and nutritional management of date orchards in the Coachella Valley and Bard, 2nd edn. California Date Commission, Indio, CA Aldrich WW, Crawford CL, Nixon RW, Reuther W (1942) Some factors affecting rate of date leaf elongation. Proc Am Soc Hort Sci 41:77–84 Alhammadi MS, Kurup SS (2012) Impact of salinity stress on date palm (Phoenix dactylifera L) - a review. In: Sharma P, Abrol V (eds) Crop production technologies. InTech, Rijeska, Croatia, pp 169–173 Aoki T, O’Donnell K, Geiser DM (2014) Systematics of key phytopathogenic Fusarium species: current status and future challenges. J Gen Plant Pathol 80:189–201 Bailey LH, Bailey EZ (1976) Hortus Third. Macmillan, New York Barreveld WH (1993) Date palm products. Bulletin 101. Agricultural Services, FAO, Rome Barrow SC (1998) A revision of Phoenix L (Palmae: Coryphoideae). Kew Bull 53:513–575

26 Beccari O (1890) Revista monografica delle specie del genere Phoenix Linn. Malesia 3(5):345–416 Carpenter JB (1981) Improvement of traditional date culture. Date Palm J 1:1–16 Carpenter JB, HS Elmer (1978) Pests and diseases of the date palm. Agriculture Handbook 527. US Dept Agr, Washington DC CCSP (2008) The effects of climate change on agriculture, land resources, water resources, and biodiversity. A Report by the U.S. Climate Change Science Program and the Subcommittee on Global Change Research. US Environmental Protection Agency, Washington DC Chevalier A (1952) Recherches sur les Phoenix africains. Rev Int Bot Appl 32:205–236 Djerbi M (1983) Diseases of the date palm. FAO, Baghdad Downer AJ, Uchida JY, Hodel DR, Elliott ML (2009) Lethal palm diseases common in the United States. HortTech 19:710–716 Dowson VHW (1982) Date production and protection with special reference to North Africa and the Near East. Technical Bulletin 35. FAO, Rome Dowson VHW, Aten A (1962) Dates: handling, processing, and packing. Plant Production and Protection Series 13. FAO, Rome El-Far AH, Oyinolye BE, Sepehrimanesh M et al (2019) Date palm (Phoenix dactylifera): novel findings and future directions for food and drug discovery. Curr Drug Disc Tech 16(1):2–10. https://doi.org/10.2174/ 1570163815666180320111937 Elliott ML (2011) First report of Fusarium wilt caused by Fusarium oxysporum f. sp. palmarum in Canary Island date palm in Florida. Plant Dis 95:356 Elliott ML, Des Jardin EA, O’Donnell K et al (2010) Fusarium oxysporum f. sp. palmarum, a novel forma specialis causing a lethal disease of Syagrus romanzoffiana and Washingtonia robusta in Florida. Plant Dis 94:31–38 FAO (2020) FAOSTAT. Food and Agriculture Organization of the United Nations, Rome. http://www.fao. org/faostat/en/#data/QC Feather TV, Ohr HD, Munnecke DE, Carpenter JB (1989) The occurrence of Fusarium oxysporum on Phoenix canariensis, a potential danger to date production in California. Plant Dis 73:78–80 Furr JR, Cook JA (1952) Nitrogen content of pinnae, fruit, and seed of Deglet Noor and Khadrawy date palms as related to nitrogen fertilization. Date Grow Inst Rep 29:13–14 Furr JR, Armstrong WW (1956) The seasonal use of water by Khadrawy date palms. Date Grow Inst Rep 33:5–7 Giblin-Davis RM (2001) Borers of palms. In: Howard FW, Giblin-Davis R, Moore D, Abad R (eds) Insects on palms. CABI, Wallingford, pp 267–314 Giblin-Davis R, Howard FW (1988) Notes on the Palmetto weevil, Rhynchophorus cruentatus (Coleoptera: Curculionidae). Proc Flor State Hort Soc 101:101–107

R. R. Krueger Glasner B, Botes A, Zaid A, Emmens J (2002) Date harvesting, packinghouse management and marketing aspects. In: Zaid A (ed) Date palm cultivation. Plant Production and Protection Paper 156. FAO, Rome, pp 177–208 Gordon TR, Martyn RD (1997) The evolutionary biology of Fusarium oxysporum. Ann Rev Phytopath 35:111– 128 Gotch T, Noack D, Axford G (2006) Feral tree invasions of desert springs. In: Abstracts, Third International Date Palm Conference, Abu Dhabi, United Arab Emirates, February 2006, p 40 Gurevich V, Lavi U, Cohen Y (2005) Genetic variation in date palms propagated from offshoots and tissue culture. J Am Soc Hort Sci 130:46–53 Gurr GM, Bertaccini A, Gopurenko D et al (2015) Phytoplasmas and their insect vectors: implications for date palm. In: Wakil W (ed) Sustainable pest management in date palm: current status and emerging challenges. Springer, Switzerland, pp 287–314 Halbert SE, Wilson SW, Bextine B, Youngblood SB (2014) Potential planthopper vectors of palm phytoplasmas in Florida with a description of a new species of the genus Omolicna (Hemiptera: Fulgoroidea). Florida Entomologist 97(1):90–97 Hodel DR (1995) Phoenix, the date palms. Palm J 122:14–36 Hodel DR, Marika MA (2016) Ohara KM (2016) The South American palm weevil: a new threat to palms in California and the Southwest. Palm Arbor 3:1–27 Jaradat AA (2001) Date palm: a tree with a taste for salt. Biosalinity News 2:5–7 Johnson D (ed) and the IUCN/SSC Palm Specialist Group (1996) Palms: their conservation and sustained utilization. IUCN, Gland, Switzerland and Cambridge, UK Kader AA, Hussein AM (2008) Harvest and postharvest handing of dates. International Center for Agricultural Research in the Dry Areas, Aleppo, Syria Krueger RR (2007) Nutritional dynamics of date palm (Phoenix dactylifera L). Acta Hort 736:177–186 Krueger R (2011) Date palm germplasm. In: Jain SM, AlKhayari J, Johnson DV (eds) Date palm biotechnology. Springer, Berlin, pp 313–336 Labanauskas CK, Nixon RW (1962) Concentrations of nutrients in pinnae of date palms in relation to an unexplained die-back of leaves in Coachella Valley, California. Date Grow Inst Rep 39:14–15 Maas EV, Hoffman GJ (1977) Crop salt tolerance current assessment. J Irrig Drain Div ASCE 103:115– 134 Malumphy C, Moran H (2007) Plant pest notice: red palm weevil Rhynchophorus ferrugineus. Central Science Laboratory, DEFRA, London 50:1–3 Martius CFP von (1836–1850) Phoeniceae. In: Historia naturalis palmarum: expositio systematica. Vol 3. F. Fleischer, Leipzig Mason SC (1915) Botanical characteristics of the leaves of the date palm used in distinguishing cultivated

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Date Palm (Phoenix dactylifera L.) Biology and Utilization

varieties. USDA Bulletin 223. US Dept Agr, Washington DC Mason SC (1925a) The minimum temperature for growth of the date palm and the absence of a resting period. J Agric Res 31:401–414 Mason SC (1925b) Partial thermoplasty of the growth center of the date palm. J Agr Res 31:415–453 Mathez F, Bliss DE (1942) The relation of leaf area to alternate bearing in the Deglet Noor palm. Date Grow Inst Rep 19:3–7 Mercier S, Louvet J (1973) Recherches sur les fusarioses. X. Une fusariose vasculaire (Fusarium oxysporum) du palmier des Canaries (Phoenix canariensis). Ann Phytopath 5:203–211 Michielse CB, Rep M (2009) Pathogen profile update: Fusarium oxysporum. Mol Plant Pathol 10:311–324 Mihi A, Tarai N, Chenchouni H (2019) Can palm date plantations and oasification be used as a proxy to fight sustainably against desertification and sand encroachment in hot drylands? Ecol Indic 105:365–375. https:// doi.org/10.1016/j.ecolind.2017.11.027 Miller W, Smith JG, Taylor N (1930) Phoenix. In: Bailey LH (ed) The standard cyclopedia of horticulture, vol 3. Macmillan, New York, pp 2592–2594 Molet T, Roda AL, Jackson LD (2011a) CPHST Pest Datasheet for Rhynchophorus ferrugineus. USDAAPHIS-PPQ-CPHST, Raleigh NC Molet T, Roda AL, Jackson LD, Salas B (2011b) CPHST Pest Datasheet for Rhynchophorus palmarum. USDAAPHIS-PPQ-CPHST, Raleigh NC Montazar A, Krueger R, Corwin D et al (2020) Determination of actual evapotranspiration and crop coefficients of California date palms using the residual of energy balance approach. Water 12(8):2253. https:// doi.org/10.3390/w12082253 Moore HE Jr (1963) An annotated checklist of cultivated palms. Prin 7:119–183 Mowry H, Dickey RD, West E (1952) Native and exotic palms of Florida. Univ Flor Bull 152. Univ Florida, Gainesville Munier P (1973) Le palmier-dattier. Laisonneuve & Larose, Paris Munier P (1974) Le probleme de l’origine du palmierdattier et l’Atlantide. Fruits 29:235–240 Nixon RW (1934) Metaxenia in dates. Proc Amer Soc Hort Sci 32:221–226 Nixon RW (1936) Metaxenia and interspecific pollinations in Phoenix. Proc Am Soc Hort Sci 33:21–26 Nixon RW (1950) Imported varieties of dates in the United States. Circular 834. US Dept Agr, Washington DC Nixon RW (1951) The date palm: “tree of life” in subtropical deserts. Econ Bot 5:274–301 Nixon RW, Carpenter JB (1978) Growing date in the United States, 3rd ed. Agric Info Bull 207. US Dept Agr, Washington DC Nixon RW, Wedding RT (1956) Age of date leaves in relation to efficiency of photosynthesis. Proc Amer Soc Hort Sci 67:265–269

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Reuther W, Crawford CL (1945) Irrigation experiments with dates. Date Grow Inst Rep 22:11–14 Reuvani O (1985) Phoenix dactylifera. In: Halevy AH (ed) Handbook of flowering. CRC Press, Boca Raton, Florida, pp 343–349 Reuvani O (1986a) Effect of left- and right-handed phyllotaxy on yield of the date palm. Acta Hort 175:257–258 Reuvani O (1986b) Date. In: Monseliese SP (ed) CRC Handbook of fruit set and development. CRC Press, Boca Raton, Florida, pp 119–144 Rygg GL (1975) Date development, handling, and packing in the United States. Agricultural Handbook 482, Agricultural Research Service, US Dept Agr Washington DC Saleh AA, Sharafaddin AH, El-Komy MH et al (2017) Fusarium species associated with date palm in Saudi Arabia. Eur J Plant Pathol 148:367–377 Salomon-Torres R, Ortiz-Uribe N, Sol-Uribe JA et al (2017) Influence of different sources if pollen on the chemical composition of date (Phoenix dactylifera L) cultivar Medjool in Mexico. Austral J Crop Sci 12 (6):1008–11015. https://doi.org/10.21475/ajcs.18.12. 06.PNE1213 Salomon-Torres R, Ortiz-Uribe N, Valdez-Salas B et al (2019) Nutritional assessment, phytochemical composition and antioxidant analysis of the pulp and seed of Medjool date grown in Mexico. Peer J 7: https://doi. org/10.7717/peerj.6821 Salomon-Torres R, Sol-Uribe JA, Valdez-Salas B et al (2020) Effect of four pollinating sources on nutritional properties of Medjool date (Phoenix dactylifera L) seeds. MDPI Agriculture 10:45. https://doi.org/10. 3390/agriculture10020045 Shabani F, Kumar L, Taylor S (2012) Climate change impacts on the future distribution of date palms: a modeling exercise using CLIMEX. PLoS ONE 7(10): e48021. https://doi.org/10.1371/journal.pone.0048021 Shabani F, Kumar L, Esmaeili A, Saremi H (2013) Climate change will lead to larger areas of Spain being conducive to date palm cultivation. J Food Agr Envir 11:2441–2446 Shabani F, Kumar L (2013b) Risk levels of invasive Fusarium oxysporum f sp in areas suitable for date palm (Phoenix dactylifera) cultivation under various climate change projections. PLoS ONE 8(12): e83404. https://doi.org/10.1371/journal.pone.0083404 Shabani F, Kumar L (2014a) sensitivity analysis of CLIMEX parameters in modeling potential distribution of Phoenix dactylifera Compilede PLoS ONE 9 (4): e94867. https://doi.org/10.1371/journal.pone. 0094867 Shabani F, Kumar L, Taylor S (2014a) Projecting date palm distribution in Iran under climate change using topography, physicochemical soil properties, soil taxonomy, land use, and climate data. Theor Appl Climatol 118:553–567 Shabani F, Kumar L, Taylor S (2014b) Suitable regions for date palm cultivation in Iran are predicted to

28 increase substantially under future climate change scenarios. J Agr Sci 152:543–557 Sullivan M (2013) CPHST Pest Datasheet for Bursaphelenchus cocophilus. USDA-APHIS-PPQ-CPHST, Raleigh, North Carolina Sudhersan C, Abo El-Nil M (1999) Occurrence of hermaphroditism in the male date palm. Palms 43 (18–19):48–50 Sun S, Liu Q, Han L et al (2018) Identification and characterization of Fusarium proliferatum, a new species of fungi that causes fungal keratitis. Nat Sci Rep 8:4859. https://doi.org/10.1038/s41598 Swingle WT (1904) The date palm and its utilization in the southwestern states. Bur Plant Ind Bull 53. US Dept Agr, Washington DC Tomlinson PB (1961) Anatomy of the monocotyledons. Clarendon Press, Oxford, II. Palmae Torres MF, Mathew LS, Ahmed I et al (2018) Genuswide sequencing supports two-locus model for sexdetermination in Phoenix. Nature Communications 9:3969. https://doi.org/10.1038/s41467-018-06375-y Tripler E, Ben-Gal A, Shani U (2007) Consequence of salinity and excess boron on growth, evapotranspiration and ion uptake in date palm (Phoenix dactylifera L, cv Medjool). Plant Soil 297:147–155 Uhl NW, Dransfield J (1987) Genera palmarum: a classification of palms based upon the work of Harold E Moore. Allen Press, Lawrence, Kansas, Jr USDA-APHIS (2010) New pest response guidelines: red palm weevil. USDA-APHIS-PPQ-Emergency and Domestic Programs–Emergency Planning, Riverdale,

R. R. Krueger Maryland. http://www.aphis.usda.gov/import_export/ plants/manuals/ USGCRP (2017) Climate Science Special Report: Fourth National Climate Assessment, Volume I. Wuebbles DJ, Fahey DW, Hibbard KA et al (eds) US Global Change Research Program, Washington DC. https://doi.org/10.7930/j0j964j6 Wattanapongsiri A (1966) A revision of the genera Rhynchophorus and Dynamis (Coleoptera: Curculionidae). Dept Agr Sci Bull Bangkok 1:1–328 Wrigley G (1995) Date palm (Phoenix dactylifera). In: Smartt J, Simmonds NW (eds) The evolution of crop plants, 2nd edn. Longman, Essex, UK, pp 399–403 Zaid A, De Wet PF (2002a) Botanical and systematic description of date palm. In: Zaid A, Arias-Jimenez EJ (eds) Date palm cultivation. Plant Production and Protection Paper 156. Rev 1. FAO, Rome, pp 1–28 Zaid A, De Wet PF (2002b) Climatic requirements of date palm. In: Zaid A, Arias-Jimenez EJ (eds) Date palm cultivation. Plant Production and Protection Paper 156. Rev 1. FAO, Rome, pp 57–72 Zaid A, De Wet PF (2002c) Pollination and bunch management. In: Zaid A, Arias-Jimenez EJ (eds) Date palm cultivation. Plant Production and Protection Paper 156. Rev 1. FAO, Rome, pp 145–175 Zaid A, De Wet PF (2002d) Date palm propagation. In: Zaid A, Arias-Jimenez EJ (eds) Date palm cultivation. Plant Production and Protection Paper 156. Rev 1. FAO, Rome, pp 73–105

2

Systematics and Evolution of the Genus Phoenix: Towards Understanding Date Palm Origins Muriel Gros-Balthazard, William J. Baker, Ilia J. Leitch, Jaume Pellicer, Robyn F. Powell, and Sidonie Bellot

Abstract

The date palm (Phoenix dactylifera L.) is an iconic crop of hot and arid regions of North Africa, the Middle East and up to northwestern India. It is a member of the genus Phoenix that constitutes a monophyletic group within

M. Gros-Balthazard (&) Center for Genomics and Systems Biology, New Yok University Abu Dhabi, Saadiyat Island, 129188 Abu Dhabi, United Arab Emirates e-mail: [email protected] M. Gros-Balthazard Institut de Recherche pour le Développement, UMR DIADE, 911 Avenue Agropolis, 34394 Montpellier, France W. J. Baker
I. J. Leitch
J. Pellicer
R. F. Powell
S. Bellot Royal Botanic Gardens Kew, TW9 3AE Richmond, United Kingdom e-mail: [email protected] I. J. Leitch e-mail: [email protected]

the Coryphoideae subfamily, in the palm family. The genus Phoenix is composed of around 14 species whose native distribution is across the South of Europe, throughout Africa, the Arabian Peninsula and South Asia. Species boundaries and relationships are poorly understood owing to the morphological similarities between the species and their ability to hybridise. In this chapter, we review the past and present distribution of Phoenix species and aspects of their reproductive biology and genome dynamics. We then summarise the current state of knowledge of the taxonomy and phylogeny of Phoenix and we outline the main challenges that have so far limited our understanding of the genus’ systematics and evolution. We conclude with a roadmap to address these challenges by combining the extraordinary resource of specimens stored in herbarium collections and the power of high throughput DNA sequencing technologies.

J. Pellicer e-mail: [email protected]

2.1

R. F. Powell e-mail: [email protected]

The date palm (Phoenix dactylifera L.) is one of 13–14 currently accepted species of the genus Phoenix (see Table 2.1 for all species author names; Fig. 2.1; Barrow 1998; WCVP 2020). It belongs to the palm family (Arecaceae, also known as Palmae, in the monocotyledon order Arecales), which comprises around 2,600 species

S. Bellot e-mail: [email protected] J. Pellicer Institut Botànic de Barcelona (IBB, CSIC-Ajuntament de Barcelona), Passeig del Migdia sn, 08038 Barcelona, Spain

Introduction

© Springer Nature Switzerland AG 2021 J. M. Al-Khayri et al. (eds.), The Date Palm Genome, Vol. 1, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-73746-7_2

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classified in 181 genera (Baker and Dransfield 2016). Palms have been important for humans throughout history, with hundreds of species being used in various ways, from timber and handicraft to folk medicine and food. Some species such as the betel nut (Areca catechu L.), the peach palm (Bactris gasipaes Kunth), the coconut (Cocos nucifera L.) and the oil palm (Elaeis guineensis Jacq.) are widely cultivated and considered to be domesticated (Clement 1992). The date palm is among the most traded of these globally important palm species and has been cultivated for more than six millennia (Tengberg 2012). For people living in hot and arid regions of North Africa and Western Asia, the date palm is one of the most precious natural resources. Its sugar-rich fruit, the date, is a source of sustenance and a traded good, while every other part of the plant can be used so that nothing goes to waste (Krueger 2021). Although the date palm is considered as the only domesticated species of the genus, all Phoenix species are used by humans, at least occasionally, for food and beverages, construction, ornamentation and/or as a remedy (Barrow 1998). For instance, the sap of many species such as Phoenix canariensis, Phoenix reclinata or Phoenix sylvestris is harvested to produce a sweet juice (Fig. 2.2; Newton et al. 2013). The central part of the stem of several Asian species, for example Phoenix rupicola, Phoenix acaulis and Phoenix loureiroi, can be dried and reduced to powder for making bread (Kulkarni and Mulani 2004). The palm heart of P. reclinata is eaten raw or boiled, and its leaf rachis is used for building fish nets (Fig. 2.2; Kinnaird 2008). Phoenix canariensis and Phoenix roebelenii are grown worldwide as ornamental palms. Leaves of Phoenix theophrasti, P. dactylifera, and P. canariensis are used as such or woven for the Catholic feasts of Palm Sunday and Assumption Sunday (Castellana 1998). The widespread use of Phoenix species gives rise to conservation issues. So far, eight species have had their conservation status formally assessed by the International Union for the Conservation of Nature (IUCN), and among them, four species are classified as “Least Concern” (Phoenix caespitosa, P. canariensis, P. loureiroi, P.

M. Gros-Balthazard et al.

reclinata), three as “Near Threatened” (Phoenix paludosa, P. rupicola, P. theophrasti) and one (Phoenix atlantica) as “Endangered” (IUCN 2020). Main threats include overexploitation, for instance in the cases of P. loureiroi (Padmanabhan and Sudhersan 1988) or P. reclinata (Kinnaird 2008), and, as for many other palms, habitat destruction (IUCN 2020). In addition, the genetic integrity of some species is jeopardised by gene flow from other species newly introduced into their range. This is notably the case of P. canariensis (Gonzales-Perez and Sosa 2009) and P. sylvestris (Newton et al. 2013), both threatened by recent introductions of date palms. Wild relatives of the date palm represent a reservoir of genetic diversity for its agricultural improvement, although this potential has been largely untapped (Gros-Balthazard 2013; Krueger 2001). Sustained efforts to conserve all Phoenix species are therefore crucial and will require a good knowledge of the systematics and evolutionary history of the genus. Phoenix is the only genus in the tribe Phoeniceae, which is part of the palm subfamily Coryphoideae, comprising 517 species classified in 47 genera (Govaerts et al. 2020). Most Coryphoideae have palmate (fan-like) leaves, but Phoenix species distinguish themselves by having pinnate (feather-like) leaves with acanthophylls (spine-like leaflets) at their base (Fig. 2.3). Despite these distinctive features, Phoenix has long been recognised as a coryphoid palm based on other morphological characters (Dransfield et al. 2008a; Uhl and Dransfield 1987). In particular, Phoenix leaves have induplicate leaf folds, a feature only found in Coryphoideae, although the way these folds develop is unique to Phoenix (Fig. 2.3; Barrow 1998). The placement of Phoenix in Coryphoideae is also supported by molecular studies, for instance, Asmussen et al. (2006). Nonetheless, its exact relationships with other coryphoid lineages remain unclear. Early molecular phylogenies yielded contradictory results, none of them being strongly supported (reviewed in Dransfield et al. 2008a). In contrast, the latest molecular studies have resolved Phoeniceae as sister to the worldwide tribe Trachycarpeae, with low to high support depending on

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Systematics and Evolution of the Genus Phoenix …

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Table 2.1 Current status of the taxonomy of Phoenix. This table lists all Phoenix species currently accepted by the World Checklist of Vascular Plant Families (WCVP 2020) and all species described since the publication of the last monograph of the genus (Barrow 1998). A discussion of the status of all other Phoenix species described can be found in Barrow (1998) Species

Current status

Type specimen (herbarium codes are in brackets)

Species treated in the last Phoenix monograph (Barrow 1998) and considered accepted by it and/or the WCVP (2020) P. acaulis Roxb.

Accepted by the WCVP (2020)

Illustration from Roxburgh (1819, plate 273)

P. andamanensis S. Barrow

Considered unplaced by the WCVP (2020)

Specimen Ellis 14189 collected in Andaman Islands in 1990 and stored at Kew herbarium (K)

P. atlantica Chev.

Accepted by the WCVP (2020) but considered unclear by Barrow 1998 and by the present study (see text)

Specimen Chevalier 45839 collected in Cape Verde in 1934 and stored in Paris herbarium (P)

P. caespitosa Chiov.

Accepted by the WCVP (2020)

Described without type. Lectotype: specimen Puccioni and Stefanini 672 (738), collected in Somalia, stored at the Centro Studi Erbario Tropicale Università degli Studi di Firenze, Italy (FT)

P. canariensis Chabaud

Accepted by the WCVP (2020)

Described without type. Lectotype: illustrations from Chabaud (1882, Figs. 66–68)

P. dactylifera L.

Accepted by the WCVP (2020)

Described without type. Lectotype: illustration from Kaempfer (1712, t. 1-2); see Fig. 2.2

P. loureiroi Kunth

Accepted by the WCVP (2020)

Described without type. Lectotype: specimen Pierre 4832 collected by Harmand in Cambodia and stored in the Herbarium Beccarianum-Malesia of Florence Herbarium (FI-B)

P. paludosa Roxb.

Accepted by the WCVP (2020)

Described without type. Lectotype: illustration from Roxburgh (1832, plate 1193)

P. pusilla Gaertn.

Accepted by the WCVP (2020)

Described without type. Lectotype: illustration from Gaertner (1788, t. 9)

P. reclinata Jacq.

Accepted by the WCVP (2020)

Described without type. Lectotype: illustration from Jacquin (1800 t. 24)

P. roebelenii O’Brien

Accepted by the WCVP (2020)

Specimen O’Brien s.n. collected in Laos in 1889 and stored in K

P. rupicola T. Anderson

Accepted by the WCVP (2020)

Specimen T. Anderson s.n. collected in India and stored in Calcutta herbarium (CAL) and K

P. sylvestris (L.) Roxb.

Accepted by the WCVP (2020)

Described without type. Lectotype: illustration from Rheede (1678–1703, plate 22–25)

P. theophrasti Greuter

Accepted by the WCVP (2020)

Specimen Greuter 7650 collected in Crete in 1966 and stored in the W. Greuter herbarium in Palermo herbarium (PAL-Gr) (continued)

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M. Gros-Balthazard et al.

Table 2.1 (continued) Species

Current status

Type specimen (herbarium codes are in brackets)

Species not treated in the last Phoenix monograph (Barrow 1998) P. chevalieri D. Rivera, S.Ríos & Obón,

Synonym of P. dactylifera according to the WCVP (2020)

Specimen D.Rivera, S.Ríos and Obón 47698 collected in Spain in 1996 and stored in Murcia herbarium (MUB)

P. iberica D. Rivera, S.Ríos & Obón

Synonym of P. dactylifera according to the WCVP (2020)

Specimen D.Rivera, S.Ríos and Obón 47696 collected in Spain in 1996 and stored in Murcia herbarium (MUB)

Fig. 2.1 Distribution of the currently recognised Phoenix species. For all species except the date palm, the presumed native distribution is presented, so that recent anthropogenic species expansion is ignored. For the date palm, we show the main area of cultivation that was

reached at the latest by the beginning of the Roman period, also ignoring recent introductions. Map by M. Gros-Balthazard, produced using distribution data from Barrow (1998), Henderson (2009) and various herbarium databases

the DNA region analysed (Asmussen et al. 2006; Baker et al. 2009; Barrett et al. 2015). Based on this assumption, the divergence of Phoenix from other coryphoids (Trachycarpeae) has been estimated to have occurred 49 ± 16 million years ago (Baker and Couvreur 2013). This age may however need a reassessment taking more

coryphoid fossils into account, for instance the one described by Matsunaga (2019). Further, within the genus, the ages of current Phoenix species are so far unknown as lineage divergence times have not yet been estimated. Due to its cultural and economic importance as well as its morphological uniqueness among

Systematics and Evolution of the Genus Phoenix …

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Fig. 2.2 Utilisation of Phoenix species. Left: P. sylvestris with a scar left by the incision of the stem for sap tapping (Mount Abu, Rajasthan) (Photo by S. Ivorra);

Right: Fish net made from P. reclinata leaf rachis (Photo by M.F. Kinnaird)

2

a

c

b

d

Fig. 2.3 Morphological specificity of the genus Phoenix within the Coryphoideae. a Pinnate leaves of a date palm (P. dactylifera). b Induplicate (V-shaped) leaflet folds of a date palm. c Acanthophylls (lowest leaflets modified as

spines) of a date palm. d Seeds from various date palm cultivars. (All photos taken in al-‘Ulā oasis, Saudi Arabia by V. Battesti)

palms, the genus Phoenix has already been extensively studied. A monograph of the genus was published relatively recently (Barrow 1998), and many studies have focused on the

morphology and anatomy of Phoenix (e.g. GrosBalthazard et al. 2016; De Mason et al. 1982; Tomlinson 1961). DNA sequence data for all the species are available in public databases and

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M. Gros-Balthazard et al.

numerous studies of the genetic diversity of single species have been carried out, not only in date palms but also, for example, in P. sylvestris (Huda et al. 2019) or P. theophrasti (Vardareli et al. 2019), along with genetic studies involving multiple species (e.g. Chaluvadi et al. 2019; Flowers et al. 2019; Pintaud et al. 2010). Despite these efforts, the taxonomy and the phylogeny of Phoenix are not fully resolved, due to the close morphological resemblance of all species (Fig. 2.4), their semi-overlapping ranges (Fig. 2.1), and their capacity to hybridise with one another. This is impeding research on the evolutionary history of the genus and the origins of the date palm, leaving many questions open, such as (1) how did Phoenix species achieve their current distribution? (2) How did the Phoenix lineage persist through past climatic changes, and will it persist through future changes? (3) How much genetic material is transferred between species? and (4) What role did genetic exchange play in the evolution and adaptation of Phoenix? In this chapter, we review the current knowledge on the biogeography, reproductive biology, taxonomy and phylogeny of Phoenix, and provide a roadmap for a better understanding of the systematics and evolution of this iconic genus.

2.2

Biogeography of the Genus Phoenix

2.2.1 Current Distribution and Habitat The ecological range of Phoenix is broad, with some species growing in constantly wet places (the rheophyte P. roebelenii) and others in dry shrublands (P. caespitosa or P. loureiroi), sometimes even in salty soils (Phoenix paludosa growing in coastal swamps). Regardless of how arid or salty their habitat is, it seems that all species need ground water to provide moisture around the roots (Barrow 1998). Current Phoenix species are naturally restricted to Afro-Eurasia, from the Canary and Cape Verde Islands, around the Mediterranean Sea, throughout Africa, the Arabian Peninsula and southern Asia across to the

Philippines (Fig. 2.1). The Cretan date palm, P. theophrasti, is one of the only two palm species native to Europe (the other being Chamaerops humilis L., the Mediterranean dwarf palm which is also in the Coryphoideae). Some species, such as the Sub-Saharan P. reclinata or South Asian P. sylvestris, have continent-wide distributions, but most are less widespread with some of them, such as Phoenix andamanensis or P. canariensis, being endemic to small islands (Fig. 2.1). Like most palms, Phoenix species are dispersed by animals who ingest the fruits and defecate the seeds (endozoochory). There are reports of bats, primates, elephants and birds eating Phoenix fruits (reviewed in Zona and Henderson 1989). Humans have disseminated many species within and outside their native range. For instance, P. sylvestris, P. theophrasti, P. rupicola and P. canariensis can be found in palm gardens of the French and Italian Riviera, and several Phoenix species have been brought to the Americas, in particular to the USA where the U.S. Department of Agriculture has led date palm breeding programs (Rivera et al. 2013; Krueger 2015). The date palm has also been intentionally dispersed, through both seedlings and offshoots, for cultivation purposes. Aside from agricultural practices, seeds have travelled with dates, and, when discarded in favourable environments, may have given rise to numerous feral populations (reviewed by Gros-Balthazard et al. 2018). This anthropogenic dispersal of Phoenix has resulted in the artificial sympatry of various species, for instance, P. dactylifera and P. sylvestris in Northwestern India (Newton et al. 2013). Whether Phoenix species naturally co-occur in the wild, and exchange genetic material through interspecific hybridisation, remains to be explored (Gros-Balthazard 2013). All current cooccurrences of the date palm with another Phoenix species could be due to human-dispersal of the former, and distinguishing natural from artificial areas of sympatry will remain difficult without the independent inference of the date palm’s native distribution (Gros-Balthazard and Flowers 2021). Aside from the date palm, the distribution ranges of many Phoenix species overlap (Fig. 2.1), but no natural sympatric areas

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Systematics and Evolution of the Genus Phoenix …

a

c

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b

d

Fig. 2.4 Selected species of the genus Phoenix. a P. dactylifera, Misfat al Abreyeen, Oman; b P. reclinata, Serengeti, Tanzania; c P. theophrasti (left)

and P. rupicola (right), San Remo, Italy; d P. loureiroi, Mount Zwekabin, Myanmar. All pictures taken by M. Gros-Balthazard

between them have been described so far. This could be due to differences in habitat that prevent species occurring in the same global region to form sympatric zones at a local scale. For instance, P. roebelenii is restricted to cliffs and riverbanks where P. loureiroi would not be found, even though they both occur in

Southeastern China. In the same way, P. caespitosa and P. reclinata both occur in southern Arabia and the Horn of Africa, but the former apparently inhabits drier places than the latter (Barrow 1998). Another example is P. paludosa, which co-occurs with P. andamanensis in the Andaman Islands and with P. loureiroi in

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Thailand, but thrives in periodically inundated estuarine coastal swamps where the two other species would not grow (Barrow 1998). Although less clear, it seems that P. pusilla, which grows in India like P. loureiroi and P. sylvestris, occurs in drier, sandier soils than these species, and only occupies the lowest parts of the altitudinal range covered by P. loureiroi. In contrast, P. sylvestris, P. rupicola and P. acaulis have adjacent distribution ranges that all overlap with the distribution of P. loureiroi in India, and all these species seem to occupy similar habitats (Barrow 1998). For instance, P. acaulis and P. loureiroi both occur in pine forests in Northern India and Nepal (Henderson 2009), but there are no reports of true sympatric zones between them so far. This may be due to unknown ecological specialisation, but it could also be that sympatric zones remain undetected, for instance, because of the morphological similarity of these species, which are often confused with each other (Henderson 2009). Further studies combining genomics, field ecology, fossil information, and species distribution modelling are required to uncover current or past natural zones of contact between Phoenix species or reasons for why they do not exist.

2.2.2 Fossil Record and Archaeological Remains Phoenix species have unique morphological features, including pinnate leaves with induplicate leaflet folds and acanthophylls at their base, and seeds with a longitudinal raphe (see Introduction; Fig. 2.2), making them easily distinguishable from other palms and thus facilitating fossil attribution to the Phoenix lineage. Nevertheless, as with all fossils, their attribution to Phoenix relies on the assumption that no other extinct lineage had Phoenix-like characteristics. Further, attribution of fossils to a particular species is especially difficult in this genus due to the close resemblance of current Phoenix species (see Sect. 4.2).

M. Gros-Balthazard et al.

The magnitude of the Phoenix fossil record has previously been difficult to estimate due to a confusion around the use of the name Phoenicites for fossilised pinnate palm leaves with reduplicate leaflet folds, and therefore not belonging to the Phoenix lineage (resolved by Read and Hickey 1972). A comprehensive review of Phoenix fossils is beyond the scope of this chapter and is already available elsewhere (Allen 2015), but we summarise the main findings below to provide a historical background to the current distribution of Phoenix species. Fossilised leaves, seeds, pollen, flowers or stems attributed with confidence to the Phoenix lineage have been found in Africa, the Americas, Asia and Europe. The oldest fossil known is a leaf sheath discovered in the Deccan Intertrappean beds of India (Bonde et al. 2000), dated to 64–67 million years ago (Ma) (Matsunaga et al. 2018). Phoenix-like palms also occurred in Europe from the Eocene onwards (56 Ma; strata ages hereafter from Cohen et al. 2019/12 unless indicated otherwise), as witnessed by their fossilised leaves, seeds, and flowers (see Barrow 1998 and references in Allen 2015). Pollen and seed fossils suggest that the ancestors of the genus occurred in Africa from the late Oligocene (23–28 Ma) onwards (based on the assumption that the fossil pollen was correctly identified, which remains unclear; Vincens et al. 2006), and certainly by the early Miocene (16–23 Ma; Pan et al. 2006). Phoenix-like fossils have also been found in North America, where flowers and seeds attest to its presence by the Middle (48.5– 52 Ma; Allen 2005) and Late (34–38 Ma) Eocene respectively (Allen 2005). A fossil leaf from the Lower Campanian (72–84 Ma) found in Montana has been tentatively attributed to Phoenix (Crabtree 1987), which would make this the oldest fossil of the genus, but its taxonomic placement is dubious due to the lack of acanthophylls (Dransfield et al. 2008a). Taken together, these fossils suggest a possible origin of the genus in India in the Late Cretaceous, at least one dispersal to the Americas, and some range expansions from India to Africa and Europe that could have been helped by the

Systematics and Evolution of the Genus Phoenix …

37

collision of India with the rest of Asia. However, some authors, apparently not aware of the existence of Indian Phoenix-like fossils, have previously suggested a possible origin of the genus in Europe (Munier 1973). Odoardo Beccari, even though he did not know of Indian Phoenix fossils, seemed to favour the hypothesis of an Indian origin of the genus, based on the current distribution and ecology of the species (reported by Popenoe 1924). The fragmentary nature of the fossil record may never allow the origin of Phoenix to be established with confidence. However, fossil-informed inferences of the historical biogeography of the whole palm family could allow alternative hypotheses about the geographic origin of the genus to be formally tested. On a more recent timescale, Phoenix remains have been excavated from many archaeological sites, either in the form of macro-remains (fragments of leaf or stem, fruits and seeds) or microremains (pollen and phytoliths). In some cases, they have been attributed to species, either based on morphological similarities or on the geographical distribution of species (i.e. remains are attributed to a given species because it is the only one that now grows in the region of the archaeological finding). We consider that most of these diagnostics should be treated with caution, given the high morphological similarity between the species (but we note the potential of morphometrics, as outlined in Sect. 4.2), especially until past species distributions have been robustly modelled. Among the archaeological remains, some belong to pre-agricultural periods and may provide information on the past distribution of Phoenix. For instance, >50,000 years-old pollen (Solecki and Leroi-Gourhan 1961) and *46,000 years-old phytoliths (Henry et al. 2011) were found in the Shanidar cave in Iraq, a region where neither the date palm nor any other Phoenix grows nowadays. In the Levantine region, remains of a *19,000 years-old burnt stem (Liphschitz and Nadel 1997) and *49,000– 69,000 years-old phytoliths were excavated (Henry et al. 2004). Those remains were

attributed to P. dactylifera, the only Phoenix growing in the region today. If species attribution is confirmed, this would indicate that the distribution of wild date palms extended into this region. On Santorini Island (Greece), fragments of a Phoenix-like leaf and an impression of a date-like fruit were recovered from a paleosol dated to 37,000 years. Both were attributed to P. theophrasti because it is the only species of the genus currently found in the region (Friedrich 1980 and Friedrich et al. 1977, cited by Rivera et al. 2014). This confirms the presence of Phoenix in the North of the Mediterranean during prehistoric times. In the Egyptian oasis of Kharga, carbonised seeds and a fossilised leaf were recovered from Pleistocene deposits and tentatively attributed to P. sylvestris (CatonThompson and Gardner 1932; Gardner 1935). The seeds need to be radiocarbon dated to ensure that they do not represent a recent contamination. However, if their archaic age is confirmed, this could suggest an ancient presence of Phoenix in Egypt, in contrast to the current assumption that the sole species growing there today, the date palm, was introduced from West Asia following its domestication (Tengberg and Newton 2016). More recent remains dating from the Neolithic period have been attributed to the date palm and are reviewed in, for example, Gros-Balthazard and Flowers (2021), Flowers et al. (2019), Tengberg (2012) and Tengberg and Newton (2016).

2

2.3

Biology of the Genus Phoenix

2.3.1 Reproductive Biology Palms display a large variety of sexual systems, with a strikingly higher proportion of dioecious species (bearing male and female flowers on separate individuals) than in angiosperms as a whole (30% and 6%, of species, respectively; Nadot et al. 2016). Illustrating this, all species of the genus Phoenix are dioecious. Initially, flower buds are bisexual but interruption of female or male organ development results in individuals

38

carrying only male or female flowers, respectively (Daher et al. 2010). Nevertheless, in some rare cases, male plants may produce female flowers (De Mason and Tisserat 1980). Sex determination in the date palm is based on a nonrecombining XY-like genomic region, where males are the heterogametic sex (Al-Dous et al. 2011; Cherif et al. 2013). This region, located at the distal end of chromosome 12, spans at least 6 Mb but contains only four genes, of which three are known to be involved in the development of reproductive organs in other plants (Hazzouri et al. 2019; Mathew et al. 2014; Torres et al. 2018). The unisexuality of Phoenix flowers is considered to be ancient. Indeed, using sexlinked microsatellite loci, Cherif et al. (2016) showed that dioecy was an ancestral character of the genus, with speciation occurring from a dioecious common ancestor. Further, fossils of Phoenix male flowers have been recorded in sediments dating back to the middle Eocene (Allen 2015; Dransfield et al. 2008a; Ogg 2004). The main agent of pollination of Phoenix is poorly understood; evidence of both anemophilous (wind) and entomophilous (insect) pollination has been reported (Barrow 1998), which is common in palm species (Henderson 1986). Evidence for wind pollination is supported by the large amount of pollen produced, the lack of a sticky pollen coat and the small size of the pollen grains (Barrow 1998). Nevertheless, female flowers do not have a large stigmatic surface to capture pollen grains transported by the wind. In addition, structures possibly resembling nectaries have been identified at the base of the ovary, supporting entomophily (Uhl and Moore 1971). Corner (1966) suggested that the large bract surrounding the inflorescence of P. sylvestris could be involved in protecting pollinating insects. Barrow (1998) noted that while many insects visit Phoenix flowers, their role as pollinators remains to be demonstrated. The only exception is for ornamental, non-native, P. canariensis from the South of France, the pollen of which was shown to be carried and deposited on the stigma by weevils (Meekijjaroenroj and Anstett 2003). In cultivation, date palms are manually pollinated. This keeps the number of

M. Gros-Balthazard et al.

male plants, which are used solely as pollen providers, as low as possible (1 male for 50–100 females), so that scarce resources (water, irrigated land and manure) can be mainly allocated to the fruit-producing female plants. The lack of specialist pollination systems in the different Phoenix species contributes to their ability to form hybrids, at least in botanical gardens and possibly also in natural environments.

2.3.2 Genome Biology To understand how Phoenix species can produce fertile hybrids and to elucidate the mechanisms by which they retain specific alleles after hybridisation events, it is essential to study their genome evolutionary dynamics. So far, there have only been a few cytogenetic studies of Phoenix, and these have mostly focused on the date palm (Dransfield et al. 2008a). All Phoenix species are assumed to be chromosomally diploid with 2n=36 chromosomes, although counts and/or karyotypes are not available for all taxa (for review, Al-Ani et al. 2010). Beal (1937) counted 36 chromosomes in 6 taxa: P. canariensis, P. hanceana var. formosanum and P. humilis (both synonyms of P. loureiroi), P. reclinata, P. sylvestris, and P. dactylifera, of which 10 cultivars were analysed, including the famous Barhee and Deglet Noor. While chromosome counts of 2n = 26 to 36 have been reported, those less than 36 are most likely incorrect, arising from chromosome nondisjunction during in vitro propagation or methodological issues (Al-Ani et al. 2010; El Hadrami et al. 2011). The genome sizes of several Phoenix species have been estimated, and their 1C-values (DNA quantity in an unreplicated gametic nucleus) are given in Table 2.2. The genome size of all species listed was obtained using flow cytometry (Pellicer and Leitch 2014). While we note that additional estimates have been reported in the literature using various bioinformatics approaches to estimate genome size from wholegenome sequence data (Al-Dous et al. 2011; Al-Mssallem et al. 2013), we have not included

2

Systematics and Evolution of the Genus Phoenix …

them here as their reliability is currently unclear (Pellicer and Leitch 2020). We have also not included genome sizes estimated using Feulgen microdensitometry due to methodological issues arising from the use of inappropriate calibration standards (Suda and Leitch 2010). The genome size of only half of the species have been studied so far (P. canariensis, P. dactylifera, P. loureiroi, P. paludosa, P. reclinata, P. roebelenii, P. rupicola, P. sylvestris and P. theophrasti), but one can already see diversity between species, although relatively low (1.5-fold, from 0.72 up to 1.08 pg). Since all species are reported to be diploid, in common with the vast majority of palms that have been studied (Barrett et al. 2019), it is likely that their most recent common ancestor was also diploid. The small genome size differences are therefore most likely the result of contrasting speciesspecific repeat dynamics and recombination processes leading to increases or decreases in the abundance of some repetitive elements, especially transposable elements. The diploid nature and relatively small chromosomes and genomes of Phoenix species facilitate their sequencing and assembly compared to many other important crops. As of today, the date palm is one of the few palms (with the oil palm, coconut, and two rattan species), and the only species from the genus Phoenix, for which a genome assembly is available (for review, Gros-Balthazard et al. 2018; latest published genome assembly, Hazzouri et al. 2019). The popularisation of long-read sequencing technologies, such as Nanopore or SMRT sequencing, and of new scaffolding technologies including 10X Genomics, Hi-C and BioNano are expected to lead to further improvements in the date palm genome assembly and to foster the generation of assemblies for the other species. This will greatly benefit our understanding of the genus Phoenix, by facilitating comparative genomics and phylogenomics (see Sect. 2.6).

39

2.4

Taxonomy

2.4.1 Taxonomic History and Number of Species Due to the cultural importance of the date palm, Phoenix has been present in the literature long before the current nomenclatural system was in place. The first Phoenix species to be mentioned in written records was P. dactylifera, as early as 4,000 years BCE in Babylonia, according to Popenoe (1924). Some accounts from the antiquity also refer to other species, especially the ones now known as P. canariensis and P. theophrasti (Pliny, cited by Barrow 1998). The type species of the genus is P. dactylifera. It was formally described by Linnaeus (1753), based on the extensive work of Kaempfer (1712), but without designating a type specimen. The illustration from Kaempfer (1712) was therefore later selected as the date palm’s lectotype, that is a type specimen recognised afterwards (Fig. 2.5; Moore and Dransfield 1979). Many Phoenix species have been described before the twentieth century without types, so lectotypes are common in the genus, and many of them are illustrations instead of herbarium specimens (Table 2.1). The first monograph of Phoenix was written by Beccari who recognised 10 species: P. acaulis Roxb., P. canariensis Hort., P. dactylifera L., P. farinifera Roxb., P. humilis Royle, P. paludosa Roxb., P. pusilla Gaertn., P. reclinata Jacq., P. rupicola T. And. and P. sylvestris Roxb. (Beccari 1890). Later, Chevalier (1952) recognised 10 species for Africa only: P. atlantica A. Chev., P. baoulensis A. Chev., P. djalonensis A. Chev. and P. dybowskii A. Chev., P. abyssinica Drude that was described after the publication of Beccari’s monograph, P. spinosa Schumach. & Thonn. that was considered a synonym by Beccari and four species already recognised by Beccari. In the second global treatment of the genus, Moore (1963) accepted 12 species, including the 10 species recognised by Beccari,

40 Table 2.2 Genome size estimates for Phoenix species. Multiple estimates for P. dactylifera were obtained from different individuals (see corresponding Sources for details)

M. Gros-Balthazard et al. Species P. canariensis

1C-value (pg)

1C-value (Mb)

0.90

880

Source Suda et al. 2005

P. dactylifera

0.89

870

Hazzouri et al. 2019

P. dactylifera

0.92

900

Hazzouri et al. 2019

P. loureiroi

0.98

958

This study, accession 2009– 3264a

P. paludosa

0.72

704

This study, accession 2004– 3708a

P. reclinata

0.94

919

This study, accession 2004– 421a

P. roebelenii

0.72

704

This study, accession 2002– 1693a

P. rupicola

1.08

1056

This study, accession 2002– 1311a

P. sylvestris

0.84

822

This study, accession 2002– 1727a

P. theophrasti

0.90

880

This study, accession 2017-400a

a Genome sizes were measured from living plants housed at the Royal Botanic Gardens, Kew using the one-step flow cytometry procedure described in Doležel et al. (2007) with slight modifications as outlined in Hazzouri et al. (2019)

but using the name P. loureirii instead of P. humilis, while Beccari considered the former to be a variety of the latter. In the same checklist, Moore also accepted P. abyssinica Drude and P. roebelenii O’Brien but did not provide a treatment of P. caespitosa Chiov. (Chiovenda 1929), or of the 4 species described by Chevalier (1952). In the most recent monograph of the genus, Barrow (1998) followed Moore (1963) in the treatment of the species described by Beccari, except for Phoenix farinifera that she synonymised under P. loureirii, (spelled P. loureiri by Barrow and now corrected to P. loureiroi because it commemorates Loureiro; J. Dransfield, pers. com.), keeping therefore 9 of the species accepted by Beccari. Barrow also synonymised all species described after Beccari’s monograph, except for P caespitosa Chiov., P. roebelenii O’Brien, P theophrasti Greuter and P. atlantica, the latter being considered by her as an unclear taxon. Together with a newly described species (P. andamanensis S. Barrow), Barrow’s monograph, therefore, recognised 13 species

(Barrow 1998). Two species endemic to the south of Spain and Morocco, namely Phoenix iberica D.Rivera, S.Ríos & Obón and Phoenix chevalieri D.Rivera, S.Ríos & Obón, were not studied by Barrow (1998) and have been synonymised to P. dactylifera based on their close morphological and/or genetic resemblance to this species (see next section) (WCVP 2020). All 13 species described by Barrow are currently accepted by the World Checklist of Vascular Plants (WCVP 2020), except for Phoenix andamanensis S.Barrow, which is considered “unplaced.” The reason is that when describing P. andamanensis, Barrow overlooked the fact that this species epithet had been used already (P. andamanensis Sander & C.F.Sander ex R.H. Pearson, currently treated as a synonym of P. paludosa by WCVP 2020) and, as a later homonym, is thus an illegitimate name (Turland et al. 2018). The nomenclatural confusion surrounding this highly distinctive species is yet to be addressed. The total number of currently accepted species has further increased to 14 because of the recognition of P. atlantica as a

2

Systematics and Evolution of the Genus Phoenix …

41

Fig. 2.5 Lectotype of Phoenix dactylifera from Kaempfer (1712), as designated by Moore and Dransfield (1979). Source: Image from Biodiversity Heritage Library, biodiversitylibrary.org

distinct species (WCVP 2020). Its genetic makeup was indeed found to differ from that of other species based on microsatellite data (Table 2.3; Henderson et al. 2006).

2.4.2 Species Delimitation and Identification Species delimitation is the process by which species boundaries are determined, leading to grouping individuals into separate entities, that is species. Species delimitation traditionally relies on the morphology and anatomy of individuals associated to geographical and ecological data. In

the last few decades, it has become increasingly common to use DNA sequencing data for taxonomic studies. In Phoenix, species boundaries have mostly been inferred from morphology and anatomy (Barrow 1998), and they have been questioned since then due to the limited morphological variation between species and their ability to hybridise (Pintaud et al. 2010). The concept of species is highly variable among taxonomists, which can lead to taxonomic studies of the same group having markedly different outcomes. In this chapter, we apply to Phoenix the unified species concept proposed by De Queiroz, which equates species with separately evolving metapopulation lineages, where a

Y

Y

N

Y

Y

Y

Y

Y

Y

P. acaulis

P. andamanensis

P. atlantica

P. caespitosa

P. canariensis

P. dactylifera

P. loureiroi

P. paludosa

P. pusilla

Fixed morphological and anatomical differences

N

N

N

?

?

N

?

N

N

Geographic isolation

?

?

?

N

N

?

N

Y

?

Ecological differences

?

?

?

N

N

N

N

?

?

Intrinsic reproductive isolation

Y

?

Y

Y

Y

?

?

?

Y

Fixed genetic differences

Y

?

Y

Y?

Y

?

?

?

Y

Monophyly

No information available (continued)

Four and nine accessions formed a clade and showed genetic differences from the other species in Barrow (1998) and Pintaud et al. (2010), respectively

Four accessions had an unresolved phylogenetic placement relative to P. sylvestris and seemed to require the inclusion of P. theophrasti to form a clade in Barrow (1998) but 20 accessions formed a clade and showed genetic differences from the other species, including P. sylvestris and P. theophrasti, in Pintaud et al. (2010). Flowers et al. (2019) identified North African date palms as hybrids of Middle Eastern date palms and P. theophrasti, which could explain the results of Barrow (1998)

Four accessions formed a clade and showed genetic differences from the other species in Pintaud et al. (2010)

No information available

Genetically isolated from the date palm based on microsatellite data (Henderson et al. 2006), but not based on genome-wide data (Gros-Balthazard et al. 2017; Flowers et al. 2019)

No information available

Two accessions formed a clade and showed genetic differences from the other species in Pintaud et al. (2010)

Comments on the genetic lines of evidence

Table 2.3 Lines of evidence underlying currently accepted (WCVP 2020) and disputed species in the genus Phoenix. ‘Y’: the species fulfils the criterion, ‘N’: the species does not fulfil the criterion, ‘?’: information on whether the species fulfils the criterion is unavailable or ambiguous. See Main text for details (Sect. 4.2)

42 M. Gros-Balthazard et al.

Y

Y

Y

Y

?

N

N

P. reclinata

P. roebelenii

P. rupicola

P. sylvestris

P. theophrasti

P. chevalieri

P. iberica

Fixed morphological and anatomical differences

Table 2.3 (continued)

?

?

?

N

N

N

N

Geographic isolation

N

N

N

N

?

Y

N

Ecological differences

N

?

N

N

?

N

N

Intrinsic reproductive isolation

N

?

Y

Y

Y

Y

Y

Fixed genetic differences

?

?

Y

Y

Y

Y

Y

Monophyly

Two accessions could not be differentiated from P. dactylifera in Gros-Balthazard et al. (2017)

No information available

Five accessions formed a clade and showed genetic differences from the other species in Pintaud et al. (2010)

Two and four accessions formed a clade and showed genetic differences from the other species in Barrow (1998) and Pintaud et al. (2010), respectively

Two and four accessions formed a clade and showed genetic differences from the other species in Barrow (1998) and Pintaud et al. (2010), respectively

Two and four accessions formed a clade and showed genetic differences from the other species in Barrow (1998) and Pintaud et al. (2010), respectively

Three and 13 accessions formed a clade and showed genetic differences from the other species in Barrow (1998) and Pintaud et al. (2010), respectively, except for one accession, possibly misidentified

Two and four accessions formed a clade and showed genetic differences from the other species in Barrow (1998) and Pintaud et al. (2010), respectively

Comments on the genetic lines of evidence

2 Systematics and Evolution of the Genus Phoenix … 43

44

metapopulation is made up of connected populations (De Queiroz 2007: 880–881). We choose this concept because it is biologically meaningful, takes the dynamism of speciation into account, and provides a framework in which to examine and evaluate all possible lines of evidence for the existence of separate evolution, regardless of the nature of the evidence. Drawing on the approach of De Queiroz (2007), we discuss below whether currently recognised and disputed Phoenix species are supported as being on separate evolutionary trajectories based on six lines of evidence pertaining to their biology, morphology, and genetic makeup (Table 2.3).

2.4.2.1 Fixed Morphological and Anatomical Differences In the last Phoenix monograph, a combination of morphological and anatomical characters (e.g. shape of the leaf crown, position of the embryo, or abundance of adaxial stomata) was used to delimit 13 Phoenix species (Table 2.3; Barrow 1998). These criteria remain valid today since there has been no further study focusing on Phoenix species delimitation based on fixed morphological differences. However, Barrow warns that her data do not differentiate P. theophrasti from P. dactylifera well, and that further research is needed (see below). The three currently disputed species, namely P. atlantica, P. chevalieri and P. iberica do not seem to be morphologically different from the date palm (Barrow 1998; Henderson et al. 2006; Rivera et al. 2008, 2014). Rivera et al. (2008, 2014) noted that the fruits of P. iberica had a shape that was intermediate between those of P. sylvestris and P. theophrasti, but we note that fruits of the latter species and of uncultivated P. dactylifera can be hard to differentiate from each other (Gros-Balthazard et al. 2016). 2.4.2.2 Geographic Isolation When assessing the existence of geographic isolation based on the current native distribution of Phoenix species (Fig. 2.1), Asian species cannot be considered geographically isolated from each-other, since their ranges overlap, even

M. Gros-Balthazard et al.

though small-scale isolation cannot be ruled out (see Sect. 2.1). Phoenix caespitosa and P. reclinata are also not isolated as they co-occur in the Horn of Africa and South Arabia. Wild Mediterranean species (P. atlantica, P. canariensis, P. chevalieri, P. iberica, P. theophrasti) are endemic to restricted areas (Fig. 2.1), but they all co-occur today with the date palm. Because the native distribution of the latter remains unknown (Gros-Balthazard and Flowers 2021), we consider the geographic isolation of the Mediterranean species unclear (Table 2.3).

2.4.2.3 Fixed Ecological Differences A comprehensive study of the ecological niches of Phoenix species is lacking and even information about their habitat is scarce (Sect. 2.1). Based on available data (reviewed in Barrow 1998), it seems that no species of Phoenix has a unique habitat, except maybe P. roebelenii, which is a rheophyte and P. paludosa, which grows near mangroves in a way that is unique in the genus according to Barrow (1998). However, even these species show ecological similarities with others, for instance with P. theophrasti, which also grows on riverbanks regularly flooded by brackish water in Crete. If considering only species that have overlapping ranges, habitat differences can be extended to P. andamanensis, P. caespitosa, P. reclinata and maybe P. pusilla (Barrow 1998). 2.4.2.4 Genetic Composition Genetic data may provide further evidence for species delimitation either because individuals from a given taxon display fixed genetic differences compared to other taxa and/or because they appear monophyletic when relationships are reconstructed (Table 2.3). Barrow (1998) performed the first genetic study of 11 Phoenix species using nuclear ribosomal 5S DNA sequences, with the goal of determining species relationships rather than species boundaries. Nonetheless, five taxa were found monophyletic in the reconstructed tree, namely P. loureiroi, P. pusilla, P. roebelenii, P. rupicola and P. sylvestris, based on 2 to 6 accessions each. Later,

Systematics and Evolution of the Genus Phoenix …

45

Pintaud et al. (2010) found that 11 species included in a phylogenetic tree constructed from microsatellite data were monophyletic based on 2 to 20 accessions each (P. acaulis, P. atlantica, P. canariensis, P. dactylifera, P. loureiroi, P. pusilla, P. reclinata, P. roebelenii, P. rupicola, P. sylvestris, P. theophrasti). These two studies are the only ones aiming at explicitly delimitating Phoenix species using genetic evidence, but other genetic studies involving multiple samples of selected species also provide clues to their genetic differentiation (Chaluvadi et al. 2019; Flowers et al. 2019; Gros-Balthazard et al. 2017). Taking all these studies into account, most recognised species appear supported by genetic data (Table 2.3), but more data are needed to support these findings, especially accessions from different populations across the geographic range of each species. As per the disputed species, genetics do not support P. iberica as evolving separately from P. dactylifera, although it is based on only two accessions (GrosBalthazard et al. 2017). The case of P. atlantica remains unclear, since after being identified as different from other species based on microsatellites (Henderson et al. 2006), it was recently shown to be genetically indistinguishable from the date palm (Gros-Balthazard et al. 2017; Flowers et al. 2019).

neither the date palm nor P. theophrasti are reproductively isolated as the latter is known to have introgressed into the genome of date palms (Flowers et al. 2019). Fertile crosses between P. canariensis or P. atlantica and date palms have also been reported in the Canary and Cape Verde Islands (González-Pérez et al. 2004; Henderson et al. 2006). In the Italian Riviera, where many Phoenix species have been introduced in city gardens, fertile hybrids have been identified based on morphological and genetic data, notably P. roebelenii  P. sylvestris and P. canariensis  P. reclinata (Gros-Balthazard and Pintaud, unpublished). Although it is tempting to conclude that all Phoenix can hybridise and produce fertile offspring, we note that fruits obtained after pollination of P. dactylifera by P. pusilla did not have fully developed seeds (Sudhersan et al. 2010). Therefore, in the absence of actual evidence of fertile hybrids for several species, we cannot conclude about their intrinsic reproductive isolation (Table 2.3). In summary, all species recognised by Barrow are supported as separately evolving lineages by both morphological and genetic evidence. This includes P. theophrasti, whose status as a separate species from the date palm was considered debatable by Barrow (1998: 553), but was since shown to be highly genetically differentiated from it (Flowers et al. 2019; Gros‐Balthazard et al. 2020). There are no post-zygotic reproductive isolation mechanisms between the Mediterranean species. On the other side, their occurrence in sub-Saharan and Asian species is largely unassessed, and the existence of prezygotic isolation mechanisms remains unclear. There are no lines of evidence supporting the recognition of P. iberica and P. chevalieri as separate species (Table 2.3). In contrast, the morphological and genetic differentiation of currently recognised species suggests that overlooked or recently lost reproductive isolation mechanisms have so far kept them on separate evolutionary trajectories. Such mechanisms could include differences in phenology or geographic isolation in the recent past or at a more local scale than the one considered here. However, it is also possible that analysing additional

2

2.4.2.5 Intrinsic Reproductive Isolation Another line of evidence for species delimitation is an intrinsic reproductive isolation, that is, the absence of interbreeding between heterospecific organisms based on intrinsic properties, as opposed to extrinsic barriers (De Queiroz 2007: 880). There is no evidence that different species of Phoenix have different pollinators (Sect. 3.1), and information about other intrinsic pre-zygotic reproductive isolation is scarce. Non-overlapping flowering times are reported for P. acaulis, P. rupicola and P. sylvestris, which have adjacent distribution ranges, but unfortunately, the flowering time of P. loureiroi, which co-occurs with these species, is unknown (Barrow 1998). Here, we consider that a given species is not reproductively isolated if it produces fertile hybrids when crossed with another species. For instance,

46

morphological and genetic data from more individuals will challenge the support for current species boundaries obtained from previous works, which were based on few accessions and genetic markers (Barrow 1998; Pintaud et al. 2010). As explained above, the case of P. atlantica is an example, since recent studies yielded contradicting results about its genetic differentiation from the date palm. Because support from other lines of evidence remains weak, more investigations are needed before the taxonomic rank of this taxon can be ascertained. Several studies focused on developing genetic or phenotypic identification criteria that could be used to assign living individuals or ancient material to currently recognised species. The shape and size of seeds has been shown to hold great potential, with some species being easily distinguishable, notably P. caespitosa or P. paludosa (Gros-Balthazard et al. 2016; Rivera et al. 2014; Terral et al. 2012). In addition, a preliminary study showed that P. theophrasti and P. dactylifera could be identified based on the morphology of their phytoliths, although the authors acknowledged that more samples were needed to fully capture intraspecific variability and hence confirm or refute the use of phytoliths for species identification in the genus (GarcíaGranero et al. 2020). Finally, genetic markers may also be used to assign samples to Phoenix species through DNA barcoding. Chloroplast sequence data (psbZ-trnfM and rpl16-rps3) have proven their utility to differentiate eight out of 13 species (Ballardini et al. 2013; Pintaud et al. 2013). Nevertheless, they failed to distinguish between P. dactylifera, P. sylvestris, P. theophrasti and P. atlantica. The recent development of multiple Phoenix whole-genome sequencing projects (see next section) should facilitate the identification of species-specific SNPs in nuclear DNA and their use for DNA barcoding. The search for species-specific markers, regardless of whether they are morphological or genetic, will only be successful if enough individuals of each species are studied so that both intra- and interspecific variation can be captured. In return, it

M. Gros-Balthazard et al.

will provide additional evidence to assess species delimitation in the genus.

2.5

Species Relationships

The relationships between Phoenix species are currently unresolved. The first attempt to infer a complete species-level phylogeny of the genus consisted of using maximum parsimony (MP) methods to analyse 15 morphological and anatomical characters and a 246–349 bp long DNA sequence from the 5S nuclear ribosomal intergenic spacer for 12 species (Barrow 1998). The phylogenetic trees obtained differed between morphological and molecular data, but they all supported a clade comprising P. dactylifera, P. theophrasti (as its sister) and P. sylvestris, which also included P. canariensis when based on morphology. We hereafter refer to the clade including the three former species as the date palm clade (Pintaud et al. 2010). This first study remained inconclusive about the relationships of the date palm clade to the other Phoenix species but provided evidence supporting P. paludosa as sister of P. roebelenii, and P. rupicola as sister of P. andamanensis (Barrow 1998). In a subsequent MP phylogeny of coryphoids based on four plastid DNA regions and including only P. canariensis, P. dactylifera and P. reclinata, the two latter were identified as sister species with 73% bootstrap support, confirming that the inclusion of P. canariensis in the date palm clade was not supported by nuclear or plastid molecular data (Dransfield et al. 2008b). The next comprehensive Phoenix phylogeny was built using neighbour joining analysis of nuclear microsatellites (and one chloroplast minisatellite) to gather more phylogenetic signal for resolving relationships between species and between individuals of a same species (Pintaud et al. 2010). The study was successful in demonstrating species monophyly (see the previous section), and it was the first to include P. atlantica, which was found to possibly be sister to P. dactylifera. However, interspecific relationships did not receive significant bootstrap

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support and were highly unstable, depending on sampling (Pintaud et al. 2010, p. 276). In a subsequent study based on two chloroplast intergenic spacers, Pintaud et al. (2013) inferred a haplotype network that supported the date palm clade of Barrow (1998) at the condition of including P. canariensis and P. rupicola in the clade in order to maintain P. theophrasti in it. The most similar species to P. dactylifera were P. atlantica and P. sylvestris. The closest species to the date palm clade was P. caespitosa, followed by P. reclinata. Two additional clades were recovered; one grouping P. pusilla, P. acaulis and P. loureiroi and one grouping P. roebelenii and P. paludosa (Pintaud et al. 2013). Each of these clades was supported by one to two single nucleotide substitutions or indels, and the relationships between them were unresolved (GrosBalthazard 2012; Pintaud et al. 2013). The most comprehensive and highly supported Phoenix phylogeny so far was made by maximum likelihood (ML) analysis of a single nuclear gene from a non-recombining, male-specific, genomic region (CYP703; Torres et al. 2018). In this tree, P. atlantica and P. theophrasti are the closest species to P. dactylifera, in agreement with Barrow (1998) and Pintaud et al. (2013), but the date palm clade would have to include all Phoenix species except P. canariensis and P. rupicola in order to include P. sylvestris, in complete disagreement with Pintaud et al. (2013), where P. canariensis and P. rupicola are included in the date palm clade while other species are more distantly related (Fig. 2.6). In 2019, the systematics of Phoenix stepped into the phylogenomic era. The first study to use more than a few DNA regions to infer the relationships of Phoenix species was based on (a) 33,505 single nucleotide polymorphisms from across the whole nuclear genomes of P. atlantica, P. dactylifera, P. sylvestris, P. theophrasti, P. canariensis and P. reclinata (Flowers et al. 2019), and (b) plastid and mitochondrial genome-wide data. The relationships recovered by Flowers et al. (2019) did not contradict previous studies, with P. canariensis, P. theophrasti and P. sylvestris branching successively around a P. atlantica-P. dactylifera clade, except that P.

reclinata branched outside of all these species, in contrast with Torres et al. 2018 (Fig. 2.6). Interestingly, in the organelle phylogenies, some African P. dactylifera samples and P. atlantica formed a clade with six P. sylvestris samples, making P. dactylifera non-monophyletic, a result already found, but less clear and with less samples, in Pintaud et al. (2013) (Fig. 2.6). A subsequent study of the plastid and mitochondrial genomes of P. roebelenii, P. canariensis, P. theophrasti, P. sylvestris and P. dactylifera recovered the same relationships (Mohamoud et al. 2019). Finally, the most recent phylogenomic study of Phoenix was based on the analysis of either all plastid genes or the full plastome of all species and yielded results in disagreement with previous studies (Fig. 2.6; Chaluvadi et al. 2019). The close relationship between P. dactylifera and P. atlantica was not recovered, and the date palm clade either, except if including many other species in it, notably P. acaulis and P. pusilla, but not the same species as in Torres et al. 2018 (Fig. 2.6). The non-monophyly of Phoenix dactylifera and P. sylvestris in some organelle phylogenies is puzzling (Flowers et al. 2019; Mohamoud et al. 2019; Pintaud et al. 2013). In these studies, the P. sylvestris samples grouping with North-African date palms were obtained from plants cultivated at USDA, California, USA. In contrast, a population-level study involving P. sylvestris sampled from the wild (as described in Newton et al. 2013) showed that they grouped outside P. dactylifera based on nuclear regions, but presented different affinities based on one plastid region: some samples grouped with MiddleEastern date palms while others displayed a singular chloroplast haplotype, with none grouping with North African date palms (Gros-Balthazard et al. 2017). Taken together, these results may indicate that two independent hybridisation events have taken place between the date palm and P. sylvestris, in which recurrent backcrosses of the hybrids with P. sylvestris led to the elimination of the date palm nuclear genome but not of its plastome, a phenomenon known as chloroplast DNA capture (Rieseberg and Soltis 1991). Alternatively, the same results could be due to

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incomplete sorting of plastome polymorphisms between the species. A wider sampling of P. sylvestris and a survey of their complete plastid and mitochondrial genomes will be required before these results can be robustly interpreted. Hybridisations between Phoenix species are likely to have happened in the past given that some species currently produce fertile hybrids (as noted above, Gros-Balthazard 2013), and Flowers et al. (2019) have recently provided genomic evidence of ancient hybridisation between P. theophrasti and the date palm, although its timing remains unclear. Unfortunately, no study has so far analysed the whole nuclear and organelle genomes of all species, preventing a global understanding of the reticulation events that may have punctuated the history of Phoenix.

2.6

A Roadmap to Improve Phoenix Systematics

Biological and technical challenges are limiting our understanding of Phoenix species interactions in the past, the phylogenetic framework in which these interactions took place, and the impact they had on the species’ genetic diversity and evolution. Many of these challenges can now be addressed, thanks to advances in (a) high throughput sequencing technologies that can maximise the amount of informative data obtained from hundreds of Phoenix samples collected in the field as well as from historical collections and (b) the development of phylogenomic methods that can accommodate large datasets, and shed new insights on population structure, and the occurrence of reticulate evolution. Nevertheless, there are still some difficulties ahead. One challenge is that the rate of molecular evolution in Phoenix may be too slow relative to the tempo of speciation events in the genus to allow a good resolution of species divergences. Traditional genetic markers used in plant phylogenetics, notably plastid regions, have been shown to evolve slowly in palms (Barrett et al. 2015), and Phoenix is probably not an exception. Nevertheless, intra-specific analyses have revealed some regions of the Phoenix nuclear

and plastid genomes that evolve sufficiently quickly for species-specific mutations to have accumulated (Pintaud et al. 2010). This suggests that deeper surveys of Phoenix genomes could provide enough information to resolve the phylogeny of the genus. The high posterior probabilities supporting the recently inferred full plastome phylogenies of the genus (Fig. 2.6; Chaluvadi et al. 2019) suggest that more data provide a better phylogenetic resolution. However, the findings could also be an artefact of the large amount of data analysed, since data that actually support the clades remain scarce. This is a general problem of phylogenomic studies (e.g. Salichos and Rokas 2013), and addressing it will require thorough analyses of the phylogenetic signal underlying each clade in the inferred species tree compared to the signal underlying alternative topologies (Dornburg et al. 2019). Another obstacle to the resolution of Phoenix relationships that has been known anecdotally for decades and recently supported by genetic evidence, is that species do exchange genetic material as a result of hybridisation (Flowers et al. 2019). Certainly, hybridisation events can mislead phylogenetic analyses, which traditionally look for a bifurcating species tree. The exact phylogenetic pattern left by reticulate evolution will depend on how much genetic material of each parent species has been retained in the hybrid, and on how much of it has been modified since the hybridisation event took place (Rieseberg and Wendell 1993). The genetic consequences of hybridisation can also be confounded by, or confused with, traces left by incomplete lineage sorting (ILS, Degnan and Rosenberg 2009), since both can lead to poorly supported clades in the species tree and conflicts between gene trees (Meng and Kubatko 2009). The development of novel methods to account for (and possibly distinguish) ILS and reticulate evolution (Flouri et al. 2020; Glémin et al. 2019; Wen et al. 2018), is now opening new opportunities for inferring species trees and/or networks of the genus, as appropriate. A final challenge faced by Phoenix systematists is the uncertainty surrounding the identity of samples used in phylogenetic studies. Indeed,

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Fig. 2.6 Summary of our knowledge of Phoenix relationships based on the three molecular studies with a comprehensive sampling of the genus. All trees were rooted with non-Phoenix outgroups. Relationships supported by less than 70% bootstrap replicates (Torres et al.

2018) or less than 0.97 posterior probability (Chaluvadi et al. 2019) are shown as unresolved. The dashed line in the leftmost tree represents two alternative positions of P. sylvestris found by Pintaud et al. (2013)

species are difficult to distinguish morphologically and the main hint for non-experts to identify most of them is the geographical origin (see Sect. 2.1). Most samples used in recent phylogenetic studies have been collected in botanic gardens, and their wild origin has not been disclosed in the publications, making it difficult to verify their identity. In addition, as mentioned previously, gardens may contain unrecognised interspecific hybrids, which, if included in phylogenies, could complicate the inference of ancient reticulation events. In this context, sampling from the wild could be a better solution, although it is challenging and expensive. A solution is to use taxonomically verified herbarium specimens already sampled from the wild, sometimes decades ago (Culley 2013). Ideally, the type specimen of each species should be sequenced, so that all other samples could be molecularly compared to the types and thereby

assigned to a species (or to a hybrid). Unfortunately, the types of half of Phoenix species consist only of illustrations (Table 2.1), preventing this approach to be applied consistently. Nevertheless, many specimens do exist which have been named by experts during the last monographic work on the genus (Barrow 1998) and could be used as references. Now that high throughput sequencing can accommodate degraded DNA, it is possible to generate large amounts of genomic data from herbarium specimens of various ages (Bieker and Martin 2018; Brewer et al. 2019). An ongoing collaboration between New York University Abu Dhabi and Royal Botanic Gardens, Kew is capitalising on this approach to produce reference genomic datasets for each species. Comparing the genomes of other samples to these references will reduce the taxonomic uncertainty surrounding them while it may also lead to changes in the

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taxonomy of the genus by shedding new light on species delimitation. Such an integrated study of Phoenix will require a close collaboration between botanists, taxonomists, phylogeneticists and population geneticists.

2.7

Conclusion and Prospects

In summary, progress in Phoenix systematics is currently impaired by our poor understanding of the relative rates of molecular evolution and species divergence, the amount of ILS, and the prevalence of both ancient and recent events of hybridisation in the genus. These challenges can be addressed by performing analyses of phylogenetic informativeness and by using methods capable of handling intra-genomic conflicts and taking reticulate evolution into account (Kapli et al. 2020). However, before any of this can be meaningfully undertaken, data will have to be generated from samples spanning the genetic diversity of each species, and sample identification and vouchering will have to be more thorough. This way, reliable pangenomes of each Phoenix species will become available to infer the spatio-temporal evolutionary framework of the genus, which will enlighten the study of critical features such as tolerance to drought and salinity (Hazzouri et al. 2020).

References Al-Ani B, Zaid A, Shabana H (2010) On the status of chromosomes in the date palm (Phoenix dactylifera L.). Acta Hort 882:253–268 Al-Dous EK, George B, Al-Mahmoud ME et al (2011) De novo genome sequencing and comparative genomics of date palm (Phoenix dactylifera). Nat Biotech 29:521–527. https://doi.org/10.1038/nbt.1860 Allen SE (2015) Fossil palm flowers from the eocene of the Rocky Mountain Region with affinities to Phoenix L. (Arecaceae: Coryphoideae). Int J Plant Sci 176:586–596. https://doi.org/10.1086/681605 Al-Mssallem IS, Hu S, Zhang X, Lin Q, Liu W, Tan J, Yu X, Liu J, Pan L, Zhang T, Yin Y (2013) Genome sequence of the date palm Phoenix dactylifera L. Nat Commun 4(1):1–9 Asmussen CB, Dransfield J, Deickmann V et al (2006) A new subfamily classification of the palm family

(Arecaceae): evidence from plastid DNA phylogeny. Bot J Linn Soc 151:15–38. https://doi.org/10.1111/j. 1095-8339.2006.00521.x Baker WJ, Couvreur TLP (2013) Global biogeography and diversification of palms sheds light on the evolution of tropical lineages. I. Historical biogeography. J Biogeog 40:274–285. https://doi.org/10.1111/j. 1365-2699.2012.02795.x Baker WJ, Dransfield J (2016) Beyond Genera Palmarum: progress and prospects in palm systematics. Bot J Linn Soc 182:207–233. https://doi.org/10.1111/ boj.12401 Baker WJ, Savolainen V, Asmussen-Lange CB et al (2009) Complete generic-level phylogenetic analyses of palms (Arecaceae) with comparisons of supertree and supermatrix approaches. Syst Biol 58:240–256. https://doi.org/10.1093/sysbio/syp021 Ballardini M, Mercuri A, Littardi C et al (2013) The chloroplast DNA locus psbZ-trnfM as a potential barcode marker in Phoenix L. (Arecaceae). Zookeys 365:71–82. https://doi.org/10.3897/zookeys.365.5725 Barrett CF, Baker WJ, Comer JR et al (2015) Plastid genomes reveal support for deep phylogenetic relationships and extensive rate variation among palms and other commelinid monocots. New Phytol 209:855–870. https://doi.org/10.1111/nph.13617 Barrett CF, McKain MR, Sinn BT et al (2019) Ancient polyploidy and genome evolution in palms. Genome Biol Evol 11:1501–1511. https://doi.org/10.1093/gbe/ evz092 Barrow S (1998) A monograph of Phoenix L. (Palmae: Coryphoideae). Kew Bull 53:513–575 Beal JM (1937) Cytological studies in the genus Phoenix. Bot Gaz 99:400–407 Beccari O (1890) Rivista monografica delle specie del genero Phoenix. Malesia 3:345–416 Bieker VC, Martin MD (2018) Implications and future prospects for evolutionary analyses of DNA in historical herbarium collections. Bot Lett 165:409– 418. https://doi.org/10.1080/23818107.2018.1458651 Bonde SD, Kumbhojkar MS, Aher RT (2000) Phoenicicaulon mahabalei gen. et sp. nov., a sheathing leaf base of Phoenix from the Deccan Intertrappean beds of India. Geophytol 29:11–16 Brewer GE, Clarkson JJ, Maurin O et al (2019) Factors affecting targeted sequencing of 353 nuclear genes from herbarium specimens spanning the diversity of angiosperms. Front Plant Sci 10:1102 Castellana R (1998) Introduction et diffusion de plantes à usages rituels en Méditerranée occidentale. In: Actes des quatrièmes Journées universitaires corses de Nice, 7–9 novembre 1998, Centre d’études corses de Nice (CECN), Nice, pp 115–127 Caton-Thompson G, Gardner EW (1932) The prehistoric geography of Kharga Oasis. Geog J 80:369–406. https://doi.org/10.1057/978-1-349-95943-3_742 Chabaud B (1882) La Phoenix canariensis. La Provence Agric Hortic Illus 19:293–297 Chaluvadi SR, Young P, Thompson K et al (2019) Phoenix phylogeny, and analysis of genetic variation

Systematics and Evolution of the Genus Phoenix …

51

in a diverse collection of date palm (Phoenix dactylifera) and related species. Plant Divers 41:330–339. https://doi.org/10.1016/j.pld.2018.11.005 Cherif E, Zehdi-Azouzi S, Crabos A et al (2016) Evolution of sex chromosomes prior to speciation in the dioecious Phoenix species. J Evol Biol. https://doi. org/10.1111/jeb.12887 Cherif E, Zehdi S, Castillo K et al (2013) Male-specific DNA markers provide genetic evidence of an XY chromosome system, a recombination arrest and allow the tracing of paternal lineages in date palm. New Phytol 197:409–415. https://doi.org/10.1111/nph.12069 Chiovenda E (1929) Pubblicazione a cura del Ministero delle colonie. Flora Somala 1:317 Chevalier A (1952) Recherches sur les Phœnix africains. Revue internationale de botanique appliquée de l’agriculture tropicale, 32ème année, bulletin n°355– 356:205–236 Clement CR (1992) Domesticated palms. Prin 36:70–78 Cohen KM, Harper DAT, Gibbard PL (2019/12) International Chronostratigraphic Chart. In: Int. Comm. Stratigr. IUGS. www.stratigraphy.org Corner EJH (1966) The Natural history of palms. Weidenfeld and Nicolson, Los Angeles Crabtree DR (1987) Angiosperms of the northern Rocky Mountains: Albian to Campanian (Cretaceous) megafossil floras. Ann Miss Bot Gard 707–747 Culley TM (2013) Why vouchers matter in botanical research. Appl Plant Sci 1:1300076. https://doi.org/10. 3732/apps.1300076 Daher A, Adam H, Chabrillange N et al (2010) Cell cycle arrest characterizes the transition from a bisexual floral bud to a unisexual flower in Phoenix dactylifera. Ann Bot 106:255–266. https://doi.org/10.1093/aob/ mcq103 De Mason DA, Tisserat B (1980) The occurrence and structure of apparently bisexual flowers in the date palm, Phoenix dactylifera L. (Arecaceae). Bot J Linn Soc 81:283–292 De Mason DA, Stolte KW, Tisserat B (1982) Floral development in Phoenix dactylifera. Can J Bot 60:1437–1446 De Queiroz K (2007) Species concepts and species delimitation. Syst Biol 56:879–886. https://doi.org/10. 1080/10635150701701083 Degnan JH, Rosenberg NA (2009) Gene tree discordance, phylogenetic inference and the multispecies coalescent. Trends Ecol Evol 24:332–340. https://doi.org/10. 1016/j.tree.2009.01.009 Doležel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2:2233–2244. https://doi.org/10.1038/ nprot.2007.310 Dornburg A, Su Z, Townsend JP (2019) Optimal rates for phylogenetic inference and experimental design in the era of genome-scale data sets. Syst Biol 68:145–156. https://doi.org/10.1093/sysbio/syy047 Dransfield J, Uhl NW, Asmussen CB et al (2008a) Genera palmarum: the evolution and classification of palms. Royal Botanic Gardens, Kew

Dransfield J, Rakotoarinivo M, Baker WJ et al (2008b) A new coryphoid palm genus from Madagascar. Bot J Linn Soc 156:79–91 Flouri T, Jiao X, Rannala B, Yang Z (2020) A bayesian implementation of the multispecies coalescent model with introgression for phylogenomic analysis. Mol Biol Evol 37:1211–1223. https://doi.org/10.1093/ molbev/msz296 Flowers JM, Hazzouri KM, Gros-Balthazard M et al (2019) Cross-species hybridization and the origin of North African date palms. Proc Nat Acad Sci U S A 116:1651–1658. https://doi.org/10.1073/pnas. 1817453116 Friedrich WL (1980) Fossil plants from Weichselian interstadials, Santorini (Greece). Thera and the Aegean World II, London Friedrich WL, Pichler H, Kussmaul S (1977) Quaternary pyroclastics from Santorini/Greece and their significance for the Mediterranean palaeoclimate. Bull Geol Soc Denmark 26:27–39 García-Granero JJ, Skoula M, Sarpaki A et al (2020) Assessment of the use of Phoenix theophrasti Greuter (cretan date palm): the ethnobotany and archaeobotany of a neglected palm. J Ethnobiol 40:101–114. https://doi.org/10.2993/0278-0771-40.1.101 Gardner EW (1935) The Pleistocene fauna and flora of Kharga Oasis. Egypt. Q J Geol Soc 91:479–518 Gaertner J (1788) De fructibus et seminibus plantarum. Stuttgart: Academiae Carolinae Glémin S, Scornavacca C, Dainat J et al (2019) Pervasive hybridizations in the history of wheat relatives. Sci Adv 5:. doi: 10.1126/sciadv.aav9188 Gonzales-Perez MA, Sosa PA (2009) Hybridisation and introgression between the endemic Phoenix canariensis and the introduced P. dactylifera in the Canary Islands. Open For Sci J 2:78–85. https://doi.org/10. 2174/1874398600902020078 González-Pérez MA, Caujapé-Castells J, Sosa PA et al (2004) Molecular evidence of hybridisation between the endemic Phoenix canariensis and the widespread P. dactylifera with random amplified polymorphic DNA (RAPD) markers. Plant Syst Evol 247:165–175. https://doi.org/10.1007/s00606-004-0166-7 Govaerts R, Dransfield J, Zona S et al (2020) World checklist of Arecaceae. Facilitated by the Royal Botanic Gardens, Kew. Published on the Internet; http://wcsp.science.kew.org/. Accessed 26 Jan 2020 Gros-Balthazard M (2013) Hybridization in the genus Phoenix: a review. Emir J Food Agric 25:831–842 Gros-Balthazard M (2012) Sur les origines, l’histoire évolutive et biogéographique du palmier-dattier (Phoenix dactylifera L.) : l’apport de la génétique et de la morphométrie. PhD dissertation, Université Montpellier II Gros-Balthazard M, Flowers JM (2021) A Brief History of the Origin of Domesticated Date Palms. In: AlKhayri JM, Jain SM, Johnson DV (eds) The date palm genome, vol 1: Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-73746-7_3. Springer, Dordrecht (this volume, Chapter 1)

2

52 Gros-Balthazard M, Galimberti M, Kousathanas A et al (2017) The discovery of wild date palms in Oman reveals a complex domestication history involving centers in the Middle East and Africa. Curr Biol 27:2211–2218. https://doi.org/10.1016/j.cub.2017.06. 045 Gros-Balthazard M, Hazzouri KM, Flowers JM (2018) Genomic insights into date palm origins. Genes (Basel) 9:1–14. https://doi.org/10.3390/genes9100502 Gros-Balthazard M, Newton C, Ivorra S et al (2016) The domestication syndrome in Phoenix dactylifera seeds: toward the identification of wild date palm populations. PLoS ONE 11: https://doi.org/10.1371/journal. pone.0152394 Gros‐Balthazard M, Battesti V, Ivorra S et al (2020) On the necessity of combining ethnobotany and genetics to assess agrobiodiversity and its evolution in crops: a case study on date palms (Phoenix dactylifera L.) in Siwa Oasis, Egypt. Evol Appl 1–23. https://doi.org/10. 1111/eva.12930 El Hadrami A, Daayf F, El Hadrami I (2011) Date palm genetics and breeding. In: Jain SM, Al-Khayri JM, Johnson DV (eds) Date palm biotechnology. Springer Science & Business Media, Dordrecht, pp 479–512 Hazzouri KM, Flowers JM, Nelson D et al (2020) Prospects for the study and improvement of abiotic stress tolerance in date palms in the post-genomics Era. 11:1–13. https://doi.org/10.3389/fpls.2020.00293 Hazzouri KM, Gros-Balthazard M, Flowers JM et al (2019) Genome-wide association mapping of date palm fruit traits. Nat Comm 10:4680. https://doi.org/ 10.1038/s41467-019-12604-9 Henderson A (2009) Palms of Southern Asia. Princeton Univ Press Henderson A (1986) A review of pollination studies in the Palmae. Bot Rev 52:221–259 Henderson SA, Billotte N, Pintaud J-C (2006) Genetic isolation of Cape Verde Island Phoenix atlantica (Arecaceae) revealed by microsatellite markers. Conserv Genet 7:213–223. https://doi.org/10.1007/ s10592-006-9128-7 Henry AG, Brooks AS, Piperno DR (2011) Microfossils in calculus demonstrate consumption of plants and cooked foods in Neanderthal diets (Shanidar III, Iraq; Spy I and II, Belgium). Proc Natl Acad Sci USA 108:486–491. https://doi.org/10.1073/pnas. 1016868108 Henry DO, Hietala HJ, Rosen AM et al (2004) Human behavioral organization in the Middle Paleolithic: were Neanderthals different? Am Anthropol 106:17– 31. https://doi.org/10.1525/aa.2004.106.1.17 Huda MN, Hasan M, Abdullah HM, Sarker U (2019) Spatial distribution and genetic diversity of wild date palm (Phoenix sylvestris) growing in coastal Bangladesh. Tree Genet Genom 15:3. https://doi.org/10. 1007/s11295-018-1310-9 IUCN 2020. The IUCN red list of threatened species. Version 2020-1. https://www.iucnredlist.org. Downloaded on 19 March 2020

M. Gros-Balthazard et al. Jacquin F von NJ, Schmidt MA (1800) Fragmenta botanica, figuris coloratis illustrata :ab anno 1800 ad annum 1809 per sex fasciculos edita/. Typis Mathiae Andreae Schmidt, typogr. Universit., Viennae, Austriae Kaempfer E (1712) Amoenitatum exoticarum politicophysico-medicarum fasciculi V. Lemgoviæ: Typis & impensis Henrici Wilhelmi Meyeri Kapli P, Yang Z, Telford MJ (2020) Phylogenetic tree building in the genomic age. Nat Rev Genet 1–17. doi:10.1038/s41576-020-0233-0 Kinnaird MF (2008) Competition for a forest palm: use of Phoenix reclinata by human and nonhuman primates. Conserv Biol 6:101–107 Krueger RR (2001) Date palm germplasm: overview and utilization in the USA. In: Proceedings of the first international conference on date palms. Al Ain: UAE University, Al Ain, pp 2–37 https://www.ars.usda. gov/research/publications/publication/?seqNo115= 124800) Krueger RR (2015) Date palm status and perspective in the United States. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date palm genetic resources and utilization, vol 1. Africa and the Americas. Springer, Dordrecht, pp 447–485 Krueger RR (2021) Date Palm (Phoenix dactylifera L.) Biology and Utilisation. In: Al-Khayri JM, Jain SM, Johnson DV (eds) The date palm genome, vol 1: Compendium of Plant Genomes,https://doi.org/10. 1007/978-3-030-73746-7_1. Springer, Dordrecht (this volume, Chapter 1) Kulkarni AR, Mulani RM (2004) Indigenous palms of India. Curr Sci 86:1598–1603 Linneaus C (1753) Species plantarum, pp 1188 Liphschitz N, Nadel D (1997) Epipalaeolithic (19,000 B. P.) charred wood remains from Ohalo II, Sea of Galilee, Israel, Mitekufat Haeven. J Isr Prehist Soc 27:5–18 Mathew LS, Spannagl M, Al-Malki A et al (2014) A first genetic map of date palm (Phoenix dactylifera) reveals long-range genome structure conservation in the palms. BMC Genom 15:285. https://doi.org/10.1186/ 1471-2164-15-285 Matsunaga K (2019) Fossils, fruits, and phylogeny: an integrative approach to understanding the evolutionary history of palms. PhD dissertation, University of Michigan Matsunaga KKS, Smith SY, Manchester SR et al (2018) Reinvestigating an enigmatic Late Cretaceous monocot: morphology, taxonomy, and biogeography of Viracarpon. Peer J 6: https://doi.org/10.7717/peerj. 4580 Meekijjaroenroj A, Anstett MC (2003) A weevil pollinating the Canary Islands date palm: between parasitism and mutualism. Naturwissenschaften 90:452– 455. https://doi.org/10.1007/s00114-003-0454-z Meng C, Kubatko LS (2009) Detecting hybrid speciation in the presence of incomplete lineage sorting using gene tree incongruence: a model. Theor Popul Biol 75:35–45. https://doi.org/10.1016/j.tpb.2008.10.004

Systematics and Evolution of the Genus Phoenix …

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Mohamoud YA, Mathew LS, Torres MF et al (2019) Novel subpopulations in date palm (Phoenix dactylifera) identified by population-wide organellar genome sequencing. BMC Genom 20:1–7. https://doi.org/10. 1186/s12864-019-5834-7 Moore HE Jr (1963) An annotated checklist of cultivated palms. Prin 7:117–184 Moore HE Jr, Dransfield J (1979) The typification of Linnaean palms. Taxon 28:59–70 Munier P (1973) Le palmier-dattier. Maisonneuve et Larose, Paris Nadot S, Alapetite E, Baker WJ et al (2016) The palm family (Arecaceae): a microcosm of sexual system evolution. Bot J Linn Soc 182:376–388. https://doi. org/10.1111/boj.12440 Newton C, Gros-Balthazard M, Ivorra S et al (2013) Phoenix dactylifera and P. sylvestris in Northwestern India: a glimpse into their complex relationships. Palms 57:37–50 Ogg JG (2004) Status of divisions of the International Geologic Time Scale. Lethaia 37:183–199. https://doi. org/10.1080/00241160410006492 Padmanabhan D, Sudhersan C (1988) Mass destruction of Phoenix loureirii in South India. Principes 32:18–123 Pan AD, Jacobs BF, Dransfield J, Baker WJ (2006) The fossil history of palms (Arecaceae) in Africa and new records from the Late Oligocene (28-27 Mya) of north-western Ethiopia. Bot J Linn Soc 151:69–81. https://doi.org/10.1111/j.1095-8339.2006.00523.x Pellicer J, Leitch IJ (2014) The application of flow cytometry for estimating genome size and ploidy level in plants. In: Besse P (ed) Molecular Plant Taxonomy. Humana Press, Totowa, NJ, pp 279–307 Pellicer J, Leitch IJ (2020) The plant DNA C-values database (release 7.1): an updated online repository of plant genome size data for comparative studies. New Phytol 226:301– 305. https://doi.org/10.1111/nph.16261 Pintaud J-C, Ludena B, Zehdi S et al (2013) Biogeography of the date palm (Phoenix dactylifera L., Arecaceae): insights on the origin and on the structure of modern diversity. Acta Hort 994:19–36 Pintaud J-C, Zehdi S, Couvreur TLP et al (2010) Species delimitation in the genus Phoenix (Arecaceae) based on SSR markers, with emphasis on the identity of the Date Palm (Phoenix dactylifera L.). In: Seberg O, Petersen G, Barfod A, Davis J (eds) Diversity, phylogeny, and evolution in the monocotyledons. Aarhus Univ Press, Arhus, Denmark, pp 267–286 Popenoe P (1924) The date-palm in antiquity. Sci Mon 19:313–325 Read RW, Hickey LJ (1972) A revised classification of fossil palm and palm-like leaves. Taxon 21:129–137 Rheede tot Drakenstein HA van Hortus (1678–1703) Indicus Malabaricus, 12 vols Rieseberg L, Wendell JF (1993) Introgression and its consequences in plants. In: Harrison RG (ed) Hybrid zones and the evolutionary process, Botany. Botany Publication and Papers., Oxford Univ Press, Oxfod, pp 70–109

Rieseberg LH, Soltis DE (1991) Phylogenetic consequences of cytoplasmic gene flow in plants. Evol Trends Plant 5:65–84 Rivera D, De Castro CO, Carreño E et al (2008) Morphological systematics of date-palm diversity (Phoenix, Arecaceae) in Western Europe and some preliminary molecular results. In: Groendijk Wilders N, Alexander C, VandenBerg RG, Hetterscheid WLA (eds) 5th International Symposium on the Taxonomy of Cultivated Plants. Wageningen, Netherlands, pp 97–104 Rivera D, Johnson D, Delgadillo J et al (2013) Historical evidence of the Spanish introduction of date palm (Phoenix dactylifera L., Arecaceae) into the Americas. Genet Res Crop Evol 60:1433–1452. https://doi.org/ 10.1007/s10722-012-9932-5 Rivera D, Obón C, García-Arteaga J et al (2014) Carpological analysis of Phoenix (Arecaceae): contributions to the taxonomy and evolutionary history of the genus. Bot J Linn Soc 175:74–122. https://doi.org/ 10.1111/boj.12164 Rivera D, Obon C, Rios S et al (1997) Las variedades tradicionales de frutales de la Cuenca del Rio Segura. Cat Etnobotanico Univ Murcia, Murcia Roxburgh W (1832) Flora Indica, or descriptions of Indian plants. Printed by the Mission Press for W, Thacker, Serampore, India Roxburgh W, Banks J (1819) Plants of the Coast of Coromandel. BHL Collec. Printed by W. Bulmer and Co. for G Nicol Bookseller, India Salichos L, Rokas A (2013) Inferring ancient divergences requires genes with strong phylogenetic signals. Nature 497:327–331. https://doi.org/10.1038/ nature12130 Solecki RS, Leroi-Gourhan A (1961) Palaeooclimatology and archaeology in the near east. Ann NY Acad Sci 95:729–739 Suda J, Leitch IJ (2010) The quest for suitable reference standards in genome size research. Cytom Part A 77A:717–720. https://doi.org/10.1002/cyto.a.20907 Tengberg M (2012) Beginnings and early history of date palm garden cultivation in the Middle East. J Arid Environ 86:139–147. https://doi.org/10.1016/j. jaridenv.2011.11.022 Tengberg M, Newton C (2016) Origine et évolution de la phoeniciculture au Moyen-Orient et en Egypte. Actes du colloque international Histoire des fruits. Pratiques des savoirs et Savoirs en pratiques. Éditions Omniscience, Toulouse, pp 83–105 Terral JF, Newton C, Ivorra S et al (2012) Insights into the historical biogeography of the date palm (Phoenix dactylifera L.) using geometric morphometry of modern and ancient seeds. J Biogeog 39:929–941 Tomlinson PB (1961) Anatomy of the monocotyledons. II. Palmae, Anat monocotyledons II Palmae Torres MF, Mathew LS, Ahmed I et al (2018) Genuswide sequencing supports a two-locus model for sexdetermination in Phoenix. Nat Comm 9:3969. https:// doi.org/10.1038/s41467-018-06375-y

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54 Turland N J, Wiersema J H, Barrie F R et al. (2018) International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code) adopted by the Nineteenth International Botanical Congress Shenzhen, China, July 2017. Regnum Vegetabile 159. Glashütten: Koeltz Botanical Books. DOI https://doi.org/10. 12705/Code.2018 Uhl NW, Dransfield J (1987) Genera palmarum: a classification of palms based on the work of Harold E. Allen Press, Lawrence KS, Moore, Jr Uhl NW, Moore HE Jr (1971) The palm gynocium. Am J Bot 58:945–992. https://doi.org/10.1002/j.1537-2197. 1971.tb10050.x Vardareli N, Doğaroğlu T, Doğaç E et al (2019) Genetic characterization of tertiary relict endemic Phoenix theophrasti populations in Turkey and phylogenetic relations of the species with other palm species

M. Gros-Balthazard et al. revealed by SSR markers. Plant Syst Evol. https:// doi.org/10.1007/s00606-019-01580-8 Vincens A, Tiercelin J-J, Buchet G (2006) New Oligocene-early Miocene microflora from the southwestern Turkana Basin palaeoenvironmental implications in the northern Kenya rift. Palaeogeogr Palaeoclimatol Palaeoecol 239:470–486. https://doi. org/10.1016/j.palaeo.2006.02.007 WCVP (2020). World checklist of vascular plants, version 2.0. Facilitated by the Royal Botanic Gardens, Kew. Published on the Internet; http://wcvp.science.kew. org/ Retrieved 26 May 2020 Wen D, Yu Y, Zhu J, Nakhleh L (2018) Inferring phylogenetic networks using PhyloNet. Syst Biol 67:735–740. https://doi.org/10.1093/sysbio/syy015 Zona S, Henderson A (1989) A review of animalmediated seed dispersal of palms. Selbyana 11:6–21

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A Brief History of the Origin of Domesticated Date Palms Muriel Gros-Balthazard and Jonathan M. Flowers

Abstract

The study of crop origins is of great interest both in the fields of evolutionary biology and applied crop research. The understanding of plant domestication involves multiple disciplines, including phylogeography, population genetics, anthropology and archaeology. In the past decade, they have prompted new discoveries on the evolutionary history of crops, including the date palm. The date palm (Phoenix dactylifera L.) is the iconic fruit crop of hot and arid regions of North Africa and West Asia. It is the keystone species of oasis agrosystems and produces sugar-rich and nutritious fruits, the dates. There are many different date cultivars each with distinctive fruit traits as well as many wild Phoenix species forming a complex of related species. Alas, a complete understanding of date palm origins remains to be elucidated. The history

M. Gros-Balthazard (&)
J. M. Flowers Center for Genomics and Systems Biology, New Yok University Abu Dhabi, NYUAD campus, 129188 Abu Dhabi, United Arab Emirates e-mail: [email protected] J. M. Flowers e-mail: [email protected] M. Gros-Balthazard Institut de Recherche pour le Développement, UMR DIADE, 911 Avenue Agropolis, 34394 Montpellier cedex 5, France

of domestication and diversification of the date palm is a puzzling question. The consequences of these processes, both genetic and morphological, are only beginning to be revealed. The genetic architecture of the domestication traits is unknown. In this chapter, we place recent advances in the fields of population genomics and archaeobotany in the context of historical views of date palm domestication. We present new models for the possible origins of this emblematic species and detail the many areas in date palm domestication research that are uncertain and would benefit from further work.

3.1

Introduction

As a prelude to modern civilisations, the advent of agriculture was a major advance in human history. The transition from hunter-gatherer societies to sedentary farming communities fostered innovations in plant cultivation and animal husbandry. From this Neolithic Revolution (Childe 1936) emerged domesticated crops, primarily cereals and pulses. While the earliest evidence of domestication (see Appendix 1 for definition) of these crops date back to around 10,000 BCE, perennial fruit crops, including major Mediterranean crops such as grapes, olives and dates, were domesticated later on, from the end of the Neolithic period through the Bronze Age (6000–3000 BCE; Janick 2005; Zohary and

© Springer Nature Switzerland AG 2021 J. M. Al-Khayri et al. (eds.), The Date Palm Genome, Vol. 1, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-73746-7_3

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Spiegel-Roy 1975). Although perennials might be the future of our food production system (Crews et al. 2018), they have been less studied than annuals, and the understanding of their evolutionary history is lagging (Gaut et al. 2015; Miller and Gross 2011). The Food & Agricultural Organization (2013) is now pressing for more studies in perennials. Recently, owing to affordable, high-throughput sequencing technologies and much more advanced statistical approaches, long-lived crops began receiving research attention. Studies of domestication are at the forefront of multiple disciplines. They not only provide insights into our past history but also keys to the adaptation of our farming system to the challenges associated with global change. An understanding of where, when and how plants were brought into cultivation are central to reconstructing the events that led to the development of modern civilisations and the expansion of human societies. Besides, evolutionary biologists recognise that crop domestication provides a framework to understand the mechanisms of evolution through the study of domestication traits and the genes that control them, the evolution of reproductive barriers and the demographic and hybridisation events that have promoted the expansion of crops (RossIbarra et al. 2007; Zeder 2017). In fact, Darwin (1859) developed his theory of evolution by natural selection to a large extent through observations of the effects of artificial selection in domesticated plants and animals. Finally, efforts focusing on modelling the demographic histories of crops and identifying the genes controlling key traits are of interest to biotechnologists and breeders interested in crop improvement (Sattar et al. 2017; Turner-Hissong et al. 2020). The date palm (Phoenix dactylifera L.) is the major perennial fruit crop in hot and arid regions of Afro-Eurasia, and among the oldest cultivated fruit crops (Zohary and Spiegel-Roy 1975). It belongs to the Arecaceae (Palmae) family and, along with 12 or 13 other interfertile species, constitutes the genus Phoenix (Barrow 1998; Gros-Balthazard et al. 2021). This iconic species holds enormous economic, symbolic and social importance throughout its traditional range of

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cultivation from Morocco in the west, across the Arabian Peninsula and to northwestern India in the east (Barrow 1998; Krueger 2021). Its sweet date fruits served as a staple of subsistence farming and source of economic prosperity dating back to the earliest civilisations of West Asia (Fuller and Stevens 2019; Tengberg 2012). Domesticated date palms probably originated in West Asia during the fourth millennium BCE, and later expanded throughout North Africa at the latest by the Roman period (Munier 1973; Nixon 1951; Tengberg 2012). Many of the earliest archaeobotanical remains are concentrated in the Gulf Region and the Tigris and Euphrates River valleys. Early civilisations in this region documented a rich history of phoeniciculture in ancient texts and left an iconographic history that dates back to the end of the fourth millennium BCE (Tengberg 2012). Fresh insight into the domestication history of date palms has come from phylogenetics and population genomics where recent work has answered a number of fundamental questions (Gros-Balthazard et al. 2018). For instance, a recent study, combining genomics and archaeometry, suggests that relictual populations (i.e. that were more widely distributed in the past) of the wild progenitor of date palms persists today in the Arabian Peninsula (Gros-Balthazard et al. 2017). Another one supports a role for introgressive hybridisation in the diversification of the crop (Flowers et al. 2019). Other recent developments include a population genetic study of ancient, germinated seeds, which provides evidence of extensive East-West exchanges of date palms at the height of the Roman Empire (Sallon et al. 2020). Despite these advances, many questions remain concerning the timing of events in the domestication history of date palms and how natural or artificial selection contributed to the domestication syndrome and trait evolution. Beyond the history of phoeniciculture, studies of date palm origins provide insights into the foundation of oasis agrosystems and how humans colonised and adapted to the hot and arid regions of North Africa and West Asia. In this chapter, we review the current understanding of the origin of the domesticated date

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A Brief History of the Origin of Domesticated Date Palms

palm from patterns in the population genetic and archaeobotanical data and highlight the present knowledge gaps. We highlight the current knowledge of date palm wild relatives, review the status of wild progenitor, outline competing models of date palm origins, emphasise the human activities responsible for date palm domestication and discuss recent studies on the genetic basis and evolution of fruit traits.

3.2

Phoenix Wild Relatives and the Wild Ancestor of Domesticated Date Palms

Phoenix dactylifera is typically considered to be a domesticated species while its wild relatives, including other species subject to human exploitation such as Phoenix sylvestris L. and Phoenix canariensis Chabaud, are generally not. What is the basis for this distinction and is it appropriate?

3.2.1 Is Date Palm a Domesticated Crop? Domestication is the process by which a wild plant is changed through artificial selection to better fit our needs (Doebley et al. 2006). The term domestication—from the Latin domesticus, meaning belonging to the house—is sometimes confused with cultivation. The cultivation of a plant corresponds to its maintenance by humans, but a plant can be cultivated without being domesticated, and cultivation does not necessarily lead to domestication. Gepts (2004) considers that the cultivation stage is a prerequisite for domestication. However, by eliminating undesirable phenotypes directly in wild populations, domestication without cultivation can occur (in situ domestication; Pickersgill 2007). There are several definitions of the term domesticated plant. Harlan (1992) defines a fully domesticated plant as one that cannot survive without human intervention. Similarly, Purugganan and Fuller (2011) consider a plant to be domesticated when it depends on humans for reproduction and survival. This definition applies

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well to domesticated cereals and legumes that have characteristics that do not allow them to survive off the field (loss of seed dispersal in particular). However, many perennial crops have evolved traits that distinguish them from their wild ancestors (the domestication syndrome; Appendix 1), and yet can survive and reproduce without human intervention (Miller and Gross 2011). Therefore, this definition is overly restrictive in the context of perennial crops as most would not be considered domesticated. In anthropology, it is human’s perception of wild and cultivated plants that legitimizes the use or not of the term domesticated plant. Here, we adopt the definition of domesticated plants proposed by Meyer et al. (2012): ‘domesticated’ refers more generally to plants that are morphologically and genetically distinct from their wild ancestors as a result of artificial selection, or are no longer known to occur outside of cultivation. By this definition, date palms are considered domesticated, though some local communities consider them to be wild in their gardens (e.g. Tuaregs of the Tassili n’Ajjer, Algeria; Battesti 2004).

3.2.2 The Wild Progenitor of Domesticated Date Palms The wild progenitor, or ancestor, of domesticated date palms, has remained unknown until recently. Prior to the application of genetic data, it was unknown whether domesticated date palms traced to a wild population of Phoenix dactylifera, an extant wild relative such as P. sylvestris or hybridisation between two or more Phoenix species (Pintaud et al. 2010; Zaid and Arias-Jiménez 1999). Independent studies have now excluded the possibility that the early domesticated population of P. dactylifera was the product of hybridisation between two wild Phoenix species and have established the identity of the progenitor. Specifically, a genetic study of Phoenix wild relatives determined that these species are all divergent from P. dactylifera and concluded that none of the known wild relatives is the direct ancestor of the domesticated species

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(Pintaud et al. 2010). Nevertheless, at least one wild relative species, namely Phoenix theophrasti Greuter, has later contributed to the diversification of the date palm during/after its diffusion (Flowers et al. 2019).

3.2.2.1 Wild Versus Feral Date Palms While searching for the wild ancestor of domesticated date palms, wild Phoenix dactylifera, a new issue arose: the difficulty to distinguish genuinely wild, ancestral populations, from feral populations. In date palms, like in many perennial fruit crops, untended stands become established either when a grove is abandoned, or when individuals escape cultivation via germination from seed. These palms, referred to as feral (Appendix 1 for definition), are not genuinely wild and may be mischaracterised as relictual populations of the ancestor of domesticated date palms due to shared phenotypic traits between the wild and the feral forms (Tengberg 2003). Untended groups of date palms can be found throughout North Africa and Western Asia, and whether they are feral or wild is unknown in most cases (Gros-Balthazard et al. 2018). Whether wild or escaped domesticates, these populations may be critical to understanding key aspects of the origin and spread of date palms, as shown by the study of feral populations scattered in the desert near Siwa oasis, Egypt (GrosBalthazard et al. 2020). Unfortunately, most studies have focused on the cultivated

Fig. 3.1 Variation in seed size and shape in Phoenix (Figure constructed by M. Gros-Balthazard)

M. Gros-Balthazard and J. M. Flowers

germplasm and these populations of uncultivated populations remain poorly understood. Distinguishing truly wild from feral populations is a hard task in perennial crops (e.g. in olives; Besnard et al. 2018) and date palms are no exception. The domestication syndrome is expected to be limited (see Sect. 3.5). Differentiating wild from domesticated palms is thus arduous, such that distinguishing wild from feral individuals may be impossible. A study based on the quantitative analysis of seed shape in Phoenix pointed out that domesticated date palms display rather elongated seeds with pointed apices, while wild relatives have smaller and rounder seeds (Fig. 3.1) (Gros-Balthazard et al. 2016). Feral date palms included in the analysis also displayed elongated seeds so that the authors proposed that wild date palms could be distinguished from feral ones based on seed shape. Seed size, on the other side, is expected to be greatly impacted by the environment. Nonetheless, we note that a total reversion to a wild phenotype may be expected after many generations, due to the canalisation process (Waddington 1960), complicating the efforts for distinguishing feral from truly wild P. dactylifera. Further research is needed to understand the feralisation process and the evolution of Phoenix fruit and seed shape under human selection versus natural environments. In the above seed morphometric study, uncultivated populations from Oman have attracted the attention of the authors as their seeds presented what is expected as a wild

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A Brief History of the Origin of Domesticated Date Palms

phenotype, that is, small and rounded seeds (Gros-Balthazard et al. 2017). A molecular study supported this hypothesis, presenting evidence that some of the uncultivated stands of Phoenix dactylifera growing in isolated areas of the Hajar Mountains of Oman are genuinely wild rather than feral palms. This provided the strongest support yet that a relictual population of the wild progenitor of domesticated date palms persists to the present-day and provides new opportunities to study the ancestor of the crop. Some of the uncultivated populations are nonetheless more likely feral or admixed, and identifying them requires evaluating the genetic and morphometrics of each specimen in the context of both wild and domesticated species.

3.2.2.2 The Native Range of the Progenitor of Domesticated Date Palms The native range of the wild progenitor of date palms is unknown. Yet, the description of the native range of a crop’s wild progenitor is a major issue when studying crop origins because domestication centres typically fall within or at the edge of this distribution range. Today, wild Phoenix dactylifera have solely been described from the Hajar Mountains of Oman although many uncultivated populations could also represent wild populations and warrant verification through genetic and morphologic analyses (Gros-Balthazard et al. 2018, 2017). Ancient evidence for wild Phoenix dactylifera can be found as archaeobotanical remains that date back to prehistoric times, before the domestication and the diffusion of domesticated date palms. Nevertheless, these finds cannot formally be identified to the species level given a lack of diagnostic criteria to distinguish Phoenix species (Gros-Balthazard et al. 2021). This ambiguity in species identification complicates the establishment of the native range of P. dactylifera. Both >50 kyr (thousand years) old pollen and ca. 46 kyr old phytoliths attributed to the date palm were found in the Shanidar cave in Iraq

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(Henry et al. 2011; Solecki and Leroi-Gourhan 1961). Also, in Iraq, date palm phytoliths were retrieved from sediments dated to ca. 10,000 BCE (Altaweel et al. 2019). Excavations in the Levantine regions yielded a *19 kyr old burnt stem and *49–69 kyr old phytoliths (Henry et al. 2004; Liphschitz and Nadel 1997) that were attributed to Phoenix dactylifera, based on it being the only Phoenix growing in the region today. Palm phytoliths have also been found in Jebel Faya, United Arab Emirates, in sediments dated to 125 kyr, although it is unclear whether they belong to date palm or Nannorrhops ritchiana (Griff.) Aitch. (Bretzke et al. 2013). In southwestern Saudi Arabia, palm phytoliths were discovered in *80 kyr deposits; these may be attributable to the date palm but the rarity in the assemblage may suggest long-distance transport by wind (Groucutt et al. 2015). In Africa, preNeolithic remains of Phoenix are almost nonexistent. The sole evidence is from the Egyptian oasis of Kharga, where carbonised seeds and a fossilised leaf of Phoenix were recovered from Pleistocene deposits (Caton-Thompson and Gardner 1932; Gardner 1935); nevertheless, only the sediment has been dated while the age of the Phoenix remains is unknown and they could represent more recent contaminating remains. Based on these considerations, it seems likely that wild Phoenix dactylifera is native to Western Asia, although the precise distribution is unknown and a larger historical distribution, covering all or parts of North Africa cannot be ruled out (see Rivera et al. 2020 for attempts to model historical distributions). Paleoclimate models that predict a Green Sahara as recently as 5000 years ago suggest that the climate was wetter and supported diverse plant communities (Tierney et al. 2017). Given that date palms are vulnerable to high humidity and excess rainfall (Barrow 1998), a wet climate during this period likely has implications for the potential distribution of the species in North Africa in the past. Such open questions about the ancient distribution of wild P. dactylifera currently hamper understanding of date palm domestication and would benefit from additional palynological

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surveys and paleovegetation and species distribution modelling.

3.2.3 Phoenix Wild Relatives: Opportunities for Discovery in Evolutionary and Crop Improvement Research Crop wild relatives consist of those phylogenetically related taxa that share a recent common ancestor with the domesticated species. Species in the Phoenix genus have weak barriers to gene flow and frequently hybridise in anthropogenic contexts (Gros-Balthazard 2013). These species are of tremendous interest both in evolutionary studies of domestication (see below) and in applied contexts. Indeed, many crop wild relatives are more tolerant of biotic and abiotic stresses and thus represent a reservoir of diversity for breeding and crop improvement (Burgarella et al. 2019; Migicovsky and Myles 2017; Zhang et al. 2017). In date palms, vanishingly little is known about stress tolerances and other traits of interest in the 13 wild relatives of Phoenix dactylifera and most of what is known is based on observations of habitat occupancy such as the preference of P. theophrasti for coastal areas and ability to survive saltwater exposure (Barrow 1998). In the past decade, many molecular studies have integrated other Phoenix species while studying date palms (i.e. Cherif et al. 2016; Flowers et al. 2019) and a few have directly focused on the genetic diversity of these date palm wild relatives (i.e. Saro et al. 2018; Vardareli et al. 2019). In this chapter, we restrict our discussion to ways in which studies of Phoenix wild relatives have informed the domestication of date palm, but there is a great need for both

M. Gros-Balthazard and J. M. Flowers

molecular studies and experimental assessment of trait diversity in these species for advances both in applied and evolutionary studies (Hazzouri et al. 2020).

3.3

Origins and Diffusion of the Date Palm

The origins of domesticated date palms have been controversial since the beginning of the nineteenth century (Tengberg 2003). Scholars have advanced different theories about the wild progenitor species and debated the location of centre(s) of origin, the number of domestication events, and how hybridisation may have contributed to the origin and diversification of the crop. Here we briefly summarise some of the most prominent ideas and discuss in more detail hypotheses that are supported by existing data.

3.3.1 Origin Hypotheses 3.3.1.1 Evidence of Early Exploitation Prior to cultivation and domestication, wild date palms had been exploited for millennia. For example, they were part of the Neanderthal diet 50,000 years ago, as evidenced by phytoliths found in dental calculus from teeth recovered in Shanidar cave, Iraq (Henry et al. 2011). The earliest evidence of date palm exploitation by modern humans has been found on two sites on the Gulf coast: Dalma Island, United Arab Emirates (Beech and Shepherd 2001) and Sabiyah, Kuwait (Parker 2010) and date back to approximately 5000 BCE (Fig. 3.2). This early evidence of exploitation in the Gulf Region has informed current views on the geographic origins of cultivation, but there remain

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A Brief History of the Origin of Domesticated Date Palms

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Fig. 3.2 A timeline of important events in the domestication history of date palms. Dates correspond to those reported in the references Sarton (1934), Zohary and Spiegel-Roy (1975), Murray (1990), Aruz (2003), Méry and Tengberg (2009), Tengberg (2012), Terral et al.

(2012), Zohary et al. (2012), Malleson (2016), Galanakis et al. (2017), Fuller and Pelling (2018), Malleson and Miracle (2018), Flowers et al. (2019). (Figure constructed by J. Flowers)

significant outstanding questions about the origins of the domesticated date palm.

the most likely origin, in keeping with available evidence on the prehistoric distribution of Phoenix dactylifera, ancient written records, archaeological remains and archaeobotanical finds (Fuller and Stevens 2019; Tengberg 2012, 2003; Zohary and Spiegel-Roy 1975).

3.3.1.2 Geographic Origins The diversity of hypotheses that have been advanced concerning the geographic origin of domesticated date palms reflect the poor understanding of both the identity and historical range of the ancestral species as detailed above. In fact, origin hypotheses have been proposed for most regions where date palms are cultivated in the Old World. Researchers have proposed geographic origin scenarios ranging from Northwest Africa in the West, to Ethiopia in the South, to the Western India in the East and many locales in between (Barrow 1998; Goor 1967; Munier 1981; Tengberg 2003). Most recent scholars, however, have considered the Gulf region to be

3.3.1.3 Number of Domestication Events Whether date palms were independently domesticated one or more times is a source of ongoing debate, as it is in the studies of many crop species, for example, in olives (Besnard et al. 2018). These controversies emerge in population genomics from challenges associated with reconstructing complex historical events from patterns of genetic ancestry and population structure and associating these patterns with human-mediated

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selection and other domestication-related activities. This problem is exacerbated in relatively poorly studied crops, including date palms. Population genetic studies of regional populations of domesticated crops often find distinct ancestries that are commonly interpreted as evidence of multiple domestication events. Choi et al. (2017) argued, however, that evidence of distinct genetic ancestries may not reflect independent de novo domestication but may instead represent single domestication with multiple origins. Multiple origins in this context refer to the number of independent ancestral gene pools from which a crop is derived irrespective of whether the ancestral populations were independently domesticated. Identifying multiple origins is relatively straightforward with population genomic data, but it is considerably more difficult to determine if different sources of genetic variation were subject to independent selection regimes that are a hallmark of multiple domestication. In the case of rice, unidirectional gene flow of functional alleles at key loci that control domesticationrelated traits provide evidence of a single origin of domestication traits. This supports a single domestication despite multiple origins apparent in the distinct genetic ancestries of subspecies of domesticated rice (Choi et al. 2017). Thus, genomic data frequently support multiple origins, but evidence of multiple domestications remains tenuous in most study systems. Domesticated date palms are geographically structured into eastern and western populations (Arabnezhad et al. 2012; Hazzouri et al. 2015; Mathew et al. 2015; Zehdi-Azouzi et al. 2015), with additional minor divisions within each of these regions (Gros-Balthazard et al. 2020; Mohamoud et al. 2019; Zango et al. 2017; ZehdiAzouzi et al. 2015). Population genetic studies have in some cases interpreted this geographic structure as evidence of multiple domestications ranging from two to four events. For example, Zehdi-Azouzi et al. (2015) and Mathew et al. (2015) proposed that the geographic structure detected between North African and West Asian date palms supports two domestications. However, the geographic structure observed in date

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palms may also be explained by a multiple origin, single domestication model. For example, it is possible that domestication-related traits were selected in the East and alleles controlling these traits were later introduced to a protodomesticated or wild population in North Africa. This scenario could account for population structure without multiple de novo domestications. Determining the number of domestication events will continue to be challenging in date palms. There is a need to continue to evaluate evidence that date palms are the product of multiple domestications or if factors such as hybridisation could be the source of the distinct genomic ancestries in geographic populations of date palm (see below) without the need to invoke multiple domestications. There is also a need to consider that domestication of date palms may not be attributable to origins from welldefined cultivation centres but is a geographically diffuse process as has been suggested for other perennial fruit crops (Miller and Gross 2011).

3.3.2 Introgressive Hybridisation The North African population of date palms is of particular interest in the context of introgressive hybridisation. This population is genetically distinct from West Asia (Arabnezhad et al. 2012), consists of multiple sub-populations (Gros-Balthazard et al. 2020; Zango et al. 2017; Zehdi-Azouzi et al. 2015), has at least 20% higher genetic diversity than populations in the Arabian Peninsula and elsewhere in West Asia (Gros-Balthazard et al. 2017; Hazzouri et al. 2015) and has distinct and deeply divergent chloro- (i.e. Pintaud et al. 2013) and mito-types (Flowers et al. 2019; Mohamoud et al. 2019) that are found at high frequency and largely restricted to North Africa. Hazzouri et al. (2015) speculated that these patterns may be at least partially explained by introgressive hybridisation with a wild relative. Evidence supporting introgression from wild Phoenix in North Africa has begun to accumulate

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(Gros-Balthazard et al. 2017, 2020; Flowers et al. 2019). Direct evidence of introgression was first reported by Flowers et al. (2019) using wholegenome resequencing of date palms and its closest wild relatives. In this study, explicit tests of admixture (e.g. ABBA-BABA and f3 tests) supported introgression between the North African population and a closely related congeneric Phoenix theophrasti, or a P. theophrasti-like population (i.e. a possibly extinct species or population that is closely related to P. theophrasti and may have been the direct source of introgressed alleles). Introgression was further supported by the segregation of theophrasti-like alleles in North Africa, population modelling that included admixture between North African date palms and P. theophrasti, haplotype sharing in introgressed genomic regions and patterns of linkage disequilibrium that is consistent with a recent history of admixture with a distant population. In addition, genome-wide estimates of population divergence supported reduced divergence in the North Africa-theophrasti comparison versus West Asia-theophrasti. Evidence of introgressive hybridisation with a Phoenix theophrasti-like population has also been implicated in a microsatellite-based study of date palms in the Siwa Oasis, Egypt. GrosBalthazard et al. (2020) surveyed more than a hundred cultivated and feral date palms and found that both shared alleles with P. theophrasti to a greater extent than samples from West Asia. Interestingly, the degree of allele sharing was higher in the samples from Siwa oasis, Egypt, than in the other samples from North Africa, and especially high in the feral accessions. Whether this intriguing pattern reflects patterns of selection favouring wild alleles in feral populations or reflects differences in demographic history and the history of hybridisation is an area of ongoing research. How much of the North African genomic ancestry traces to Phoenix theophrasti or the theophrasti-like population? Estimates of P. theophrasti ancestry in North African date palms was estimated at 5–18% (Flowers et al. 2019). The upper bound of this estimate is

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remarkably consistent with an estimate of ancestry from an unsampled ghost population (18%) in North African date palms (Gros-Balthazard et al. 2017). The signature of introgression is strongest in cultivars from the Maghreb that have 15–18% of their genomic ancestry that traces to P. theophrasti, while cultivars from Egypt and Sudan showed a smaller (5%) ancestry fraction. It is unknown whether this is because western date palms experienced higher or more recent gene flow from a theophrasti-like population or whether the theophrasti-like ancestry of eastern African varieties has been diluted by introgression from Asian cultivars. Phoenix theophrasti has a present-day distribution limited to Crete and the Aegean Sea region (Boydak 2019; Vardareli et al. 2019), but once may have had a broader geographic range in the Eastern Mediterranean (Fuller and Stevens 2019; Kislev et al. 2004). The fact that the current range of P. theophrasti does not overlap with the range of date palm remains a source of uncertainty concerning the geography of introgressive hybridisation between these species (Flowers et al. 2019). Questions related to the geographic context of hybridisation, the age of the introgression event (s), whether hybridisation was human-mediated, or the product of natural events are the subject of continuing work. These questions are currently being addressed through expanded surveys of genomic variation, studies of ancient DNA, and population genetic modelling. Other areas of active investigation include studies of other Phoenix wild relatives to determine the extent to which hybridisation with additional species may have contributed to the diversity of date palms (Pintaud et al. 2010).

3.3.3 Models of Domesticated Date Palm Origins We have outlined patterns in the population genetic, archaeobotanical and other sources of data that are consistent with some models for the origins of domesticated date palms and

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inconsistent with others. Here we expand on geographic models of date palm domestication and highlight sources of evidence that are either consistent or inconsistent with each model. We outline the simplest models that could account for current data.

3.3.3.1 Expansion Model Prior to the availability of genetic data, the preferred model for date palm origins proposed a domestication centre in West Asia followed by range expansion (Tengberg 2003). Under this model, the current range of cultivation in the Old World—from North Africa to Northwestern India—resulted from westward dispersal from a domestication centre in the east via trade routes traversing the Sahara (Munier 1973) or sea-faring routes across the Mediterranean (Nixon 1951) and any population of wild Phoenix that may have inhabited North Africa was replaced without admixture. Key elements of this model are supported by archaeobotanical and archaeological remains. For example, various sources support a thriving date palm culture in ancient Mesopotamia and the Upper Gulf Region by the early Bronze Age (late fourth/early third millennia BCE) (Tengberg 2012) (Fig. 3.2). By contrast, there is limited evidence of ancient date palm cultivation or wild Phoenix remains in North Africa. The earliest evidence of cultivation dates to the New Kingdom, mid-second millennium BCE, in the Nile River Valley (Popenoe 1924; Tengberg and Newton 2016), and from the beginning of the first millennium BCE in Fezzan in modern-day Libya (Pelling 2005) (Fig. 3.2). Prior to that, there are only sparse remains in Egypt (reviewed in Gros-Balthazard et al. 2020; e.g. Giza, 2700– 2100 BCE; Malleson 2016; Malleson and Miracle 2018) and no reliable remains recorded further west (Flowers et al. 2019). The earliest remains in the Maghreb date to much later, first appearing at the Roman settlement of Volubilis (Morocco) in the Classical Period (ca. 400–100 BCE; Flowers et al. 2019; Fuller and Pelling 2018) (Fig. 3.2). The expansion model is the simplest way to account for the absence of wild Phoenix remains in North Africa and the

M. Gros-Balthazard and J. M. Flowers

disparity in ages of remains in North Africa and the Upper Gulf/Eastern fringe of the Fertile Crescent (Tengberg 2012). This expansion model is attractive in its simplicity, but population genetic studies indicate a more complex history. Arguably, the two most difficult patterns to reconcile with a simple expansion is the higher nucleotide diversity in the western population and evidence of introgression in North Africa. The difference in diversity is inconsistent with a population genetic bottleneck that presumably would have accompanied the founding of a new population on the African continent (Hazzouri et al. 2015), while introgression is inconsistent with a simple expansion. Despite this, there is evidence of a westward expansion from genetic data that support gene flow from West Asia to North Africa. For example, asymmetrical gene flow into North Africa has been proposed to account for the low to moderate frequency of the eastern cpDNA and Y-chromosome haplotypes in North Africa, but the near absence of the western cpDNA and Ychromosome haplotypes in the Arabian Peninsula and elsewhere in West Asia (Cherif et al. 2013; Hazzouri et al. 2015; Mathew et al. 2015; Zehdi-Azouzi et al. 2015). Second, GrosBalthazard et al. (2017) and Flowers et al. (2019) estimated that *82% of North African ancestry can be traced to West Asian date palm. While there are alternative explanations for these patterns, the simplest explanation is the gene flow of eastern alleles into North Africa at a time of geographic expansion of the crop.

3.3.3.2 Leaky Expansion Model The leaky expansion model includes the westward movement of West Asian date palm into North Africa as proposed by the expansion model but also proposes admixture with a Phoenix wild relative such as P. theophrasti or a theophrasti-like population. Although two independent reports support hybridisation between date palm and P. theophrasti (Flowers et al. 2019; Gros-Balthazard et al. 2020), details of the nature of hybridisation are unclear and it is presently difficult to distinguish among various competing scenarios (Fig. 3.3). The disjunct

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Fig. 3.3 Models of date palm population history. (a) A model where West Asian date palms and Phoenix theophrasti hybridised in an unknown location in the Eastern Mediterranean followed by expansion to Africa from the hybrid source. (b) A model where a resident African P. theophrasti-like population hybridised with West Asian date palm following range expansion. Introgressive hybridisation between P. dactylifera and resident populations of P. theophrasti in Greece and Turkey may not have occurred. (c) A model similar to (a) but

including hybridisation with a third unknown source of variation. (d) A model that invokes both the expansion of West Asian date palms and transport of P. theophrasti to North Africa where hybridisation occurred. Pie charts illustrate the genomic ancestry of a population. Thick arrows represent migration. X indicates introgressive hybridisation. Thin arrows point to the product of introgressive hybridisation. (Figure constructed by J. Flowers)

present-day distributions of cultivated date palms and P. theophrasti add uncertainty to the geography of hybridisation in the past. For example, one possibility is that introgression occurred prior to range expansion of the West Asian crop outside of Africa, where the two species ranges may have once come into contact (Fig. 3.3a; Flowers et al. 2019). Phoenix dactylifera X P. theophrasti hybrid populations from Epidaurus, Peloponnese (Flowers et al. 2019) and possibly Turkey (Boydak and Barrow 1995) represent extant examples of such admixed populations. An alternative model incorporates the expansion of West Asian date palms to North Africa but proposes that the introgressive hybridisation occurred in situ with a resident theophrasti or theophrasti-like population (Fig. 3.3b). This model could explain theophrasti introgression signatures in North Africa. However, the North African populations is segregating highfrequency cpDNA and mtDNA haplotypes that

are not shared with Phoenix theophrasti (hence the reference to “theophrasti-like”) and found at very low frequency elsewhere ( ATA) that acts as a recessive loss-of-function mutation. These two mutations in VIRESCENS support a model that accounts for much of the variation in fruit colour. Taken together, the relatively uniform yellow fruit colour in wild stands of the closest relatives of date palm (Phoenix sylvestris, P. theophrasti), the diversity in date palm fruit colour (Jaradat and Zaid 2004), and multiple independent mutations in date palm VIRESCENS and parallelism with oil palm (Hazzouri et al. 2015) suggest that fruit colour was selected during date palm domestication. Sugar composition is a prominent trait that varies among date palm cultivars. Date palms

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deposit large concentrations of sugar during fruit development with as much as 80–85% in the form of sucrose at the fresh, or khalal, stage (Chao and Krueger 2007). At the onset of ripening, many varieties such as Khalas invert sucrose to reducing sugars to the extent that there is little, or no sucrose retained in the tamar (dry) stage. Other varieties such as Sukkari retain sucrose in high concentrations in the dry stage. Hazzouri et al. (2019) reported a quantitative trait locus (QTL) on linkage group 14 that controls variation in this trait in date palm fruits. They found that within the QTL is a cluster of cell wall invertases and an alkaline/neutral invertase and reported what appear to be multiple deletions in this region including the homozygous deletion of a cell wall invertase in many of the sucrose-type varieties. This suggests that sucrose-rich varieties may have evolved via the selection on loss-offunction alleles at the invertase locus. A similar finding was reported in a subsequent independent study (Malek et al. 2020). Amorós et al. (2014) surveyed sugar composition and other compounds in developing fruits of date palms and their Phoenix wild relatives. Phoenix dactylifera were the only Phoenix species surveyed to deposit large amounts of sucrose in khalal stage fruit, whereas only trace amounts were reported for P. loureiroi Kunth, P. canariensis, Phoenix roebelenii O’Brien and Phoenix reclinata Jacq. This suggests that the process of sucrose deposition in date palms is unique to domesticated date palm and further suggests a two-phase model for the origin of the sweet sucrose-rich varieties. In the first stage, date palms were selected by early farmers to increase the deposition of sucrose in the early stages of fruit development perhaps to offset the acidic taste at the fresh stage. In the second stage, the sukkary-type varieties were selected to retain sucrose throughout the ripening process. Future research on the fruit colour and sugar QTL regions will focus on the origins of the alleles and the selective forces (e.g. soft or hard sweeps) that may be operating at these loci.

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3.6

M. Gros-Balthazard and J. M. Flowers

Conclusions and Prospects

The last decade has seen tremendous progress in the study of date palm domestication and the origins of the crop. Yet, despite these gains, there remain many significant outstanding questions in many areas of research. There is a need for extensive population genetic modelling of population history to estimate the ages of key events such as the expansion of the crop and the timing of hybridisation and to distinguish various competing models of population history. Genetic studies of key traits, including those that control the domestication syndrome, and those that underlie traits that differ among varieties would greatly enrich understanding of domestication particularly if coupled with population genomic studies of the origins of alleles and the history and type of selection acting on these genes. Finally, while a handful of studies have begun to apply genetic and genomic methods to Phoenix wild relatives, studies of domestication would benefit from studies of traits such as offshoot production, fruit traits such as gene expression and metabolomic variation during fruit development in both date palms and their wild relatives. Such studies would provide important insight into the genes controlling the domestication syndrome and shed light on the origins and diversity of fruit traits that are valued by consumers and enthusiasts worldwide.

Term

Definition

Cultivar

Clonally propagated named types (i.e. Khalas or Deglet Noor) are formally referred to as “cultivars” in the literature (informally as “varieties”), although local categorisation practices at the origin of given palm names may be more complex (Battesti 2013, Battesti et al. 2018, GrosBalthazard et al. 2020). True-totype cultivars refer to palms that are indeed exclusively propagated through offshoots under a single name. On the other side, ethnovarieties are groups of multiple clonal lines of palms, displaying the same form and the same dates according to farmers. Other given names (local categories) may refer to groups of palms having a heterogeneous combination of genes and also heterogeneous morphologies, but are assigned a common name because of a shared characteristic (example: khalt, meaning palms growing from a seed in the Maghreb)

Cultivation

The activities leading to the production of food or other services from plants. The cultivation of date palm, phoeniciculture, involves only limited cultivation practices (see Sect. 3.4)

Domestication

The process by which a wild crop is modified genetically and phenotypically by human activities that impact its life cycle (reproduction, propagation, selection)

Domestication syndrome

The phenotypic traits in a domesticated species that distinguish it from a wild species. Individual genotypes may have a mix of wild traits and domesticated traits

Feral

Feral date palms are individuals originating from domesticated date palms but growing without human intervention during the life cycle. Feral populations may arise either when a cultivated population is abandoned, or when individuals escape from cultivation by (continued)

Appendix 1 and Terminology Term

Definition

Artificial selection

The process where either desirable genotypes (under conscious selection) or higher fitness genotypes (under unconscious selection) increase in frequency owing to human activity in an anthropogenic context

Center of domestication

The geographic region where a crop was first domesticated. In perennial crops, domestication may be geographically diffuse and lacking distinct centres (continued)

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Term

Definition scattering seeds outside the fields in a natural environment that is conducive to population settlement and expansion. Although they look wild, they are not considered as genuinely wild (see Wild)

Introgression

The transfer of genetic material from one population or species to another

Wild

An adjective referring to a population or species of plant that has not been domesticated. The term is often appropriately applied to the relatives or progenitor (i.e. ancestor) of a domesticated crop, but wrongly applied to uncultivated populations without consideration if the ancestry of the population traces to a relative (or ancestor) or the domesticated crop (see Feral)

References Alcántara JM, Rey PJ (2003) Conflicting selection pressures on seed size: evolutionary ecology of fruit size in a bird-dispersed tree. Olea europaea. J Evol Biol 16(6):1168–1176 Altaweel M, Marsh A, Jotheri J et al (2019) New insights on the role of environmental dynamics shaping southern Mesopotamia: from the pre-Ubaid to the early Islamic period. Iraq 81:23–46 Amorós A, Pretel MT, Almansa MS et al (2009) Antioxidant and nutritional properties of date fruit from Elche grove as affected by maturation and phenotypic variability of date palm. Food Sci Tech Int 15(1):65–72 Arabnezhad H, Bahar M, Mohammadi HR, Latifian M (2012) Development, characterization and use of microsatellite markers for germplasm analysis in date palm (Phoenix dactylifera L.). Sci Hort 134:150–156 Aruz J, Wallenfels R (2003) Art of the first cities: the third millennium B.C. from the Mediterranean to the Indus. Metropolitan Museum of Art, New York Balick MJ (1984) Ethnobotany of palms in the Neotropics. Adv Econ Bot 1:9–23 Barrow S (1998) A monograph of Phoenix L. (Palmae: Coryphoideae). Kew Bull 53:513–575 Battesti V (2004) Odeur sui generis, Le subterfuge dans la domestication du palmier dattier (Tassili n’Ajjer, Algérie). In: Bonte P, Brisebarre A-M, Helmer D, Sidi Maamar H (eds) Anthropozoologica — Domestications animales: dimensions sociales et symboliques

71

(Hommage à Jacques Cauvin). Publications Scientifiques du Muséum, Paris, pp 301–309 Battesti V (2013) L’agrobiodiversité du dattier (Phoenix dactylifera L.) dans l’oasis de Siwa (Égypte): entre ce qui se dit, s’écrit et s’oublie. Rev d’ethnoécologie 4 Battesti V, Gros-Balthazard M, Ogéron C et al (2018) Date Palm Agrobiodiversity (Phoenix dactylifera L.) in Siwa Oasis, Egypt: Combining Ethnography, Morphometry, and Genetics. Hum Ecol 46:529–546 Beech M, Shepherd E (2001) Archaeobotanical evidence for early date consumption on Dalma Island, United Arab Emirates. Antiq 75(287):83–89 Besnard G, Terral J-F, Cornille A (2018) On the origins and domestication of the olive: a review and perspectives. Ann Bot 121(3):385–403 Bolmgren K, Eriksson O (2010) Seed mass and the evolution of fleshy fruits in angiosperms. Oikos 119 (4):707–718 Boydak M (2019) A new subspecies of Phoenix theophrasti Greuter (Phoenix theophrasti Greuter subsp. golkoyana Boydak) from Turkey. Forestist 69 (2):133–144 Boydak M, Barrow S (1995) A new locality for Phoenix in Turkey: Gölköy-Bödrum. Prin 39(3):117–122 Bretzke K, Armitage SJ, Parker AG et al (2013) The environmental context of Paleolithic settlement at Jebel Faya, Emirate Sharjah, UAE. Quat Int 300:83– 93 Brown B, Aaron M (2001) The politics of nature. In: Smith J (ed) The rise of modern genomics, 3rd edn. Wiley, New York, pp 234–295 Burgarella C, Barnaud A, Kane NA et al (2019) Adaptive introgression: an untapped evolutionary mechanism for crop adaptation. Front Plant Sci 10:4 Caton-Thompson G, Gardner EW (1932) The prehistoric geography of Kharga Oasis. Geogr J 80(5):369–406 Chao CT, Krueger RR (2007) The date palm (Phoenix dactylifera L.): overview of biology, uses and cultivation. HortSci 42(5):1077–1082 Charbonnier J (2013) La maîtrise du temps d’irrigation au sein des oasis alimentées par des aflâj. Rev d’Ethnoécol 4 Cherif E, Zehdi S, Castillo K et al (2013) Male-specific DNA markers provide genetic evidence of an XY chromosome system, a recombination arrest and allow the tracing of paternal lineages in date palm. New Phytol 197(2):409–415 Chevalier A (1952) Les palmiers du littoral atlantique du Sud du Maroc et les faux-dattiers des palmeraies de Marrakech, de Tiznit et du Sous. Comp Rend Acad Sci 234:171–173 Chevrollier F (2016) From Cyrene to Gortyn. Notes on the relationships between Crete and Cyrenaica under Roman domination (1st c. BC to 4th c. AD) In: Francis JE, Kouremenos A (eds) Roman Crete: new perspectives. Oxbow Books, Philadelphia, pp 11–26 Childe VG (1936) Man makes himself. Watts & Co, London

72 Choi JY, Platts AE, Fuller DQ et al (2017) The rice paradox: multiple origins but single domestication in Asian Rice. Mol Biol Evol 34(4):969–979 Crews TE, Carton W, Olsson L (2018) Is the future of agriculture perennial? Imperatives and opportunities to reinvent agriculture by shifting from annual monocultures to perennial polycultures. Glob Sustain 1:E11 Darwin C (1859) On the origins of species by means of natural selection. Murray, London Doebley JF, Gaut BS, Smith BD (2006) The molecular genetics of crop domestication. Cell 127(7):1309– 1321 Dowson VHW, Aten A (1962) Dates, handling, processing and packing. Food and Agriculture Organization, Rome Flowers JM, Hazzouri KM, Gros-Balthazard M et al (2019) Cross-species hybridization and the origin of North African date palms. Proc Nat Acad Sci USA 116(5):1651–1658 Food and Agriculture Organization (2013) Perennial crops for food security: proceedings of the FAO Expert Workshop; 28–30 August 2013, Rome, Italy. In: Batelo C, Wade L, Cox S et al (eds) Biodiversity & ecosystem services in agricultural production systems. Food and Agriculture Organization, Rome Fuller D, Pelling R (2018) Plant economy: archaeobotanical studies. Volubilis après Rome. Brill, Leiden, Netherlands, pp 349–368 Fuller DQ (2018) Long and attenuated: comparative trends in the domestication of tree fruits. Veg Hist Archaeobot 27:165–176 Fuller DQ, Stevens CJ (2019) Between domestication and civilization: the role of agriculture and arboriculture in the emergence of the first urban societies. Veg Hist Archaeobot 28:263–282 Galanakis Y, Tsitsa E, Günkel-Maschek U (2017) The Power of images: reexamingthe wall paintings from the throne room at knossos. Annu Br Sch Athens112:47–98. https://doi.org/10.1017/ S0068245417000065 Gardner EW (1935) The Pleistocene fauna and flora of Kharga Oasis. Egypt. J Geol Soc London 91:479–518 Gaut BS, Díez CM, Morrell PL (2015) Genomics and the contrasting dynamics of annual and perennial domestication. Trends Genet 31(12):709–719 Gepts P (2004) Plant domestication as a long-term selection experiment. Plant Breed Rev 24(2):1–44 Goor A (1967) The history of the date through the ages in the Holy Land. Econ Bot 21:320–340 Gros-Balthazard M (2013) Hybridization in the genus Phoenix: a review. Emir J Food Agric 25(11):831–842 Gros-Balthazard M, Galimberti M, Kousathanas A et al (2017) The discovery of wild date palms in Oman reveals a complex domestication history involving centers in the Middle East and Africa. Curr Biol 27 (14):2211–2218 Gros-Balthazard M, Hazzouri KM, Flowers JM (2018) Genomic insights into date palm origins. Genes (Basel) 9(10):502

M. Gros-Balthazard and J. M. Flowers Gros-Balthazard M, Newton C, Ivorra S et al (2016) The domestication syndrome in Phoenix dactylifera seeds: toward the identification of wild date palm populations. PLoS ONE 11(3): Gros-Balthazard M, Battesti V, Ivorra S et al (2020) On the necessity of combining ethnobotany and genetics to assess agrobiodiversity and its evolution in crops: a case study on date palms (Phoenix dactylifera L.) in Siwa Oasis, Egypt. Evol Appl 13(8):1818–1840 Gros-Balthazard M, Baker WJ, Leitch IJ, Pellicer J, Powell RF, Bellot S (2021) Systematics and Evolutionof the Genus Phoenix: TowardsUnderstanding Date Palm Origins. In: Al-Khayri JM, Jain SM, Johnson DV (eds) The date palm genome, vol 1: Compendium of Plant Genomes, https://doi.org/10. 1007/978-3-030-73746-7_2. Springer, Dordrecht (this volume, Chapter 2) Groucutt HS, White TS, Clark-Balzan L et al (2015) Human occupation of the Arabian Empty Quarter during MIS 5: evidence from Mundafan Al-Buhayrah, Saudi Arabia. Quat Sci Rev 119:116–135 Harlan JR (1992) Crops & man. American Society of Agronomy, Madison, WI, USA Hazzouri KM, Flowers JM, Visser HJ et al (2015) Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop. Nat Comm 6:8824 Hazzouri KM, Gros-Balthazard M, Flowers JM et al (2019) Genome-wide association mapping of date palm fruit traits. Nat Comm 10:4680 Hazzouri KM, Flowers JM, Nelson D, Lemansour A, Masmoudi K, Amiri K (2020, Mar 17) Prospects for the study and improvement of abiotic stress tolerance in date palms in the post-genomics era. Front Plant Sci 11:293. https://doi.org/10.3389/fpls.2020.00293 Henry AG, Brooks AS, Piperno DR (2011) Microfossils in calculus demonstrate consumption of plants and cooked foods in Neanderthal diets (Shanidar III, Iraq; Spy I and II, Belgium). Proc Nat Acad Sci USA 108 (2):486–491 Henry DO, Hietala HJ, Rosen AM et al (2004) Human behavioral organization in the Middle Paleolithic: were Neanderthals different? Am Anthro 106(1):17– 31 Janick J (2005) The origin of fruits, fruit growing and fruit breeding. Plant Breed Rev 25:255–320 Jaradat AA, Zaid A (2004) Quality traits of date palm fruits in a center of origin and center of diversity. Food Agr Envirt 2:208–217 Johnson DV, Al-Khayri JM, Jain SM (2013) Seedling date palms (Phoenix dactylifera L.) as genetic resources. Emir J Food Agric 25(11):809–830 Kennedy J (2008) Pacific bananas: complex origins, multiple dispersals? Asian Perspect 47(1):75–94 Kislev ME, Hartmann A, Galili E (2004) Archaeobotanical and archaeoentomological evidence from a well at Atlit-Yam indicates colder, more humid climate on the Israeli coast during the PPNC period. J Archaeol Sci 31(9):1301–1310

3

A Brief History of the Origin of Domesticated Date Palms

Krueger RR (2021) Date Palm (Phoenix dactylifera L.) Biology and Utilisation. In: Al-Khayri JM, Jain SM, Johnson DV (eds) The date palm genome, vol 1: Compendium of Plant Genomes, https://doi.org/10. 1007/978-3-030-73746-7_1. Springer, Dordrecht (this volume, Chapter 1) Landsberger B (1967) The date palm and its by-products according to the cuneiform sources. Archiv für Orientforschung. Beihefte 17, Weidner, Graz (1967) Liphschitz N, Nadel D (1997) Epipalaeolithic (19,000 B. P.) charred wood remains from Ohalo II, Sea of Galilee. Israel. J Isr Prehist Soc 27:5–18 Malek JA, Mathew S, Mathew L et al (2020) Deletion of beta-fructofuranosidase (invertase) genes is associated with sucrose content in date palm fruit. Plant Direct 4 (5): Malleson C (2016) Informal intercropping of legumes with cereals? A re-assessment of clover abundance in ancient Egyptian cereal processing by-product assemblages: archaeobotanical investigations at Khentkawes town, Giza (2300–2100 BC). Veg Hist Archaeobot 25:431–442 Malleson C, Miracle R (2018) Giza botanical database. https://doi.org/10.6078/M7JH3J99 Mathew LS, Seidel MA, George B et al (2015) A genome-wide survey of date palm cultivars supports two major subpopulations in Phoenix dactylifera. G3 5(7):1429–1438 McKey D, Elias M, Pujol B et al (2010) The evolutionary ecology of clonally propagated domesticated plants. New Phytol 186(2):318–332 Méry S, Tengberg M (2009) Food for eternity? The analysis of a date offering from a 3rd millennium BC grave at Hili N, Abu Dhabi (UAE). J Archaeol Sci 36 (9):2012–2017 Meyer RS, DuVal AE, Jensen HR (2012) Patterns and processes in crop domestication: an historical review and quantitative analysis of 203 global food crops. New Phytol 196(1):29–48 Migicovsky Z, Myles S (2017) Exploiting wild relatives for genomics-assisted breeding of perennial crops. Front Plant Sci 8:460 Miller AJ, Gross BL (2011) From forest to field: perennial fruit crop domestication. Amer J Bot 98(9):1389–1414 Mohamoud YA, Mathew LS, Torres MF et al (2019) Novel subpopulations in date palm (Phoenix dactylifera) identified by population-wide organellar genome sequencing. BMC Genom 20:498 Munier P (1981) Origine de la culture du palmier-dattier et sa propagation en Afrique. Notes historiques sur les principales palmeraies africaines. Fruits 36(9):531– 556 Munier P (1973) Le palmier-dattier. G.-P, Maisonneuve et Larose, Paris Nixon RW (1951) The date palm: “tree of life “ in the subtropical deserts. Econ Bot 5:274–301 Parker AG (2010) Palaeoenvironmental evidence from H3, Kuwait. In: Carter RA, Crawford HEW (eds) Maritime interactions in the Arabian Neolithic: the

73

evidence from H3, As-Sabiyah, an Ubaid-related site in Kuwait. Brill, Boston, pp 189–212 Pelling R (2005) Garamantian agriculture and its significance in a wider North African context: the evidence of the plant remains from the Fazzan project. J North Afr Stud 10(3–4):397–412 Peyron G (2000) Cultiver le palmier-dattier. Cirad, France Pickersgill B (2007) Domestication of plants in the Americas: insights from mendelian and molecular genetics. Ann Bot 100(5):925–940 Pintaud J-C, Ludena B, Aberlenc-Bertossi et al (2013) Biogeography of the date palm (Phoenix dactylifera L., Arecaceae): insights on the origin and on the structure of modern diversity. Acta Hort 994:19–38 Pintaud J-C, Zehdi S, Couvreur TLP et al (2010) Species delimitation in the genus Phoenix (Arecaceae) based on SSR markers, with emphasis on the identity of the date palm (Phoenix dactylifera L.). In: Seberg O, Petersen G, Barfod A, Davis J (eds) Diversity, phylogeny, and evolution in the monocotyledons. Aarhus Univ Press, Arhus, Denmark, pp 267–286 Popenoe P (1924) The date-palm in antiquity. Sci Mon 19 (3):313–325 Purugganan MD, Fuller DQ (2011) Archaeological data reveal slow rates of evolution during plant domestication. Evolution (NY) 65(1):171–183 Rivera D, Abellán J, Palazón JA et al (2020) Modelling ancient areas for date palms (Phoenix species: Arecaceae): Bayesian analysis of biological and cultural evidence. Bot J Linn Soc 193(2):228–262 Ross-Ibarra J, Morrell PL, Gaut BS (2007) Plant domestication, a unique opportunity to identify the genetic basis of adaptation. Proc Nat Acad Sci USA 104 (Suppl 1):8641–8648 Roué M, Battesti V, Césard N, Simenel R (2015) Ethnoecology of pollination and pollinators: knowledge and practice in three societies. Rev d’Ethnoécol 7 Sallon S, Cherif E, Chabrillange N et al (2020) Origins and insights into the historic Judean date palm based on genetic analysis of germinated ancient seeds and morphometric studies. Sci Adv 6(6):eaax0384 Saro I, García-Verdugo C, González-Pérez MA et al (2018) Genetic structure of the Canarian palm tree (Phoenix canariensis) at the island scale: does the ‘island within islands’ concept apply to species with high colonisation ability? Plant Biol J 21(1):101–109 Sarton G (1934) The artificial fertilization of date-palms in the time of Ashur-Nasir-Pal B.C. 885–860. Isis 21 (1):8–13 Sattar MN, Iqbal Z, Tahir MN et al (2017) CRISPR/Cas9: a practical approach in date palm genome editing. Front Plant Sci 8:1469 Solecki RS, Leroi-Gourhan A (1961) Palaeoclimatology and archaeology in the Near East. Ann NY Acad Sci 95(1):729–739 Tengberg M (2012) Beginnings and early history of date palm garden cultivation in the Middle East. J Arid Envir 86:139–147

74 Tengberg M (2003) Research into the origins of date palm domestication. In: Emirates Center for Strategic Studies and Research (ed) The date palm: from traditional resource to green wealth, Abu Dhabi, pp 51–64 Tengberg M, Newton C (2016) Origine et évolution de la phoeniciculture au Moyen-Orient et en Egypte. In: Ruas M-P (ed) Des fruits d’ici et d’ailleurs: regards sur l’histoire de quelques fruits consommés en Europe. Éditions Omniscience, Toulouse Terral J-F, Newton C, Ivorra S et al (2012) Insights into the historical biogeography of the date palm (Phoenix dactylifera L.) using geometric morphometry of modern and ancient seeds. J Biogeog 39(5):929–941 Tierney JE, Pausata FSR, deMenocal PB (2017) Rainfall regimes of the Green Sahara. Sci Adv 3: Turner-Hissong SD, Mabry ME, Beissinger TM et al (2020) Evolutionary insights into plant breeding. Curr Opin Plant Biol 54:93–100 Vardareli N, Doğaroğlu T, Doğaç E et al (2019) Genetic characterization of tertiary relict endemic Phoenix theophrasti populations in Turkey and phylogenetic relations of the species with other palm species revealed by SSR markers. Plant Syst Evol 305:415– 429 Waddington CH (1960) Experiments on canalyzing selection. Genet Res 1:140–150

M. Gros-Balthazard and J. M. Flowers Zaid A, Arias-Jiménez EJ (1999) Date palm cultivation. Food and Agricultural Organization of the United Nations, Rome Zango O, Cherif E, Chabrillange N et al (2017) Genetic diversity of southeastern Nigerien date palms reveals a secondary structure within western populations. Tree Genet Genomes 13:75 Zeder MA (2017) Domestication as a model system for the extended evolutionary synthesis. Interface Focus 7 (5) Zehdi-Azouzi S, Cherif E, Guenni K et al (2016) Endemic insular and coastal Tunisian date palm genetic diversity. Genetica 144(2):181–190 Zehdi-Azouzi S, Cherif E, Moussouni S et al (2015) Genetic structure of the date palm (Phoenix dactylifera) in the old world reveals a strong differentiation between eastern and western populations. Ann Bot 116(1):101–112 Zhang H, Mittal N, Leamy LJ et al (2017) Back into the wild: apply untapped genetic diversity of wild relatives for crop improvement. Evol Appl 10(1):5–24 Zohary D, Hopf M, Weiss E (2012) Domestication of plants in the old world, 3rd edn. Oxford Univ Press, Oxford, UK Zohary D, Spiegel-Roy P (1975) Beginnings of fruit growing in the old world. Science 187(4174):319–327

Part II Biodiversity and Molecular Identification

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Genome Conformity of In Vitro Cultures of Date Palm Sherif F. El-Sharabasy, Ehab M. B. Mahdy, and Hesham S. Ghazzawy

Abstract

Current knowledge suggests that not all genomic information transmits across subsequent generations. Variability in date palm genomes comes from the infidelity of DNA replication and segregation of unequal chromosomes. DNA lesions in nature are also the source of genomic alteration. Additionally, exposure of explants to external factors such as UV light and chemical mutagens can result in a new useful mutation. Biological cell systems deal naturally with the numerous DNA damages to maintain the date palm genome integrity by evolving several response systems such as checkpoint responses to various DNA damage types. Checkpoints are a well-known control mecha-

nism in the plant cell cycle and respond to DNA replication breaks of dsDNA and diverse other types of DNA damage. Varied evidence indicates that genomic instability is probably the key reason for mutagenesis and the main factor in releasing new desirable mutants against abiotic stress. Thus, understanding how date palm tissues are regulated to maintain their genomic stability is offundamental importance. The range of variation is a selective characteristic resulting from the biological systems across date palm tissue culture. This chapter highlights the causes and sources of genomic instability, genetic alterations, and genetic behavior across date palm tissue cultures.

4.1 S. F. El-Sharabasy (&)
H. S. Ghazzawy Central Laboratory of Date Palm Research and Development (CLDPRD), Agricultural Research Centre (ARC), 9 Gamaa St., 12619, Giza, Egypt e-mail: [email protected]; [email protected] E. M. B. Mahdy National Gene Bank (NGB), Agricultural Research Centre (ARC), 9 Gamaa St., 12619, Giza, Egypt e-mail: [email protected]; [email protected] H. S. Ghazzawy Date Palm Research Center of Excellence, King Faisal University, Al-Ahsa 31982, Saudi Arabia e-mail: [email protected]; [email protected]

Introduction

Tissue culture approaches established for the control of morphogenesis using diverse attributes have an effect on date palm varieties. Tissue culture allows the study of the basic features of cell differentiation and applies various modern aspects of plant genetics and improvement, e.g., virus-free regeneration, genome editing and conservation. In recent years, we have witnessed the use of tissue culture approaches in breeding programs, especially with long lifespan plants such as the date palm. The fundamental challenge is to micropropagate plantlets that are genetically identical to their source. Variants often result in somaclonal variation (Krishna et al. 2016; Leva

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et al. 2012). These somaclones can differ in their morphological, biochemical, cytological, and molecular attributes with in vitro propagation (Bhatia et al. 2011; Leva and Petruccelli 2012; Leva et al. 2012). Tissue culture has exhibited great progress toward sustainable utilization in date palm. Consequently, the behavior of in vitro-propagated plantlets should be assessed after the juvenile stages under in vitro conditions. Various markers are used to detect somaclones (Soliman et al. 2009). Molecular markers are the most reliable methods for their early detection. Studies on genetic fidelity have been carried out extensively in date palm (Al-Khateeb et al. 2019; Krishna et al. 2016; Zehdi-Azouzi et al. 2015) improvement among breeders. In vitro date palm micropropagation allows identification of the genetic and physiological pathways. These are (1) control of metabolism, (2) resistance to various stresses, (3) range of genetic stability, and (4) effects of reproduction. Genetic stability is achieved either with the structural integrity of the genome or with epigenetic changes. Therefore, genetic instability extends to several genetic variation levels (Krishna et al. 2016), including cytological and molecular variations. This heterogeneity also comes at a cost to the utilization of the inherent vulnerability of the unstable genome. Genetic stability is affected naturally by the level of variability that occurs, and the processes of DNA repair. This activity will increase somaclonal variation and the loss of gene functions (Krishna et al. 2016). This chapter describes the causes and sources of genetic instability and genetic behavior across tissue culture processes. We highlight molecular marker techniques for detecting genetic instability stability, in vitro-regenerated date palms, and cryopreserved germplasm.

4.2

Genome Situation and Limitations

Date palm (Phoenix dactylifera L.; 2n = 36) is a dioecious evergreen tree capable of living over 100 productive years and nearly >2000 varieties

are recorded around the world (Johnson 2011). The available use of genomic information provides a significant motivation for date palm genetic improvement and sustainable utilization to detect novel genes and regulatory elements (Unamba et al. 2015). The genome size is 658– 772.3 Mb in length and assembled 58–60% of the genome (Al-Dous et al. 2016; Al-Mssallem et al. 2013; Racchi et al. 2014). The assembly consists of 82,354 scaffolds with an N50 of 329.9–897.2 kb and a maximum scaffold size of B4.5 Mb (Al-Mssallem et al. 2013). The assembled sequences cover about 90% of the genome and contain >42,000 genes (Hazzouri et al. 2020). Full-genome assemblies of plastids revealed 158,462 bp and 715,001 bp for mitochondria (Fang et al. 2012). This assembly allows researchers to enable discoveries and characterize active pathways during the development and maturity of date palm (Al-Dous et al. 2016; Fang et al. 2012; Hazzouri et al. 2020). Date palm embryogenic calli transcriptome reading reveals about 50,852,300 paired-end reads have been made recently with *87% reading alignment (43,987,250 reads) and 53,250 transcripts. Reading alignments showed a total of 2584 transcripts involved through various metabolic pathways. Transcripts involve many classes such as APETALA2 (AP2), auxin-responsive factors (ARFs), leafy cotyledon (LEC), late embryogenesis-abundant protein (LEA), mitogenactivated protein kinase (MAPK), somatic embryogenesis receptor kinase (SERK), small auxin up RNA-like (SAUR-like) auxin-responsive family and WRKY and WUSCHEL (WUS) transcription factors (Naganeeswaran et al. 2020). Previous studies on genomic sequences demonstrated that the date palm experiences genome duplication, either ancient wholegenome duplication or massive segmental duplications (Hazzouri et al. 2020). Studies on genetic diversity show that date palm genes related to resistance to biotic/abiotic stress and sugar metabolism enrich loci of chromosomes where the density of single-nucleotide polymorphisms (SNP) is relatively low (Jombart and Ahmed 2011; Hazzouri et al. 2015). There are more than 7 million single-nucleotide

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polymorphisms in date palms (Al-Mssallem et al. 2013; Hazzouri et al. 2015, 2020) based on whole-genome resequencing of >70 cultivars. Somaclonal variation limits their uses, especially when explants are conserved through in vitro plant regeneration. Somaclonal variation results in inducing mutagenesis and is influenced by many factors and features. The in vitro stabilization results in true-to-type plants across the establishment of tissue culture (El-Hadrami et al. 2011). When tissue culture induces genetic variation, it is not desirable (Kim et al. 2015). The variation amount expected varies with these features. In vitro regenerants must be genetically identical to the maternal explant; lack of understanding this has resulted in disastrous consequences in attempts to produce true-to-type plants. Numerous works describe the variability observed in in vitro plant regenerants (Chaluvadi et al. 2014; El-Dawayati et al. 2012a, b; Kumar et al. 2010a,b). These alterations are regenerated from a new modification that interferes with media on the genome, e.g., chimeras and offtypes. The use of free off-types or genetic offtypes depends on the technique used throughout the in vitro proliferation. The in vitro culture variation affects directly several interacting factors on the multiplied cells, as discussed and explained below. Murashige (1974) described three types of cell differentiation in vitro, which define early for proliferates and are based on the cell-tissue organizations (Bairu et al. 2011; Panis et al 2011; Senula et al. 2018; Skirvin et al. 1994). These are: (a) Organized tissues. These include tissues that form buds and organ cultures, which keep the organizational structure of the individual cells, organs and tissue of a plant. The organized culture closely resembles in vivo vegetative propagation. If the organized tissues do not break, the identical progeny will grow up as in the original plant material. (b) Nonorganized tissue. This comes from tissue isolated from an organized explant, induced dedifferentiation divisions, or stimulating

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callogenesis. The initial explant, in this case, is chimera tissue. The callus disperses clumps of cell aggregates and single-cell results referred to as suspension culture; a nonorganized tissue structure is promoted. High concentrations of plant growth regulators in the medium can induce this growth. Here, genetic stability is often low. (c) Mixed tissue. This is a mediate and combining of previous types. Isolated explants are first dedifferentiated by division, often from tissue which is rapidly developed. Here, we must consider that organized tissue can develop from nonorganized cultures, either through special techniques or spontaneously. Finally, in all cases, the progeny is often not identical to the initial explant. Protocols of in vitro proliferating can lead to genetic change and induce the mutation applied to many species. Molecular markers assist both in documenting genetic variability and sorting out the confusion in the pedigree of many varieties.

4.3

Causes of Genomic Instability

The regeneration method also maintains the inheritance of desired characteristics. Genetic instability is undesirable, especially from the industrial production point of view, but may enrich the gene pool (Kim et al. 2015). The causes of genetic instability involve several features and factors, such as DNA methylation, chromosomal abnormalities, and point mutations (El-Hadrami et al. 2011; Krishna et al. 2016). Studies of these in vitro-derived variants have shown that they may be due to point mutation through genetic events or DNA methylation. They may also occur as a result of chromosomal mutations or ploidy changes. These phenomena often lead to irreversible epigenetic and pleiotropic variants called chimeras. Genetic instability is an essential component of improvement, especially in date palm variations which can introduce promising new traits

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(Al Kaabi et al. 2007; Jain 2007, 2012; Krishna et al. 2016). Variants derive from genetic changes, DNA methylation or epigenetic changes. Somaclones have additional advantages in the efforts of breeders to select a new desired characteristic, especially against biotic/abiotic stresses. The rate of somaclonal variation is related to the genotype and the length of the proliferation process. Basing upon the selective agent, in vitro selection can be done at any phase during in vitro culture. The selection method often depends on the advanced control of the cultivation technique, the ease of application, and the adequacy of the selective agent to induce genetic variation. Several off-types like dwarfism and low fruit set are produced at high rates across date palm tissue culture. These phenomena always take a few years after proliferation to become apparent due to their difficult detection during earlier stages. However, the development of molecular markers has possibly made them detectable earlier and accurately to eliminate them from the mass industrial production (Al Kaabi et al. 2007; Jain 2012). These variants can be investigated further to enrich the genetic pool to improve date palm.

4.4

Variations of in Vitro Plants

Genomic variations come from either the original explant or exposure to an exogenous factor during in vitro culturing and are often heritable (ElHadrami et al. 2011). Nonorganized explants make it possible to obtain variability. The variation involves a dedifferentiation of tissue under defined conditions and subsequent organogenesis and regeneration. In vitro proliferation is mainly dependent on four steps: initiation of explants, multiplication, organ elongation, and adaptation. Each step is based on the factor(s) to pass on successfully. Initiation of an explant is the main step across date palm tissue culture because inducing the somaclonal variation is only associated with the formation of callogenesis (Abahmane 2011). The advantage of direct organogenesis is to use low

growth regulator concentrations to avoid the callus phase. The relationship between them induces many laboratories to try to avoid extreme callus development at any prefoliation stage. Callus initiation in vitro, which is probably analogous to wound responses observed in vivo, occurs readily on the cut or exposed surface of the explant in contact with the medium. However, the variation is not limited to callus regenerants. Several reviews have reported considerable variation in phenotypic and genotypic variations observed in regenerants from all forms of cell culture.

4.4.1 Factors Influencing Somaclonal Variation The source of somaclonal variation is still being debating, and the frequency of genetic changes in vitro is uncertain. Variability is conditioned by the source of the maternal genotype, kind of explant, number of subcultures, and other factors as detailed below.

4.4.1.1 Growth Regulators Growth regulators can induce variation across date palm tissue culture (Khierallah and Hussein 2013). The growth regulators with somaclonal variation are still controversial and ambiguous (Fouad 2019). Auxins control developmental processes, including adventitious root formation, cell elongation, and somatic embryogenesis. Some auxins such as 2,4-D are effective in inducing variations. Cytokinins regulate cell division, callus formation, cell extinction, and shoot morphogenesis. The frequency of variation increases with higher levels of concentration (Roshanfekrrad et al. 2017; Solangi et al. 2020). High concentrations can change the frequency of chromosomal abnormalities and ploidy changes compared to point mutations. Explant excising itself is a process that stimulates the wound responses that promote growth regulators. Date palm genotypes vary in terms of their response to these growth regulators. Various growth regulators have been used to induce

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callogenesis in date palm tissue culture (AlKhayri and Al-Bahrany 2012; Hassan et al. 2013; Roshanfekrrad et al. 2017; Solangi et al. 2020). Solangi et al. (2020) described the best balance of auxin/cytokinin concentrate in date palm tissue culture. Abscisic acid (ABA) retards development of somatic embryos and can also optimize proliferating synchronization (AlKhayri and Al-Bahrany 2012). SghaierHammami et al. (2010) demonstrated that ABA leads to an increase in the rate of proliferation and protein content in somatic embryos. Calli were produced by micropropagation of immature leaf explants on a medium containing 2,4-D (Gueye et al. 2009). Others induced growth of embryogenic callus and somatic embryos (Roshanfekrrad et al. 2017). Cytokinins and activated charcoal improved the somatic embryogenesis of date palm cultivars (Hassan et al. 2013). A high concentration of plant-grown regulars (PGRs) can induce callogenesis in date palm tissue culture (Mazri et al. 2018). Besides, natural additives can induce variations because they are affected by plant growth and physiological activities (Ghazzawy and ElSharabasy 2019). This was evidenced in several experiments performed on date palm (Al-Khayri 2010, 2011a; Ghazzawy and El-Sharabasy 2019; Hegazy et al. 2009; Khierallah and Hussein 2013). Organic additives, such as yeast extract, coconut milk, casein hydrolysate, and pineapple extract, are now used instead of plant growth regulators. Utilization of natural additives may reduce the possibility of genetic instability in date palm tissue culture (Ghazzawy and El-Sharabasy 2019). Tryptone, yeast extract, casein hydrolysate, and pineapple extract influence the production of date palm cv. Malakaby somatic embryogenesis and callogenesis (Al-Khayri 2010, 2011a; Hegazy et al. 2009). Coconut water promotes somatic embryogenesis, rather than casein hydrolysate, and provides genetic stability (Khierallah and Hussein (2013). Organic additives can successfully increase callus growth. Casein hydrolysate and coconut water can increase somatic embryogenesis (Al-Khayri 2010, 2011a; Hegazy et al. 2009).

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4.4.1.2 Cultivars The variability exhibited in vitro depends on the response of the concentrate to genotype and age. The variable rate is not similar for all cultivars. The response between date palm cultivars in variability also could have a genetic basis where cultivars vary in terms of their response to growth regulators. Some cultivars show highly excessive variation, but others are fairly stable. Regenerants procured from older tissue exhibit prior existing variability more than younger tissue. The age of the explant is associated with the variation that exists in it. A few variations may be the result of pre-existing natural mutations, chimeras, or cultural conditions. However, it is difficult to clarify the massive rate of variability observed in cultivars only a few years ago. Many date palm cultivars have proliferated remarkably well, such as Deglet Noor (Bouguedora et al. 2017; Sghaier-Hammami et al. 2010); Sakkoty (Hassan et al. 2013); Najda (Mazri et al. 2017); Khalas (Hassan et al. 2013); Kanthali (Uddin and Titov 2007); Kheneizi (Kurup et al. 2014); Shamia (Hassan et al. 2013); Medjool (Hassan et al. 2013; Mazri et al. 2018; Roshanfekrrad et al. 2017); Bartamuda (Abo El Fadl 2014; Hassan et al. 2013); Sagai (Alansi et al. 2018); Barhee (Hassan et al. 2013); Takerbucht (Bouguedora et al. 2017); and Malakaby (Hassan et al. 2013). Some genotypes may produce trueto-type plants, whereas others are more prone to variability. Most cultivars of the date palm still need to be analyzed for their capacity for in vitro proliferation. Among those having a desired trait (s) there have been observed a certain degree of recalcitrance (Gueye et al. 2009; Zouine et al. 2005). According to Jain (2007), the choice of genotype is critical to limit the appearance of somaclonal variants. 4.4.1.3 Ploidy Level In vitro regeneration capability is a heritable trait. The response to variations occurs as changes in ploidy and chromosomes among Phoenix spp. and within cultivars. Somaclonal variation to obtain the isolation of callus tissue is different in chromosome numbers among regenerated plants.

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Induced undifferentiated cells across in vitro conditions can change genetically in the chromosome number, polysomy or ploidy level, due to irregular cells across stimulating calli, including endoreduplication, mitosis, and DNA amplification. The high rate of retroelement and the sensitivity of a growth regulator often cause variability during the proliferation of date palm in vitro (El-Hadrami et al. 2011; Jain 2007, 2012). Changes in chromosome numbers vary from 26 to 36 (El-Hadrami et al. 2011). Changes can affect cytological features, including polysomy, polyploidy, chromosome breakage, or nucleolar heterochromatin aggregation. The explant source (protoclone, gametoclone, somaclone) is a positive variability source (El-Hadrami et al. 2011). Despite cytological approaches that have witnessed more progress, it is still useful but insufficient to detect date palm variants.

4.4.1.4 Explant Sources Explants show unequal variability. Variations may be due to chimera tissue, or tissue having potential mutation. Different explants of date palm have been achieved successfully in many experiments such as meristems (Baharan and Mohammadi 2018; Jain 2007; Kurup et al. 2014; Mazri et al. 2017; Saptari and Sumaryono 2018); apical buds (Khierallah and Hussein 2013); cuttings and axillary buds (Jain 2007); embryos (Khierallah and Hussein 2013; SghaierHammami et al. 2010); leaf explants (Baharan and Mohammadi 2018; Gueye et al. 2009; Jain 2007; Saptari and Sumaryono 2018) and inflorescence and flower explants (Abdelaziz et al. 2019; Solangi et al. 2020; Zayed 2017). The explant origin is affected by genetic stability (Abdelaziz et al. 2019; Khierallah and Hussein 2013; Solangi et al. 2020). It is still quite problematic to obtain calli from certain genotypes to initiate date palm callogenesis using immature leaf explants (Gueye et al. 2009). Most observations hold that methylation constitutes the main factor controlling variations during date palm tissue culture (El-Hadrami et al. 2011; Jain 2007). Differences in protein content and function have been studied by Sghaier-Hammami et al. (2010).

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Inflorescence and flower explants are the preferred source (Abul-Soad et al. 2017; Mirani et al. 2019; Solangi et al. 2020; Zayed 2017) as compared to bud, shoot-tips, root, and leaf explants (Baharan and Mohammadi 2018; Kurup et al. 2014; Mazri et al. 2017; Saptari and Sumaryono 2018). This is due to saving time and lower efforts to regenerate date palm clones. Several reports on the unveiling of the entire genome may shed light on some of those questions (Solangi et al. 2020).

4.4.1.5 Duration of Tissue Culture The duration of tissue culture is still a key factor to induce somaclonal variation (El-Mageid 2019). Clones must be replaced with another explant taken from the original mother plant to maintain genetic stability. New cultures established commercially and older ones must be eliminated on schedule to ensure continuity (Jain 2012; Mirani 2019). Therefore, most laboratories regularly sample their tissue cultured ex vitro plants in the field or greenhouse to ensure that their characteristics are still maintained stably and with a lower number of subcultures (Mirani 2019; Zayed 2020) from an explant. Conserving germplasm is necessary such as in a repository; cultures can be stored for an extended period in sealed containers under very low temperatures in liquid nitrogen (−196 °C). Minimizing the duration of micropropagation contributes to maintaining identical proliferation of in vitro cultures (Jain 2011, 2012; El-Mageid 2019). In contrast, culture duration can be a good source of desirable variants (Kim et al. 2015). Therefore, some subcultures will induce callogenesis (ElMageid 2019; Zayed 2020). During tissue culture some osmotic stresses may occur widely in the case of long culture incubation under an adverse condition of chemical and physical stresses (El-Dawayati et al. 2012a, b). Some studies suggest avoiding callogenesis when the date palm tissue culture is subjected to less than 11 subcultures (Mirani et al. 2019). Previous reports have demonstrated that the PEG (Bekheet 2011; Bekheet et al. 2005; Puente-Garza et al. 2017) and high concentrations of sugars such as sucrose, mannitol and

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sorbitol (El-Dawayati et al. 2012a, b) have the potential to create osmotic stress. Those acting as osmotic regulators or growth retardants cause osmotic stresses in plant culture media. Many attempts have shown the avoidance of subculture numbers to prevent callus induction across date palm tissue culture (El-Mageid 2019; Jain 2012; Zayed 2020). Jain (2007, 2011, 2012) pointed to the number of subculture cycles which are useful to lower or limit the appearance of somaclones after the step of plant proliferation. Eshraghi et al. (2005) reported the occurrence of genetic variation reaching the sixth generation. The maximum genetic change found that the variation is about 17%, with an average rate of 3.4% per multiplication cycle (El-Hadrami et al. 2011; Jain 2011, 2012). The in vitro regeneration process mainly occurs due to the de novo effects across differentiated cell proliferation from culturing the explant to tissue regeneration.

4.4.1.6 Proliferation Rate A high micropropagation rate exhibits variants among cultures. Excessive rates of proliferated cultures manifest more variability frequencies than those grown at moderate flows. A variation rate of 5% is acceptable. In general, the frequency can be 30–50 plants/month. However, this rate leads to a higher variability rate; but this decreases to 4 plants/month. The frequency of genetic variation associated with proliferation systems varies by 1–3% for the culture of shoots and 10% (per culture cycle) for somatic embryoids; in either case, tissue may reflect the variation existing in the maternal donors. ABA increased the proliferation frequency and protein content in date palm somatic embryos (El-Hadrami et al. 2011; Sghaier-Hammami et al. 2010). 4.4.1.7 Exposure to Pressure Subjecting in vitro cultures to a specific pressure will result in greater variability rates. Sometimes tissue culturists do utilize in vitro proliferation as the direct-selection, and useful tool, for single gene traits than for those with multigenic control, instead of traditional breeding programs.

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Exposure to a specific pressure in vitro is to select cell lines with distinctive characteristics like resistance or tolerance to biotic/abiotic stress (es) (El-Dawayati et al. 2012a, b), herbicides and particular chemical compounds (Mirani et al. 2019; Zayed 2020). As a useful tool for plant improvement, the use of these schemes is of long-term value; the selected trait must be notably expressed with all genes expressed in the same manner at the whole plant level. Scientists suggest the conditions of in vitro culturing must be so unnatural for cultured tissues that the most rapidly growing cells survive because of repeated subculturing (El-Dawayati et al. 2012a, b; Zayed 2020). Comprehensive observation is needed to determine which of the newly planted cultures, especially in cell suspension cultures, grow slowly, and produce clumps. The growth rate of suspension culture accelerates with time, and the size of clumps decreases (El-Dawayati et al. 2012a, b). Many studies have been reviewed and attempted (El-Dawayati et al. 2012a, b; Mirani et al 2019; Puente-Garza et al. 2017; Zayed 2020) to ascertain adverse conditions of chemical and physical stresses. High concentrations of sugars (El-Dawayati et al. 2012a, b) have induced potential osmotic stresses because they stimulated osmotic regulators or growth retardants. Mirani et al. (2019) exposed the date palm tissue culture to 11 subcultures. Polyethylene glycol (PEG) lowers hyperhydration and enhances maturation and the germination of somatic embryos. PEG is utilized as a selection agent or cryoprotectant solution. PEG accumulated in the proline acts as an osmotic stress indicator (El-Sharabasy et al. 2008). Also, PEG stimulates in vitro drought stress and an observed extensive modification of somatic embryogenesis. Casein hydrolysate and coconut water stimulate date palm somatic embryogenesis and embryonic calli (Al-Khayri 2010, 2011a; El-Hadrami et al. 2011; Hegazy et al. 2009). Coconut water promoted somatic embryogenesis more than casein hydrolysate (Khierallah and Hussein 2013).

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4.4.1.8 Culture Conditions Irregularities in genomes often occur during the period that cells are away from the stabilization and control influences of an intact organism and placed in an in vitro unnatural condition. The control systems that regulate a plant are interrupted due to contact with the excised explant in preparation for placement in media (Jain 2012). As evidence, callus naturally results in excised tissue before growing and elongating into an organ. All stimulators and additives mediated these consequences, then led to variants. Al-Khateeb (2006) and El-Hadrami et al. (2011) indicated that adding PEG-8000 to 60 g L-1 sucrose media induces stress conditions during date palm cv. Sukary embryo formation and development.

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3.4% per multiplication cycle (El-Hadrami et al. 2011; Jain 2012). Most somaclonal variations manifest an epigenetic or physiological basis and are therefore reversible. Identification and early detection of these off-types will avoid occurring abnormalities and shortcomings in the future. It must associate morphological attributes with molecular markers for the complementary date palm characterization and evaluation (El-Sharabasy and Rizk 2019; Rizk and El-Sharabasy 2008). El-Sharabasy and Rizk (2019) completed an atlas of the date palm in Egypt. Also, a descriptor for date palm is provided by Rizk and El-Sharabasy (2008).

4.4.3 Genetic and Epigenetic Changes 4.4.2 Phenotypic Changes Phenotypic variation is principally expressed by changes in morphological characteristics, which can, for instance, lead to growth habit, production, uniformity, and tolerance to biotic/abiotic stresses. Variability has been observed in most cases in individual varieties of date palm (ElHadrami et al. 2011; Mirani et al. 2019; ElSharabasy and Rizk 2005). Observed morphological changes include abnormalities in leaves (Fig. 4.1), inflorescences (Fig. 4.2), dwarfism, reduction or enhancement in plant vigor, time of flowering, failed pollination, leaf bleaching, deformed offshoots, fruit quality and resistance to disease (Fig. 4.3) in different countries where dates are grown. Dwarfism and fruit-set failure were observed, and they cause extreme economic losses. AlWasel (2001) reported that the in vitro-derived dwarf plants did not achieve more than 25% in height, and changes in leaf morphology occurred at a rate of 10–50%, and 1–3% for albino and leaf variegation, based on the cultivars proliferated. Eshraghi et al. (2005) reported the occurrence of genetic variation carried to the sixth generation. Genetic change variation accumulates at a rate of about 17%, with an average of

Somaclonal variation may be due to DNA methylation, chromosome abnormalities, or point mutations. Changes deriving from in vitro cultures are almost heritable, transmitted by meiosis and often irreversible. Abnormalities in chromosomes are common across tissue culture and affect genetic improvement of date palm. Somaclones are conducive to change by exposure to mutagenic agents (Mirani et al. 2020; Rathore et al. 2020); different types of mutations determine the basis for somaclonal variation (Mirani et al. 2020). Numerous discussions continue considering genetic and epigenetic changes, which occur with in vitro culture and derived regenerates. Genetic changes including in recombinant DNA, aneuploidy, polyploidy and cytological abnormalities, have been assessed for regenerants (Bhatia et al. 2011; Borchetia et al. 2009; Kumar et al. 2010a,b). The genetic stability of regenerants compared to their maternal origin have been assessed by Leva and Petruccelli (2012) and Leva et al. (2012). The genetic fidelity of in vitro-propagated plants cannot be detected easily by any current technique (Mallón et al. 2010) and this issue is gaining importance with emphasis on changes at the genetic level. The use of different

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a

b

c

d

Fig. 4.1 Abnormalities in leaf morphology. a Asymmetric growth and pinnae rolling, b Abnormality in the color of pinnae; the plant goes to the yellow-greenish color of leaflets and then later back to green, c Color changes in

some leaflets and/or parts of leaflets, d Asymmetric growth in a single side of leaflets. (Photos by S. F. ElSharabasy and R. M. Rizk)

methodologies is necessary to assess (Peredo et al. 2009) and predict genetic identity with a high probability. Characterization and evaluation using morphological, physiological, and cytological must be employed to profile regenerants. These methods, which are consuming more time and effort, are dependent upon characteristics that can be

affected by the process of in vitro proliferation. As well, it is difficult to predict the probability of genetic-symmetric regenerants or asymmetric regenerants. Exogenous cytokinin changes occur in the global DNA methylation and chromosomal number levels and methylation at specific sites for somatic embryogenesis-derived oil palm

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b

a

c

Fig. 4.2 Abnormalities in in vitro-derived plants show changes occurred in the fruit set. a No fruit set, b Failure to set fruit, c Multiple carpels, compared with a single carpel in the middle. (Photos by S. F. El-Sharabasy and R. M. Rizk)

(Elaeis guineensis Jacq; Jaligot et al. 2004) at a higher rate than derived in date palm. Several biological functions have correlated to 5-methylcytosine in DNA. Due to these, changes in date palm development have resulted from the inhibition of DNA methyltransferase. This enzyme involves DNA methylation in cell programming and differentiation. The type and concentration of growth regulators influence 5methylcytosine. It should determine the level of

genetic variation. Storing in vitro explants should prevent inducing somaclonal variation. It is necessary to develop a reliable diagnostic molecular marker set to detect, as early as possible, derived variations such as SNP, and reveal a temporary immersion system for multiplication and regeneration. Mirani et al. (2020) demonstrated a simple PCR-based method for the screening of date palm somaclones to establish the application of transposon-based DNA markers for identification.

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a

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Fig. 4.3 Abnormalities occurred in the crown. a Asymmetry in young leaves, b Inversion of the crown toward the ground, c Plant vigor is very extremely compact,

referred to as a plunged (immersed) crown. (Photos by S. F. El-Sharabasy and R. M. Rizk)

4.4.4 In Vitro Variant Marker Detection

date palm’s long-life span and a lack of germplasm descriptions, progress in date palm improvement has been slow (Chaluvadi et al. 2014; El-Sharabasy and Rizk 2019; Rizk and ElSharabasy 2008). Mirani et al. (2019) revised phenotypic attributes, derived from immature inflorescence explants during tissue culture of cvs. Kashuwari and Gulistan. Various methodologies detect somaclones, including proteomics (De Carvalho et al. 2014;

To date, markers to detect variants are quite limited, except for morphological attributes which are in use. That may be because they are cheap and easy to use visually. Changes are naturally a product of plant genome plasticity across the mechanism of suitable adaptation to new environmental conditions. As a result of the

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Gurevich et al. 2005; Tan et al. 2016); cytogenetics (Soliman et al. 2009); epigenetics (Ho et al. 2013); and genomics (Ting et al. 2013; Tranbarger et al. 2012). Biochemical approaches were used to differentiate regenerants by Khierallah et al. (2014). The analyses of isozyme and peroxidase did not reflect polymorphism in date palm cultivars (Khierallah et al. 2014). Abnormalities were detected among in vitro plants by Gurevich et al. (2005). Abnormalities in date palm cv. Barhee are shown in Fig. 4.4 (Soliman et al. 2009). Information of variability among cultivars was clearly detected by several DNA markers (Fig. 4.5) involving representational difference analysis (RDA) technology (Vorster et al. 2002); restriction fragment length polymorphism (RFLP); randomly amplified polymorphic DNA (RAPD) (Bader et al. 2007; Khierallah et al. 2014; Modi et al. 2017; Othmani et al. 2010; Saker et al. 2006; Shair et al. 2016); amplified fragment length polymorphism (AFLP) (Rhouma Chatti et al. 2011; Saker et al. 2006), simple sequence repeats (SSR) (Al-Faifi et al. 2016, 2017; Arabnezhad et al. 2012; Chaluvadi et al. 2014, 2019; Elmeer et al. 2019; Hamwieh et al. 2010; Kumar et al. 2010b; Racchi and Camussi 2014, 2018; Salomon-Torres et al. 2017; Yusuf et al. 2015); inter-simple sequence repeats (ISSR) (Abd-Alla 2010; Adawy et al. 2002; Ben Saleh and El-Helaly 2003; Saha et al. 2016; Zehdi

et al. 2004); start codon targeted (SCoT); random amplified microsatellite polymorphism (RAMPO) (Soumaya et al. 2011); and single strand conformational polymorphism (SSCP) (Abd-Alla 2010; Gurevich et al. 2005). Many genes have detected variations through somatic embryogenesis in date palms (Al-Harrasi et al. 2018; Hazzouri et al. 2020; Osorio-Guarín et al. 2019; Rikek et al. 2019). Those include protein receptor-like kinases [somatic embryogenesis receptor kinase (SERK) and CLAVATA1 (CLV1)]; mitogen-activated protein kinase (MAPK); transcription factors [WUSCHEL (WUS), APETALA2/ethylene-responsive factor (AP2/ERF), PICKLE (PKL), AINTEGUMENTA (ANT) and WRKY]; extracellular proteins [arabinogalactan protein (AGP); germin-like protein (GLP); embryogenic cell protein (ECP) and late embryogenesis-abundant protein (LEA)] and glutathione S-transferase (GST). Advanced technologies are now routinely used in date palm, including genome sequencing and SNP (Fu et al. 2017) and transposon-based DNA markers (Mirani et al. 2020). The date palm has a unique metabolism of fruit development and ripening. Therefore, Yang et al. (2010) and Al-Mssallem et al. (2013) exploited large-scale genomic and transcriptomic attributes which have profoundly paved the way for studying the genome within the genus Phoenix. A road map to future studies will further clarify the domestication history of this iconic crop (Gros-

Fig. 4.4 Abnormality in tissue culture-derived cv. Barhee regenerants profiled by SDS-PAGE. M = Marker, 1 = Normal, 2 = Excessive of vegetative growth,

3 = Habituation excessive of vegetative growth, 4 = Dwarfism, 5 = Habituation of dwarfism, and 6 = Apical bent. (Source: Soliman et al. 2009)

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Balthazard et al. (2018). These techniques will in the future shed light on and result in avoiding failure to detect abnormalities in cultures easily, but to help to select what cultivar is the best for in vitro production of true-to-type plants with highinterest quality. The SERK-protein family, as a somatic embryogenesis receptor kinase, is affected by the expression of PhSERKL-gene. The PhSERKL genes depress nonembryogenic tissue rather than embryogenic callus (Rekik et al. 2015), using RACE-PCR during the establishment of date palm somatic embryogenic competence

acquisition and globular embryo formation of in vitro proliferation. The RAPD technique is still reportedly useful for studying genetic variation in date palm and could be relevant to determine genetic instability of in vitro-derived plants at early stages (AlKhalifah and Askari 2007; Ali et al. 2007; Bader et al. 2007; Hamza et al. 2012; Hussam and Khierallah 2013). RDA technology diagnoses variability in regenerates and detects genomic loss, rearrangements, amplification, point mutations, and pathogenic organisms between two genomes.

Fig. 4.5 Polymorphism in in vitro-derived date palm cv. Barhee detected by ISSR profile. M = Marker, 1 = Normal, 2 = Excessive of vegetative growth, 3 = Habituation

excessive of vegetative growth, 4 = Dwarfism, 5 = Habituation of dwarfism, and 6 = Apical bent. (Source: Soliman et al. 2009)

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Previous reviews pointed to a frequency of genetic variation of less than 3% in confirming the genetic stability of in vitro-derived date palms (Ramanovsky 2010). Two thousand genes vary between normal and dwarf expression (Ramanovsky 2010), the most common traits resulting from in vitro-derived date palms. Genes that biosynthesize gibberellins, auxins, expansins, brassinosteroids, and WRKY-transcription factors, expressed differentiation. Around 21 of these genes were verified by quantitative reverse transcriptase PCR. The low genetic variation of variegated (yellow leaf) palms observed by AFLP band patterns (Gurevich et al. 2005) was due to several mutations not easily detected by variegation. Another set of markers, DNA fingerprints, were utilized and applied to Tunisian germplasm. RFLP and RAPD can identify date palm cultivars. The RFLP technique tags the traits and can distinguish date palm populations (Gurevich et al. 2005). The ISSR technique permits the detection of polymorphisms in cultivars of date palms (AbdAlla 2010). ISSR is a useful and powerful technique applied to many species to detect polymorphisms without prior information about the DNA sequences. This technique can successfully identify date palm cultivars (Abd-Alla 2010; Zehdi et al. 2004, 2012). Zehdi et al. (2004, 2012) assessed genetic variability using ISSR among a set of Tunisian date palm cultivars. Abd-Alla (2010) has proved that the ISSR marker is a reliable method to assess genetic stability. On that subject, the absence or loss of polymorphism regenerated in vitro plants may contribute to three hypotheses. First, the generated plants are conservative forms or less prone to genetic changes because of their low gene expression. Second, genetic variation can probably be induced during tissue culture, but the molecular markers used cannot detect the variant. That approach does not employ more molecular techniques to assess genetic variation because the number of primers used does not cover the genome. Finally, epigenetic variations have been induced through date palm tissue culture and

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reviewed by Baránek et al. (2010, 2015) and Ho et al. (2013). Previous studies reported that DNA methylation changes sometimes occur at any phase of in vitro culture (Baránek et al. 2015; Ho et al. 2013). Variants may contribute to epigenetic variations such as DNA methylation (Baránek et al. 2010, 2015); histone modification; and chromatin re-modulation (Vanyushin 2006, 2014; Vanyushin and Ashapkin 2006, 2011) that cannot identify variants by using molecular genetic analysis. Regenerants coming from an off-type, e.g., mantled off-type of oil palm, using the DNAmethylate process, have low levels of fruit set and supernumerary carpels (Jaligot et al. 2014; Ong-Abdullah et al. 2015). Nevertheless, these regenerants go directly for alleviation during their maturation because most are reversible. The dwarfism type of cv. Medjool compared with the normal cultivar is due to their process of DNA methylation. If cytosine is methylated, it affects gene expression (Ong-Abdullah et al. 2015, 2016). The methylation-sensitive amplified polymorphism method can analyze the DNA variability of date palm compared with the mother plants. It may be related to gene expression during offshoot development (Fang and Chao 2006, 2007). Further, no significant difference in DNA methylation was found among normal and mantled clones in the EgDEF1 gene (Jaligot et al. 2014). Therefore, the EgDEF1 genes are involved in identifying mantled phenotypes of oil palm (Ong-Abdullah et al. 2015). Earlier assessment will allow selecting a desirable genotype and avoid the risk of resulting somaclonal variations (Kim et al. 2015).

4.5

Genetic Stability Under Cryopreservation

Cryopreservation is, in general, a new technology with the initial protocols developed in the 1980s, based upon freeze-induced dehydration. In the early 1990s, a new set of vitrificationbased protocols became available (Engelmann 2004, 2009). Because of ultra-low temperature applications such as liquid nitrogen, biological

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specimens can be stored and conserved for an extended period with guaranteed genetic stability. However, molecular damage can still occur at ultra-low temperatures (Engelmann 2009). A more recent technique, encapsulation dehydration, allows exposure of the explants to rigorous treatments. Due to the detrimental or lethal moisture content of explants, the high cryoprotectant and desiccation concentrations should be pre-treated to reduce moisture content. Because of the harm of exposure, protectants must occur during rapid exposure to low temperatures such as liquid nitrogen (Engelmann 2009; Mahdy 2018; Sayed et al. 2017), thus avoiding lethal intracellular ice crystallization. In the dioecious date palm, seed propagation results in variable offspring of either gender, so it is not a feasible method to regenerate true-to-type cultivars (El-Dawayati et al. 2012a, b). Additionally, date palm germplasm is difficult to store for a long period using conventional means and has high potential loss risk. Monitoring the genetic fidelity of cryopreserved specimens in longterm storage is particularly important, especially as the maintenance of archive collections with hundreds of accessions is very expensive (ElDawayati et al. 2012a, b; Engelmann 2009; Jain 2011). Any occurred change not observed in young plants may be expressed later in mature trees. Variability in date palm resulting from genetic or epigenetic changes is a well-known phenomenon of in vitro procedures, and especially with the subcultures of multiplication. Cryogenic applications for date palm germplasm provide good long-term storage and conservation (Jain 2011). It features blocking the thread enzymes and avoids the subculturing limits that threaten genetic and epigenetic changes. Cryogenics is the most appropriate technology and cost-effective method for long-term conservation of date palm genetic resources (Bekheet 2011; Bekheet and Taha 2005), because it does not lead to changes at the morphological, cytological or genetic levels, as evidenced and noted by Harding (2004). Date palm cryopreservation is governed by several factors, i.e., explant size, cryoprotectant concentration, moisture content, and thawing (El-Dawayati

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et al. 2012a, b; Jain 2011). The success of cryopreservation applications depends on the recovery of intact revival plants (El-Dawayati et al. 2012a, b). Minireviews shed light on the difference and significance of additives as cryoprotectants. Nagy (2010) presented a hands-on summary by listing preservation methods used for various tissue and discussed all the approaches used for drying/freezing techniques and fluid/buffered solution and practical aspects. Several cryoprotectants including dimethyl sulfoxide (DMSO) (El-Dawayati et al. 2012a, b); glycerol, polyethylene glycol (PEG) (Fouad 2019; Puente-Garza et al. 2017); sorbitol, mannitol, and sucrose (El-Dawayati et al. 2012a, b; El-Hadrami et al. 2011; Fouad 2019; Jain 2011) have been used to cryopreserve oil palm germplasm. However, few date palm varieties have been minireviewed. There is a need to specify the optimum cryoprotectant components (Ellis et al. 2006) for each genotype. Some genetic instability results from the DMSO (up to 10%) cryoprotectant acted with chromatin and nucleic acids. DMSO cryoprotectant inhibits DNA synthesis, changes the secondary DNA and RNA structure and generates mitotic irregularities. The variation of embryogenic lines is attributed to the callus treatment with 10% DMSO/30 min. No evidence of genetic instability appeared. Cryopreservation eliminates tissue-bearing genetic change. Scientists point to the changes within clonal genetics which found that the observed genetic instability was presumably a result of repeated in vitro subculturing rather than the manipulations of cryopreservation itself. Similar studies of cowpea plants by Sayed et al. (2017) and Mahdy (2018) assessed the effect of DNA integrity at completely controlled humidity under various storage conditions using SSRs and SCoT markers. It is notably, on an agarose gel, that the stored DNA samples with a protectant additive showed no detectable degradation. Without a protectant, conserved samples will be affected by the degradation and damaging of DNA. The success of cryopreservation, which stabilizes germplasm genetically, may develop problems either directly or at the wounded

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surfaces of cells which are prone to endanger the genetic stability of regeneration. Therefore, it must avoid and retard the formation callus at the earliest stages possible. El-Dawayati et al. (2012a, b) reported that the date palm callusprotectant of sucrose at 15 °C/8 mon was effective. The effect of date palm embryogenic culture on cvs. Bartamoda and Sakkoty, conserved at 5 ° C in darkness, was investigated by El-Ashry et al. (2013). The dark condition prevented the proliferation of embryogenic cultures. Genetic stability using RAPD markers revealed that the surviving plantlets were relatively stable genetically. Date palm cv. Bartamoda has a higher similarity than cv. Sakkoty. Another investigation by Alansi et al. (2017) reported that the embryogenic calli-derived plantlets of cvs. Sagai and Khalas treated with liquid nitrogen as a cryopreservation tool for testing genetic fidelity of ISSR showed similarity to their donor plants. There was no evidence of any genetic change on the level of morphological, cytological, biochemical, and molecular techniques. Integrity can indicate that genetic stability is the norm (Harding 2004). This may be due to the decrease of studies in which genetic stability of date palm exists for only a few cultivars, with appropriate genetic marker systems available. Therefore, genetic markers (Abd-Alla 2010; Kumar et al. 2010a,b; Othmani et al. 2010) must employ observations of morphological attributes, transgene integration and expression analysis, isoenzyme markers or cytology analyses (Jokipii et al. 2004; Ryynänen and Aronen 2005).

4.6

Genetic Behavior of in Vitro Plants

Tissue culture, on a large scale, provides the needs of sustainable date palm production (AbulSoad and Mahdi 2010). Date palm in vitro manipulation can produce elite offshoots quickly and help to overcome the low regeneration of a mother plant over a year. The behavior of date palm regenerants is of different importance during in vitro proliferation compared to nursery

establishment. Some abnormalities (Mirani et al. 2019) can be due to environmental and management factors. Date palm variants do occur during in vitro culturing. The variability range within in vitro-derived offshoots is a selective trait controlled by the internal mechanism regulating its growth and evolution, in which the genotype is expressed (Mirani et al. 2019, 2020). Genetic stability during tissue culture is affected by many factors (Al-Khateeb et al. 2019). For instance, errant copies of a gene produced during mitosis, rendered silent by methylation, aberrations in chromosomes, recognition signals at each stage of evolution, and as well the DNA recombination rate can be affected (El-Hadrami et al. 2011; El-Mageid 2019). Additionally, the mechanism of genetic stabilization could score the number of correlations between controlling signals (El-Mageid 2019). DNA stability using RAPD during in vitro proliferation points to the frequency of somaclonal variation that scores at less than 3%, confirming genetic stability (Saker et al. 2006). Genes of gibberellins, auxins, expansins, brassinosteroids, and WRKY-transcription biosynthesis pass in a way that is not the norm or as the variant (Ramanovsky 2010). DNA methylation was observed in the dwarf type of cv. Medjool compared to the normal cultivar (Ong-Abdullah et al. 2015, 2016). Cytosine methylation is an essential attribute in gene expression if it is methylated. The methylationsensitive amplified polymorphism (MSAP) technique analyzes the DNA variability of a date palm compared with the mother plant (Baránek et al. 2010, 2015). This also may relate to gene expression during offshoot development (Fang and Chao 2007, 2019). Another set of markers, DNA amplification fingerprinting, was initially utilized and applied to Tunisian germplasm. RFLP and RAPD markers are exploited frequently in the identification of date palm cultivars. The RFLP marker tags important economic attributes in date palm (Arunachalam 2011). The RFLP technique can detect polymorphism between date palm variants using the cDNA probe of a zinc finger protein motif. These markers can analyze rare and extinct ancient date palms.

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Changes regenerated through in vitro culture are observed. These alterations may be rearrangements at the genetic and epigenetic levels that occur during the intermediate stages in an in vitro plant, including point mutations, mobile element activity, chromosomal structure and number, number of gene copies, and DNA methylation. These changes can result in problems of genetic stability in vitro-regenerated plants and prevent or limit the effectiveness of treatments such as in vitro plantations or genetic transformation (Bairu et al. 2011). The precise mechanisms of these variations are still being debated. Polyploidization comes from chromosomal abnormalities, the fusion of nuclei, endomitosis, and endoreduplication (ElHadrami et al. 2011). The number of chromosomes arising as observed in various date palm cultivars may be a factor. Genetic instability in the oil palm was demonstrated through DNA methylation and specific genes (Fang and Chao 2006; Jaligot et al. 2004, 2014). Cryopreservation causes a decrease in DNA methylation and the activation of silent retrotransposons (Al-Mssallem et al. 2013). Reduced methylated DNA contributes to the instability of the genome that inhibits and blocks the normality of plant growth development throughout in vitro proliferation plants and transgenic plants, which are increasing tenfold in the copy number of retrotransposon families, e.g., Tto1, Tto2, and Tto3 (Al-Mssallem et al. 2013).

4.7

Maintenance of Genomic Stability

As a large-scale plant culture process, micropropagation carries many risks (Jain 2011). Variants may be off-type and not identical genetically to the mother plant. Generally, date palm proliferated in vitro is through two main methods. One, somatic embryogenesis. This is in vitro production of regenerants in a short time and at a high rate of propagation. Variants appear later with the possibility of mutations and abnormalities occurring in growing vitro plants in the nursery and field. Several researchers have

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investigated this subject, including Al-Khayri (2011), Alansi et al. (2018), Hassan et al. (2013), Khierallah and Hussein (2013), and Roshanfekrrad et al. (2017). The second method is organogenesis. This method yields in vitro plantlets highly identical in their genetic and vegetative characteristics to the mother plant (Hegazy and Aboshama 2010; Mazri et al. 2018; Zayed 2017). This method has success in the initiation of vegetative buds (Abahmane 2011). Hence, direct organogenesis lowers the use of growth regulator concentrations and consequently precludes callus formation. Date palm tissue culture by organogenesis has been achieved using various meristematic explants (Al-Khateeb 2006). Direct organogenesis is promising for mass production (Hegazy and Aboshama 2010; Hegazy et al. 2009; Mazri et al. 2018; Zayed 2017). The effects of external factors on multiplication such as growth regulators have been investigated. Meristemic explants are used as an effective and rapid method of micropropagation (Zayed 2017). However, somaclonal variation may seriously result from and limit the broader utility condition of in vitro proliferation (Hegazy and Aboshama 2010; Mazri et al. 2018). Many factors such as explant source, culturing time, number of subcultures, plant growth regulators, genotypes, and media components can induce variations (Hegazy and Aboshama 2010). It is necessary to determine genetic conformity to assess the quality of regenerants and the domain of their commercial usage. In this context, molecular markers can detect variability and confirm genetic fidelity over in vitro micropropagation (Garg et al. 2011). Sources of genetic variation are very numerous. In tissue culture, these variations can result from exposure of explants to exogenous stresses or contribute to the tissue itself mutating. In this context, genetic changes may be generated from the disorder of DNA replication or damaged DNA molecules (Bouwman and Jonkers 2012; Ghosal and Chen 2013). DNA damage pathways are disorganized, arising from mutagenesis and genomic instability (Chatterjee and Walker 2017; Howard et al. 2015; Wolters and Schumacher 2013). DNA damage

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responding (DDR) is a result of promoting tissue to exhibit DNA lesions. DNA repair is a prime active pathway through the cell cycle stages (Chatterjee and Walker 2017). These are (1) base excision repair (BER), (2) nucleotide excision repair (NER), (3) mismatch repair (MMR), (4) homologous recombination (HR), and (5) non-homologous end joining (NHEJ) (Howard et al. 2015; Rybaczek and KowalewiczKulbat 2013). This response requires sufficient time to allow DNA repair of pathways to remove the damage. Damages can be repaired via reversal and interstrand cross-link (ICL) repair (Enoiu et al. 2012; Ho et al. 2011). Repair processes keep tissue genetically stable. Some damage categories are the substrate for DNA damage tolerance pathways (Chatterjee and Walker 2017; Howard et al. 2015). In callogenesis, tissue is activated to remove cells with extensive genome instability and explains the low number of in vitro-derived variants among regenerants. Checkpoints react to various types of DNA damage, e.g., DNA replication and DNA breakage (Ben-Yehoyada et al. 2009; Huang et al. 2010; Wu et al. 2006). Another prime source of change is cytological variation, such as the segregation of unequal chromosomes (El-Hadrami et al. 2011; Puizina et al. 2004). Fortunately, cellular systems have several ways to deal with such agents leading to a modification in DNA constituents. Previous clues pointing to genome instability are probably a cause for formatting carcinogenesis (Ben-Yehoyada et al. 2009; Chatterjee and Walker 2017; Huang et al. 2010; Rybaczek and Kowalewicz-Kulbat 2013; Wu et al. 2006). As well, it may derive from aging, disease, mutation, etc. Therefore, the study of genome instability is of fundamental importance to understand how cells naturally regulate the entirety of their biological processes and conserve the stability of DNA constituents. Minireviews have covered various aspects of mechanisms underlying maintaining genome stability (Hong et al 2010; Lukas et al. 2011). The failure of DNA repair or irreparable DNA damage (Chatterjee and Walker 2017) that can occur is an essential agent for stimulating genome instability. Double-strand breaks can be

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repaired by either homologous recombination or non-homologous end-joining pathways under normal conditions (House et al. 2014; Howard et al. 2015; Price and D’Andrea 2013; Vignard et al. 2013). The gene of somatic embryogenesis receptor kinase-like (SERKL) cDNA, designatedPhSERKL, was isolated using RACE-PCR across date palm tissue culture (Rekik et al. 2015). This type of protein shares the whole of the SERK-family characteristic domains involving five leucine-rich repeats, one proline-rich region motif, a transmembrane domain, and kinase domains. The authors proposed that the level of PhSERKL expression is lower in nonembryogenic tissue and organs than in embryogenic callus.

4.8

Conclusions and Prospects

Several recent methods of date palm micropropagation have shown progress. However, the fundamental challenge of in vitro production is regenerants bearing a variable attribute, viz donors. Note that new technologies, e.g., genome editing, represent the next generation of the Green Revolution, which allows the generation of new promising varieties bearing a desirable genetic characteristic. The risk of genetic instability through tissue culture is very high. Applying somaclonal variations will increase undoubled exploitation. They are beneficial tools to rescue or recover materials of interest in which the mechanism of complex polygenic traits is still unknown. Genetic fidelity can be screened using various approaches. The use of molecular markers to assess the genetic conformity of regenerants may be inadequate. No further documentation is available marking progress on the genetic integrity of regenerants and preservatives, the loss of date palm genetic resources and no instance of the use of available domesticated Phoenix and other species. Therefore, we hope that more research is conducted on date palm genetic studies by applying novel technologies to detect in vitro regenerants. These may suggest that the number

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of molecular markers used should be increased to achieve more precise consequences. Moreover, it should lead to new technologies that are low in cost and provide rapid detection.

References Abahmane L (2011) Date palm micropropagation via organogenesis. In: Jain SM, Al-Khayri JM, Johnson DV (eds) Date palm biotechnology. Springer, Dordrecht, pp 69–90 Abd-Alla MM (2010) Genetic stability on Phoenix dactylifera var karama produced in vitro. N Y Sci J 3:70–75 Abdelaziz AM, Soliman SSA, Ahmed TAS et al (2019) Micropropagation of zaghlol and barhy date palm cultivars using immature female inflorescence explants: effect of growth regulators balance. Zagazig J Agric Res 46(6A):2023–2035. https://doi. org/10.21608/zjar.2019.51922 Abo El Fadl R (2014) effect of silicon on somatic embryogenesis and shoot of dry date palm (Phoenix dactylifera L.) cv. bartamuda Egypt J Desert Res 64:65–82. https://doi.org/10.21608/ejdr.2014.5810 Abul-Soad AA, Emara KS, Abdallah AS, Mahdi SM (2017) Somatic embryogenesis in Phoenix dactylifera L. using floral bud explants. Acta Hort 13–28. https:// doi.org/10.17660/ActaHortic.2017.1187.2 Abul-Soad AA, Mahdi SM (2010) Commercial production of tissue culture date palm (Phoenix dactylifera L.) by inflorescence technique. J Genet Eng Biotech 8:39–44 Adawy S, Hussein E, El-Khishin D et al (2002) Genetic variability studies and molecular fingerprinting of some Egyptian date palm (Phoenix dactylifera L.) cultivars: II- RAPD and ISSR profiling. Arab J Biotech 5(2):225–236 Al Kaabi HH, Zaid A, Ainsworth C (2007) Plant-off-types in tissue culture-derived date palm (Phoenix dactylifera L) plants. Acta Hort 736:267–281 Alansi S, Al-Qurainy F, Khan S et al (2017) Genetic fidelity testing in regenerated plantlets of cryopreserved and non-cryopreserved cultivars of Phoenix dactylifera L. Pak J Bot 49:2313–2320 Alansi S, Al-Qurainy F, Nadeem M et al (2018) Efficient micropropagation via somatic embryogenesis of potential cultivar sagai of Phoenix dactylifera L. Pak J Bot 50(6):2251–2258 Al-Dous EK, George B, Al-Mahmoud ME et al (2016) De novo genome sequencing and comparative genomics of date palm (Phoenix dactylifera). Nat Biotech 29:521–527 Al-Faifi SA, Migdadi HM, Algamdi SS et al (2016) Development, characterization and use of genomic SSR markers for assessment of genetic diversity in some Saudi date palm (Phoenix dactylifera L.)

95 cultivars. Elect J Biotech 21:18–25. https://doi.org/ 10.1016/j.ejbt.2016.01.006 Al-Faifi SA, Migdadi HM, Algamdi SS et al (2017) Development of genomic simple sequence repeats (SSR) by enrichment libraries in date palm. In: AlKhayri JM, Jain SM, Johnson DV (eds) Date palm biotechnology protocols Vol II. Humana Press, New York, pp 315–337. https://doi.org/10.1007/978-14939-7159-6_24 Al-Harrasi I, Al-Yahyai R, Yaish MW (2018) Differential DNA methylation and transcription profiles in date palm roots exposed to salinity. PLoS ONE 13: https:// doi.org/10.1371/journal.pone.0191492 Ali TA, Jubrail JM, Jasim AM (2007) The use of RAPDs for the detection of genetic stability of regenerated plantlets of barhee palm in Iraq. Acta Hort 736:127– 134. https://doi.org/10.17660/ActaHortic.2007.736.11 Al-Khalifah NS, Askari E (2007) Early detection of genetic variation in date palms propagated from tissue culture and offshoots by DNA fingerprinting. Acta Hort 736:105–112. https://doi.org/10.17660/ ActaHortic.2007.736.8 Al-Khateeb AA (2006) Role of cytokinin and auxin on the multiplication stage of date palm (Phoenix dactylifera L.) cv. sukary. Biotech 5:349–352. https://doi.org/10.3923/biotech.2006.349.352 Al-Khateeb SA, Al-Khateeb AA, Sattar MN et al (2019) Assessment of somaclonal variation in salt-adapted and non-adapted regenerated date palm (Phoenix dactylifera L). Fresen Environ Bull 28(5):3686–3695 Al-Khayri JM (2010) Somatic embryogenesis of date palm (Phoenix dactylifera L.) improved by coconut water. Biotech (Faisalabad) 9:477–484. https://doi. org/10.3923/biotech.2010.477.484 Al-Khayri JM (2011a) Basal salt requirements differ according to culture stage and cultivar in date palm somatic embryogenesis. Amer J Biochem Biotechnol 7:32–42. https://doi.org/10.3844/ajbbsp.2011.32.42 Al-Khayri JM (2011b) Influence of yeast extract and casein hydrolysate on callus multiplication and somatic embryogenesis of date palm (Phoenix dactylifera L.). Sci Hort 130:531–535. https://doi. org/10.1016/j.scienta.2011.07.024 Al-Khayri JM, Al-Bahrany AM (2012) Effect of abscisic acid and polyethylene glycol on the synchronization of somatic embryo development in date palm (Phoenix dactylifera L.). Biotech 11:318–325. https://doi.org/ 10.3923/biotech.2012.318.325 Al-Mssallem IS, Hu S, Zhang X et al (2013) Genome sequence of the date palm Phoenix dactylifera L. Nat Comm 4:2274. https://doi.org/10.1038/ncomms3274 Al-Wasel ASA (2001) Phenotypic comparison of tissue culture-derived and conventionally propagated date palm (Phoenix dactylifera L.) cv. barhee trees. I. vegetative characteristics. J King Saud Univ 13:65–73 Arabnezhad H, Bahar M, Mohammadi HR, Latifian M (2012) Development, characterization and use of microsatellite markers for germplasm analysis in date palm (Phoenix dactylifera L.). Scient Hort 134:150– 156. https://doi.org/10.1016/j.scienta.2011.11.032

96 Arunachalam V (2011) Date palm. In: Arunachalam V (ed) Genomics of cultivated palms. Elsevier Inc, Amsterdam, pp 49–59 Bader SM, Baum M, Khierallah HSM, Choumane W (2007) The use of RAPDs technique for the detection of genetic stability of date palm plantlets derived from in vitro culture of inflorescence. J Edu Sci 20(2):149–159 Baharan E, Mohammadi PP (2018) Induction of direct somatic embryogenesis and callogenesis in date palm (Phoenix dactylifera L.) using leaf explants. BioTech 99(3):197–203. https://doi.org/10.5114/bta.2018.77480 Bairu MW, Aremu AO, Van Staden J (2011) Somaclonal variation in plants: causes and detection methods. Plant Growth Reg 63:147–173. https://doi.org/10. 1007/s10725-010-9554-x Baránek M, Čechová J, Raddová J et al (2015) Dynamics and reversibility of the DNA methylation landscape of grapevine plants (Vitis vinifera) stressed by in vitro cultivation and thermotherapy. PLoS ONE 10: https:// doi.org/10.1371/journal.pone.0126638 Baránek M, Křižan B, Ondrušíková E, Pidra M (2010) DNA-methylation changes in grapevine somaclones following in vitro culture and thermotherapy. Plant Cell Tiss Organ Cult 101:11–22. https://doi.org/10. 1007/s11240-009-9656-1 Bekheet SA (2011) In vitro conservation of date palm germplasm. In: Jain SM, Al-Khayri JM, Johnson DV (eds) Date palm biotechnology. Springer, Netherlands, Dordrecht, pp 337–360 Bekheet SA, Taha HS, El-Bahr MK (2005) Preservation of date palm cultures using encapsulated somatic embryos. Arab J Biotech 8:319–328 Ben Saleh M, El-Helaly R (2003) Using ISSR to investigate the similarities and dissimilarities between some Tunisian date palm (Phoenix dactylifera L.) cultivars. In: International Conference on Date Palm. King Saud University, Qaseem, Kingdom of Saudi Arabia, pp 16–19 Ben-Yehoyada M, Wang LC, Kozekov ID et al (2009) Checkpoint signaling from a single DNA interstrand crosslink. Mol Cell 35(5):704–715 Bhatia R, Singh KP, Sharma TR, Jhang T (2011) Evaluation of the genetic fidelity of in vitropropagated gerbera (Gerbera jamesonii Bolus) using DNA-based markers. Plant Cell Tiss Organ Cult 104:131–135. https://doi.org/10.1007/s11240-0109806-5 Borchetia S, Das SC, Handique PJ, Das S (2009) High multiplication frequency and genetic stability for commercialization of the three varieties of micropropagated tea plants (Camellia spp.). Sci Hort 120:544– 550. https://doi.org/10.1016/j.scienta.2008.12.007 Bouguedora N, Si-Dehbi F, Fergani K et al (2017) Somatic embryogenesis in date palm (Phoenix dactylifera L.) “deglet nur” and “takerbucht” cultivars. Int J Plant Rep Biol 9(1):43–48. https://doi.org/10. 14787/ijprb.2017 9.1.43-48 Bouwman P, Jonkers J (2012) The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance. Nat Rev Canc 12(9):587–598

S. F. El-Sharabasy et al. Chaluvadi SR, Khanam S, Aly MA, Bennetzen JL (2014) Genetic diversity and population structure of native and introduced date palm (Phoenix dactylifera) germplasm in the United Arab Emirates. Trop Plant Biol 7(1):30–41. https://doi.org/10.1007/s12042-0149135-7 Chaluvadi SR, Young P, Thompson K et al (2019) Phoenix phylogeny, and analysis of genetic variation in a diverse collection of date palm (Phoenix dactylifera) and related species. Plant Divers 41:330–339. https://doi.org/10.1016/j.pld.2018.11.005 Chatterjee N, Walker GC (2017) Mechanisms of DNA damage, repair and mutagenesis. Environ Mol Mutagen 58(5):235–263. https://doi.org/10.1002/em.22087 De Carvalho SR, Carmo LST, Luis ZG et al (2014) Proteomic identification of differentially expressed proteins during the acquisition of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.). J Proteom 104:112–127 El-Ashry AA, Shaltout AD, El-Bahr MK et al (2013) In vitro preservation of embryogenic cultures of two Egyptian dry date palm cultivars at darkness and low temperature conditions. J Appl Sci Res 9:1985–1992 El-Dawayati MM, Abd El-Bar OH, Zaid ZE, Zein El-Din AFM (2012a) In vitro morpho-histological studies of newly developed embryos from abnormal malformed embryos of date palm cv. gundila under desiccation effect of polyethylene glycol treatments. Ann Agri Sci 57(2):117–128. https://doi.org/10.1016/j.aoas.2012. 08.005 El-Dawayati MM, Zaid ZE, El-Sharabasy SF (2012b) Effect of conservation on steroids contents of callus explants of date palm cv. sakkoti. Aust J Basic Appl Sci 6(5):305–310 El-El-Hadrami A, Daayf F, Elshibli S et al (2011) Somaclonal variation in date palm. In: Jain SM, AlKhayri JM, Johnson DV (eds) Date palm biotechnology. Springer, Netherlands, Dordrecht, pp 183–203 Ellis D, Skogerboe D, Andre C et al (2006) Implementation of garlic cryopreservation techniques in the national plant germplasm system. Cryo Lett 27:99– 106 El-Mageid IS (2019) Evaluation of genetic stability by using protein and ISSR markers during callus development stage of some date palm (Phoenix dactylifera L.) cultivars under effect of 2,4-D and picloram. Middle East J Appl Sci 9(2):483–493 Elmeer K, Mattat I, Malki AA et al (2019) Assessing genetic diversity of shishi date palm cultivars in Saudi Arabia and Qatar using microsatellite markers. Int J Hort Sci Tech 6:1–9. https://doi.org/10.22059/ijhst. 2019.271662.267 El-Sharabasy S, Rizk R (2019) Atlas of date palm in Egypt. FAO, Egypt El-Sharabasy SF, Wanas WH, Al-Kerdany AY (2008) Date palm in vitro screening to drought tolerance using isozymes. Arab J Biotech 11:263–272 El-Sharabasy SS, Rizk RM (2005) Morphological diversity of date palm (Phoenix dactylifera L.) in Egypt soft date palm cultivars. Mans Hort J 30(11)7001–7027

4

Genome Conformity of In Vitro Cultures of Date Palm

Engelmann F (2004) Plant cryopreservation: progress and prospects. In vitro Cell Dev Biol Plant 40:427–433. https://doi.org/10.1079/IVP2004541 Engelmann F (2009) 3. Plant germplasm cryopreservation: progress and prospects. Cryobiol 59:370–371. https://doi.org/10.1016/j.cryobiol.2009.10.017 Enoiu M, Jiricny J, Scharer OD (2012) Repair of cisplatin-induced DNA interstrand crosslinks by a replication-independent pathway involving transcription-coupled repair and translesion synthesis. Nucl Acids Res 40(18):8953–8964 Eshraghi P, Zarghami R, Ofoghi H (2005) Genetic stability of micropropagated plantlets in date palm. J Sci 16:311–315 Fang J, Chao CT (2006) Methylation sensitive amplification polymorphism in date palms and their offshoots. Hort Sci 41:994A–994. https://doi.org/10. 21273/HORTSCI.41.4.994A Fang J-G, Chao CT (2007) Methylation-sensitive amplification polymorphism in date palms (Phoenix dactylifera L.) and their off-shoots. Plant Biol 9:526–533. https://doi.org/10.1055/s-2007-964934 Fang Y, Wu H, Zhang T et al (2012) A complete sequence and transcriptomic analyses of date palm (Phoenix dactylifera L.) mitochondrial genome. PLoS One 7(5):e37164 Fouad HAM (2019) effect of some treatments to improve growth of tissue culture plantlets of date palm at the rooting stage and acclimatization stage. PhD thesis, Faculty of Agriculture, Cairo University, Egypt Fu Y, Yang M, Zeng F, Biligetu B (2017) Searching for accurate marker-based prediction of an individual quantitative trait in molecular plant breeding. Front Plant Sci 8:1182 Garg R, Patel RK, Jhanwar S et al (2011) Gene discovery and tissue-specific transcriptome analysis in chickpea with massively parallel pyrosequencing and web resource development (WOA). Plant Phys 156:1661– 1678 Ghazzawy H, El-Sharabasy SF (2019) Effect of natural additives as coconut milk on the shooting and rooting media of in vitro barhi date palm (Phoenix dactylifera L.). In: By-products of palm trees and their applications. Mater Res Proc 11:186–192. http://doi.org/10. 21741/9781644900178-13 Ghosal G, Chen J (2013) DNA damage tolerance: a double-edged sword guarding the genome. Trans Canc Res 2(3):107–129 Gros-Balthazard M, Hazzouri K, Flowers J (2018) Genomic insights into date palm origins. Genes 9:502. https://doi.org/10.3390/genes9100502 Gueye B, Morcillo F, Collin M et al (2009) Acquisition of callogenic capacity in date palm leaf tissues in response to 2,4-D treatment. Plant Cell Tiss Organ Cult 99:35–45. https://doi.org/10.1007/s11240-0099573-3 Gurevich V, Lavi U, Cohen Y (2005) Genetic variation in date palms propagated from offshoots and tissue culture. J Amer Soc Hort Sci 130:46–53. https://doi. org/10.21273/JASHS.130.1.46

97 Hamwieh A, Farah J, Moussally S et al (2010) Development of 1000 microsatellite markers across the date palm (Phoenix dactylifera L.) genome. Acta Hort 269–277. https://doi.org/10.17660/ActaHortic.2010. 882.29 Hamza H, Benabderrahim MA, Elbekkay M et al (2012) Investigation of genetic variation in Tunisian date palm (Phoenix dactylifera L.) cultivars using ISSR marker systems and their relation with fruit characteristics. Turk J Biol 36:449–458. https://doi.org/10. 3906/biy-1107-12 Harding K (2004) Genetic integrity of cryopreserved plant cells: a review. Cryo Lett 25:3–22 Hassan MM, Ibrahim IA, Ebrahim MKH, Komor E (2013) Improvement of somatic embryogenesis and plant regeneration of seven date palm (Phoenix dactylifera L.) cultivars: effect of cytokinins and activated charcoal. J Appl Hort 15(1):26–31. https:// doi.org/10.37855/jah.2013.v15i01.05 Hazzouri KM, Flowers JM, Nelson D et al (2020) Prospects for the study and improvement of abiotic stress tolerance in date palms in the post-genomics era. Front Plant Sci 11:293. https://doi.org/10.3389/fpls. 2020.00293 Hazzouri KM, Flowers JM, Visser HJ et al (2015) Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop. Nat Comm 6:8824. https://doi.org/10.1038/ncomms9824 Hegazy A, Nasr MI, Ibrahim IA, El-Bastawissy HH (2009) Micropropagation of date palm cv. malakaby through somatic embryogenesis 3-effect of tryptone, yeast extract, casein hydrolysate and pineapple extract. J Agric Chem Biotech 34:1629–1644. https://doi.org/ 10.21608/jacb.2009.90293 Hegazy AE, Aboshama HM (2010) An efficient novel pathway discovered in date palm micropropagation. Acta Hort 882:167–176. https://doi.org/10.17660/ ActaHortic.2010.882.18 Ho TV, Guainazzi A, Derkunt SB et al (2011) Structuredependent bypass of DNA interstrand crosslinks by translesion synthesis polymerases. Nucl Acids Res 39 (17):7455–7464 Ho WK, Ooi SE, Mayes S et al (2013) Methylation levels of a novel genetic element, EgNB3 as a candidate biomarker associated with the embryogenic competency of oil palm. Tree Genet Genom 9(4):1099–1107 Hong JP, Byun MY, An K et al (2010) OsKu70 is associated with developmental growth and genome stability in rice. Plant Phys 152:374–387 House NC, Koch MR, Freudenreich CH (2014) Chromatin modifications and DNA repair: beyond doublestrand breaks. Front Genet 5:296 Howard SM, Yanez DA, Stark JM (2015) DNA damage response factors from diverse pathways, including DNA crosslink repair, mediate alternative end joining. PLoS Genet 11(1): Huang M, Kim JM, Shiotani B et al (2010) The FANCM/FAAP24 complex is required for the DNA interstrand crosslink-induced checkpoint response. Mol Cell 39(2):259–268

98 Hussam SM, Khierallah NHH (2013) The role of coconut water and casein hydrolysate in somatic embryogenesis of date palm and genetic stability detection using RAPD markers. Res Biotech 4:20–28 Jain SM (2007) Recent advances in date palm tissue culture and mutagenesis. Acta Hort 736:205–211 Jain SM (2011) Prospects of in vitro conservation of date palm genetic diversity for sustainable production. Emir J Food Agric 23:110–119 Jain SM (2012) Date palm biotechnology: current status and prospective-an overview. Emir J Food Agric 24:386–399 Jaligot E, Beulé T, Baurens F-C et al (2004) Search for methylation-sensitive amplification polymorphisms associated with the “mantled” variant phenotype in oil palm (Elaeis guineensis Jacq.). Genome 47:224– 228. https://doi.org/10.1139/g03-085 Jaligot E, Hooi WY, Debladis E et al (2014) DNA methylation and expression of the EgDEF1 gene and neighboring retrotransposons in mantled somaclonal variants of oil palm. PLoS ONE 9: Jokipii S, Ryynänen L, Kallio PT et al (2004) A cryopreservation method maintaining the genetic fidelity of a model forest tree, Populus tremula L.  Populus tremuloides Michx. Plant Sci 166:799–806. https://doi.org/10.1016/j.plantsci.2003. 11.017 Jombart T, Ahmed I (2011) Adegenet 1.3–1: new tools for the analysis of genome-wide SNP data. Bioinform 27:3070–3071. https://doi.org/10.1093/bioinformatics/ btr521 Khierallah HSM, Al-Sammarraie SKI, Mohammed HI (2014) Molecular characterization of some Iraqi date palm cultivars using RAPD AND ISSR markers. Asian J Sci Res 4:490–503 Kim H, Kim S-T, Kim S-G, Kim J-S (2015) Targeted genome editing for crop improvement. Plant Breed Biotech 3:283–290. https://doi.org/10.9787/PBB. 2015.3.4.283 Krishna H, Alizadeh M, Singh D et al (2016) Somaclonal variations and their applications in horticultural crops improvement. 3 Biotech 6:54. https://doi.org/10.1007/ s13205-016-0389-7 Kumar N, Modi AR, Singh AS et al (2010a) Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay. Phys Mol Biol Plants 16(2):207–213 Kumar N, Singh AS, Modi AR et al (2010b) Genetic stability studies in micropropagated date palm (Phoenix dactylifera L.) plants using microsatellite marker. J For Environ Sci 26(1):31–36 Kurup S, Aly M, Lekshmi G, Tawfik N (2014) Rapid in vitro regeneration of date palm (Phoenix dactylifera L.) cv. kheneizi using tender leaf explant. Emir J Food Agric 26:539–543. https://doi.org/10.9755/ejfa.v26i6. 18051 Leva AR, Petruccelli R (2012) Monitoring of cultivar identity in micropropagated olive plants using RAPD and ISSR markers. Biol Plant 56:373–376. https://doi. org/10.1007/s10535-012-0102-6

S. F. El-Sharabasy et al. Leva AR, Petruccelli R, Rinaldi LMR (2012) Somaclonal variation in tissue culture: a case study with olive. In: Leva A, Rinaldi L (eds) Recent advances in plant in vitro culture. IntechOpen, London, UK pp:123– 150. http://dx.doi.org/10.5772/50367 Lukas J, Lukas C, Bartek J (2011) More than just a focus: the chromatin response to DNA damage and its role in genome integrity maintenance. Nat Cell Biol 13 (10):1161–1169 Mahdy EMB (2018) Genetical studies on DNA storage and preservation on some accessions of cowpea plant. PhD thesis, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt Mallón R, Rodríguez-Oubiña J, González ML (2010) In vitro propagation of the endangered plant Centaurea ultreiae: assessment of genetic stability by cytological studies, flow cytometry and RAPD analysis. Plant Cell Tiss Organ Cult 101:31–39. https://doi.org/10.1007/ s11240-009-9659-y Mazri MA, Belkoura I, Meziani R et al (2017) Somatic embryogenesis from bud and leaf explants of date palm (Phoenix dactylifera L.) cv. najda. 3 Biotech 7:58. https://doi.org/10.1007/s13205-017-0676-y Mazri MA, Meziani R, Belkoura I et al (2018) A combined pathway of organogenesis and somatic embryogenesis for an efficient large-scale propagation in date palm (Phoenix dactylifera L.) cv. mejhoul. 3 Biotech 8:215. https://doi.org/10.1007/s13205-0181235-x Mirani AA, Teo CH, Abul-Soad AA et al (2019) Phenotypic reversion of somaclonal variants derived from inflorescence of date palm (Phoenix dactylifera L.) in the open field trials. Sarhad J Agri 35:719–726. https://doi.org/10.17582/journal.sja/2019/35.3.719. 726 Mirani AA, Teo CH, Markhand GS (2020) Detection of somaclonal variations in tissue cultured date palm (Phoenix dactylifera L.) using transposable elementbased markers. Plant Cell Tiss Org Cult 141:119–130. https://doi.org/10.1007/s11240-020-01772-y Modi A, Gajera B, Subhash N, Kumar N (2017) Evaluation of clonal fidelity of micropropagated date palm by random amplified polymorphic DNA (RAPD) In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date palm biotechnology protocols. Vol. 2. Humana Press, NY, pp 81–89. https://doi.org/10.1007/978-1-49397159-6_8 Murashige T (1974) Plant propagation through tissue cultures. Ann Rev Plant Physiol 25:135–166. https:// doi.org/10.1146/annurev.pp.25.060174.001031 Naganeeswaran S, Fayas TP, Rajesh MK (2020) Dataset of transcriptome assembly of date palm embryogenic calli and functional annotation. Data Brief 31: https:// doi.org/10.1016/j.dib.2020.105760 Nagy ZT (2010) A hands-on overview of tissue preservation methods for molecular genetic analyses. Org Divers Evol 10:91–105. https://doi.org/10.1007/ s13127-010-0012-4 Ong-Abdullah M, Ordway JM, Jiang N et al (2015) Loss of karma transposon methylation underlies the

4

Genome Conformity of In Vitro Cultures of Date Palm

mantled somaclonal variant of oil palm. Nature 525:533–537 Ong-Abdullah M, Ordway JM, Jiang N et al (2016) Tissue culture and epigenetics. Planter 92:741–749 Osorio-Guarín JA, Garzón-Martínez GA, DelgadilloDuran P et al (2019) Genome-wide association study (GWAS) for morphological and yield-related traits in an oil palm hybrid (Elaeis oleifera x Elaeis guineensis) population. BMC Plant Biol 19:533. https://doi. org/10.1186/s12870-019-2153-8 Othmani A, Rhouma S, Bayoudh C et al (2010) Regeneration and analysis of genetic stability of plantlets as revealed by RAPD and AFLP markers in date palm (Phoenix dactylifera L.) cv. deglet nour. Int Res J Plant Sci 1:48–55 Panis B, Piette B, Andre E et al (2011) Droplet vitrification: the first generic cryopreservation protocol for organized plant tissues? Int Symp Cryopreserv Hortic Species 908:157–163 Peredo EL, Arroyo-García R, Revilla MÁ (2009) Epigenetic changes detected in micropropagated hop plants. J Plant Phys 166:1101–1111. https://doi.org/10.1016/ j.jplph.2008.12.015 Price BD, D’Andrea AD (2013) Chromatin remodeling at DNA double-strand breaks. Cell 152(6):1344–1354 Puente-Garza CA, Meza-Miranda C, Ochoa-Martínez D, García-Lara S (2017) Effect of in vitro drought stress on phenolic acids, flavonols, saponins, and antioxidant activity in Agave salmiana. Plant Physiol Biochem 115:400–407. https://doi.org/10.1016/j.plaphy.2017. 04.012 Puizina J, Siroky J, Mokros P et al (2004) Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11dependent genome fragmentation during meiosis. Plant Cell 16:1968–1978 Racchi M, Bove A, Turchi A et al (2014) Genetic characterization of Libyan date palm resources by microsatellite markers. 3 Biotech 4:21–32 Racchi ML, Camussi A (2014) Date palms of Al-Jufrah oasis: genetic fingerprinting of local cultivars and pollinators. In: Dies palmarum, Sanremo, 5– 7/12/2013, Centro Palme Sanremo, pp. 1–9 Racchi ML, Camussi A (2018) The date palms of Al Jufrah-Libya: a survey on genetic diversity of local varieties. J Agric Envir Intern Dev 122(1):161–184 Ramanovsky A (2010) Molecular analysis of somaclonal variants in date palm (Phoenix dactylifera L.). BarIlan University. Ramat Gan, Israel Rathore MS, Patel PR, Siddiqui SA (2020) Callus culture and plantlet regeneration in date palm (Phoenix dactylifera L.): an important horticultural cash crop for arid and semi-arid horticulture. Physiol Mol Biol Plants 26(2):391–398. https://doi.org/10.1007/s12298019-00733-w Rekik I, Drira N, Grubb CD, Elleuch A (2015) Molecular characterization and evolution studies of a SERK like gene transcriptionally induced during somatic embryogenesis in Phoenix dactylifera L. cv. deglet nour. Genetika 47:323–337

99 Rhouma Chatti S, Baraket G, Dakhlaoui Dkhil S et al (2011) Molecular research on the genetic diversity of Tunisian date palm (Phoenix dactylifera L.) using the random amplified microsatellite polymorphism (RAMPO) and amplified fragment length polymorphism (AFLP) methods. Afr J Biotech 10:10352– 10365. https://doi.org/10.5897/AJB10.2242 Rikek I, Chaâbene Z, Kriaa W et al (2019) Transcriptome assembly and abiotic related gene expression analysis of date palm reveal candidate genes involved in response to cadmium stress. Compa Biochem Physiol Part C Toxicol Pharmacol 225: https://doi.org/10. 1016/j.cbpc.2019.108569 Rizk RM, El-Sharabasy SF (2008) A descriptor for date palm (Phoenix dactylifera l.) characterization and evaluation in gene banks. Plant Genet Res Newsl 150:42–44 Roshanfekrrad M, Zarghami R, Hassani H et al (2017) Effect of AgNO3 and BAP on root as a novel explant in date palm (Phoenix dactylifera cv. medjool) somatic embryogenesis. Pak J Biol Sci. 20(1):20–27. https://doi.org/10.3923/pjbs.2017.20.27 Rybaczek D, Kowalewicz-Kulbat M (2013) Relation of the types of DNA damage to replication stress and the induction of premature chromosome condensation. In: Chen C (ed) New research directions in DNA repair. Intech Open, pp 231–248. https://doi.org/10.5772/ 54020 Ryynänen L, Aronen T (2005) Genome fidelity during short- and long-term tissue culture and differentially cryostored meristems of silver birch (Betula pendula). Plant Cell Tiss Organ Cult 83:21–32. https://doi.org/ 10.1007/s11240-005-3396-7 Saha S, Adhikari S, Dey T, Ghosh P (2016) RAPD and ISSR based evaluation of genetic stability of micropropagated plantlets of Morus alba L. variety S-1. Meta Gene 7:7–15. https://doi.org/10.1016/j.mgene. 2015.10.004 Saker MM, Adawy SS, Mohamed AA, El-Itriby HA (2006) Monitoring of cultivar identity in tissue culture-derived date palms using RAPD and AFLP analysis. Biol Plant 50:198é204. https://doi.org/10. 1007/s10535-006-0007-3 Salomon-Torres R, Ortiz-Uribe N, Villa-Angulo C et al (2017) Assessment SSR markers used in analysis of genetic diversity of date palm (Phoenix dactylifera L.). Plant Cell Biotech Mol Biol 18:269–280 Saptari RT, Sumaryono (2018) Somatic embryogenesis from shoot tip of date palm (Phoenix dactylifera L.). E-Journal Menara Perkebunan. 86(2):81–90. https:// doi.org/10.22302/iribb.jur.mp.v86i2.313 Sayed AIH, El-Shaer HFA, El-Halwagi A, Mahdy EMB (2017) Comparative study of DNA preservation under various conditions on local Egyptian cowpea germplasm. Int J Life Sci Pharma Res 3:64–70. https://doi. org/10.25141/2471-6782-2017-5.0064 Senula A, Büchner D, Keller ER, Nagel M (2018) An improved cryopreservation protocol for Mentha spp. Based on Pvs3 as the cryoprotectant. Cryo Lett 39:345–353

100 Sghaier-Hammami B, Jorrín-Novo JV, Gargouri-Bouzid R, Drira N (2010) Abscisic acid and sucrose increase the protein content in date palm somatic embryos, causing changes in 2-DE profile. Phytochem 71:1223– 1236. https://doi.org/10.1016/j.phytochem.2010.05. 005 Shair OH, Askari E, Khan PR (2016) Genetic and anatomical analysis of normal and abnormal flowers of date palm cultivar ‘barhy’ derived from offshoot and tissue culture. Pak J Bot 48(3):1061–1065 Skirvin RM, Mcpheeters KD, Norton M (1994) Sources and frequency of somaclonal variation. HortSci 29:1232–1237 Solangi N, Abul-Soad AA, Markhand GS et al (2020) Comparison among different auxins and cytokinins to induce date palm (Phoenix dactylifera L.) somatic embryogenesis from floral buds. Pakist J Bot 52:1243–1249. https://doi.org/10.30848/PJB2020-4 (30) Soliman KA, Rizk RM, El-Sharabasy SS (2009) Genetic analysis of abnormalities in tissue culture-derived date palm (Phoenix dactylifera L.), barhi cultivars. Egypt J Biotech 33:122–134 Soumaya RC, Ghada B, Sonia DD et al (2011) Molecular research on the genetic diversity of Tunisian date palm (Phoenix dactylifera L.) using the random amplified microsatellite polymorphism (RAMPO) and amplified fragment length polymorphism (AFLP) methods. Afr J Biotech 10(51):10352–10365 Tan HS, Liddell S, Ong-Abdullah M et al (2016) Differential proteomic analysis of embryogenic lines in oil palm (Elaeis guineensis Jacq.). J Proteom 143:334–345 Ting NC, Jansen J, Nagappan J et al (2013) Identification of QTLs associated with callogenesis and embryogenesis in oil palm using genetic linkage maps improved with SSR markers. PLoS ONE 8(1): Tranbarger TJ, Kluabmongkol W, Sangsrakrul D et al (2012) SSR markers in transcripts of genes linked to post-transcriptional and transcriptional regulatory functions during vegetative and reproductive development of Elaeis guineensis. BMC Plant Biol 12:1 Uddin SN, Titov S (2007) Somatic embryogenesis of Musa sp. cv. kanthali using floral bud explants. J Plant Sci 2(1):35–44. https://doi.org/10.3923/jps.2007.35.44 Unamba CIN, Nag A, Sharma RK (2015) Next generation sequencing technologies: the doorway to the unexplored genomics of non-model plants. Front Plant Sci 6:1074 Vanyushin BF (2006) DNA methylation in plants. In: Doerfler W, Böhm P (eds) DNA methylation: basic mechanisms. Springer-Verlag, Berlin, pp 67–122 Vanyushin BF (2014) Epigenetics today and tomorrow. Russ J Genet Appl Res 4:168–188. https://doi.org/10. 1134/S2079059714030083 Vanyushin BF, Ashapkin VV (2006) DNA methylation in plants. Curr Top Microbio Immun 301:67–122. https://doi.org/10.1007/3-540-31390-7_4

S. F. El-Sharabasy et al. Vanyushin BF, Ashapkin VV (2011) DNA methylation in higher plants: past, present and future. Biochim Biophys Acta Gene Regul Mech 1809:360–368. https://doi.org/10.1016/j.bbagrm.2011.04.006 Vignard J, Mirey G, Salles B (2013) Ionizing-radiation induced DNA double-strand breaks: a direct and indirect lighting up. Radiother Oncol 108(3):362–369 Vorster B, Kunert K, Cullis C (2002) Use of representational difference analysis for the characterization of sequence differences between date palm varieties. Plant Cell Rep 21:271–275. https://doi.org/10.1007/ s00299-002-0501-9 Wolters S, Schumacher B (2013) Genome maintenance and transcription integrity in aging and disease. Front Genet 4:19 Wu L, Multani AS, He H et al (2006) Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres. Cell 126:49–62 Yang M, Zhang X, Liu G et al (2010) The complete chloroplast genome sequence of date palm (Phoenix dactylifera L.). PLoS one 5:e12762. https://doi.org/10. 1371/journal.pone.0012762 Yusuf AO, Culham A, Aljuhani W et al (2015) Genetic diversity of Nigerian date palm (Phoenix dactylifera) germplasm based on microsatellite mMarkers. Int J Bio-Sci Bio-Tech 7:121–132. https://doi.org/10. 14257/ijbsbt.2015.7.1.12 Zayed EMM (2017) Direct organogenesis and indirect somatic embryogenesis by in vitro reversion of mature female floral buds to a vegetative state. In: Clifton NJ (ed) Methods in molecular biology, vol. 1637:47–59. doi: https://doi.org/10.1007/978-1-4939-7156-5-5 Zayed ZE, El-Dawayati MM, Hussien FA, Saber TY (2020) Enhanced in vitro multiplication and rooting of date palm cv. yellow maktoum by zinc and copper ions. Plant Archiv 20(1):517–528 Zehdi S, Cherif E, Rhouma S et al (2012) Molecular polymorphism and genetic relationships in date palm (Phoenix dactylifera L.): the utility of nuclear microsatellite markers. Sci Hort 148:255–263. https://doi.org/10.1016/j.scienta.2012.10.011 Zehdi S, Sakka H, Rhouma A et al (2004) Analysis of Tunisian date palm germplasm using simple sequence repeat primers. Afr J Biotech 3:215–219. https://doi. org/10.5897/AJB2004.000-2040 Zehdi-Azouzi S, Cherif E, Moussouni S et al (2015) Genetic structure of the date palm (Phoenix dactylifera) in the old world reveals a strong differentiation between eastern and western populations. Ann Bot 116(1):101–112. https://doi.org/10.1093/aob/mcv068 Zouine J, El Bellaj M, Meddich A et al (2005) Proliferation and germination of somatic embryos from embryogenic suspension cultures in Phoenix dactylifera. Plant Cell Tiss Organ Cult 82:83–92. https://doi. org/10.1007/s11240-004-6914-0

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Date Palm Genetic Identification and Improvement Utilizing Molecular Markers and DNA Barcoding Ehab M. B. Mahdy and Sherif F. El-Sharabasy

Abstract

The level of variation in date palm cultivars and among Phoenix species, and their structure of DNA molecules can play a beneficial role in the efficient utilization of germplasm resources. The background of evolutionary gene flow and diversity of cultivars are essential factors for these variabilities. Investigating the diversity has been utilized successfully in date palm genetic identification and improvement, a rapid increase in date palm genome sequences and DNA barcoding in date palm varieties. Furthermore, it is significant to verify the material obtained and analyze genetic diversity. Identification of genotypes is even related highly to individual palms. Date palm improvement can be achieved by recent and novel platforms such as clustered regularly interspaced short palin-

E. M. B. Mahdy (&) National Gene Bank (NGB), Agricultural Research Centre (ARC), 9 Gamaa St., P.O. Box 12619, Giza, Egypt e-mail: [email protected]; [email protected] S. F. El-Sharabasy Central Laboratory of Date Palm Research and Development (CLDPRD), Agricultural Research Centre (ARC), 9 Gamaa St., P.O. Box 12619, Giza, Egypt e-mail: [email protected]; [email protected]

dromic repeats (CRISPR), competitive allele specific PCR (KASP) markers and targeting induced local lesions in genomes (TILLING). We highlight the most useful various molecular markers and DNA barcoding that can be utilized. This chapter provides information on molecular marker techniques based on detection methods. DNA barcoding is included for different applications on date palm genetic identification and improvement.

5.1

Introduction

Recent years have witnessed considerable progress and interest in utilizing molecular markers in plant genetics and crop improvement. Genetic markers are an observed variable in a sequence of a gene or a fragment of the chromosome. The objective concerning using a novel marker is to facilitate and maximize breeding for sustainable utilization (Jamil et al. 2020; Poczai et al. 2013). To do this, the first improvement attempts were done by applying a strategy of phenotypic-based selection. This approach consumes considerable time with plants having a long lifespan. Phenotypic-based markers are not reliable because they are used only for external attributes. These markers are visual observations (ElSharabasy and Rizk 2005; Rizk and ElSharabasy 2008). Cytological markers investigate genetic variation illustrating the origin and classification between and within species. The

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third sort are biochemical markers that can analyze by specific criteria (Khierallah et al. 2014), which are, as a rule, tissue- and organ-specific. They are affected by various extraction methodologies, plant tissue and different plant growth stages (Al-Jibouri and Adham 1990; Munshi and Osman 2010). These markers allow us to investigate genetic diversity, phylogenetic relationships of species and gender determination (AlYahyai and Al-Khanjari 2008; Qacif et al. 2007). However, these markers are limited due to proteins that are not transmitted to new progeny. Those markers are products of the interactions between gene expression and the effect of abiotic factors (Eissa et al. 2009; Qacif et al. 2007). Therefore, scientists have tended to use DNAbased markers due to this dependence on polymorphism in DNA sequences among individuals. These markers are completely accurate and diverse in the techniques used, which are also widely applicable to several species. The application of molecular markers can be a challenge in date palms due to their complex and large genome. Besides, the date palm genomes are heterogeneous because of the process of pollination and reproduction (Bekheet and ElSharabasy 2015; Sattar et al. 2017). Additionally, the proliferation of seed propagation is still prominent in the countryside of Arabian countries. Despite numerous cultivars of date palm being grown across the world, many are neglected and underutilized and may be lost forever without attention to their conservation. For more understanding, variability in dates is a necessary basis for effectively utilizing available variation to benefit users. The complementarity among traditional and modern markers must be captured to improve new desired types (Perez-deCastro et al. 2012; Tazeb et al. 2018). Molecular markers have been utilized for date palm improvement but face overall challenges. This chapter presents an overview of the approaches of DNA-based markers that are applied potentially for date palm improvement and genetic diversity. We highlight the utilization of DNA taxonomy and molecular markers for the

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sustainable uses of date palm improvement and to better understand phylogeny.

5.2

Overview of Molecular Markers and Ideal Genetic Markers

Genetic markers play a significant role in the field of genetic improvement. These markers can be classified into two categories: (1) the classic category that includes morphological, biochemical and cytological markers and (2) an advanced category that consists of genetic markers. Genetic markers are closely related to molecular markers, which investigate polymorphisms exhibited among different individual sequences. A sequence is a gene or DNA sequence with a known chromosome location controlling a particular attribute. The polymorphism acts on the molecular changes such as insertion, deletion, point mutation, duplication and translocation that do not necessarily affect gene(s) activity. Therefore, molecular markers should be ideally codominant, distributed throughout the genome, highly reproducible and with a high level of polymorphism (Mondini et al. 2009). Molecular markers are classified into different groups based on the mode of gene action, method of detection and mode of transmission (Semagn et al. 2006). Types of molecular markers have generally developed and applied in different disciplines and activities in various crops. Therefore, genetic markers which are ideally applicable should have the following characteristics: (a) No detrimental effect on phenotype (b) Codominant in expression (c) Single copy (d) Economical to use (e) High in polymorphism (f) Assay easily (g) Multi-functional (h) Available for unrestricted use (i) Genome-specific, especially for polyploids (j) Can be multiplexed (k) Amenable to automation

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5.2.1 Non-PCR-Based Markers These markers are based on sequence-specific cutting using restriction enzymes. Many restriction enzymes have been cloned and are available. This sequence-specific cutting is a key tool in molecular biology.

5.2.1.1 Restriction Fragment Length Polymorphism (RFLP) RFLP was the first technological development of molecular markers used in the 1980s, which was based on hybridization. The RFLP methodology is based on targeting a recognition site(s) by restriction enzymes. These enzymes will not cut if a point mutation occurs in an RFLP pattern. The resulting variations lead to the gain or loss of recognition sites, resulting in RFLP patterns (Madhumati 2014). RFLPs are relatively small and codominant. Polymorphism among individuals results from InDels, point mutations, translocations, duplications and/or inversions.

5.2.2 PCR-Based Markers Various molecular marker techniques have been developed and improved upon since Kary Mullis developed the polymerase chain reaction (PCR) technique in 1983 (Mullis et al 1986; Mullis and Faloona 1987). This technique works by amplifying a small quantity of DNA extracted from any living organism. Primers play a vital role in the sensitivity and efficiency of PCR which is dependent on different key factors.

5.2.2.1 Random Amplified Polymorphic DNA (RAPD) This technique was developed independently by Williams et al. (1990) and Welsh and Mcclelland 1990a,b). Polymorphism is based on changes in primer-binding sites in the DNA sequence. Amplification occurs during the PCR when the two hybridization sites are similar to each other and in the opposite direction. RAPD is a dominant marker system.

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5.2.2.2 Amplified Fragment Length Polymorphism (AFLP) The AFLP technique combines the RFLP with PCR technologies, due to the limitations present in the RAPD and RFLP techniques (Vos et al. 1995), in which the DNA is digested and then the PCR is performed (Ewens 1999; Lynch and Walsh 1998). AFLP markers are cost-effective, and there is no need for prior sequence information. Genomic DNA is digested by a pair of restriction enzymes (normally a frequent cutter and a rare cutter). It leads to the amplification of only those fragments which have been cut by these cutters. Adaptors are short, enzyme-specific DNA sequences generally used for finishing an unknown DNA sequence (Vos et al. 1995). PCR products were visualized by agarose/ polyacrylamide gel stained with AgNO3 or by autoradiography (Madhumati 2014). 5.2.2.3 Simple Sequence Repeats (SSR) or Microsatellites SSRs are a class of short tandem repetitive DNA nucleotides of 1–6 nucleotides (Tautz 1989) that occur on average every 6–7 kb (Cardle et al. 2000) and simple sequence length polymorphisms (Schlotteröer et al. 1991). They are also called microsatellites (Litt and Luty 1989). SSRs are abundant in the genome of a taxon (Beckmann and Weber 1992), chloroplast (Provan et al. 2001) and mitochondria (Rajendrakumar et al. 2007). Also, SSRs exist in protein-coding genes and expressed sequence tags (ESTs) (Morgante et al. 2002; Rekik et al. 2015). They represent lesser tandem repeats for each locus with a high level of polymorphism (Zane et al. 2002), because of the occurrence of various numbers of repeats in microsatellite regions (Kalia et al. 2011) that may be due to slippage of single-strand DNA, recombination of doublestrand DNA, retrotransposons and mismatches. Because adventitious SSR markers, which are codominant, have high reproducibility and are abundant in the genome, they are used in various plant disciplines (Kalia et al. 2011; Tautz 1989).

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5.2.2.4 Random Amplified Microsatellite Polymorphisms (RAMP) RAMP markers have a wide distribution in the genome. This includes a radiolabeled SSR primer consisting of a 5 anchor and a 3 repeat, which amplify genomic DNA in the absence or presence of RAPD primers. Therefore, RAMP markers (Wu et al. 1994) overcome the deficiency of RAPD and SSR markers. RAMP primers keep the anchored primers, compared to RAPDs, with a 10–15 °C higher temperature, to help the efficient annealing temperature. These markers are cost-effective and reflect higher polymorphism. Therefore, they have been successfully applied in molecular characterization (Salazar et al. 2014). 5.2.2.5 Sequence-Related Amplified Polymorphism (SRAP) For the amplification of open reading frames (ORFs), SRAP markers were developed by Li and Quiros (2001). The primers used are 17–18 nucleotides long with CCGG sequence in the forward primer and AATT in the reverse primer. The PCR product is visualized through autoradiography and scored by the presence or absence of a band. SRAPs are dominant, simple and efficient, with applications in several fields (Salazar et al. 2014). As dominant markers, they have been successfully used to investigate the genetic variations within and among taxa (Uzun et al. 2009). 5.2.2.6 Inter Simple Sequence Repeat (ISSR) ISSR markers were developed by Zietkiewicz et al. (1994) to amplify the distance between two identical DNA oppositely oriented microsatellite repeats regions. Primers may be di-, tri-, tetra- or pentanucleotide repeats, with a size of 15–30 bases. ISSR primers may be unanchored (Gupta et al. 1994) or anchored at the 30–50 end and having 1–4 degenerate bases. These extend through the flanking regions. Products of ISSR are 200–2000 bp long and visualized by means of agarose or PAGE. They are dominant markers (Tsumura et al. 1996; Zietkiewicz et al. 1994),

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simple, less reproducible and need no prior knowledge of DNA sequences.

5.2.2.7 Transposable Elements (Transposons) Mobile DNA sequences changing their locations in the genome are called transposons or transposable elements. These elements were discovered in maize by Barbara McClintoch (Finnegan 1989; Grzebelus 2006). The transposable elements can be broken down into two transposon classes: Class I: Retrotransposons (commonly called copy-and-paste elements or retro-elements) propagate via RNA-intermediate and generate an additional new copy in the genome. Most transposon markers utilize this class due to their repetitive DNA, constituting about 50–60% of the whole plant genome (Grzebelus 2006; Kumar 2004; Kumar and Bennetzen 1999; Kumar and Hirochika 2001; Kumar et al. 1997; Watts et al. 2016; Witte et al. 2001). Based on structure and transposition cycle, retrotransposons can be further classified into two subclasses, which are distinguished based on the presence/absence of long terminal repeats (LTRs) at their ends: (a) LTR retrotransposons (LINE; long interspersed nuclear elements), (b) Non-LTR retrotransposons (SINE; short interspersed nuclear elements). LTRs are an essential subclass because of their coverage in almost 70% of the plant genome (Kumar et al. 1997; Pearce et al. 1996; Shirasu 2000). They do not code for any protein but instead contain the promoter and terminators for transcription, such as ORFs, POL and GAG. These positions provide the basis for primer binding sites in many techniques. LTR retrotransposons are further classified by the root of the encoded gene sequence into Ty1-copia and Ty3-gypsy retrotransposons (Roy et al. 2015). Class II: DNA transposons (commonly called cut-and-paste elements) are simply excised from the site of the donor by moving to the novel position. They are further divided into subclasses: (1) terminal inverted repeats (TIRs) and

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(2) non-TIRs (Moisy et al. 2014; Sabot and Schulman 2006; Schulman and Wicker 2013). As transposons, these elements have been distributed abundantly and widely over the genome. Therefore, they are an important source for the development and progress of molecular markers (Kalendar 2011; Kalendar et al. 2011; Moisy et al. 2014). The following are some important retrotransposon-based molecular markers. Inter-retrotransposon amplified polymorphism (IRAP) IRAP and REMAP are retrotransposon markers described by Kalendar et al. (1999) to create DNA fingerprints (Moisy et al. 2014). IRAPs target two adjacent LTRs varying in size (100–5000 bp) through the application of primers. The orientation of LTR sequences can be tail-to-tail, head-to-head or head-to-tail (Poczai et al. 2013). Only a single primer is necessary to create IRAP products from tail-to-tail and head-to-head. Both 5’ and 3’ primers are needed for head-to-tail LTR primers to amplify the intervening genomic DNA. Retrotransposon microsatellite amplification polymorphisms (REMAP) REMAP is an important retrotransposon-based marker commonly exploited by polymorphisms among regions of anchored SSR and LTR sequences to analyze the genetic diversity. Primers are chosen for the loci of the microsatellite because of avoiding slippage among individual microsatellite motifs (Roy et al. 2015). This technique can be regarded as a modified or extended version of the ISSR technique. IRAP and REMAP have been utilized separately and together to study genetic diversity because they result in reproducible banding profiles (Kalendar and Schulman 2006). Retrotransposon-based insertion Polymorphism (RBIP) The RBIP technique, a molecular marker developed by Flavell et al. (1998), was achieved via a primer of 3’ and 5’ end insertion flanking regions to detect polymorphism among LTR sites (Agarwal et al. 2008). The retrotransposon insertion sites result in the typing of a single locus compared to other retrotransposonbased markers (Fan et al. 2014).

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Inter-primer binding site (iPBS) Amplification The iPBS technique was developed by Kalendar et al. (2010) to overcome the key problem where there is no prior genome sequence information of LTRs, by utilizing the PBS region of retrotransposons that are shared by LTR elements with 18 nucleotides complementary to a limited set of tRNA. To obtain such information, cloning and sequencing of LTR were performed. Recently, iPBS markers have been utilized for the identification of genetic variation and associations among plants (Baloch et al. 2015; Kalendar and Schulman 2014; Kalendar et al. 2011). Inter-SINE amplified polymorphism (ISAP) The marker ISAP was designed for potato by Seibt et al. (2012) and based on the lack of LTR motifs. ISAP markers are based on the amplification of sequences among adjacent short, interspersed elements (SINE) and require extensive prior genomic information about SINE elements. This method may prove to be extremely specific; however, ISAP may not become very popular in plant genetics. Specific amplification polymorphism (SSAP) Waugh et al. (1997) developed this technique, which is quite similar to the AFLP procedure, whereas no prior information of LTR elements is required. The procedure is based upon ligation, the short double-stranded adaptor ligation to the restricted DNA fragments which are digested by an infrequent restriction enzyme paired with a frequent one. Then, selective amplification with a retrotransposon-specific primer, and paired with either a rare or a frequent site adaptor primer. Ty1-copia and Ty3gypsy retrotransposons are very commonly used (Fan et al. 2014).

5.2.2.8 Cleaved Amplified Polymorphic Sequences (CAPS) CAPS were originally called RFLP-PCR markers because they are based upon amplification of the target DNA by PCR and then the digestion of the products by restriction enzymes (Jarvis et al. 1994; Michaels and Amasino 1998). To design their primers, prior genomic sequence

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information is required. CAPS markers can find polymorphisms either alone or combined with other markers such as SCAR, AFLP or RAPD (Agarwal et al. 2008). CAPS, as a codominant marker, are multipurpose, e.g., genotyping, genetic mapping, genetic diversity and identification studies (Spaniolas et al. 2006; Wang and Roberts 2006; Weiland and Yu 2003).

5.2.2.9 Sequence-Characterized Amplified Regions (SCAR) SCAR markers were first developed by Paran and Michelmore (1993) for downy mildew resistance genes in lettuce. These markers are mostly applied to physical mapping (Bhagyawant 2016) due to their being specific, highly reproducible, codominant and monolocus markers. Their procedures are based on the purification of PCR products followed by a SCAR primer design (Kiran et al. 2010) to detect the target DNA polymorphism and then investigate their sequences. These sequences are utilized in the synthesis of specific SCAR primers (Bhagyawant 2016). 5.2.2.10 Sequence-Based Markers Sequencing technology is a more novel processing technique in which nucleotide bases and their orientation are identified along the DNA/RNA (França et al. 2002). The fact that hybridization-based markers are polymorphic and less reliable has revolutionized plant breeding and improvement. Sanger Method Sanger technology was the first method of sequencing devised by Sanger and Coulson (1975). The basic Sanger method is that DNA molecules in single strands, which have variability in the length of single nucleotides, can be separated using PAGE. In the earliest studies, T4 DNA polymerase and DNA polymerase, I of Escherichia coli were exploited in this method and then loaded on acrylamide gels. Sanger and colleagues developed a new sequencing technique by polymerization of enzymes to overcome the limitations via the old technology (Sanger and Coulson 1975; Sanger et al. 1977). This method requires a low amount of DNA and has high reproducibility, but it

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detects fewer polymorphisms below the species level (França et al. 2002). Pyrosequencing Pyrosequencing is a synthesis principle-based sequencing technique (Hyman 1988) whereas phosphate is identified by DNA synthesis (Agah 2004; Mashayekhi and Ronaghi 2007; Ronaghi 2001). The primers used are hybridized with a single-stranded DNA template and then combined with specific enzymes (Gharizadeh et al. 2006). Deoxynucleoside triphosphates use the reaction for four cycles. Pyrophosphate (PPi) is continuously released when the reaction begins with nucleic acid polymerization. The DNA polymerase activity is monitored by this technique. Pyrosequencing is classified into two types: solid phase sequencing and liquid phase sequencing. Next-Generation Sequencing (NGS) The demand for extensive throughput sequencing technology revolutionized and led to the development of NGS. This technique can create several hundreds of millions, to hundreds of billions of DNA bases per run (Ansorge 2009; Shendure and Ji 2008). The more successful development of NGS is provided by commercial services like Illumina HiSeq (Bentley et al. 2008), Roche 454 FLX Titanium (Thudi et al. 2012) and Ion Torrent PGM (Rothberg et al. 2011). NGSs regenerate the whole genome more precisely (Deschamps et al. 2012). Several characteristics of NGSs are (1) more accurate, (2) lower in cost, (3) ease of detecting SNPs, (4) detection of the diversity within and between species, (5) construction of genome maps, (6) construction of genome-wide association studies (GWAS) (Elshire et al. 2011), and (7) strengthen the field of metagenomics (Mardis 2008, 2013). Genotyping by Sequencing (GBS) GBSs were developed in the Buckler Lab, Cornell University, USA, under the Illumina platform to investigate the high-resolution association in maize. Today it successfully uses numerous techniques and is applied to many other species with large, complex, highly diverse genomes (Elshire et al. 2011). GBS has several desirable characteristics including lower cost, creation of a great magnitude of SNPs (Beissinger et al. 2013), less handling of samples, ease of application in

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several fields (Poland and Rife 2012) and requires less PCR (Davey et al. 2011). Two types of GBS based on the use of ion PGM systems are (a) Digestion methods: The method is based upon the digestion of DNA by one or two specific restriction enzymes before the ligating of adapters. This method is mainly exploited in marker-assisted selection (MAS) programs for the identification of new markers. (b) PCR-based methods: Specific PCR-primers are exploited to identify a fragment of interest as a complete set of SNPs for a genome section. Single-nucleotide Polymorphism (SNP) SNP is a change in a single base pair occurring in coding or non-coding regions of genes or between two genes (intergenic region) with different frequencies (Graham 2011) that may be insertion/deletions (InDel), transitions or transversions due to their abundance and frequency in genome plants and animals. The frequency of SNP in plants occurs every 100– 300 bp (Graham 2011; Ortiz 2010; Xu et al. 2012). This change in the smallest unit can provide numerous simple markers. To detect SNP, a large number of SNP methods and assays have been developed based on different techniques of detection platforms, and allelic discrimination such as RFLP and CAPS markers. Primer extension, invasive cleavage, oligonucleotide ligation and allelespecific hybridization are the most important, based on different molecular mechanisms (Sobrino and Carracedo 2005; Sobrino et al. 2005). Various types of high-throughput genotyping platforms can be made more attractive for genotyping by these techniques (Agarwal et al. 2008). Diversity Array Technology (DArT) DArT is a remarkable technique for the genotyping of polymorphic loci found over the genome. This technology is a highly reproducible microarray hybridization technology; no previous sequence information is required to detect the interest loci (Jaccoud 2001; Wenzl et al. 2004). DArT is a

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useful technology for use in the detection of polymorphic loci due to its high throughput, high reproducibility and it needs only a low amount of DNA. Polymorphic markers in genotyping arrays are commonly used for genotyping (Huttner et al. 2005; Wenzl et al. 2004).

5.3

Applications of Molecular Markers and DNA Barcodes in Palms

Molecular markers play a key and important role in different disciplines and fields involving the conservation of genetic resources, evolution, phylogenetics and the identification of promising cultivars. These markers serve and solve the unique problems facing date palm breeders and technologists, which are not solvable through conventional methodologies.

5.3.1 Palm Evolution and Phylogeny Evolution combines geographical with morphological changes among Phoenix species. Molecular biology provides information concerning genetic makeup to obtain full information about the phylogeny and evolution of date palm and its congeners. The potential markers are applied based on the availability of genome sequence data, the stability of genetic nature and the simplicity of use (Dong et al. 2012). The origin of the date palm (Phoenix dactylifera L.) is still obscure, along with its wild relatives such as P. sylvestris Roxb. and P. theophrastii Greuter. All Phoenix species are dioecious and freely hybridize, which generates much variation within/between palms. This variability results from continuous multiplication by seeds; most date palms are propagated via seeds, as occurs in Lower Egypt (Riad 1993). The integrity between date palm phylogeny and evolution should be able to differentiate morphological characterization with biochemical and molecular markers. Many reports have appeared on morphological attributes to distinguish and study the relationship among Phoenix species.

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The high-throughput phenotyping platform (HTPP) is still the most comprehensive evaluation of morphological attributes, which makes available precise information for traits of interest. El-Sharabasy and Rizk (2019) compiled the Atlas of the Date Palm in Egypt. In addition, an earlier descriptor for date palm was published by Rizk and El-Sharabasy (2008). Phoenix iberica D. Rivera, S. Rios & Obón, resembles in vegetative characters, cvs. Medjool or Barhee of P. dactylifera, but its fruits are intermediate between P. theophrastii and P. sylvestris. The Barbary palm (P. chevalier D. Rivera, S. Ríos & Obón) resembles P. canariensis Chabaud in that leaves are not glaucous or waxy but are differentiated with very long feathery leaflets and less stiff acanthophyllia as compared with those of dactylifera. (Haddouch 1996). Phoenix atlantica A. Chev. in the Cape Verde Islands, tamareira in Portuguese, has a thick trunk, short acanthophylls and small fruits. Another point, introgression by wild species of date palm gene pools have been proposed as a source of diversity (Flowers et al. 2019). Biologists make great efforts to find target genes to analyze the variation at which loci may affect phenotypic attributes. GWAS provides analysis for important cases and questions of date palm evolution and build phylogenetic trees to enable quantitative tree comparison of date palm species. Ge et al. (2020) presented a visual analysis system and comparison of phylogenetic trees for evolutionary genome-wide analysis. However, those did not enable them to find loci that affect their attributes. The relationships among lipid content and fatty acid composition are predominantly important factors in palm chemotaxonomy. Fatty acids in seeds reflect the genetic drift that may occur in the phylogeny of palms. Several are recognized as new sources of oils for multiple uses. A recent review on palm seed and fruit lipid composition has been provided by Guerin et al. (2020) relative to date palm phylogeny. Unsaturated fatty acids appear to provide an adaptive benefit in the coldest environments by sustaining storage lipids in liquid form throughout germination. A group of C2H2 zinc finger transcription factors belong to a

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plant-specific protein of indeterminate domain (IDD). However, some of the IDD genes and their molecular evolution in the subfamily are unknown in detail or function; they have the most roles in various features during plant development. IDD transcriptions could significantly be employed when differentiated in number, expression and C-terminal throughout the trait evolution of plant growth. Prochetto and Reinheimer (2020) increased a correlation structure of IDD-transcriptors to identify the genes owing to plant development and evolution. In this context, genetic analyses can strongly support those studies. Genetic analyses play a large role in distinguishing and classifying species. Identification of Phoenix atlantica was achieved by microsatellite and minisatellite analysis (Henderson et al. 2006). Microsatellite analysis can separate P. dactylifera and P. canariensis, and their putative hybrids in the Canary Islands (González-Pérez et al. 2004). Barrow (1998) reported a cluster among P. dactylifera, P. theophrastii and P. sylvestris with low resolution, using the intergenic spacer region of 5S DNA units. Molecular studies need to assess and state the genetic relationships within and among date palm cultivars and other relatives. Rivera et al. (2008) compared P. dactylifera cultivars from Africa and the Near East with their relatives using nuclear microsatellite polymorphism, polymorphic ITS regions and chloroplast microsatellite patterns. Mohamoud et al. (2019) proposed that the earliest proliferation of dactylifera transpired independently in three discrete regions. Their data contribute to an understanding of the origins of dactylifera. There is consensus that date palms have a long history of domestication and cultivation.

5.3.2 Genetic Diversity Assessment The total number of date palm trees grown across the world is more than 100 million and more than 2000 cultivars have been named (Al-Mssallem 1996; Govaerts and Dransfield 2005; Zaid and Arias-Jiménez 2002; Zehdi-Azouzi et al. 2016). Several neglected and underutilized date palms

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may be lost if conservation action is not taken (Akhtar et al. 2014). Conservation of date palm has become a vital element in sustainable utilization, crop improvement and food security, especially in Arab nations because of the loss of habitat, low known genetic resources and the limited number of conventional conservation methods for the maintenance of its genetic resources (Akhtar et al. 2014). Each germplasm accession has its unique genetic makeup represented at the level of coding sequences. Also, molecular markers that have a unique level of polymorphism to utilize in genetic diversity. This feature may be because of the limitations in primer/genotype number, the range of marker reliability and amplification of interest used in the analyses (Al-Dous et al. 2011). Date palm conservation addresses the identification, description, documentation of the agrobiodiversity and the systematic relationships of cultivars/species growing across the world and exhibiting hundreds of morphological traits. The characterization of date palm cultivars provides more information about the status of in situ/ex situ conservation and sustainable utilization to apply to future accounts of the biodiversity of date palms (Al-Dous et al. 2011; Enan and Ahemad 2014, 2016). These need to support germplasm collection, characterization, evaluation, documentation, establishment and distribution by rapid developments in plant genetics and

biotechnology (Bekheet 2011; Bekheet and ElSharabasy 2015; Bekheet and Taha 2013). Generally, the methods fall under the following categories: in situ conservation, barely applied until now and ex situ conservation, including procedures of slow growth via in vitro techniques; cryopreservation; gene bank fields botanic gardens; seed banks and DNA banks (Bekheet 2011; Engelmann 2004, 2009; Jain 2011, 2012). DNA storage and preservation have developed as one of the emerging ex situ techniques for germplasm conservation. A DNA bank is a particular type of plant genetic resource collection that preserves and distributes DNA molecules and provides associated information (Mahdy 2018; Sayed et al. 2017). These methods are more helpful to utilize the diversity for the improvement within and among Phoenix taxa. For this, we must strongly recommend applying the modern and progressive different methods of in situ and ex situ conservation; and the useful complementary of both approaches to facilitate the use of such genetic resources in date palm improvement and add new value to the existing collections. Date palm characterization and evaluation should incorporate the discriminatory power of morphological characterization with biochemical and molecular markers (Fig. 5.1). Several phenology studies have been performed (ElSharabasy and Rizk 2019; Rizk and ElSharabasy 2008) to utilize resources from date

Fig. 5.1 Polymorphism of two ISSR markers (HB-12 and HB-08) for nine date palm cultivars. M = DNA Ladder (100 bp), 1 = Samani, 2 = Zaghloul, 3 = Bent-

Eisha, 4 = Hayani, 5 = Orebi, 6 = Om El-Ferakh, 7 = Amhat, 8 = Selmi, 9 = Barhi. Source Eissa et al. 2009)

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palm gene pools. Attaha et al. (2013) and Saha et al. (2013) exploited the variations in protein patterns on SDS-PAGE to identify the degree of similarity and differences among 17 seedling cultivars grown in the Basrah, Iraq region. Their results showed that protein pattern differences were in band number, location, thickness and density leading to variations in protein quantity and quality among the studied strains. On the other hand, information derived from genetic resources were detected and screened (Fig. 5.2) by several DNA-based markers involving (AFLP) amplified fragment length polymorphism (Ibrahim et al. 2014); (RFLP) restriction fragment length polymorphism, (RAPD) randomly amplified polymorphic DNA (Emoghene et al. 2015; Hussein et al. 2005; Ibrahim et al. 2014; Marsafari and Mehrabi (2013); SSR simple sequence repeats (Abdulla and Gamal 2010; Arabnezhad et al. 2012; Bodian et al. 2014; Elmeer et al. 2019; Hamwieh et al. 2010; Hussein et al. 2005a,b; Ibrahim et al. 2014; Fig. 5.2 Polymorphism based on RAPD analysis of the semidry date palm in Egypt. M = DNA ladder (100 bp), 1 = Karamat, 2 = Sewi, 3 = Holow Ghanem, 4 = Agua, 5 = Saidi, 6 = Barhi, 7 = Aglani, 8 = Amri, 9 = Ashbeer, 10 = Deglet Noor. Source Soliman et al. (2006)

E. M. B. Mahdy and S. F. El-Sharabasy

Khierallah et al. 2014; Kumar et al. 2010; Marsafari and Mehrabi 2013; Mirbahar et al. 2014; Racchi and Camussi 2018; Yusuf et al. 2015) and SCoT start codon targeted polymorphism (Soumaya et al. 2020). Emoghene et al. (2015) identified genetic variation and phylogenetic relationships among Nigerian date palm germplasm via RAPD markers to screen for genetic polymorphisms. The status of date palm genetic resources in Nigeria, introduced from Namibia and Israel, were used as part of Nigerian collections from 1981 to 1990, cataloged from various ecogeographical systems (Ataga et al. 2012). Racchi and Camussi (2018) surveyed the genetic diversity of date palm genetic resources growing in the AlJufrah oases, Libya to understand their genetic history. They investigated the polymorphism via SSR markers of a plastid minisatellite that discriminates between African and Middle Eastern varieties. Data have shown that the western chorotype was similar to that reported for

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Tunisian cultivars, greater than those in Algeria, Morocco and Mauritania. Moroccan germplasm from the Figuig oasis was exploited by Bodian et al. (2014) to analyze the genetic variabilities among 128 date palm genotypes using SSRs. The molecular variance analysis showed 59% of variability among germplasms. Yusuf et al. (2015) investigated the genetic diversity and geographical patterns of 14 cultivars from Nigeria and Saudi Arabia using microsatellite markers. Ibrahim et al. (2014) characterized the genetic diversity of 3 date palm cultivars collected from the El-Kharga and Dakhleh oases, Egypt, based on morphological variability and molecular markers by RAPD, SSR and AFLP technologies. The results indicated that each cultivar has its genetic makeup at the level of the coding sequence. Coding and non-coding genes have been utilized in genetic diversity among genomes. Soumaya et al (2020) ascertained the effectiveness of SCoT markers in estimating genetic diversity in date palm genotyping. Advanced technologies are now routinely used in date palm studies, such as genome sequencing and SNPs. Al-Mssallem et al. (2013) illustrated that the date palm has a unique metabolism that promotes fruit development and ripening. Large-scale genomic and transcriptomic data have paved the way for further genomic studies between dactylifera and other relatives of the Arecaceae. Date palm genomewide association is superior to marker-assisted recurrent selection and phenotypic selection in terms of gain per unit cost and required time (Wong and Bernardo 2008). Various studies are reportedly advantageous for genome-wide use (Al-Dous et al. 2016; AlMssallem et al. 2013; Dussert et al. 2013; Singh et al. 2013; Xiao et al. 2017). Genome-wide association is a potential platform for improving date palms and performs with different sizes of populations and markers (Hazzouri et al. 2019, 2020; Jamil et al. 2020; Nakaya and Isobe 2012).

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5.3.3 Date Palm Gender Determination Determining sex in dioecious species is usually identified with the physical separation by different mechanisms related to the species (AlKhalifah et al. 2012; Atia and Adawy 2015; Kharb and Mitra 2017; Pipatchartlearnwong et al. 2019; Siljak-Yakovlev et al. 1996). Sexually propagated dioecious date palms present problems in determining their sex in the early stage of life development. Hence, population stands of equal male to female ratio for fruit production are inefficient. Genetic improvement is limited because the date palm requires several years to reach the reproductive stage and reveal gender. Determining gender has been effective in identifying the factors of genetic and epigenetic diversity, which are involved in determining the sex of an individual date palm (Atia and Adawy 2015; Rizk et al. 2006). Efforts have been made for decades to determine gender in date palm, but most were unsuccessful, based on a wide range of methodologies that include cytology (Siljak-Yakovlev et al. 1996), biochemistry (Rizk et al. 2006: see Fig. 5.3) and molecular markers (Al-Ameri et al. 2016a; Heikrujam et al. 2015: see Fig. 5.4). Conventional breeding of date palms is extremely laborious due to the long juvenile phase. Research has focused on methodologies that were simple and accurate in the differentiation of gender. The system of date-XY chromosomes helps to locate the Y chromosome-specific DNA markers which identify the male palms. The first extensive and successful effort using a cytological marker differentiated the heterochromatin region of chromosomes using chromomycin A3 stain of date palm root-tips as a new tool for sex determination (Siljak-Yakovlev et al. 1996). In this context, PCR-based markers have been utilized (Intha and Chaiprasart 2018) to determine the gender of date palms at the earliest

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the hypothesis of palm gender determination. The SRY-date gene in the dactylifera Y-chromosome region was amplified and identified by Solliman et al. (2019). GBS platform is an essential tool for better understanding the pathways of date palm sex chromosomes. Date cv. Khalas was sequenced successfully using the GBS platform (Zhao et al. 2012). Malek (2014) invented methods to analyze DNA or RNA from date palm plant tissue, germplasm or seed for the presence of (i) a nucleic acid sequence or genotype that identifies the sex of the plant, tissue, germplasm or seed or (ii) a molecular marker in linkage disequilibrium with the nucleic acid sequence or genotype. Also disclosed were kits for identifying male and female date palm plants before flowering, methods of breeding a date palm plant and a method of planting a date palm seed of known sex. It is possible to distinguish between male and female date palm trees with at least 90% accuracy using these methods. Fig. 5.3 Protein profile using SDS-PAGE for female (lane 1) and six male genotypes (lanes 2–7) of date palm cv. Zaghloul. Source Rizk et al. (2006)

5.3.4 Identification Among Cultivars and Species

stage. Date palm gender can be identified by molecular markers, including AFLP markers (Pipatchartlearnwong et al. 2019); RAPD (Kichaouibr and Ayesh 2013; Mohsenzadeh and Pasalari 2010; Pipatchartlearnwong et al. 2019; Younis et al. 2008); SCoT (Zhao et al. 2012; Pipatchartlearnwong et al. 2019); ISSR (AlAmeri et al. 2016a; Hamama et al. 2003; Younis et al. 2008); CDDP (Atia and Adawy 2015); SCAR (Al-Ameri et al. 2016b; Al-Qurainy et al. 2018; Dhawan et al. 2013; Kharb and Mitra 2017); ITAP (Zhao et al. 2012); ILP (Pipatchartlearnwong et al. 2019) and SSR markers (Cherif et al. 2013; Hussein 2015; Maryam et al. 2016; Pipatchartlearnwong et al. 2019). De novo transcriptome sequencing of the palmyra palm (Borassus flabellifer L.) has been used according to Pipatchartlearnwong et al. (2019). Atia and Adawy (2015) reported that a set of sex-specific PCR-based markers exposed

Recent advancements in molecular markers and DNA barcoding offer more opportunities to identify the genetic diversity among cultivars and within Phoenix species. Genetic markers determine the polymorphic variability and classification of date palm genetic materials (Jamil et al. 2020; Sallon et al. 2020). It is necessary to increase the identification of date palm varieties with high yield and to precisely correlate and relate genotypes and phenotype (Furbank and Tester 2011). Various studies have identified date palm cultivars, such as the investigation by Carreño et al. (2020) in Spain. Many molecular markers such as SSRs and SNPs have been exploited to differentiate date palm cultivars and investigate date palm diversity (Jamil et al. 2020). Some studies have used transcriptomes to analyze the genes involved in identification of other palms. The oil content of oil palm (Elaeis guineensis Jacq.) is an essential attribute associated with several transcript

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Fig. 5.4 Gender determination of seven date palm cultivars using RAPD profile. DNA ladder with 100 bp; Lanes 1–7 refer to cultivars: Sakaote (female), Dagana

(male), Malkabi (male), Sakoty (male), Bertamoda (female), Malakabi (female), Dagana (female) Source Solomon et al. (2008)

elements, e.g., synthesis of fatty acids, metabolism of plastid carbon and plastid transporters (Bourgis et al. 2011). These transcript-elements belonging to the WRI1 transcription factor were 57-fold higher, compared to Phoenix dactylifera. Enzymes of triacylglycerol-assembly express in palm species. The comparative sequencing studies of Bourgis et al. (2011) could provide more insights into the pattern of gene expression among at least two species lacking information in the genome.

Currently, some studies have used DNA barcodes to investigate the species of Phoenix. Diverse molecular markers have efficiently assessed the genetic diversity date palm cultivars in Egypt (Abd El-Azeem et al. 2011; Ibrahim et al. 2014; Saker et al. 2006); Oman (AlRuqaishi et al. 2008); Morocco (Bodian et al. 2014; Sedra 2013; Sedra et al. 1998); Saudi Arabia (Al-Khalifah and Askari 2007; AlKhalifah et al. 2012); Tunisia (Zehdi et al. 2005, 2012); and Sudan (Elshibli and

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Korpelainen 2008). These studies have helped to identify markers suitable for the identification of date palm varieties. The high-throughput phenotyping platform (HTPP) system is attained through the comprehensive evaluation of morphological traits that make the availability of precise information for the attribute(s) of interest (Finkel 2009). Studies in HTPP identified cultivars of date palms and among Phoenix species (Eissa et al. 2009; ElSharabasy and Rizk 2019; Haddouch 1996; Rizk and El-Sharabasy 2008). On the other hand, high-throughput genotyping platforms have shown more continuous advancement and development in molecular marker technologies from RFLP to SNPs and diversity in technologies of array-based markers. These studies have also permitted the identification of molecular markers suitable for identifying date palm varieties. In this context, genetic analyses allow for easy identification among Phoenix species, such as P. atlantica (Henderson et al. 2006). Microsatellite markers are effective to distinguish among P. dactylifera, P. canariensis and P. atlantica, and also their putative hybrids in the Canary Islands (González-Pérez et al. 2004). Barrow (1998) compared P. dactylifera, P. theophrastii and P. sylvestris using the intergenic spacer region of 5S DNA units. Studies of molecular markers are critical to assess the genetic relationships among date palm species and their relatives. Rivera et al. (2008) compared dactylifera cultivars from Africa and the Near East with related Phoenix species using nuclear microsatellite polymorphism, polymorphic ITS regions and chloroplast microsatellite patterns. Various molecular markers have been commonly employed to construct the linkage mapping and identification within and among Phoenix taxa. The platform of genomewide association studies has been used to estimate genetic diversity in palms. Despite the importance of cultivated palms, they still fall behind other important crop genetic resources. Recent projects have sequenced several genomes of date palms (Al-Dous et al. 2011; Al-Mssallem et al. 2013; Zhang et al. 2012) and the discrimination of the putative palm sex chromosome

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(Zhao et al. 2012). These resources expand information for further genetic identification in the palm. Abu-Afifeh (2018) identified ten unknown Egyptian seedling date palms originated in Mut, Al-Dakhla Oasis, Egypt, as a part of the Palm Development Program in Arab Countries, determining their genetic background distance using DNA taxonomy technology as well as AFLP, SSR, ISSR and RAPD markers.

5.4

Suitability Utilization to Improve Date Palm

Introgression of molecular markers has developed new techniques in which some genes of interest are transferred from date palm variants to new promising ones. This occurs using an appropriate method, e.g., marker-assisted selection (MAS), to transit a selected desirable attribute from exotic germplasm into a proliferated cultivar. MAS has played a significant role in the usage of wild genes. Mapping by quantitative trait loci (QTL) and other markers on breeding should contribute physical and genetic maps for the accurate detection of candidate genes.

5.4.1 Genetic and QTL Mapping Most attributes are naturally polygenic and quantitative and are controlled by multiple genes on the chromosome(s)and referred to as QTLs (Al-Dous et al. 2011; Dhingani et al. 2015). QTLs are used to locate such genes of interest (Ting et al. 2018; Zhang et al. 2016). Genetic mapping is the main area of research that uses molecular markers to identify a target gene and the distance between genes (Al-Mssallem et al. 2013; Singh et al. 2013; Ting et al. 2018; Zhang et al. 2016). The combination of the integrity of the strategy of polymorphic markers from phenotyping to genotyping data has been used by Al-Dous et al. (2011), Al-Mssallem et al. (2013), and Hazzouri et al. (2019). The HTPP system has been widely used to distinguish among morphological attributes (Fig. 5.5; El-Sharabasy and Rizk 2019; Rekis and Laiadi 2020; Rizk and El-

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Fig. 5.5 Fruits and seeds of some soft date palm cultivars in Egypt. 13 = Hayani, 14 = Halawi, 15 = Oreebi, 16 = Om El-Ferakh, 17 = Amhat,

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18 = Selmi, 19 = Kapoushi, 20 = Sofer El Domin, and 21 = Beid El Gamal. Source El-Sharabasy and Rizk (2005)

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Sharabasy 2008; Salem et al. 2008). On the other hand, more advanced molecular markers are also being used, such as polymorphic markers, which are essential tools of crop breeding (Angaji 2009; Singh et al. 2020). A genetic map can select two different parents having allelic variations in the trait of interest. An association map displays the relatedness of molecular markers with a specific trait in which it is statistically covariant among the polymorphism in a marker of the trait (Hazzouri et al. 2020). Therefore, association mapping identifies allele numbers because of the availability of genetic variations with background and phenotypic data that can be utilized (Zhang et al. 2016). Association mapping is presently inflexible and costly due to the protracted juvenile stage of the date palm. An alternative approach is to map target attributes at an earlier stage of growth (Hazzouri et al. 2019; Romero Navarro et al. 2017) through tissue culture. Due to the long lifespan of date palms, linkage mapping is not useful to study the linkage in the palm. These limitations of QTL mapping can be overcome by introducing linkage-disequilibrium-based association mapping (Gupta et al. 2005). More than 150 markers are used to construct the linkage maps (Jeennor and Volkaert 2014; Ting et al. 2018). Several of these markers vary according to the studies, and directly to the genome size of species. QTL mapping has finally utilized new strategies for palm improvement. From this, MAS has been discussed potentially in the oil palm (Jeennor and Volkaert 2014; Rance et al. 2001; Seng et al. 2016; Ting et al. 2018; Ukoskit et al. 2014). The development of an oil palm RFLPmarker map is complicated; the marker-based QTL mapping population consists of 84 trees segregated by the influence of shell thickness. Various genetic analyses have been exploited on date palms viz. microsatellite and genome-wide technology. Novel works on date palm have been sequenced (Al-Dous et al. 2011; Al-Mssallem et al. 2013) and identified putative sex chromosomes (Torres et al. 2016; Zhao et al. 2012). These sources provide a substantial basis for the progression of genetic QTLs.

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Lack of genetic resources may have led to many challenges to providing the full form of date palm genetic mapping. The deficiency of generating the draft genome sequence led to a new tool to overcome the various problems in biotechnology, genetic improvement, sex determination and growth development in palms (Hazzouri et al. 2020). Mathew et al. (2014) provided the first genetic map using a modified GBS approach (Elshire et al. 2011) to overcome challenges due to the lack of genetic resources for date palm and comparing it to other commercial palms. Identification of a locus is critical to assess the population makeup. The use of an applicable novel technology will be a suitable platform to scan genetic diversity (Mathew et al. 2015). Using these SNPs, in combination with 188 anchor SNPs and 123 microsatellites, Bai et al. (2018) constructed a linkage map of oil palm containing 10,023 markers covering 16 chromosomes. Oil content of the fruit is selected using genome-wide identification (Bai et al. 2017). Modern technologies such as GWAS and SNPs provide numerous choices of desirable molecular markers in screening genetic variation and evolution of Phoenix populations (Hazzouri et al. 2019, 2020). High-density QTL-mapping will contribute to the target attributes and hasten date palm genetic improvement (Gyawali et al. 2019). The date palm genome size is approximately 550–650 Mbp long (Malek 2010). The high-density SNP and SSR-based genetic maps on oil palm have improved coverage of the genome compared to AFLP and SSR markers (Ting et al. 2014). Therefore, these novel techniques will be more advantageous for genetic mapping.

5.4.2 Association Mapping of Date Palms Association mapping is the covariance between the polymorphisms in the marker and the target trait. Association mapping identifies allele numbers and the availability of more genetic variations with huge backgrounds (Hazzouri et al.

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2015). The characteristics of association mapping provide greater resolution with a higher number of recombination processes (Flowers et al. 2019). Compared to linkage map limitations, these are overcome by the introduction of linkage disequilibrium-based association mapping (Flowers et al. 2019; Gupta et al. 2005; Hazzouri et al. 2015, 2019; Mackay and Powell 2007; Myles et al. 2009; Yamada and Skøt 2010) which are known as a gametic phase disequilibrium and describe nonrandom (nonequal) association of alleles at different loci in a population (Flowers et al. 2019; Hazzouri et al. 2020). The correlation coefficient and the disequilibrium coefficient are two widely used statistical methods for measuring linkage disequilibrium. Various applications of linkage disequilibrium (LD) and association mapping in crop plants are reported (Ersoz et al. 2007; Myles et al. 2009). In date palm, the linkage disequilibrium is a necessary measure (Flowers et al. 2019). GWAS led to the interest genes and mutations in comparing fruit color and fruit sugar (Hazzouri et al. 2019, 2020). Investigations of associated mapping patterns have rapidly become the preferred method to improve palms with complex morphological attributes. Notable, various factors are responsible for the significance of linkage disequilibria, such as mutations, epistasis, size of the population, selection re-arrangement, and autogamy. LD decreases with a higher rate of mutation, recombination, gene conversion, and recurrent mutations (Khan and Korban 2012; Mackay and Powell 2007). Generally, association mapping falls into two categories as follows:

been used to develop many tightly linked genes into functional markers (Lau et al. 2015). Association mapping yields a complete understanding of linkage disequilibrium and genome-wide association (Hamblin et al. 2011). Many reviews have distinguished among date palm varieties (Mathew et al. 2015). Association mappings of candidate-genes have been utilized for date palm improvement (Pintaud et al. 2013). Detecting the variations among date palm populations is an essential factor in association mapping (Hazzouri et al. 2015, 2020). Also, it qualifies the high-resolution mapping of candidate genes. The evidence for the level of breeding in date palm may permit generation of a map of the homozygosity of genes (Hazzouri et al. 2015, 2019; Hildebrandt et al. 2009). Gan et al. (2018) undertook high-density genetic mapping of oil palm via the first use of the DArTseq genotyping platform, using SNP and SSR markers to construct a genetic map of oil palm populations. A gene of acyl-ACP thioesterase B-group is involved in the biosynthesis of fatty acids and is related to palmitic acid in oil palm (Xia et al. 2019). Overexpression triggers a significant increase in the content of palmitic acid in mesocarp tissue. Polymorphisms of domestication divergence of date palm trees have been reported. Hazzouri et al. (2015) described polymorphisms of different mutations for vital agronomic attributes. A copia-like retrotransposon insertion variation in the R2R3 MYB-like orthologue of the VIRESCENS gene is associated with the trait of fruit color (Hazzouri et al. 2019).

5.4.2.1 Candidate Gene-Based Association Mapping Candidate gene-based association mapping is a useful technique when candidate genes are directly or indirectly affected by target attributes with known biological functions (Sehga et al. 2016). It requires the detection of SNPs between varieties and within specific genes. Resequencing of amplicons is the simplest method for investigating the gene(s) of interest. The candidate-gene technique has been used successfully for the characterization and cloning of QTLs. It has also

5.4.2.2 Genome-Wide Association Study (GWAS) Many organizations have commercially developed GWAS platforms to study the genetics of variations in nature and to target desirable attributes. GWAS can examine a tiny block in size in which the diversity of QTLs allows a costeffective method with high throughput (Atwell et al. 2010). Palms are the most cultivated trees used for association mapping. GWAS platforms are utilized in many reviews and projects to improve

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date palm. GBS technology was used on 70 Phoenix cultivars for a diverse set of fruit and leaves. Mathew et al. (2015) classified the cultivars into two loci of the gene pool from North Africa and the Arabian Gulf, in terms of the history of agriculture. GWAS platforms provide the basis for the best understanding of palm genetic resources. GWAS mapping produces complete information on LD and genome-wide throughput. Several reports have differentiated within and among Phoenix taxa (Mathew et al. 2015; Pintaud et al. 2013). Detecting variations in date palm gene pools using GWAS platforms is conducive to high-resolution mapping of candidate genes and homozygosity of genes (Hamblin et al. 2011; Hazzouri et al. 2019, 2020). In a recent study, Hazzouri et al. (2019) reported an improved long-read genome assembly for dactylifera:772.3 Mb in length, with contig 897.2 Kb and performed GWAS of the sex discriminating region and 21 fruit attributes. The attribute of fruit color is associated with the R2R3-MYB transcription factor VIRESCENS gene, whereas, in other species, MYB transcription factors and invertase are concerned with sugar composition and fruit color. Therefore, the importance of analogous evolution in the Phoenix variations has been proved (Hazzouri et al. 2019). In oil palm, Wong and Bernardo (2008) concluded that GW favors selection based on MAS and HTPP in terms of time and cost; especially with those species having a relatively small population size and long generation intervals. MAS and large-scale proliferation of elite dura and pisifera parents of oil palm generated the new commercial tenera palms with higher O/DM potential (Teh et al. 2016). Teh et al (2016) made selections-based fundamentally upon the short range of LD distance with long breeding sets and heterogeneous breeding populations due to the correlation of 80 loci with O/DM and the existence of three main signals. Twelve candidate-genes responsible for plant architecture and yield were determined using GWAS (Osorio-Guarín et al. 2019). Therefore, the associated markers may be interesting, treasured sources for the progress of

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MAS in palm improvement. The choice of MAS for gene-selection permits the identification of alleles and to select a desirable tree. These sources can provide a substantial basis for the progression of genetic QTL. Bai et al. (2017, 2018) constructed a high-density linkage mapping using 188 anchor SNPs and 123 microsatellites, which can accelerate date palm genetic improvement.

5.4.3 Marker-Assisted Selection (MAS) The marker-assisted selection approach avoids the difficulties related to environmental plant breeding, including tolerance to environmental extremes (Kumar et al. 2011). Therefore, breeders typically choose MAS to identify alleles across subsequent progenies and desired individuals across the segregations (Das et al. 2017). The importance of MAS schemes used are (1) marker-assisted backcrossing, (2) gene pyramiding, (3) marker-assisted recurrent selection, and (4) genomic selection. Marker-assisted backcrossing is the oldest method of the backcrossing methods. Its efficiency is enhanced when improved molecular are used. Long-read sequencing of cv. Barhee BC4 (Mathew et al. 2014) represents an integrated genetic map to place contigs of 18 linkage groups (Hazzouri et al. 2019). This sequence is available (Hazzouri et al. 2019) at the Date Palm Genome Hub website (https://datepalmgenomehub. abudhabi.nyu.edu). Marker-assisted recurrent selection is a simple method in which molecular markers are applied to morphological attribute(s) of interest. In this technique, the selection-based improvement should apply to species characteristics with long generation intervals, small population size, and long lifespan (Wong and Bernardo 2008). Marker-assisted gene pyramiding is used to improve resistance to biotic stresses by selecting two or more genes and involves assembling a pyramid of desired genes in various crops (Ye and Smith 2008). The microbiomes of date palm leaves and roots are diverse.

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Gammaproteobacteria and Alphaproteobacteria proteobacteria are dominant in the date palm rhizosphere owing to ecosystems (Mosqueira et al. 2019). Plant growth promoters motivate the solubilization of inorganic phosphate, nitrogen fixation, the production of siderophores, phytohormones, 1-aminocyclopropane-1-carboxylate deaminase, and exopolysaccharide that form symbiotic associations with dates (Hazzouri et al. 2019). Under drought-like conditions, inoculation of date palm roots with endophytes encourages growth (Cherif et al. 2015). On the other hand, genes with nucleotide-binding site/leucine-rich repeats, NBS-LRR (McHale et al. 2006), receptor-like kinases, RLKs (Shiu and Bleecker 2001) and receptor-like proteases, RLPs (Kruijt et al. 2005) are the date palm candidates of gene susceptibility to bayoud disease (Fusarium oxysporum; Hazzouri et al. 2015, 2019; Sedra 2011). A more advanced type of marker-assisted selection called genomic-selection was first developed by Meuwissen et al. (2001). It can predict the value of candidate genes according to the estimating of genomic-based breeding back to the distribution of high-density markers throughout the genome. In genome selection, genetic markers are utilized and selected in such a way that all QTLs in a linkage disequilibrium have at least a single marker (Goddard and Hayes 2007). Genomic selection and high-throughput phenotyping have revolutionized genetic improvement programs by their selection accuracy (Ingvarsson and Street 2011). A high-throughput phenotyping platform gives a precise comprehensive measurement of plant attributes, which provides accurate information about the traits of interest (Finkel 2009). Similarly, the development in the field of highthroughput phenotyping platforms has made it possible to obtain precise data for various complex traits. Genome-wide SNPs enable rapid efforts toward the nature of genetic and phenotypic diversity, and to be applicable to agronomic improvement in date palm (Hazzouri et al. 2020; McClure et al. 2014). The chloroplast genomes of date palms are similar to tobacco with little rearrangement and gene loss or gain. A date palm chloroplast

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genome based on pyrosequencing was sequenced completely by Yang et al. (2010). The highthroughput sequencing quickly identified varieties which are diverse in their chloroplastgenomes (Singh et al. 2020; Yarra et al. 2020). Transcriptomic analysis of cpDNA provides evidence for finding regulatory mechanisms of transcription and translation in chloroplasts (Yang et al. 2010). The date palm responses toward heat and drought stress are improved (Safronov et al. 2017). Currently, improvements of date palm have been limited by the absence of high-resolution mapping and heterozygosity of varieties and cultivars. Complementary between the genetic map, by adding novel technologies, must be achieved (Das et al. 2018; Singh et al. 2020). Date palm improvement contributes to several fields owing to abiotic/biotic stresses. It needs to map more complete sets of genes, which will detect the candidate genes with genetic mapping and other recent technologies (Daccord et al. 2017; Yarra et al. 2020).

5.4.4 Functional Markers Functional markers have been applied successfully in breeding for agronomic attributes and disease resistance in crop plants. Functional markers are related directly to alleles and can also select alleles in a population more efficiently because of their polymorphism of loci that cause variability in phenotypic attributes. However, most markers are reached more effectively through MAS (Bernardo et al. 2015). After more advancement of next-generation sequencing (NGS) technology, single nucleotide polymorphism (SNP) markers have become the marker of choice and applied in genotyping for various crops. Date palm breeding using MAS has uncovered candidate genes. Also, the target attributes contribute to other monocotyledonous and dicotyledonous plants (Ho et al. 2007). SNPs have intravarietal polymorphisms. Most SNPs are in genes having vital functions. Based on RNA-sequencing data, Yang et al. (2010) found 18 polycistronic transcription units and 3 highly

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expression-biased genes-atpF, trnA-UGC and rrn23. Bhore et al. (2010) reported the annotation of the American oil palm Elaeis oleifera (Kunth) Cortés. The beta-carotene hydroxylase gene is a domain in the fatty acid hydroxylase superfamily. The gene SHELL (Sh), which is of codominant monogenic inheritance and encodes a transcriptional activator factor homologous to the gene SEEDSTICK (Singh et al. 2013), plays a functional role in Elaeis guineensis production. This gene is responsible for endocarp formation in oil palm. These methodologies are helpful to determine date palms by fruit type in the nursery and pre-nursery stages 24 months before production begins (Reyes et al. 2015), thereby reduced the time and area used in oil palm breeding programs. Using RACE-PCR, Rekik et al. (2015) isolated a somatic embryogenesis receptor kinase-like (SERKL) cDNA designated PhSERKL gene expressed during the establishment of date palm somatic embryogenic competence acquisition and globular embryo formation by in vitro proliferation. This kind of protein shares the whole of SERK-family characteristic domains involving five leucine-rich repeats, one proline-rich region motif, a transmembrane domain, and kinase domains. They proposed that the expression level of PhSERKL was lower in non-embryogenic tissue and organs than in embryogenic callus (Rikek et al. 2019). KASPTM (Kompetitive Allele Specific PCR) is a recent multiplexed technique used for genotyping. It is a homogenous technology and a platform of fluorescence-based genotyping. Therefore, it produces allele-specific extension and the generation of signals and transfer of fluorescence resonance energy (Allen et al. 2011). The KASP platform is classified and polymorphed in many crops (Su et al. 2018). Its applicability to date palm genotyping will play a significant role in the breeding, genetic resources collection, progress of sequencing technology, and increasing of SNP tags (Lister et al. 2013; KBiosciences, www.kbioscience.co.uk).

E. M. B. Mahdy and S. F. El-Sharabasy

5.5

Targeting Induced Local Lesions iN Genome (TILLING)

TILLING is a nontransgenic technique of reverse genetics. Several methods, including array-based technologies, enzymatic mismatch cleavage, high-performance liquid chromatography, and mass spectrometry, can identify mutants by a standard mutagen-like ethyl methanesulfonate (Kurowska et al. 2011). The TILLING technique allows a higher rate of point mutation. Bioinformatics tools such as project aligned-related sequences and evaluate SNPs (PARSESNP) are then applied and annotated for these mutants. TILLING is easily applicable to any species and not affected by the size of the genome and/or the level of ploidy in a species. High throughput TILLING precisely identifies new alleles at lower cost and in less time (McCallum et al. 2000). In this context, TILLING by sequencing (TbyS) is a high-throughput platform to mutagenize TILLING populations. The TbyS platform will speed up studies of plant functional genomics by a rapid increase in genome-editing (Kumar et al. 2017). TILLING was first established in Arabidopsis thaliana (McCallum et al. 2000) and utilized successfully in various crops such as sorghum (Nida et al. 2012); soybean (Li et al. 2017); oats (Chawade et al. 2010); Brassica spp. (Harloff et al. 2011; Wang et al. 2008); wheat (Rawat et al. 2012; Slade et al. 2005; Uauy et al. 2009); and peanut (Knoll et al. 2011). Genome sequencing applied the TILLING technique to date palm genetic improvement and use against abiotic and biotic stresses. The TILLING platform enables early detection of mutations that have occurred in various date palm improvement programs like tissue culture, long-term conservation, and breeding against osmotic stress. Several known genes are involved in somatic embryogenesis in date palms (Hazzouri et al. 2020; Osorio-Guarín et al. 2019). These genes include protein receptor-like kinases [somatic embryogenesis receptor kinase (SERK) and

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CLAVATA1 (CLV1)]; mitogen-activated protein kinase (MAPK); transcription factors [WUSCHEL (WUS), APETALA2/Ethyleneresponsive factor (AP2/ERF), PICKLE (PKL), AINTEGUMENTA (ANT) and WRKY]; extracellular proteins [arabinogalactan protein (AGP), germin-like protein (GLP); embryogenic cell protein (ECP) and late embryogenesis-abundant protein (LEA)]; and glutathione S-transferase (GST). Future research will focus on variations in resistance to abiotic and biotic stress of date palm. The investigations on diverse other relatives such as oil palm have been readily exploited for more application in somatic embryogenesis and intra- and interspecific crosses (GrosBalthazard 2013; Gros-Balthazard et al. 2017).

5.6

Genomic Editing (CRISPR)

CRISPR-Cas9 technology is a recent advancement and progression in genome-editing characterized by its simplicity, ease of use, competency, versatility, and cleaved methylated loci (Hsu et al. 2013; Lozano-Juste and Cutler 2014). Genome editing is a useful supplementary tool for genetic improvement using genetic modifications in the genome of interest. CRISPR is a genome-editing technique applied successfully in various plants (Feng et al. 2013). The tools of genome editing such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9 have accelerated the editing of plant genomes (Sattar et al. 2017). Among palms, most reports involve oil palm genome editing. A few reviews on date palm have been published of CRISPR/Cas9 concerning metal tolerance (Chaâbene et al. 2017) and tolerance to cadmium and chromium stresses (Chaâbene et al. 2018) by the targeting genes Pdpcs and Pdmt. However, different genes can combine desirably for date palm using sgRNAs that point to multiplex genome editing and to effective genome editing in several other species such as oil palm (Aprilyanto et al. 2019; Yarra et al. 2019, 2020). Applying Cas9 can be a challenge in genome editing of the date palm because its genome is highly heterogeneous, has

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a high polymorphic rate of SNPs and is ultimately genetically unstable (Sattar et al. 2017). The genes isoflavone reductase (IFR) and methallothionine-like protein (MT) were edited by Budiani et al. (2018) to carry a sequence of gene recognition (sgRNA) constructed and transformed into oil palm calli using Agrobacterium tumefaciens. By alternating the palmitoyl acyl thioesterase (PATE) gene, responsible for high palmitic acid content in oil palm, an efficacy test was conducted by Aprilyanto et al. (2016). They designed the sgRNA, which follows with CRISPR/Cas9 plasmid construction containing optimized sgRNA and Agrobacterium-mediated transformation. They concluded that the design of sgRNA could effectively guide Cas9 to cleave exon-1 of the oil palm PATE gene. Aprilyanto et al. (2019) attempted to develop a plasmid CRISPR/Cas9 containing four sgRNAs to allow multiple-gene editing in the genome of oil palm by finding the optimum 20-nt guides from four gene regions and joined later with a promoter and tracrRNA fragment to construct a 472 bp module. CRISPR/Cas9 improved oil yield and reviewed the methods for in vitro-mediated propagation and genetic transformation of oil palm (Yarra et al. 2019). Date palm genome sequencing empowers genetic improvement (Singh et al. 2013), the editing of interest genes against biotic and abiotic stresses (Singh et al. 2020; Yarra et al. 2020) and excludes the cultivars with low quality from markets (Hani et al. 2020).

5.7

DNA Barcode Utilization

Tautz et al. (2003) produced the first DNA barcode markers. They are efficient in the discrimination of a candidate gene barcode, based on a locus that would ideally be the same for all kingdoms (CBOL Plant Working Group 2009). It is also called DNA-taxonomy. Efforts to find a robust locus-barcode for the kingdom Plantae have made some progress but remains quite a challenge. Therefore, various DNA genes of the plastid genome (erbcL, matK, rpoB, rpoC1,

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trnH-psbA, ycf1) and nuclear genome (ITS) have been suggested for application to the various taxa. The first step is determining which marker can best be used to publicize DNA barcoding (CBOL Plant Working Group 2009). There are two general categories of potential use of DNA barcodes: plant taxonomy and plant systematics (CBOL Plant Working Group 2009). However, these markers have shown to be promising tools to assess biodiversity, the evolution of taxa, identification, plant systematics, and plant populations as a genetic delimitation of biological species. Interspecific and intraspecific relationships are still potential high-resolution tools in systematics at the generic and species levels. Chloroplast DNA (cpDNA) has extremely conserved regions in species (CBOL Plant Working Group 2009). Therefore, it can be classified into three functional classes in two main categories: (a) Non-coding regions. These regions are a key source of data for molecular systematics, phylogenetics, and DNA barcodes for species (Shew et al. 2007). It includes introns and intergenic spacers. (b) Coding regions. These regions consist of protein-coding genes. The first attempts at DNA barcodes in palms were done by Jeanson et al. (2011) and generated 92% success in species identification using matK, rbcL, and ITS2. Markers of cpDNA were used for the identification of species (Al-Qurainy et al. 2011; Chen et al. 2010; CBOL Plant Working Group 2009; Enan and Ahmed 2014, 2016; Hani et al. 2020; Pintaud et al. 2013). Several loci of cpDNA such as rbcL, rpoB, rpoC1, psbA-trnH spacer, nrITS2, psbZ, trnFM, and matK have been tested for DNA barcoding (Abu-Afifeh 2018; Chen et al. 2010; Enan and Ahmed 2014, 2016; Hani et al. 2020; Hollingsworth et al. 2011; Kress et al. 2007; Lahaye et al. 2008). Some recent studies have used DNA barcodes to investigate Phoenix species. Several molecular markers efficiently assess the genetic diversity in date palm cultivars such as in Egypt (Abd El-

E. M. B. Mahdy and S. F. El-Sharabasy

Azeem et al. 2011; Ibrahim et al. 2014; Saker et al. 2006); Oman (Al-Ruqaishi et al. 2008); Morocco (Bodian et al. 2014; Sedra 2013; Sedra et al. 1998); Saudi Arabia (Al-Khalifah and Askari 2007; Al-Khalifah et al. 2012); Tunisia (Zehdi et al. 2005, 2012); and Sudan (Elshibli and Korpelainen 2008). These studies have helped to identify markers suitable for the identification of date palm varieties. A data matrix using rpoB and psbA-trnH has shown constant and variable regions resulted from three major groups of eight date cultivars (a) 58% BS, (b) 64% Bs, and (c) 67% BS. The economically most important cultivar Ajwa showed similarity with Khodary and Sefri (AlQurainy et al. 2011). Forty date palm cultivars were analyzed by Jeanson et al. (2011). Various genes, including four DNA markers, three plastids encoded (matK, rbcL, psbA-trnH), and one molecular encoded (nrITS2), were tested for segregation among species. The combination of three markers (matK, rbcL, nrITS2) results in 92% species discrimination. The two core markers suggested by CBOL-PWG, rbcL and matK, have a low species discrimination rate. Kress et al. (2009) demonstrated that DNA barcodes revamp the robust community phylogeny by engaging a supermatrix scheme for species in situ. Pintaud et al. (2013) examined various origins of the Phoenix dactylifera gene pools using cpDNA barcoding and DNA microsatellites. A validation to identify dactylifera populations must be considered. The data show no sign of interspecific hybridization. The importance of intraspecific dactylifera gene flow was due to the geographic structure of the agrobiodiversity. A cpDNA region with a length of 700 bp compared with the minisatellite was developed as a DNA barcode and sited among psbZ and trnFM. The psbZ and trnFM were sequenced and analyzed for 136 individuals representing all Phoenix species except P. andamanesis S. Barrow. The repetition minisatellite showed 2–7 of 12 bp motifs. Phoenix reclinata and P. canariensis had species-specific haplotypes. Polymorphisms originated in the flanking sequences, together with homopolymers, insertion/deletion,

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and substitutions (Ballardini et al. 2013). Barcoding of cpDNA is a useful marker to identify and assess the genetic diversity of a species. Wild relatives could be discriminated against based upon the information of mutations occurring in the trnL region (Bakker et al. 2000). Minireviews were done to identify date palm cultivars by Naeem et al. (2014), Enan and Ahamed (2016), Abu-Afifeh (2018), and Hani et al. (2020). Discrimination among cultivars is essential to exclude the quality of date palm cultivars in markets (Hani et al. 2020). In this context, Hani et al (2020) stated that the psbAtrnH loci are not appropriate for DNA barcoding in date palm cultivars because they are not useful for the phylogenetic relationships at the taxonomic level. The loci of rbcL, matK, rpoC1, and ycf5 identified successfully unknown Egyptian seedling date palm cultivars based on their genetic background and genetic distance (AbuAfifeh 2018). Regions of matK and rbcL reveal potential discriminatory powers in the discrimination of the palm family (Naeem et al. 2014). Both dactylifera and sylvestris showed no variation due to the close relationship. The matK alone or with rpoC1 are constructed similarity trees among 11 dactylifera cultivars (Enan and Ahamed 2014). Hani et al (2020) stated matK, rbcL, ycf5 can be utilized for barcoding and defining the genetic variation between date palm cultivars. Finally, DNA barcoding has the potential to discriminate among species of Arecaceae and cultivars. To achieve a complete assessment, it must necessarily involve morphological attributes with DNA barcoding using more loci (Naeem et al. 2014). DNA taxonomy constructs a potential discriminator of a phylogenetic tree within interspecies and intraspecies of Phoenix. Several studies on Arecaceae reported that the rbcL is the most used loci as a potential power of DNA-barcode-based marker (Abu-Afifeh 2018; Hani et al. 2020; Naeem et al. 2014). However, these studies are insufficient and still lack information about the relationships among intraspecies.

5.8

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Conclusions and Prospects

There has been rapid and steady advancement and development in molecular marker technologies since the 1980s. A diversity of technologies of array-based markers in current use have made a large change in the movement to meet the challenges of the traditional methods and easily applied to various aspects of palms. Better utilization of these molecular markers in recent years is due to the considerable number of research publications each year. Another major challenge is that a proportion of these studies fail to influence practical-level breeding. Many neglected and underutilized taxa of Phoenix may be lost if conservation measures are not taken. Each germplasm accession has its unique genetic structure at the level of coding sequences. The sequence of the coding and noncoding genes indicates the genetic diversity among genomes. The lack of information about economic returns and agricultural attributes, and the unavailability of genetic resources, limits sustainable utilization and the development of functional diagnostic markers to apply and benefit Phoenix tree crops. The fundamental reason for the slow release of new technologies on date palm genomes is the decrease in the number of skilled researchers and specialized laboratories. In the coming years, the scientific world will achieve progress through innovations in molecular markers and DNA barcoding technologies to more fully exploit the various biological attributes of the genus Phoenix. Finally, candidate genes have to be deeply rooted to target the analyses of gene functions in the future. Projects on date palm improvement should concentrate on the loci of most interest, such as gender discrimination and genotyping. These attributes represent the main obstacles to date palm production. Also, there is a large gap in exploiting the availability of date palm genetic resources. These resources must be employed for sustainable utilization by which more studies focus on date palm phylogeny, evolution,

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breeding and genetics. Molecular markers have made noteworthy progress but are still insufficient and not fully utilized. If applied well, these efforts will uncover new options and choices to overcome obstacles. The approaches of date palm improvement need to be more robust in terms of genetic mapping and genotyping. Date palm breeding can be achieved through the incorporation of recent and novel platforms such as the CRISPER platform, KASP Markers, and TILLING technology.

References Abd El-Azeem R, Hashem M, Hemeida A (2011) Identification and genetic similarity analysis of date palm (Phoenix dactylifera L.) collected from different regions in Siwa oasis morphological traits and molecular markers. Egypt J Genet Cytol 40:281–300. https:// doi.org/10.21608/ejgc.2011.10793 Abdulla M, Gamal O (2010) Investigation on molecular phylogeny of some date palm (Phoenix dactylifera L.) cultivars by protein, RAPD and ISSR markers in Saudi Arabia. Austral J Crop Sci 4:23–28 Abu-Afifeh AA (2018) Molecular characterization of some date palm genotypes in Egypt. PhD thesis, Faculty of Agriculture, Ain Shams Univ, Cairo, Egypt Agah A (2004) A multi-enzyme model for pyrosequencing. Nucl Acid Res 32:e166–e166. https://doi.org/10. 1093/nar/gnh159 Agarwal M, Shrivastava N, Padh H (2008) Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep 27:617–631. https://doi. org/10.1007/s00299-008-0507-z Akhtar W, Rasheed AW, Shinwari ZK et al (2014) Genetic characterization of different Pakistani date palm varieties. Pak J Bot 46:2095–2100 Al-Ameri AA, Al-Qurainy F, Gaafar ARZ et al (2016a) Molecular identification of sex in Phoenix dactylifera using inter simple sequence repeat markers. Biomed Res Int 2016:1–5. https://doi.org/10.1155/2016/ 4530846 Al-Ameri AA, Al-Qurainy F, Gaafar ARZ et al (2016b) Male specific gene expression in dioecious Phoenix dactylifera (date palm) tree at flowering stage. Pak J Bot 48:131–135 Alansari MS, Al-kazaz AKA, Khierallah HS (2014) Assessment of genetic distance among some Iraqi date palm cultivars (Phoenix dactylifera L.) using randomly amplified polymorphic DNA. Iraqi J Sci 55 (4B):1833–1843 Al-Dous EK, George B, Al-Mahmoud ME et al (2011) De novo genome sequencing and comparative genomics

E. M. B. Mahdy and S. F. El-Sharabasy of date palm (Phoenix dactylifera). Nature Biotech 29:521–527. https://doi.org/10.1038/nbt.1860 Al-Jibouri AAM, Adham KM (1990) Biochemical classification of date palm male cultivars. J Hort Sci 65:725–729 Al-Khalifah NS, Askari E (2007) Early detection of genetic variation in date palms propagated from tissue culture and offshoots by DNA fingerprinting. Acta Hort 736:105–112. https://doi.org/10.17660/ ActaHortic.2007.736.8 Al-Khalifah NS, Askari E, Shanavas Khan AE (2012) Molecular and morphological identification of some elite varieties of date palms grown in Saudi Arabia. Emir J Food Agric 24:456–461 Al-Khateeb SA, Al-Khateeb AA, Beltagi HS, Muhammad NS (2019) Genotypic variation for drought tolerance in three date palm (Phoenix dactylifera L.) cultivars. Fresenius Environ Bull 28:4671–4683 Allen AM, Barker GL, Berry ST et al (2011) Transcriptspecific, single-nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat (Triticum aestivum L.). Plant Biotech J 9(9):1086– 1099 Al-Mssallem IS, Hu S, Zhang X et al (2013) Genome sequence of the date palm Phoenix dactylifera L. Nat Comm 4:2274. https://doi.org/10.1038/ncomms3274 Al-Mssallem IS (1996) Date palm (Phoenix dactylifera L.), vol 7. Encyclopedia Works Publishing & Distribution, Riyadh, Saudi Arabia Al-Qurainy F, Al-Ameri AA, Khan S et al (2018) SCAR marker for gender identification in date palm (Phoenix dactylifera L.) at the seedling stage. Int J Genom 2018:1–6. https://doi.org/10.1155/2018/3035406 Al-Qurainy F, Khan S, Al-Hemaid FM et al (2011) Assessing molecular signature for some potential date (Phoenix dactylifera L.) cultivars from Saudi Arabia, based on chloroplast DNA sequences rpoB and psbAtrnH. Int J Mol Sci 12:6871–6880. https://doi.org/10. 3390/ijms12106871 Al-Ruqaishi IA, Davey M, Alderson P, Mayes S (2008) Genetic relationships and genotype tracing in date palms (Phoenix dactylifera L.) in Oman, based on microsatellite markers. Plant Genet Res 6:70–72. https://doi.org/10.1017/S1479262108923820 Al-Yahyai R, Al-Khanjari S (2008) Biodiversity of date palm in the Sultanate of Oman. Afr J Agric Res 3:389–395 Angaji SA (2009) QTL mapping: a few key points. Int J Appl Res Nat Prod 2:1–3 Ansorge WJ (2009) Next-generation DNA sequencing techniques. New. Biotech 25:195–203. https://doi.org/ 10.1016/j.nbt.2008.12.009 Aprilyanto V, Darmawan C, Subroto AP et al (2016) Genome editing for improving of oil quality through implementation of CRISPR/Cas9 system. Ann Tech Rep 3:1–10 Aprilyanto V, Darmawan C, Utomo C, Liwang T (2019) Development of CRISPR/Cas9 plasmid for multiple

5

Date Palm Genetic Identification and Improvement …

sites genome editing in oil palm (Elaeis guineensis Jacq.). In: 1st int conf bioinformatics, biotechnology and biomedical engineering. AIP Publishing, pp 1–6 Arabnezhad H, Bahar M, Mohammadi HR, Latifian M (2012) Development, characterization and use of microsatellite markers for germplasm analysis in date palm (Phoenix dactylifera L.). Scient Hort 134:150– 156. https://doi.org/10.1016/j.scienta.2011.11.032 Ataga CD, Mohammed AH, Yusuf AO (2012) Status of date palm (Phoenix dactylifera L.) genetic resources in Nigeria. Int J Life Sci Pharma Res 2(2):46–51 Atia AMM, Adawy SS (2015) Novel set of sex-specify PCR-based markers reveals new hypothesis of sex differentiation in date palm. J Plant Sci 3:150–161 Attaha AHM, Mhoder TY, Abd A-KM (2013) Protein pattern of Phoenix dactylifera seeded strains grown in Basrah region using cluster and principal component analysis. J Plant Sci 2. https://doi.org/10.5539/jps. v2n2p149 Atwell S, Huang YS, Vilhjálmsson BJ et al (2010) Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines. Nature 465:627– 631 Bai B, Wang L, Lee M et al (2017) Genome-wide identification of markers for selecting higher oil content in oil palm. BMC Plant Biol 17:93. https:// doi.org/10.1186/s12870-017-1045-z Bai B, Wang L, Zhang YJ et al (2018) Developing genome-wide SNPs and constructing an ultra highdensity linkage map in oil palm. Scient Rep 8:691. https://doi.org/10.1038/s41598-017-18613-2 Bakker FT, Culham A, Gomez-Martinez R et al (2000) Patterns of nucleotide substitution in angiosperm cpDNA trnL (UAA)-trnF (GAA) regions. Mol Biol Evol 17:1146–1155. https://doi.org/10.1093/ oxfordjournals.molbev.a026397 Ballardini M, Mercuri A, Littardi C et al (2013) The chloroplast DNA locus psbZ-trnfM as a potential barcode marker in Phoenix L. (Arecaceae). Zoo Keys 365:71–82. https://doi.org/10.3897/zookeys.365.5725 Baloch FS, Alsaleh A, De Miera LES et al (2015) DNA based iPBS-retrotransposon markers for investigating the population structure of pea (Pisum sativum) germplasm from Turkey. Biochem Syst Ecol 61:244–252. https://doi.org/10.1016/j.bse.2015.06. 017 Barrow S (1998) A revision of Phoenix. Kew Bull 53:513–575 Beckmann JS, Weber JL (1992) Survey of human and rat microsatellites. Genom 12:627–631. https://doi.org/ 10.1016/0888-7543(92)90285-Z Beissinger TM, Hirsch CN, Sekhon RS et al (2013) Marker density and read depth for genotyping populations using genotyping-by-sequencing. Genet 193 (4):1073–1081. https://doi.org/10.1534/genetics.112. 147710 Bekheet SA (2011) In vitro conservation of date palm germplasm. In: Jain SM, Al-Khayri JM, Johnson DV

125

(eds) Date palm biotechnology. Springer, Netherlands, Dordrecht, pp 337–360 Bekheet SA, El-Sharabasy SF (2015) Date palm status and perspective in Egypt. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date palm genetic resources and utilization. Springer, Netherlands, Dordrecht, pp 75– 123 Bekheet SA, Taha HS (2013) Complementary strategy for conservation of date palm germplasm. Global J Biodiv Sci Manag 3:96–107 Bentley DR, Balasubramanian S, Swerdlow HP et al (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53– 59. https://doi.org/10.1038/nature07517 Bernardo A, Wang S, Amand PS, Bai G (2015) Using next generation sequencing for multiplexed traitlinked markers in wheat. PLoS ONE 10(12): https:// doi.org/10.1371/journal.pone.0143890 Bhagyawant SS (2016) RAPD-SCAR markers: an interface tool for authentication of traits. J Biosci Med 04:1–9. https://doi.org/10.4236/jbm.2016.41001 Bhore SJ, Kassim A, Loh CY, Shah FH (2010) Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library. Bioinfo 5:104–122. https:// doi.org/10.6026/97320630005104 Bodian A, Nachtigall M, Frese L et al (2014) Genetic diversity analysis of date palm (Phoenix dactylifera L.) cultivars from Morocco using SSR markers. J Biodiv Bioprosp Dev 01:1–8. https://doi.org/10. 4172/2376-0214.1000126 Bourgis F, Kilaru A, Cao X et al (2011) Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning. Proc Nat Acad Sci USA 108:12527– 12532. https://doi.org/10.1073/pnas.1106502108 Budiani A, Putranto RA, Riyadi I et al (2018) Transformation of oil palm calli using CRISPR/Cas9 system: toward genome editing of oil palm. IOP Conf Ser Earth Environ Sci 183(1): https://doi.org/10.1088/ 1755-1315/183/1/012003 Cardle L, Ramsay L, Milbourne D et al (2000) Computational and experimental characterization of physically clustered simple sequence repeats in plants. Genet 156:847–854 Carreño E, Rivera D, Obón C et al (2020) What are candits? Study of a date palm landrace in Spain belonging to the western cluster of Phoenix dactylifera L. Genet Res Crop Evol. https://doi.org/10.1007/ s10722-020-00973-w CBOL Plant Working Group (2009) A DNA barcode for land plants. Proc Nat Acad Sci USA 106:12794– 12797 Chaâbene Z, Hakim IR, Rorat A et al (2017) Copper toxicity and date palm (Phoenix dactylifera) seedling tolerance: monitoring of related biomarkers. Environ Toxicol Chem 37:797–806. https://doi.org/10.1002/ etc.4007CrossRefPubMedGoogleScholar

126 Chaâbene Z, Rorat A, Hakim I et al (2018) Insight into the expression variation of metal-responsive genes in the seedling of date palm (Phoenix dactylifera). Chemosph 197:123–134. https://doi.org/10.1016/j. chemosphere.2017.12.146 Chen S, Yao H, Han J, Liu C (2010) Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS ONE 5: Cherif E, Zehdi S, Castillo K et al (2013) Male-specific DNA markers provide genetic evidence of an XY chromosome system, a recombination arrest and allow the tracing of paternal lineages in date palm. New Phytol 197:409–415. https://doi.org/10.1111/nph. 12069 Daccord N, Celton JM, Linsmith G et al (2017) Highquality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat Genet Adv 49:1099–1106. https://doi.org/10.1038/ng. 3886 Das G, Patra JK, Baek KH (2017) Insight into MAS: a molecular tool for development of stress resistant and quality of rice through gene stacking. Front Plant Sci 8:985. https://doi.org/10.3389/fpls.2017.00985 Davey JW, Hohenlohe PA, Etter PD et al (2011) Genomewide genetic marker discovery and genotyping using next-generation sequencing. Nat Rev Genet 12:499– 510. https://doi.org/10.1038/nrg3012 Deschamps S, Llaca V, May GD (2012) Genotyping-bysequencing in plants. Biol 1:460–483. https://doi.org/ 10.3390/biology1030460 Dhawan C, Kharb P, Sharma R et al (2013) Development of male-specific SCAR marker in date palm (Phoenix dactylifera L.). Tree Genet Genom 9:1143–1150. https://doi.org/10.1007/s11295-013-0617-9 Dhingani RM, Umrania VV, Tomar RS et al (2015) Introduction to QTL mapping in plants. Ann Plant Sci 4(4):1072–1079 Dong W, Liu J, Yu J et al (2012) Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding. PLoS ONE 7: https://doi.org/10.1371/journal.pone. 0035071 Eissa E, Abd El-Razek A, El-Sharabasy S, Rizk R (2009) Morphological and molecular genetic characterization of soft date palm (Phoenix dactylifera L.) cultivars in Egypt. Egypt J Genet Cytol 38:269–284 Elmeer K, Mattat I, Al-Malki A et al (2019) Assessing genetic diversity of shishi date palm cultivars in Saudi Arabia and Qatar using microsatellite markers. Int J Hort Sci Tech 6:1–9 El-Sharabasy SF, Rizk RM (2005) Morphological diversity of date palm (Phoenix dactylifera L.) in Egypt soft date palm cultivars. Mansoura Hort J 30(11):7001– 7027 El-Sharabasy SF, Rizk RM (2019) Atlas of date palm in Egypt. FAO, Egypt Elshibli S, Korpelainen H (2008) Microsatellite markers reveal high genetic diversity in date palm (Phoenix dactylifera L.) germplasm from Sudan. Genet

E. M. B. Mahdy and S. F. El-Sharabasy 134:251–260. https://doi.org/10.1007/s10709-0079232-8 Elshire RJ, Glaubitz JC, Sun Q et al (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS ONE 6: https://doi.org/ 10.1371/journal.pone.0019379 Emoghene BO, Asemota O, Idu M, Eke CR (2015) Molecular characterization of some date palms in Nigeria using RAPD markers. J App Biol Biotech 3:26–30. https://doi.org/10.7324/JABB.2015.3505 Enan RM, Ahmed A (2016) Cultivar-level phylogeny using chloroplast DNA barcode psbK-psbI spacers for identification of emirati date palm (Phoenix dactylifera L.) varieties. Genet Mol Res 15(3). https://doi.org/ 10.4238/gmr.15038470 Enan MR, Ahmed A (2014) DNA barcoding based on plastid matK and RNA polymerase for assessing the genetic identity of date (Phoenix dactylifera L.) cultivars. Genet Mol Res 13(2): 3527–3536. https:// doi.org/10.4238/2014.February.14.2 Engelmann F (2004) Plant cryopreservation: progress and prospects. Vitro Cell Dev Biol Plant 40:427–433. https://doi.org/10.1079/IVP2004541 Engelmann F (2009) Plant germplasm cryopreservation: progress and prospects. Cryobiol 59:370–371. https:// doi.org/10.1016/j.cryobiol.2009.10.017 Ersoz ES, Yu J, Buckler ES (2007) Applications of linkage disequilibrium and association mapping in crop plants. In: Varshney RK, Tuberosa R (eds) Genomics-assisted crop improvement. Springer, Dordrecht, pp 97–119 Ewens WJ (1999) Genetics and analysis of quantitative traits. Am J Hum Genet 11:798–800 Fan F, Cui B, Zhang T et al (2014) LTR-retrotransposon activation, IRAP marker development and its potential in genetic diversity assessment of masson pine (Pinus massoniana). Tree Genet Genom 10:213–222. https:// doi.org/10.1007/s11295-013-0677-x Feng Z, Zhang B, Ding W et al (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23:1229–1232. https://doi.org/10.1038/cr.2013.114 Finkel E (2009) With ‘phenomics’, plant scientists hope to shift breeding into overdrive. Science 325:380–381. https://doi.org/10.1126/science.325_380 Finnegan DJ (1989) Eukaryotic transposable elements and genome evolution. Trends Genet 5:103–107. https:// doi.org/10.1016/0168-9525(89)90039-5 Flavell AJ, Knox MR, Pearce SR, Ellis THN (1998) Retrotransposon-based insertion polymorphisms (RBIP) for high throughput marker analysis. Plant J 16:643–650. https://doi.org/10.1046/j.1365-313x. 1998.00334.x Flowers JM, Hazzouri KM, Gros-Balthazard M et al (2019) Cross-species hybridization and the origin of North African date palms. Proc Nat Acad Sci USA 116:1651–1658. https://doi.org/10.1073/pnas. 1817453116 França LTC, Carrilho E, Kist TBL (2002) A review of DNA sequencing techniques. Quar Rev Biophys

5

Date Palm Genetic Identification and Improvement …

35:169–200. https://doi.org/10.1017/ S0033583502003797 Furbank RT, Tester M (2011) Phenomics - technologies to relieve the phenotyping bottleneck. Trends Plant Sci 16:635–644. https://doi.org/10.1016/j.tplants.2011.09. 005 Gan ST, Wong WC, Wong CK et al (2018) High density SNP and DArT-based genetic linkage maps of two closely related oil palm populations. J App Genet 59:23–34. https://doi.org/10.1007/s13353-017-0420-7 Ge T, Lu Y, Lu K et al (2020) VEGA: visual comparison of phylogenetic trees for evolutionary genome analysis (ChinaVis 2019). J Vis 23:523–537. https://doi. org/10.1007/s12650-020-00635-0 Gharizadeh B, Herman ZS, Eason RG et al (2006) Largescale pyrosequencing of synthetic DNA: a comparison with results from Sanger dideoxy sequencing. Electroph 27:3042–3047. https://doi.org/10.1002/elps. 200500834 Goddard ME, Hayes BJ (2007) Genomic selection. J Anim Breed Genet 124:323–330 González-Pérez M, Caujapé-Castells J, Sosa P (2004) Molecular evidence of hybridisation between the endemic Phoenix canariensis and the widespread P. dactylifera with random amplified polymorphic DNA (RAPD) markers. Plant Syst Evol 247. https:// doi.org/10.1007/s00606-004-0166-7 Govaerts R, Dransfield J (2005) World checklist of palms. Roy Bot Gardens, Kew Graham J (2011) Book review. Molecular plant breeding By Y. Xu. CABI Wallingford, UK. Expl Agric 47:173. https://doi.org/10.1017/S0014479710000669 Gros-Balthazard M (2013) Hybridization in the genus Phoenix: a review. Emir J Food Agric 25:831–842. https://doi.org/10.1093/jhered/esy064 Gros-Balthazard M, Galimberti M, Kousathanas A et al (2017) The discovery of wild date palms in Oman reveals a complex domestication history involving centers in the middle east and Africa. Curr Biol 27:2211.e8–2218.e8. https://doi.org/10.1016/j.cub. 2017.06.045 Grzebelus D (2006) Transposon insertion polymorphism as a new source of molecular markers. J Fruit Ornam Plant Res 14:21–29 Guerin C, Serret J, Montúfar R et al (2020) Palm seed and fruit lipid composition: phylogenetic and ecological perspectives, Ann Bot 125(1, 2):157–172. https://doi. org/10.1093/aob/mcz175 Gupta M, Chyi Y-S, Romero-Severson J, Owen JL (1994) Amplification of DNA markers from evolutionarily diverse genomes using single primers of simplesequence repeats. Theor Appl Genet 89:998–1006. https://doi.org/10.1007/BF00224530 Gupta PK, Rustgi S, Kulwal PL (2005) Linkage disequilibrium and association studies in higher plants: present status and future prospects. Plant Mol Biol

127

https://doi.org/10.1007/s11103-00557:461–485. 0257-z Gyawali A, Shrestha V, Guill KE et al (2019) Single-plant GWAS coupled with bulk segregant analysis allows rapid identification and corroboration of plant-height candidate SNPs. BMC Plant Biol 19:412. https://doi. org/10.1186/s12870-019-2000-y Haddouch M (1996) Present situation and development prospects for the date palm sector in Morocco. Opti Méditerr 88:63–79 Hamama L, Cornee N, Leclerc V et al (2003) Date palm (Phoenix dactylifera) offshoot identification by PCRISSR markers. Acta Hort 616:453–457. https://doi. org/10.17660/ActaHortic.2003.616.70 Hamblin MT, Buckler ES, Jannink J-L (2011) Population genetics of genomics-based crop improvement methods. Trends Genet 27:98–106 Hamwieh A, Farah J, Moussally S et al (2010) Development of 1000 microsatellite markers across the date palm (Phoenix dactylifera L.) genome. Acta Hort 882:269–277. https://doi.org/10.17660/ActaHortic. 2010.882.29 Hani HA, Gadalla EG, Haggag S et al (2020) Identification of some cultivars of Egyptian date palm (Phoenix dactylifera L.) using DNA barcoding. Plant Arch 20:1807–1813 Hazzouri KM et al (2015) Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop. Nat Comm 6:8824. https://doi.org/10. 1038/ncomms9824 Hazzouri KM, Flowers JM, Nelson D et al (2020) Prospects for the study and improvement of abiotic stress tolerance in date palms in the post-genomics era. Front Plant Sci 11:293. https://doi.org/10.3389/fpls. 2020.00293 Hazzouri KM, Gros-Balthazard M, Flowers JM et al (2019) Genome-wide association mapping of date palm fruit traits. Nat Comm 10:4680. https://doi.org/ 10.1038/s41467-019-12604-9 Heikrujam M, Sharma K, Prasad M, Agrawal V (2015) Review on different mechanisms of sex determination and sex-linked molecular markers in dioecious crops: a current update. Euphy 201:161–194. https://doi.org/ 10.1007/s10681-014-1293-z Henderson SA, Billotte N, Pintaud J-C (2006) Genetic isolation of Cape Verde Island Phoenix atlantica (Arecaceae) revealed by microsatellite markers. Conserv Genet 7:213–223. https://doi.org/10.1007/ s10592-006-9128-7 Hildebrandt F, Heeringa SF, Rüschendorf F et al (2009) A systematic approach to mapping recessive disease genes in individuals from outbred populations. PLoS Genet 5: Ho C-L, Kwan Y-Y, Choi M-C et al (2007) Analysis and functional annotation of expressed sequence tags (ESTs) from multiple tissues of oil palm (Elaeis

128 guineensis Jacq.). BMC Genom 8:381. https://doi.org/ 10.1186/1471-2164-8-381 Hollingsworth PM, Graham SW, Little DP (2011) Choosing and using a plant DNA barcode. PLoS ONE 6: Hsu PD, Scott DA, Weinstein JA et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotech 31:827–832. https://doi.org/10.1038/ nbt.2647 Hussein EHA, Adawy SS, Ismail SE, El-Itriby HA (2005) Molecular characterization of some Egyptian date palm germplasm using RAPD and ISSR markers. Arab J Biotech Jan 8:83–98 Hussein MA (2015) Determination of sex-specific DNA markers for date palm (Phoenix dactylifera L.) grown in Egypt utilizing nuclear microsatellite markers. J Agric Chem Biotech 6:237–246. https://doi.org/10. 21608/jacb.2015.48402 Huttner E, Wenzl P, Akbari M et al (2005) Diversity arrays technology: a novel tool for harnessing the genetic potential of orphan crops. In: Serageldin I, Persley GJ (eds) Discovery to delivery: bioVision Alexandria 2004, Proc 2004 conf world biological forum. CABI Publishing, UK, pp 145–155 Hyman ED (1988) A new method of sequencing DNA. Anal Biochem 174:423–436. https://doi.org/10.1016/ 0003-2697(88)90041-3 Ibrahim IA, Hashem MH, Hemeida AA et al (2014) Characterization of genetic diversity of date palm (Phoenix dactylifera L.) cultivars collected from New Valley governorate (El-Kharga and Dakhleh) based on morphological variability and molecular markers. Life Sci J 11:879–889 Ingvarsson PK, Street NR (2011) Association genetics of complex traits in plants. New Phytol 189:909–922 Intha N, Chaiprasart P (2018) Sex determination in date palm (Phoenix dactylifera L.) by PCR based marker analysis. Sci Hort 236:251–255. https://doi.org/10. 1016/j.scienta.2018.03.039 Jaccoud D (2001) Diversity arrays: a solid-state technology for sequence information independent genotyping. Nucl Acid Res 29:25e–25. https://doi.org/10. 1093/nar/29.4.e25 Jain SM (2011) Prospects of in vitro conservation of date palm genetic diversity for sustainable production. Emir J Food Agric 23:110–119 Jain SM (2012) Date palm biotechnology: current status and prospective - an overview. Emir J Food Agric 24:386–399 Jamil S, Shahzad R, Kanwal S et al (2020) DNA fingerprinting and population structure of date palm varieties grown in Punjab Pakistan using simple sequence repeat markers. Inter J Agric Biol 23:943– 950. https://doi.org/10.17957/IJAB/15.1373 Jarvis P, Lister C, Szabo V, Dean C (1994) Integration of CAPS markers into the RFLP map generated using recombinant inbred lines of Arabidopsis thaliana. Plant Mol Biol 24:685–687. https://doi.org/10.1007/ BF00023565

E. M. B. Mahdy and S. F. El-Sharabasy Jeanson ML, Labat J-N, Little DP (2011) DNA barcoding: a new tool for palm taxonomists? Ann Bot 108:1445–1451. https://doi.org/10.1093/aob/mcr158 Jeennor S, Volkaert H (2014) Mapping of quantitative trait loci (QTLs) for oil yield using SSRs and genebased markers in African oil palm (Elaeis guineensis Jacq.). Tree Genet Genom 10:1–14. https://doi.org/10. 1007/s11295-013-0655-3 Kalendar R (2011) The use of retrotransposon-based molecular markers to analyze genetic diversity. Ratar Povrt 48:261–274. https://doi.org/10.5937/ ratpov1102261K Kalendar R, Antonius K, Smýkal P, Schulman AH (2010) iPBS: a universal method for DNA fingerprinting and retrotransposon isolation. Theor Appl Genet 121:1419–1430 Kalendar R, Flavell AJ, Ellis THN et al (2011) Analysis of plant diversity with retrotransposon-based molecular markers. Hered 106:520–530. https://doi.org/10. 1038/hdy.2010.93 Kalendar R, Grob T, Regina M et al (1999) IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques. Theor Appl Genet 98:704– 711. https://doi.org/10.1007/s001220051124 Kalendar R, Schulman AH (2006) IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nat Prot 1:2478–2484. https://doi.org/10.1038/nprot. 2006.377 Kalendar R, Schulman AH (2014) Transposon-based tagging: IRAP, REMAP, and iPBS. In: Besse P (ed) Molecular plant taxonomy. Humana Press, Totowa, NJ, Methods in molecular biology, pp 233– 255 Kalia RK, Rai MK, Kalia S et al (2011) Microsatellite markers: an overview of the recent progress in plants. Euphy 177:309–334. https://doi.org/10.1007/s10681010-0286-9 Khan MA, Korban SS (2012) Association mapping in forest trees and fruit crops. J Exp Bot 63:4045–4060. https://doi.org/10.1093/jxb/ers105 Kharb P, Mitra C (2017) Early sex identification in date palm by male-specific sequence-characterized amplified region (SCAR) markers. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date palm biotechnology protocols, vol II. Humana Press. New York, NY, pp 199–207 Khierallah HSM, Al-Sammarraie SKI, Mohammed HI (2014) Molecular characterization of some Iraqi date palm cultivars using RAPD and ISSR markers. J Asian Sci Res 4:490–503 Kichaouibr A, Ayesh M (2013) Genotyping and identification of six date palm (Phoenix dactylifera L.) cultivars of the Gaza Strip by random amplification of polymorphic DNA. Emir J Food Agric 25:916. https:// doi.org/10.9755/ejfa.v25i11.15918 Kiran U, Khan S, Mirza KJ et al (2010) SCAR markers: a potential tool for authentication of herbal drugs. Fitoterapia 81:969–976. https://doi.org/10.1016/j. fitote.2010.08.002

5

Date Palm Genetic Identification and Improvement …

Kress WJ, Erickson DL, Jones FA et al (2009) Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proc Nat Acad Sci 106:18621–18626. https://doi.org/10.1073/pnas. 0909820106 Kruijt M, De Kock MJ, De Wit PJ (2005) Receptor-like proteins involved in plant disease resistance. Mol Plant Pathol 6:85–97 Kumar A (2004) Retrotransposons and their contributions to plant genome and gene evolution. Encyclopedia of plant and crop science. CRC Press, Boca Raton FL, pp 1–5 Kumar A, Bennetzen JL (1999) Plant retrotransposons. Ann Rev Genet 33:479–532. https://doi.org/10.1146/ annurev.genet.33.1.479 Kumar A, Hirochika H (2001) Applications of retrotransposons as genetic tools in plant biology. Trends Plant Sci 6:127–134. https://doi.org/10.1016/S1360-1385 (00)01860-4 Kumar A, Pearce SR, McLean K et al (1997) The Ty1copia group of retrotransposons in plants: genomic organisation, evolution, and use as molecular markers. Genetica 100(1–3):205–217 Kumar APK, McKeown PC, Boualem A et al (2017) TILLING by sequencing (TbyS) for targeted genome mutagenesis in crops. Mol Breed 37:14. https://doi. org/10.1007/s11032-017-0620-1 Kumar K, Amaresan N, Bhagat S et al (2011) Isolation and characterization of rhizobacteria associated with coastal agricultural ecosystem of rhizosphere soils of cultivated vegetable crops. World J Microbiol Biotech 27:1625–1632. https://doi.org/10.1007/s11274-0100616-z Kumar N, Singh AS, Modi AR et al (2010) Genetic stability studies in micropropagated date palm (Phoenix dactylifera L.) plants using microsatellite marker. J Forest Sci 26:31–36 Kurowska M, Daszkowska-Golec A, Gruszka D et al (2011) TILLING - a shortcut in functional genomics. J Appl Genet 52:371–390. https://doi.org/10.1007/ s13353-011-0061-1 Lahaye R, Van der Bank M, Bogarin D, Warner J (2008) DNA barcoding the floras of biodiversity hotspots. Proc Nat Acad Sci USA 105:2923–2928 Lau WCP, Rafii MY, Ismail MR et al (2015) Review of functional markers for improving cooking, eating, and the nutritional qualities of rice. Front Plant Sci 6:832. https://doi.org/10.3389/fpls.2015.00832 Li G, Quiros CF (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theor Appl Genet 103:455–461. https://doi.org/10.1007/s001220100570 Lister DL, Jones H, Jones MK et al (2013) Analysis of DNA polymorphism in ancient barley herbarium material: validation of the KASP SNP genotyping platform. Taxon 62(4):779–789

129

Litt M, Luty JA (1989) A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am J Hum Genet 44:397–401 Lozano-Juste J, Cutler SR (2014) Plant genome engineering in full bloom. Trends Plant Sci 19:284–287. https://doi.org/10.1016/j.tplants.2014.02.014 Lynch M, Walsh B (1998) Genetics and analysis of quantitative traits, vol 1. Sinauer, Sunderland, MA Mackay I, Powell W (2007) Methods for linkage disequilibrium mapping in crops. Trends Plant Sci 12:57–63. https://doi.org/10.1016/j.tplants.2006.12. 001 Madhumati B (2014) Potential and application of molecular markers techniques for plant genome analysis. Int J Pure App Biosci 2:169–188 Mahdy EMB (2018) Genetical studies on DNA storage and preservation on some accessions of cowpea plant. Al-Azhar University, Gaza, Egypt Malek JA (2010) Next generation DNA sequencing applied to the date palm tree (Phoenix dactylifera). Acta Hort 882:249–252. https://doi.org/10.17660/ ActaHortic.2010.882.27 Malek JA (2014) Genetics of gender discrimination in date palm. U.S. Patent Application No. 14/008,012 Mardis ER (2008) Next-generation DNA sequencing methods. Ann Rev Genom Hum Genet 9:387–402. https://doi.org/10.1146/annurev.genom.9.081307. 164359 Mardis ER (2013) Next-generation sequencing platforms. Ann Rev Anal Chem 6:287–303. https://doi.org/10. 1146/annurev-anchem-062012-092628 Marsafari M, Mehrabi AA (2013) Molecular identification and genetic diversity of Iranian date palm (Phoenix dactylifera L.) cultivars using ISSR and RAPD markers. Austral J Crop Sci 7:1160–1166 Maryam, Jaskani MJ, Awan FS et al (2016) Development of molecular method for sex identification in date palm (Phoenix dactylifera L.) plantlets using novel sex-linked microsatellite markers. 3 Biotech 6:22. https://doi.org/10.1007/s13205-015-0321-6 Mashayekhi F, Ronaghi M (2007) Analysis of read length limiting factors in pyrosequencing chemistry. Anal Biochem 363:275–287. https://doi.org/10.1016/j.ab. 2007.02.002 Mathew LS, Seidel MA, George B et al (2015) A genome-wide survey of date palm cultivars supports two major subpopulations in Phoenix dactylifera. G3 (5):1429–1438. https://doi.org/10.1534/g3.115. 018341 Mathew LS, Spannagl M, Al-Malki A et al (2014) A first genetic map of date palm (Phoenix dactylifera) reveals long-range genome structure conservation in the palms. BMC Genom 15:285. https://doi.org/10.1186/ 1471-2164-15-285 McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeting induced local lesions in genomes

130 (TILLING) for plant functional genomics. Plant Physiol 123:439–442. https://doi.org/10.1104/pp.123. 2.439 McClure K, Sawler J, Gardner KM et al (2014) Genomics: a potential panacea for the perennial problem. Am J Bot 101:1780–1790 McHale L, Tan X, Koehl P, Michelmore RW (2006) Plant NBS-LRR proteins: adaptable guards. Genome Biol 7:212 Meuwissen TH, Hayes BJ, Goddard ME (2001) Prediction of total genetic value using genome-wide dense marker maps. Genet 157:1819–1829 Michaels SD, Amasino RM (1998) A robust method for detecting single-nucleotide changes as polymorphic markers by PCR. The Plant J 14:381–385. https://doi. org/10.1046/j.1365-313X.1998.00123.x Mirbahar AA, Markhand GS, Khan S, Abul-Soad AA (2014) Molecular characterization of some Pakistani date palm (Phoenix dactylifera L.) cultivars by RAPD markers. Pak J Bot 46(2): 619–625 Mohamoud YA, Mathew LS, Torres MF et al (2019) Novel subpopulations in date palm (Phoenix dactylifera) identified by population-wide organellar genome sequencing. BMC Genom 20:498. https://doi.org/10. 1186/s12864-019-5834-7 Mohsenzadeh S, Pasalari H (2010) Identification of a male-specific RAPD marker in Phoenix dactylifera. J Hort Sci Biotech 85:144–146. https://doi.org/10. 1080/14620316.2010.11512645 Moisy C, Schulman AH, Kalendar R et al (2014) The Tvv1 retrotransposon family is conserved between plant genomes separated by over 100 million years. Theor Appl Genet 127:1223–1235. https://doi.org/10. 1007/s00122-014-2293-z Mondini L, Noorani A, Pagnotta M (2009) Assessing plant genetic diversity by molecular tools. Diver 1:19– 35. https://doi.org/10.3390/d1010019 Morgante M, Hanafey M, Powell W (2002) Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes. Nat Genet 30:194–200. https://doi.org/10.1038/ng822 Mosqueira MJ, Marasco R, Fusi M et al (2019) Consistent bacterial selection by date palm root system across heterogeneous desert oasis agroecosystems. Sci Rep 9:4033. https://doi.org/10.1038/s41598-019-40551-4 Mullis K, Faloona F, Scharf S et al (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp, Quant Biol 51:263–273 Mullis K, Faloona F (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–350 Munshi A, Osman G (2010) Investigation on molecular phylogeny of some date palm (Phoenix dactylifera L.) cultivars by protein, RAPD and ISSR markers in Saudi Arabia. Austral J Crop Sci. 4(1):23–28 Myles S, Peiffer J, Brown PJ et al (2009) Association mapping: critical considerations shift from genotyping to experimental design. Plant Cell 21:2194–2202. https://doi.org/10.1105/tpc.109.068437

E. M. B. Mahdy and S. F. El-Sharabasy Naeem A, Khan AA, Cheema HMN et al (2014) DNA barcoding for species identification in the Palmae family. Genet Mol Res 13(4):10341–10348. https:// doi.org/10.4238/2014.December.4.29 Nakaya A, Isobe SN (2012) Will genomic selection be a practical method for plant breeding? Ann Bot 110:1303–1316. https://doi.org/10.1093/aob/mcs109 Ortiz R (2010) Molecular plant breeding. Crop Sci 50:2196–2197. https://doi.org/10.2135/cropsci2010. 12.0004br Osorio-Guarín JA, Garzón-Martínez GA, DelgadilloDuran P et al (2019) Genome-wide association study (GWAS) for morphological and yield-related traits in an oil palm hybrid (Elaeis oleifera x Elaeis guineensis) population. BMC Plant Biol 19:533. https://doi. org/10.1186/s12870-019-2153-8 Paran I, Michelmore RW (1993) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor Appl Genet 85:985–993. https://doi.org/10.1007/BF00215038 Pearce SR, Pich U, Harrison G et al (1996) TheTy1-copia group retrotransposons of Allium cepa are distributed throughout the chromosomes but are enriched in the terminal heterochromatin. Chromos Res 4:357–364. https://doi.org/10.1007/BF02257271 Perez-de-Castro AM, Vilanova S, Canizares J et al (2012) Application of genomic tools in plant breeding. Curr Genom 13:179–195. https://doi.org/10.2174/ 138920212800543084 Pintaud J-C, Ludeña B, Aberlenc-Bertossi F et al (2013) Biogeography of the date palm (Phoenix dactylifera L., Arecaceae): insights on the origin and on the structure of modern diversity. Acta Hort 994:19–38. https://doi.org/10.17660/ActaHortic.2013.994.1 Pipatchartlearnwong K, Juntawong P, Wonnapinij P et al (2019) Towards sex identification of asian palmyra palm (Borassus flabellifer L.) by DNA fingerprinting, suppression subtractive hybridization and de novo transcriptome sequencing. Peer J 7:e7268. https://doi. org/10.7717/peerj.7268 Poczai P, Varga I, Laos M et al (2013) Advances in plant gene-targeted and functional markers: a review. Plant Meth 9:6. https://doi.org/10.1186/1746-4811-9-6 Poland JA, Rife TW (2012) Genotyping-by-sequencing for plant breeding and genetics. The Plant Genome 5:92–102. https://doi.org/10.3835/plantgenome2012. 05.0005 Prochetto S, Reinheimer S (2020) Step by step evolution of indeterminate domain (IDD) transcriptional regulators: from algae to angiosperms. Ann Bot 126(1):85– 101. https://doi.org/10.1093/aob/mcaa052 Provan J, Powell W, Hollingsworth PM (2001) Chloroplast microsatellites: new tools for studies in plant ecology and evolution. Trends Ecol Evol 16:142–147. https://doi.org/10.1016/S0169-5347(00)02097-8 Qacif N, Baaziz M, Bendiab K (2007) Biochemical investigations on peroxidase contents of male and female inflorescences of date palm (Phoenix dactylifera L.). Sci Hort (Amsterdam) 114:298–301

5

Date Palm Genetic Identification and Improvement …

Racchi ML, Camussi A (2018) The date palms of Al Jufrah-Libya: a survey on genetic diversity of local varieties. J Agric Envir Int Dev 112(1):161–184 Rajendrakumar P, Biswal AK, Balachandran SM et al (2007) Simple sequence repeats in organellar genomes of rice: frequency and distribution in genic and intergenic regions. Bioinfo 23:1–4. https://doi.org/10. 1093/bioinformatics/btl547 Rance KA, Mayes S, Price Z et al (2001) Quantitative trait loci for yield components in oil palm (Elaeis guineensis Jacq.). Theor Appl Genet 103:1302–1310. https://doi.org/10.1007/s122-001-8204-z Rekik I, Drira N, Grubb D, Elleuch A (2015) Molecular characterization and evolution studies of a SERK like gene transcriptionally induced during somatic embryogenesis in Phoenix dactylifera L cv Deglet Nour. Genetika 47:323–337. https://doi.org/10.2298/ GENSR1501323R Rekis A, Laiadi Z (2020) Interrelationships between some vegetative components and fruit quality of algerian date palm. Agric Sci Tech 12(1):77–81. https://doi. org/10.15547/ast.2020.01.014 Reyes PA, Ochoa JC, Montoya C et al (2015) Development and validation of a bi-directional allele-specific PCR tool for differentiation in nurseries of dura, tenera and pisifera oil palms. Agron Colomb 33:5–10. https:// doi.org/10.15446/agron.colomb.v33n1.47988 Riad M (1993) The date palm sector in Egypt. In: Ferry M, Greiner D (eds) Le palmier dattier dans l’agriculture d’oasis des pays méditerranéens. Opti Méditerr, Zaragoza, Spain, pp 45–53 Rikek I, Chaâbene Z, Kriaa W et al (2019) Transcriptome assembly and abiotic related gene expression analysis of date palm reveal candidate genes involved in response to cadmium stress. Compa Biochem Physiol Part C Toxicol Pharmacol 225: https://doi.org/10. 1016/j.cbpc.2019.108569 Rivera D, Obón de Castro C, Carreño E et al (2008) Morphological systematics of date-palm diversity (Phoenix, Arecaceae) in western Europe and some preliminary molecular results. Acta Hort 799:97–104. https://doi.org/10.17660/ActaHortic.2008.799.11 Rizk RM, El-Sharabasy SF (2008) A descriptor for date palm (Phoenix dactylifera L.) characterization and evaluation in gene banks. Plant Genet Res Newsl 150:42–44 Rizk RM, El-Sharabasy SF, Soliman, KS (2006) Characterization and evaluation of six males date palm (Phoenix dactylifera L.) genotypes in Egypt. Proc 1st Botan Gard Strat Conf, Giza. 1(1):287–299 Romero Navarro J, Willcox M, Burgueño J et al (2017) A study of allelic diversity underlying flowering-time adaptation in maize landraces. Nat Genet 49:476–480. https://doi.org/10.1038/ng.3784 Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3–11. https://doi.org/10. 1101/gr.11.1.3

131

Rothberg JM, Hinz W, Rearick TM et al (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475:348–352. https://doi. org/10.1038/nature10242 Roy NS, Choi J-Y, Lee S-I, Kim N-S (2015) Marker utility of transposable elements for plant genetics, breeding, and ecology: a review. Genes Genom 37:141–151. https://doi.org/10.1007/s13258-0140252-3 Sabot F, Schulman AH (2006) Parasitism and the retrotransposon life cycle in plants: a hitchhiker’s guide to the genome. Hered 97:381–388. https://doi. org/10.1038/sj.hdy.6800903 Safronov O, Kreuzwieser J, Haberer G et al (2017) Detecting early signs of heat and drought stress in Phoenix dactylifera (date palm). PLoS ONE 12: https://doi.org/10.1371/journal.pone.0177883 Saker MM, Adawy SS, Mohamed AA, El-Itriby HA (2006) Monitoring of cultivar identity in tissue culture-derived date palms using RAPD and AFLP analysis. Biol Plant 50:198–204. https://doi.org/10. 1007/s10535-006-0007-3 Salazar JA, Rasouli M, Moghaddam RF et al (2014) Lowcost strategies for development of molecular markers linked to agronomic traits in Prunus. Agric Sci 5 (5):430–439. https://doi.org/10.4236/as.2014.55044 Salem AOM, Rhouma S, Zehdi S et al (2008) Morphological variability of mauritanian date-palm (Phoenix dactylifera L.) cultivars as revealed by vegetative traits. Acta Bot Croat 67:81–90 Sallon S, Cherif E, Chabrillange N et al (2020) Origins and insights into the historic judean date palm based on genetic analysis of germinated ancient seeds and morphometric studies. Sci Adv 6:eaax0384 Sanger F, Coulson AR (1975) A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol 94(3):441–448 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Nat Acad Sci 74:5463–5467. https://doi.org/10.1073/pnas.74. 12.5463 Sattar MN, Iqbal Z, Tahir MN et al (2017) CRISPR/Cas9: a practical approach in date palm genome editing. Front Plant Sci 8:1469. https://doi.org/10.3389/fpls. 2017.01469 Sayed AIH, El-Shaer HFA, El-Halwagi A, Mahdy EMB (2017) Comparative study of DNA preservation under various conditions on local Egyptian cowpea germplasm. Int J Life Sci Pharma Res 3:64–70. https://doi. org/10.25141/2471-6782-2017-5.0064 Schlotteröer C, Amos B, Tautz D (1991) Conservation of polymorphic simple sequence loci in cetacean species. Nature 354:63–65. https://doi.org/10.1038/354063a0 Schulman AH, Wicker T (2013) A field guide to transposable elements. Plant transposons and genome dynamics in evolution. Wiley-Blackwell, Oxford, UK, pp 15–40

132 Sedra MH (2011) Molecular markers for genetic diversity and bayoud disease resistance in date palm. In: Jain SM, Al-Khayri JM, Johnson DV (eds) Date palm biotechnology. Springer pp, 533–550. https://doi.org/ 10.1007/978-94-007-1318-5_25 Sedra MH (2013) Genetic diversity analysis, moroccan cultivar genotyping and rapid screening for bayoud disease resistance in date palm using molecular techniques. Acta Hort 271–285. https://doi.org/10. 17660/ActaHortic.2013.994.27 Sedra MH, Lashermes P, Trouslot P et al (1998) Identification and genetic diversity analysis of date palm (Phoenix dactylifera L.) varieties from Morocco using RAPD markers. Euphy 103:75–82. https://doi. org/10.1023/A:1018377827903 Sehga D, Singh R, Rajpal VR (2016) Molecular breeding for sustainable crop improvement. Springer International Publishing, Cham Seibt KM, Wenke T, Wollrab C et al (2012) Development and application of SINE-based markers for genotyping of potato varieties. Theor Appl Genet 125:185–196. https://doi.org/10.1007/s00122-012-1825-7 Semagn K, Bjørnstad Å, Ndjiondjop MN (2006) An overview of molecular marker methods for plants. Afr J Biotech 5:2540–2568. https://doi.org/10.5897/ AJB2006.000-5110 Seng T-Y, Ritter E, Mohamed Saad SH et al (2016) QTLs for oil yield components in an elite oil palm (Elaeis guineensis) cross. Euphy 212:399–425. https://doi. org/10.1007/s10681-016-1771-6 Shendure J, Ji H (2008) Next-generation DNA sequencing. Nat. Biotech 26:1135–1145. https://doi.org/10. 1038/nbt1486 Shew J, Lickfy BE, Schilling EE (2007) Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms. Am J Bot 94(3):275–288 Shirasu K (2000) A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion. Genome Res 10:908–915. https://doi.org/ 10.1101/gr.10.7.908 Shiu SH, Bleecker AB (2001) Plant receptor-like kinase gene family: diversity, function, and signaling. Sci STKE 2001, re22 Siljak-Yakovlev S, Cerbah M, Sarr A et al (1996) Chromosomal sex determination and heterochromatin structure in date palm. Sex Plant Reprod 9:127–132. https://doi.org/10.1007/BF02221391 Singh A, Roychowdhury R, Singh T et al (2020) Improvement of crop’s stress tolerance by gene editing CRISPR/Cas9 system. In: Roychowdhury R, Choudhury S, Hasanuzzaman M, Srivastava S (eds) Sustainable agriculture in the era of climate change. Springer, Cham. https://doi.org/10.1007/978-3-030-45669-624 Singh R, Low E-TL, Ooi LC-L et al (2013a) The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK. Nature 500:340–344. https://doi.org/10.1038/nature12356 Singh R, Ong-Abdullah M, Low ET et al (2013b) Oil palm genome sequence reveals divergence of

E. M. B. Mahdy and S. F. El-Sharabasy interfertile species in old and new worlds. Nature 500:335–339. https://doi.org/10.1038/nature12309 Sobrino B, Brión M, Carracedo A (2005) SNPs in forensic genetics: a review on SNP typing methodologies. Foren Sci Int 154:181–194. https://doi.org/10. 1016/j.forsciint.2004.10.020 Sobrino B, Carracedo A (2005) SNP typing in forensic genetics: a review. In: Clifton NJ (ed) Methods in molecular biology. 297:107–126. https://doi.org/10. 1385/1-59259-867-6:107 Soliman K, Rizk RM, El-Sharabasy SF (2006) Genetic polymorphism of semi-dry date palm (Phoenix dactylifera L.) cultivars in Egypt. J Biotech 22:261– 273 Solliman ME, Mohasseb HA, Al-Khateeb AA et al (2019) Identification and sequencing of date-SRY Gene: a novel tool for sex determination of date palm (Phoenix dactylifera L.). Saudi J Biol Sci 26:514–523. https:// doi.org/10.1016/j.sjbs.2017.08.002 Soumaya R, Sarra C, Maha M et al (2020) Gene-targeted markers to assess genetic diversity and population structure within Tunisian Phoenix dactylifera L. cultivars. Silvae Genet 69:29–35. https://doi.org/10. 2478/sg-2020-0005 Spaniolas S, May ST, Bennett MJ, Tucker GA (2006) Authentication of coffee by means of PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis. J Agric Food Chem 54:7466–7470. https://doi.org/10. 1021/jf061164n Su TB, Li PR, Yang JJ et al (2018) Development of costeffective single nucleotide polymorphism marker assays for genetic diversity analysis in Brassica rapa. Mol Breed 38(4):42 Tautz D (1989) Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucl Acid Res 17:6463–6471. https://doi.org/10.1093/nar/ 17.16.6463 Tautz D, Arctander P, Minelli A et al (2003) A plea for DNA taxonomy. Trends Ecol Evol 18:70–74. https:// doi.org/10.1016/S0169-5347(02)00041-1 Tazeb A, Box PO, Markos D (2018) Molecular marker techniques and their novel applications in crop improvement: a review article. Global J Mol Sci 13 (1):1–16. https://doi.org/10.5829/idosi.gjms.2018.01. 16 Teh C-K, Ong A-L, Kwong Q-B et al (2016) Genomewide association study identifies three key loci for high mesocarp oil content in perennial crop oil palm. Sci Rep 6:19075. https://doi.org/10.1038/srep19075 Thudi M, Li Y, Jackson SA et al (2012) Current state-ofart of sequencing technologies for plant genomics research. Brief Funct Genom 11:3–11. https://doi.org/ 10.1093/bfgp/elr045 Ting N-C, Jansen J, Mayes S et al (2014) High density SNP and SSR-based genetic maps of two independent oil palm hybrids. BMC Genom 15:309. https://doi.org/ 10.1186/1471-2164-15-309 Ting N-C, Mayes S, Massawe F et al (2018) Putative regulatory candidate genes for QTL linked to fruit

5

Date Palm Genetic Identification and Improvement …

traits in oil palm (Elaeis guineensis Jacq.). Euphy 214. https://doi.org/10.1007/s10681-018-2296-y Torres MF, Mathew LS, Purchase C et al (2016) Mapping and sequencing of sex determination genes in Phoenix dactylifera. In: Qatar foundation annual research conference proceedings Vol. 2016 Issue 1. Hamad bin Khalifa University Press. https://doi.org/10.5339/ qfarc.2016.EEPP2787 Tsumura Y, Ohba K, Strauss SH (1996) Diversity and inheritance of inter-simple sequence repeat polymorphisms in douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica). Theor Appl Genet 92:40–45. https://doi.org/10.1007/BF00222949 Uauy C, Paraiso F, Colasuonno P et al (2009) A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol 9:115. https://doi.org/10.1186/1471-2229-9-115 Ukoskit K, Chanroj V, Bhusudsawang G et al (2014) Oil palm (Elaeis guineensis Jacq.) linkage map, and quantitative trait locus analysis for sex ratio and related traits. Mol Breed 33(2): 415–424. https://doi. org/10.1007/s11032-013-9959-0 Uzun A, Yesiloglu T, Aka-Kacar Y et al (2009) Genetic diversity and relationships within Citrus and related genera based on sequence related amplified polymorphism markers (SRAPs). Sci Hort 121(3):306–331. 2https://doi.org/10.1016/j.scienta.2009.02.018 Vos P, Hogers R, Bleeker M et al (1995) AFLP: a new technique for DNA fingerprinting. Nucl Acid Res 23:4407–4414 Wang C, Roberts PA (2006) Development of AFLP and derived CAPS markers for root-knot nematode resistance in cotton. Euphy 152:185–196. https://doi.org/ 10.1007/s10681-006-9197-1 Watts A, Kumar V, Bhat SR (2016) Centromeric histone H3 protein: from basic study to plant breeding applications. J Plant Biochem Biotech 25:339–348. https://doi.org/10.1007/s13562-016-0368-4 Waugh R, McLean K, Flavell AJ et al (1997) Genetic distribution of bare–1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol Gen Genet (MGG) 253:687–694. https://doi.org/10.1007/ s004380050372 Weiland JJ, Yu MH (2003) A cleaved amplified polymorphic sequence (CAPS) marker associated with root-knot nematode resistance in sugar beet. Crop Sci 43:1814–1818. https://doi.org/10.2135/cropsci2003. 1814 Welsh J, Mcclelland M (1990) genomes using PCR with arbitrary primers. Nucl Acid Res 18(24):7213–7218. https://doi.org/10.1093/nar/18.24.7213 Wenzl P, Carling J, Kudrna D et al (2004) Diversity arrays technology (DArT) for whole-genome profiling of barley. Proc Nat Acad Sci 101:9915–9920. https:// doi.org/10.1073/pnas.0401076101 Williams JGK, Kubelik AR, Livak KJ et al (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl Acid Res 18:6531– 6535. https://doi.org/10.1093/nar/18.22.6531

133

Witte C-P, Le QH, Bureau T, Kumar A (2001) Terminalrepeat retrotransposons in miniature (TRIM) are involved in restructuring plant genomes. Proc Nat Acad Sci 98:13778–13783. https://doi.org/10.1073/ pnas.241341898 Wong CK, Bernardo R (2008) Genome-wide selection in oil palm: increasing selection gain per unit time and cost with small populations. Theor Appl Genet 116:815–824. https://doi.org/10.1007/s00122-0080715-5 Wu K, Jones R, Danneberger L, Scolnik PA (1994) Detection of microsatellite polymorphisms without cloning. Nucl Acid Res 22:3257–3258. https://doi.org/ 10.1093/nar/22.15.3257 Xia W, Luo T, Dou Y et al (2019) Identification and validation of candidate genes involved in fatty acid content in oil palm by genome-wide association analysis. Front Plant Sci 10. https://doi. org/10.3389/fpls.2019.01263 Xu Y, Lu Y, Xie C et al (2012) Whole-genome strategies for marker-assisted plant breeding. Mol Breed 29:833–854. https://doi.org/10.1007/s11032-0129699-6 Yamada T, Skøt L (2010) Allelic diversity for candidate genes and association studies: methods and results. In: Huyghe C (ed) Sustainable use of genetic diversity in forage and turf breeding. Springer, Dordrecht, pp 391– 396 Yang M, Zhang X, Liu G et al (2010) The complete chloroplast genome sequence of date palm (Phoenix dactylifera L.). PLoS One. 5: e12762. https://doi.org/ 10.1371/journal.pone.0012762.t005 Yarra R, Cao H, Jin L et al (2020) CRISPR/Cas mediated base editing: a practical approach for genome editing in oil palm. 3 Biotech 10:306. https://doi.org/10.1007/ s13205-020-02302-5 Yarra R, Jin L, Zhao Z, Cao H (2019) Progress in tissue culture and genetic transformation of oil palm: an overview. Int J Mol Sci 20:5353. https://doi.org/10. 3390/ijms20215353 Ye G, Smith KF (2008) Marker-assisted gene pyramiding for inbred line development: basic principles and practical guidelines. Int J Plant Breed 2:1–10 Younis RAA, Ismail OM, Soliman SS (2008) Identification of sex-specific DNA markers for date palm (Phoenix dactylifera L.). Res J Agric Biol Sci 4:278– 284 Yusuf AO, Culham A, Aljuhani W et al (2015) Genetic diversity of nigerian date palm (Phoenix dactylifera) germplasm based on microsatellite markers. Int J BioSci Bio-Tech 7:121–132. https://doi.org/10.14257/ ijbsbt.2015.7.1.12 Zaid A, Arias-Jiménez EJ (2002) Date Palm Cultivation. FAO plant production and protection paper 156 Rev. 1, Rome Italy Zane L, Bargelloni L, Patarnello T (2002) Strategies for microsatellite isolation: a review. Mol Ecol 11:1–16. https://doi.org/10.1046/j.0962-1083.2001.01418.x

134 Zehdi-Azouzi S, Cherif E, Guenni K et al (2016) Endemic insular and coastal Tunisian date palm genetic diversity. Genetica 144:181–190. https://doi.org/10.1007/ s10709-016-9888-z Zehdi S, Cherif E, Rhouma S et al (2012) Molecular polymorphism and genetic relationships in date palm (Phoenix dactylifera L.): the utility of nuclear microsatellite markers. Sci Hort 148:255–263. https://doi.org/10.1016/j.scienta.2012.10.011 Zehdi S, Trifi M, Billotte N et al (2005) Genetic diversity of tunisian date palms (Phoenix dactylifera L.) revealed by nuclear microsatellite polymorphism. Hered 141:278–287. https://doi.org/10.1111/j.16015223.2004.01855.x Zhang G, Pan L, Yin Y et al (2012) Large-scale collection and annotation of gene models for date palm (Phoenix

E. M. B. Mahdy and S. F. El-Sharabasy dactylifera, L.). Plant Mol Biol 79:521–536. https:// doi.org/10.1007/s11103-012-9924-z Zhang P, Zhong K, Tong H et al (2016) Association mapping for aluminum tolerance in a core collection of rice landraces. Front Plant Sci 7:1415. https://doi. org/10.3389/fpls.2016.01415 Zhao Y, Williams R, Prakash CS, He G (2012) Identification and characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.). BMC Plant Biol 12:237. https://doi.org/10.1186/1471-222912-237 Zietkiewicz E, Rafalski A, Labuda D (1994) Genome fingerprinting by simple sequence repeat (SSR)anchored polymerase chain reaction amplification. Genom 20:176–183

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DNA Fingerprinting of Date Palm Pollen Sources and Their Relevance to Yield and Fruit Traits Mohamed M. S. Saleh, Esam A. M. Mostafa, Nagah E. Ashour, and Samy A. A. Heiba

Abstract

The date palm (Phoenix dactylifera L.), 2n=36, is a dioecious long-lived monocotyledonous plant. It is a dioecious plant with male and female flowers borne on separate palms. In commercial plantations, artificial pollination is essential for economical yield, since the female inflorescences are hand pollinated with selected pollen from males. It is generally known that pollen from different sources affects the yield and fruit quality. The pollen sources influences the size and shape of the seeds which is known as the xenia effect, and directly affects fruit set, yield and fruit physical and chemical characteristics referred to as the metaxenia effect. Many experiments have shown that no constant trend is detected for the genetic relationship

between the pollen sources and commercial female cultivars. There is a clear relationship between them due to the ecogenetics (gene-environment interactions) for both the selected pollen source and the commercial female cultivar. The phylogenetic relationships among date palms can be investigated through fingerprinting analysis using RAPD. The RAPD–PCR can detect the genetic variability and the relationships among date palms, and also estimate the expression of similarity using the Dice coefficient. PCR is an enzymatic reaction, so the outcome can be significantly influenced by the quality and concentration of template DNA, PCR component concentrations and PCR cycling conditions.

6.1 M. M. S. Saleh (&)
E. A. M. Mostafa
N. E. Ashour Pomology Department, Agricultural and Biological Division, National Research Centre, Cairo, Egypt e-mail: [email protected] E. A. M. Mostafa e-mail: [email protected] N. E. Ashour e-mail: [email protected] S. A. A. Heiba Genetics and Cytology Department, Genetic engineering and Biotechnology Division, National Research Centre, Cairo, Egypt e-mail: [email protected]

Introduction

The date palm (Phoenix dactylifera L.) is known as one of the world’s oldest cultivated plants. The date palm belt stretches from the Indus Valley in the east to the Atlantic Ocean in the west. Date palms can grow in very hot and dry environments and is fairly tolerant of salty and alkaline soils. Date palms need a long, extremely warm summer with little rain and very low humidity, but with plenty of underground water or surface irrigation, from pollination to harvest. (Zaid and De Wet 2002). Date palm is a dioecious plant where artificial pollination is necessary for good economic yield,

© Springer Nature Switzerland AG 2021 J. M. Al-Khayri et al. (eds.), The Date Palm Genome, Vol. 1, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-73746-7_6

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since male palms serve only as pollen sources. These males are typically highly variable in the sense that their growth, vigor, spathe characteristics, and pollen quality differ greatly. There is general opinion among date growers that the success of fruit production and fruit traits depend primarily on pollination success. There are several factors that influence pollination, such as the source and viability of pollen grains, the successful time for artificial pollination, repeated pollination processes and environmental factors such as wind and rainfall (Mohamed 2010). The source of pollen is considered a critical and limiting factor in the characteristics of palm production and fruit quality. Over the past century, the effect of pollen from different sources of readily discernible characteristics on seeds and fruits was noted during the period immediately following fertilization. These direct or immediate effects are known as xenia and have been recognized in many species. (Denney 1992). Pollen from various male palms are commonly known to affect yield and fruit quality, known as the metaxenia effect (Swingle 1928). This phenomenon has been studied by many researchers and results have shown that in addition to influencing the size and shape of the seed, pollen grains from different males have a direct influence on the fruit setting, yield, physical and chemical characteristics of the fruit and exhibited metaxenic effects depending on the female cultivar (El-Salhy et al. 2010; Mostafa et al. 2016; Shaheen et al. 1989a, b). Xenia/metaxenia has been suggested primarily to increase the yield of maize (Weingartner et al. 2002) and many fruit crops, including pecans, pistachios, and avocados (Robbertse et al. 1996; Sedgley and Griffin 1989), as well as date palms (Merwad et al. 2015; Mustafa et al. 2014; Nixon 1928; Samy et al. 2015; Shaheen et al. 1989a, b). On the other hand, many studies show that no constant trend is detected for the genetic relationship between the pollenizers and the commercial female cultivars, while there is a clear relationship between them due to ecogenetics. The use of DNA markers provides a powerful tool to certify the identity of the cultivars through the variety of fingerprints at the seedling stage.

M. M. S. Saleh et al.

Random amplified polymorphic DNA (RAPD) markers can be used for fast screening of variations in the nuclear genome. RAPD markers are used to characterize the germplasm in the date palm. Several researchers have demonstrated that DNA markers are effective in detecting relationships between various genotypes and showed the connection between yield traits and molecular traits. The RAPD approach seems to be an effective tool to examine genetic variation and to recognize different varieties (Merwad et al. 2020; Mostafa et al. 2016, 2018; Mustafa et al. 2014; Samy et al. 2015; Sarrwy et al. 2014). Recently, RAPD and (inter-simple sequence repeats) ISSR analyses were employed to identify an unknown date palm cultivar grown in the Matrouh Governorate, Egypt (Moghaieb et al. 2010). The main objective of this chapter is to describe the relevance of using DNA fingerprinting of date palm pollen sources and their relationship to yield and fruit traits, as well as the genetic relation between the pollen sources and the commercial female cultivars of date palms and the effect of xenia and metaxenia on yield and fruit traits. And finally, to identify the relationship between the pollen sources and commercial female cultivars due to ecogenetics.

6.2

Pollen Sources and the Relation to Xenia and Metaxenia

The genus Phoenix is made up of 14 species naturally distributed in the Old World. This genus includes the date palm, Phoenix dactylifera L., cultivated for its fruits, and other species cultivated for food, ornamental and religious purposes (Gros-Balthazard 2013). During the period immediately after fertilization, the effect of pollen from different sources of readily discernible characteristics on seeds and fruits was noted for over a century. It was found that the pollen of the date palm (Phoenix dactylifera L.) and other species of Phoenix have a direct effect on the size, shape and color of the seeds, as well as on the size of the fruit, the rate of fruit growth and the maturation period of the female cultivars. This direct effect

6

DNA Fingerprinting of Date Palm Pollen Sources …

on the male parent’s development of the date fruit is specific and conclusive and varies from the individual male used to fertilize the female flowers, with each male having roughly the same effect on the fruit of all varieties and having the same effect over different years. This direct effect of pollen on parts of the seed and fruit outside the embryo and endosperm is termed metaxenia (Swingle 1928). Metaxenia is described as having an effect on purely maternal tissue, rather than on parts resulting from syngamy (Swingle 1928). These effects include changes in the scale, shape, and timing of growth, as well as the chemical properties of the date fruit that were found to be precocious as a result of fertilization by various pollens to suggest a hormonal cause for metaxenic effects in the date palm (Nixon 1935; Osman et al. 1974; Swingle 1928). Denney (1992) indicated that it is possible to assign differential effects of metaxenia size in date palm, leading to various concentrations in one or more of the three hormones, most closely linked to fruit development: auxins, cytokinine and/or gibberellic acid. He also stated that smaller fruits (seed + pericarp) have lower levels of hormones and larger fruits higher hormone levels. In order to achieve metaxenic effects in date palm fruit cultivars, Abbas et al. (2014) investigated the impact of two pollen sources, namely male cvs. Ghannami Akhdar and Khikri Adi, on some aspects of fruit production with regard to IAA concentration changes during fruit development such as in cv. Hillawi. Figure 6.1 illustrates the relationship between the type of pollen and the period per week after pollination at free IAA levels. It is obvious that at weeks 5, 7 and 10, Khikri Adi pollen generated fruit had significantly (p = 0.05) higher levels of IAA in fruit compaction, produced by Ghannami Akhdar pollen. However, there was no significant difference in free IAA levels between fruits produced by the two types of pollen at weeks 13, 15, and 17. Metaxenia, unlike xenia, cannot be explained by pollen-induced genetic elements (chromosomes), as no such chromosomes exist in the tissue that displays the direct parental influence. The simplest and most likely explanation of metaxenia is that the embryo or endosperm, or

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both, secrete hormones or analogous soluble substances that spread into the mother plant tissue to support the seed and fruit and have a particular impact on these tissues that varies depending on the specific pollen source used to fertilize the embryo and endosperm. On the other hand, Olfati et al. (2010) reported that xenia is the effect of male parent genes on fruit or seed development. They added that xenia could be used to identify the best parents of pollinators to decrease the period of fruit development and increase yield in mixed cultivar plantings. They also stated that metaxenia is the influence of pollen on the form of the fruit and other properties.

6.3

Molecular Studies

Molecular studies can be used to determine the phylogenetic tree and relationships among different varieties of date palms. Randomly amplified polymorphic DNA (RAPD) markers are of particular interest (Welsh and McClelland 1990; Williams et al. 1990). DNA profiles based on arbitrary primed PCR are both time- and cost-effective.

6.3.1 DNA-Fingerprint Similarity DNA-fingerprint similarity is generally defined as the fraction of shared bands. Similarity of DNA signatures is increasingly being used to make inferences regarding rates of genetic variability within and between natural populations. The similarity index, the average fraction of mutual restriction fragments, is considered to provide upwardly skewed estimates of population homozygosity, but almost unbiased estimates of the average identity-in-state for random pairs of individuals (Lynch 1990). Fingerprinting analysis using RAPD, simple sequence repeats (SSR) and single nucleotide polymorphic (SNP) have been used to investigate the phylogenetic relationships among date palms. Genome sequencing of many date palm cultivar is an advantage of next-generation sequencing technologies (Al-Dous et al. 2011; Al-Mssallem et al. 2013; Hazzouri et al. 2015). Genomic data

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M. M. S. Saleh et al.

Fig. 6.1 Interaction between pollen type and number of weeks from pollination during growth and production of date palm fruit cv. Hillawi on concentration of IAA. Every point represents the mean three replicates. Source Abbas et al. (2014)

have proved to be very useful biomarkers in plant genome analysis and breeding genome sequencing for many date palm cultivars.

6.3.2 Molecular Studies Several molecular studies have determined phylogenetic trees and relationships among different varieties of date palms. Short sequence repeat (SSR) and single nucleotide polymorphism (SNP) are very useful for the study and reproduction of plant genomes. Billotte et al. (2004) established nuclear SSR markers and examined the genetic diversity of the date palm; Hamwieh et al. (2010) tested SSRs throughout the whole date palm genome. Al-Mssallem et al. (2013) noted that the varieties in north Africa differed significantly from those in the Middle East, based on the SNPs of 11 date palm varieties. In this concern, Mustafa et al. (2014) investigated the impact of various pollen sources on yield, fruit characteristics, and the phylogenetic similarities with Amhat cv. using RAPD– PCR marker RAPD in Egypt. They stated that the 6 date palm accessions were screened with 6 RAPD primers, which developed multiple band profiles with a number of amplified DNA fragments having a range of 6–10 (Fig. 6.2). The total number of fragments produced by the 6 primers was 45 with 7.5 as fragments/primer averages (Table 6.1). The number of polymorphic fragments, though, had a range of 4–9. With priming OP-O14, a maximum

number of 10 amplicons was amplified, and the minimum number of fragments (4) was amplified with priming OP-O13. With primers OP-O14, the highest number of polymorphic bands (9) was obtained and exhibited the highest percentage of polymorphism (90%). They added that the total number of polymorphic bands obtained by 6 successful primers was 37 polymorphic from 45 bands with 82.22% polymorphism. Sarrwy et al. (2014) studied the influence of different pollen grain sources on yield and fruit quality of Sewi cv. date palm and its phylogenetic relationships using RAPD markers. They detected, as shown in Fig. 6.3 and Table 6.2, that the number of bands with the 9 primers revealed a total of 52 bands, among which 36 bands were polymorphic with an average 69.23% of polymorphism for the studied primers. The number of polymorphic bands ranged from 4 with primer OP -A02, OP-013, and OP-014 to 8 bands with primer OP-A07 with an average 5.67 bands/primer. Samy et al. (2015) studied the phylogenetic map among 3 pollen sources and their effect on fruit setting, yield and quality of cvs. Zaghloul and Samani, using the RAPD–PCR technique and found that 9 primers revealed 56 fragments, where 39 were polymorphic bands with a ratio of 69.64% polymorphism and 17 were monomorphic bands with a ratio of 30.36%, as shown in Fig. 6.4 and Table 6.3. The products of 9 RAPD primers represented showed that the number of total bands for each primer varied from 5

6

DNA Fingerprinting of Date Palm Pollen Sources …

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Fig. 6.2 Example of 6 date palm cultivars RAPD–PCR banding patterns amplified with 6, 10-mer random primers OPB07, Ms= 100-bp ladder. Source Mustafa et al. (2014)

Table 6.1 The total number of bands, monomorphic bands, polymorphic bands, band frequency mean, and polymorphism percentage of the 6 date palm cultivars using RAPD–PCR Primer name

Total number of bands

Monomorphic bands

Polymorphic bands

Mean of band frequency

Polymorphism %

OP-A02

8

1

7

0.542

87.50

OP-B07

8

2

6

0.667

75.00

OP-B10

6

2

4

0.639

66.667

OP-O10

7

1

6

0.524

85.714

OP-O13

6

1

5

0.667

83.333

OP-O14

10

1

9

0.567

90.00

Total

45

8

37

0.601

82.222

Average

7.5

1.33

6.17

–

–

Source Mustafa et al. (2014)

fragments of primers (OPA-04) and (OPO-14) to 7 fragments for (OPA-07, OPO-10, OPO-13, OP O-19), with an average of 6.22 bands/primer with polymorphism 85.71, 71.42 and 57.14, respectively. While the primers (OPA-02 and OPB-10) gave 6 bands with percentage of 66.67% polymorphism, primer OPB-07 also detected 6 bands where 1 was monomorphic and 5 were polymorphic bands, with 83.33% polymorphism. Mostafa et al. (2016) examined the impact of metaxenia on fruit yield and the relationship among some date palm pollen sources and 2 female cultivars using molecular RAPD markers. They reported that the DNA of the 5 date palm accessions was screened for 9 RAPD primers; these produced multiple band profiles with a number of amplified DNA amplicons ranging from 4 to 8

bands (Table 6.4, Fig. 6.5). There were 55 fragments, a total number of which the 9 primers formed with a rate of 6.11 fragments/primer. While, the number of polymorphic fragments varied between 4 and 8. The OPB-07 and OP-O14 primers amplified a maximum of 8 bands, while the lowest number of fragments (4) was amplified using the OPB-10 and OPO-19 primers. The OPB07 primer was used to detect the highest polymorphism ratio (87.5%). Also, Mostafa et al. (2018) investigated the production, fruit properties and genetic diversity of 4 Egyptian date palm cultivars and 5 different pollen sources by molecular markers. The results in Table 6.5 and Fig. 6.6 showed that 6 primers (primers code and sequence) gave 46 alleles, 11 of which were monomorphic and 35

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M. M. S. Saleh et al.

Fig. 6.3 RAPD–PCR banding patterns of 6 date palm cultivars:- (1) Sewi, (2) Noubaria, (3) Giza, (4) Aswan, (5) Rasheed, (6) New Valley, amplified with the 9, 10-mer random primers:- (a) OP-A02, (b) OP-A04, (c) OP-A07,

(d) OP-B07, (e) OPB10, f() OP-O10, (g) OP-O13, (h) OP- O14, (i) OP-O19; MS = 100-bp ladder of nine primers. Source Sarrwy et al. (2014)

Table 6.2 Total number of bands, monomorphic bands, polymorphic bands, and polymorphism of the six date palm cultivars for nine primers using RAPD-PCR No.

Primer code

Sequence(5–3′)

Monomorphic bands

Polymorphic bands

Total bands

Polymorphism %

A

OP-A02

CAGGCCCTTC

2

4

6

66.67

B

OP-A04

AATCGGGCTG

2

3

5

60.00

C

OP-A07

GAAACGGGTG

1

7

8

87.50

D

OP-B07

GGTGACGCAG

2

5

7

71.42

E

OP-B10

CTGCTGGGAC

2

4

6

66.67

F

OP-O10

TCAGAGCGCC

3

4

7

57.14

G

OP-O13

GTCAGAGTCC

1

3

4

75.00

H

OP-O14

AGCATGGCTC

1

3

4

75.00

I

OP-O19

CAATCGCCGT

2

3

5

60.00

–

–

Total

16(30.77%)

36(69.23%)

52(100%)

69.23%

Source Sarrwy et al. (2014)

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DNA Fingerprinting of Date Palm Pollen Sources …

141

Fig. 6.4 Description of RAPD–PCR banding patterns of 5 five date palm cultivars amplified with the 9 random 10-mer primers; OPO-19; MS = 100-bp 9 primers ladder. Source Samy et al. (2015) Table 6.3 Total number, monomorphic, polymorphic band and percentage of polymorphism as revealed on five date palm genotypes by nine RAPD primers Primer

Total bands

Monomorphic bands

Polymorphic bands

Unique bands

% polymorphism

OPA-02

6

2

4

1

66.67

OPA-04

5

2

3

1

60

OPA-07

7

1

6

1

85.71

OPB-07

6

1

5

1

83.33

OPB-10

6

2

4

1

66.67

OPO-10

7

2

5

0

71.42

OPO-13

7

3

4

1

57.14

OPO-14

5

2

3

0

60

OPO-19

7

2

5

1

71.42

Total bands

56(100%)

17(30.36%)

39(69.64%)

7(12.5%)

69.64%

Source Samy et al. (2015) Table 6.4 Total number, monomorphic, polymorphic of bands and polymorphism percentage as revealed of five date palm cultivars using nine RAPD primers Primer

Total bands

Monomorphic bands

Polymorphic bands

% polymorphism

OPA-02

6

3

3

50

OPA-04

5

2

3

60

OPA-07

7

3

4

57.14

OPB-07

8

1

7

87.5

OPB-10

4

1

3

75

OPO-10

7

2

5

71.43

OPO-13

6

1

5

83.33

OPO-14

8

4

4

50

OPO-19

4

1

3

75

Total bands

55(100%)

18(32.73%)

37(67.27%)

(67.27%)

Source Mostafa et al. (2016)

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M. M. S. Saleh et al.

Fig. 6.5 DNA amplification fragments for 5 date palm cultivars with the RAPD–PCR technique:- (1) Sewi, (2) Amhat, (3) Giza, (4) Aswan, and (5) New Valley, using

OPA-02, OPA-04, OPA-07, OPB-07, OPB-10, OPO-13, OPB-14 and OPO-19 primers, M= 100 bp ladder. Source Mostafa et al. (2016)

polymorphic alleles with a polymorphic percentage of 76.08% and molecular weight ranged from 170 to 2005 bp. These findings were observed in 9 Egyptian date palm genotypes which were examined using RAPD–PCR technique. On the primers levels they noted that 6 different alleles were revealed due to primer OPA02, 4 of them were polymorphic and 2 were monomorphic alleles while, primer OP-A04 gave 10 alleles, 1 was monomorphic and 9 bands were polymorphic with percentage of 90% and the bands ranged from 2005 to 224 bp. Moreover, 7 different alleles were detected due to the third primer (OP-B07), ranged from 970 to 350 bp with 71.43% polymorphism. Primer OP-O10 showed 9 bands, 3 were monomorphic and 6 polymorphic alleles with a polymorphism ratio of 66.67% and these ranged from 1765 to 170 bp. In addition, 6 and 8 different bands of OP-O14 and OP-O19 primers

were identified, with polymorphic percentages of 50 and 100%, respectively (Table 6.5).

6.3.3 PCR—Amplification of RAPD Randomly amplified polymorphic DNA (RAPD) markers are particularly of interest. DNA profiles are both time- and cost-effective based on arbitrary primed PCR (Welsh and McClelland 1990; Williams et al. 1990). DNA fragments from the PCR amplification of random genomic DNA segments with single arbitrary nucleotide sequence primers are RAPD markers.

6.3.3.1 How It Works Unlike conventional PCR analysis, RAPD does not involve any precise knowledge of the target organism’s DNA sequence: the same 10-mer primers will or will not amplify a DNA fragment, depending on the positions complementing the

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DNA Fingerprinting of Date Palm Pollen Sources …

143

Table 6.5 Total alleles, monomorphic alleles, polymorphic alleles, and polymorphism of nine date palm genotypes for six primers using RAPD–PCR techniques No

Code of primer

Sequence (5`!3`)

Alleles range

Total alleles

Monomorphic alleles

Polymorphic alleles

Polymorphism %

a

OP-A02

CAGGCCCTTC

801:242

6

2

4

66.67

b

OP-A04

AATCGGGCTG

2005:224

10

1

9

90.00

c

OP-B07

GGTGACGCAG

970:350

7

2

5

71.43

d

OP-O10

TCAGAGCGCC

1765:170

9

3

6

66.67

e

OP-O14

AGCATGGCTC

1059:278

6

3

3

50.00

f

OP-O19

CAATCGCCGT

Total

958:230

8

0

8

100

2005:170

46

11

35

76.08%

Source Mostafa et al. (2018)

primer’s sequence. For example, if the primers annealed too far apart or 3’ ends of the primers do not face each other, no fragment is formed. Therefore, if a mutation that was previously complementary to the primer occurred at the site of the template DNA, a PCR product would not have been created, resulting in a different pattern of amplified DNA segments on the gel.

6.3.3.2 RAPD Limitations (a) Almost all RAPD markers are dominant, i.e., they are not distinguishable if a DNA fragment is repeated from a locus that is heterozygous (1 copy) or homozygous (2 copies). Codominant RAPD markers are only rarely recognized as different-sized fragments of DNA amplified from the same locus. (b) PCR is an enzymatic reaction, so the outcome may be greatly affected by the quality and concentration of template DNA, PCR component concentrations and PCR cycling conditions. Thus, the RAPD technique is notoriously dependent on the laboratory and involves reproducibility of carefully constructed laboratory protocols. (c) The differences between the primer and the prototype can lead to complete absence of the PCR product, as well as a mere reduction of the number of the product. The RAPD results can also be difficult to understand.

6.3.3.3 RAPD and Differential Display Analysis Primers that are used in the differential display analysis are synthesized in MWG Biotech (NS3 and ITS1) according to White and Castillo (1990). RAPD is carried out using at least five random primers according to Welsh et al. (1992). Sambrook et al. (1989) found that the amplification reaction was performed in a 25 ll reaction mixture containing 2 ll of genomic DNA, 3 ll of primers, 2.5 ll of a 10X Taq DNA polymerase reaction buffer, 1.5 units of Taq DNA polymerase, and 200 mm of dNTPs each. A DNA Thermo Cycler (PTC-100 PCR version 9.0-USA) used the following PCR software. The first stage consisted of initial denaturation at 94 °C for 5 min followed by 35 cycles at 94 °C for 30 s, 42 °C for 90 s for annealing temperature, 72 °C for 90 s and final extension at 72 °C for 2 min. In the 1X TAE buffer, RAPD– PCR products were isolated into 1.5% agarose gels and then detected by ethidium bromide staining. Genetic variation was studied using RAPD markers among 43 date palm accessions including 37 accessions from Morocco and 6 cultivars from Iraq and Tunisia. Pre-screening of 123 primers on 4 genotypes allowed 19 primers to be selected which revealed polymorphism and yielded reproducible results. All 43 genotypes

144

M. M. S. Saleh et al.

Fig. 6.6 Banding patterns of nine date palm cultivars using six RAPD primers and five pollenizers:- (1) Giza, (2) Aswan, (3) New Valley, (4) Noubaria, (5) Rasheed;

and 4 females:- (6) Sewi, (7) Amhat, (8) Zaghloul, (9) Samany; M= 100 bp DNA ladder. Source Mostafa et al. (2018)

studied could be distinguished by their band patterns. Therefore, RAPD technology seemed rather successful in recognizing date palm accessions. Genetic distance dependent on RAPD has been used to assess the relationships among accessions. The grouping association that cluster analysis established was very small. However, related varieties are grouped together, morphologically. Among the date palm varieties grown in Morocco, a fairly low polymorphism and a lack of obvious organization were observed. This may be linked to the manner in which the Moroccan date palm germplasm was introduced and preserved, which included

minimal exchange of foundations between cultivars, and the periodic creation of new recombinant cultivars following sexual reproduction (Sedra et al. 1998). Samy et al. (2015), using molecular markers as modern breeding approaches, estimated the genetic relationships among the studied date palms and their effect on improving the fruit indispensable to achieve date palm genetic improvement, considering it is lengthy and dioeciously by nature. In this concern, RAPD is successful and very important to explain genetic diversity. Thus, tools should enhance the relationship between

6

DNA Fingerprinting of Date Palm Pollen Sources …

by Amhat and New Valley pollen sources (0.526), while the lowest genetic similarity (0.368) was detected between cv. Amhat and the Aswan pollen source. UPGMA cluster testing was performed to graphically represent the genetic differences among 6 date palm accessions (Fig. 6.7). The dendrogram obtained was divided into two main clusters; one cluster included the three accessions of cv. Amhat to Giza, Aswan and Rasheed, while the other main cluster contained two subclusters, New Valley and Noubaria. Also, Sarrwy et al. (2014) studied the influence of different pollen sources on yield and fruit quality of cv. Sewi and their phylogenetic relationships using RAPD markers. They reported that the proximity matrix (Table 6.7) distinguished the relationships among 5 males and cv. Sewi; cv. Rasheed pollen source was the best male with 0.792, and the second was Noubaria (0.618), while the last one was New Valley with 0.333 r–value. RAPD data were used to generate a UPGMA dendrogram (Fig. 6.8). The obtained dendrogram showed that the six date palm cultivars formed two main clusters. The first one included three different pollen sources (Aswan, Rasheed, Noubaria) and the second cluster included Giza, New Valley, and Sewi. The results obtained by analysis of molecular variance (AMOVA) analysis supported genetic relationships among six genotypes in two clusters. The phylogenetic map between three different pollen sources and both Zaghloul and Samani

molecular markers and fruit characteristics. This may improve the efficiency of selection of date palm cultivars produced from sexual reproduction.

6.3.3.4 The Development of LocusSpecific, Codominant RAPD Markers (a) The polymorphic marker band of RAPD is isolated from the gel. (b) The PCR reaction amplifies it. (c) The PCR product is sequenced and cloned. (d) The sequenced characterized amplified region marker (SCAR) is a new longer, specialized primer that is built for the DNA sequence.

6.4

Phylogenetic Tree

A phylogenetic, or evolutionary, tree is a branching diagram that shows the evolutionary relationships among different biological species or other entities based on similarities and differences in their physical or genetic properties. Mustafa et al. (2014) estimated the genetic similarities among different pollen sources and cv. Amhat in Egypt. They reported that the estimated genetic similarities, as shown in Table 6.6, had a range 0.368–0.632. The highest genetic similarity (0.632) was observed between cv. Amhat and Noubaria pollen source followed

Table 6.6 Proximity matrix between cv. Amhat and 5 male pollen sources

Case

145

Matrix File Input Amhat

Noubaria

New Valley

Giza

Rasheed

Amhat

1

Noubaria

0.632

1

New Valley

0.526

1

1

Giza

0.421

0.474

0.579

1

Rasheed

0.474

0.632

0.737

0.211

1

Aswan

0.368

0.526

0.632

0

0.158

Source Mustafa et al. (2014)

Aswan

1

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M. M. S. Saleh et al.

Fig. 6.7 Dendrogram using average linkage (among groups) of date palm cultivars 1 female and 5 males 2, 3, 4, 5, 6. Source Mustafa et al. (2014)

Table 6.7 Proximity matrix for six date palm cultivars

Sewi

1.000

Noubaria

0.618

1.000

Giza

0.455

0.727

1.000

Aswan

0.509

0.122

0.165

1.000

Rasheed

0.792

0.182

0.636

0.118

1.000

New Valley

0.333

0.591

0.227

0.773

0.682

1.000

CASE

Sewi

Noubaria

Giza

Aswan

Rahseed

New Valley

Source Sarrwy et al. (2014)

Fig. 6.8 Dendrogram using average linkage (between groups) for six cultivars: - (1) Sewi, (2) Noubaria, (3) Giza, (4) Aswan, (5) Rasheed,) 6) New Valley. Source Sarrwy et al. (2014)

female date palms were studied by Samy et al. (2015). They reported that the genetic relationships among the five accessions were estimated in terms of similarity using the Dice coefficient. As shown in Table 6.8, the phylogenetic tree revealed high values between New Valley and both Zaghloul and Samani (0.731 and 1.00, respectively), while it revealed medium relations between Rasheed pollen source and both Zaghloul and Samani (0.402 and 0.667, respectively). Also the pollen source of El Noubaria revealed medium value for relationships (0.447) with Zaghloul whereas El Noubaria with Samani recorded the lowest value (0.282).

RAPD markers were used to describe 5 date palm genotypes grown in different regions of Egypt and more assays with regard to polymorphic detection analysis by UPGMA of the dendrogram in (Fig. 6.9). This phylogenetic tree divided these genotypes into 2 subclusters, the first includes Rasheed, New Valley and Samani while the second cluster includes El Noubaria and Zaghloul genotypes. Mostafa et al. (2016) examined the relation among some date palm pollenizers and 2 female cultivars using RAPD molecular markers. The results are shown in a proximity matrix (Table 6.9) that the relationships revealed a

6

DNA Fingerprinting of Date Palm Pollen Sources …

Table 6.8 Proximity matrix between two females (Zaghloul and Samani) with three males from Rasheed, El Noubaria, and New Valley

Case

147

Matrix File Input Rasheed

El Noubaria

New Valley

Zaghloul

Samani

Rasheed

1

…..

…..

…..

…..

El Noubaria

0.632

1

…..

…..

…..

New Valley

0.526

1

1

…..

…..

Zaghloul

0.402

0.447

0.731

1

…..

Samani

0.667

0.282

1

0.211

1

Source Samy et al. (2015)

Fig. 6.9 Dendrogram using average linkage (between groups) of date palm cultivars two females Zaghloul and Samani with three males Rasheed, El Noubaria, and New Valley. Source Samy et al. (2015)

medium value (0.454) between cv. Sewi and the pollen source New Valley and a low value (0.135) between Sewi and Aswan (pollen source). Furthermore, the relationship between cv. Sewi and the Giza pollen source was 0.195. On the other side, the phylogenetic tree revealed a high relationship (1.000) between cv. Amhat and the Giza pollen source. The lowest relationship (0.250) was detected between cv. Amhat and the New Valley pollen source. In addition, the ratio between the cv. Amhat female cultivar and the Aswan pollen source was 0.979. The relationships exhibited two clusters, the first containing two subclusters, the first comprising Amhat and Giza, while the second subcluster only includes Aswan. The second cluster included Sewi and New Valley cultivars. These results are shown in Fig. 6.10.

Table 6.9 Proximity matrix of five Egyptian date palm genotypes

Case

Sewi

Also, Mostafa et al. (2018) researched the production, fruit properties and genetic diversity of four Egyptian date palm cultivars and five separate pollen sources using molecular markers. They found the Giza pollen source to be genetically closer to cv. Sewi cv. (98.5%) and to cv. Zaghloul cv., while the lowest value was found between the Giza pollen source and Amhat (42.9%), due to the similarity and dissimilarity between the 5 pollen sources and the 4 female Egyptian cultivars. The Noubaria pollen source commonly showed with cv. Samany the lowest value (9.5%). On the other hand, the average similarity value was observed among cv. Sewi and both New Valley and Rasheed pollen sources, with 52.4%, 57.1%, respectively, and 52.4% between cv. Amhat and Noubaria the pollen source (Table 6.10).

Amhat

Giza

Aswan

Sewi

….

Amhat

0.000

….

Giza

0.195

1.000

….

Aswan

0.135

0.979

0.596

….

New Valley

0.454

0.250

0.149

0.244

Source Mostafa et al. (2016)

New Valley

….

148

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Fig. 6.10 Dendrogram using average (between groups) linkage for five cultivars of Egyptian date palm: - (1) Sewi, (2) Amhat, (3) Giza, (4) Aswan, (5) New Valley. Source Mostafa et al. (2016) Table 6.10 Similarity and dissimilarity among four female Egyptian date palm cultivars and five pollenizers Case

Matrix File Input

Giza

1

Aswan

0.619

1

NewValley

0.667

0.429

1

Noubaria

0.952

0.333

0.571

1

Rasheed

0.619

0.195

0.619

0.429

1

Sewi

0.985

0.286

0.524

0.143

0.571

1

Amhat

0.429

0.19

0.714

0.524

0.122

0.571

Zaghloul

0.81

0.381

0.429

0.238

0.762

0.19

0.762

1

Samany

0.667

0.143

0.762

0.095

0.143

0.429

0.143

0.524

Giza

Aswan

Valley

Noubaria

Rasheed

Sewi

Amhat

Zaghloul

Samany

1 1

Source Mostafa et al. (2018)

Fig. 6.11 Dendrogram of five pollenizers and four female Egyptian cultivars. Source Mostafa et al. (2018)

The dendrogram was divided into two clusters among five pollen sources and four female date palms. Rasheed, Amhat, Aswan, Noubaria and Samany were in the first cluster, while Sewi, Zaghloul, New Valley and Giza were in the second cluster (Fig. 6.11).

6.5

Other Phylogenetic Analyses

(a) Williams et al. (1990) used amplified polymorphic DNA (RAPD) markers for rapid screening of nuclear genome variations. While Pritchard et al. (2000) used multilocus genotype data to analyze the population structure.

6

DNA Fingerprinting of Date Palm Pollen Sources …

(b) Principal component analysis (PCA) of SNP genotypes was performed using EIGENSTRAT for 62 cultivars (Price et al. 2006). (c) The RAPD approach appears to be a reliable technique to examine the genetic diversity of date palm germplasm, and many oligonucleotide primers are now being used for possible genetic diversity analysis of date palm and variable variety recognition. The holes or incomplete data are excluded if the coverage of the web is less than 90%. (d) The constructed phylogenetic tree was visualized with EvolView, an online visualization tool for phylogenetic tree (Zhang et al. 2012). (e) The phylogenetic tree was developed by MEGA using all SNP sites of the varieties (NJ 1000 boot strap process, version 6.06) (Tamura et al. 2013).

6.6

Relevance of Pollen Sources to Yield and Fruit Traits

There is a common agreement among date palm growers that fruit production success depends mainly on pollination efficiency. Date palm cultivars are divided into three main groups according to their fruit moisture content, i.e., soft: more than 30% humidity, semidry: from 20–30% and dry cultivars: less than 20%. The pollen grain source is considering a critical and limiting factor for palm production and fruit characteristics. Many investigators have shown that pollen grains from different male date palms have a direct impact on fruit set, yield and fruit physical and chemical properties, metaxenia, in addition to the their effects on the size and shape of the seed, xenia (El-Hammady et al. 1977; ElSalhy et al. 1997; Mothew et al. 1975; Munir et al. 2020; Mustafa et al. 2014; Soliman et al. 2020).

149

6.6.1 Fruit Set and Yield The first indication of expected yield of fruit crops can be estimated by the obtained values of fruit set and fruit retention. In some date, palm cultivars, greater fruit set is due to the compatibility of the male as a pollenizer with the commercial female cultivar. In this concern, Sarrwy et al. (2014) pollinated Sewi female date palms (semidry type) with pollen grains from five different Egyptian regions (Giza, Aswan, New Valley, Noubaria, Rasheed). They found that pollenizers from the Raseed district gave the highest fruit set and fruit retention values, followed by Noubaria, Aswan, Giza, and finally New Valley (Table 6.11). They reported that yield of Sewi female date palm recorded higher bunch weight and yield per palm due to pollenizers from Rasheed and Noubaria, in both studied seasons, respectively, comparrf with the other pollenizer sources. They added that the genetic relationships reached high and medium values (0.792 and 0.618) among cv. Sewi cultivar and both Rasheed and Noubaria pollenizers. The lower bunch weight and yield per palm of Sewi were recorded from New Valley and Giza pollenizers which recorded lower genetic relationships (0.333 and 0.455, respectively) between them and cv. Sewi. Pollenizers from different locations were used to pollinate cv. Amhat (soft type) as studied by Mustafa et al. (2014), who found that pollen from Noubaria recorded the highest fruit set and fruit retention values, also achieved the highest yield of bunch weight and palm yield with high genetic relationship (63.2%) them compared with the other pollenizer sources (Table 6.12). They added that Amhat female palm recorded the lowest fruit set and fruit retention when pollinated with Aswan pollenizer which related by the lowest genetic relationship (36.8%), while the lowest yield was recorded due to Giza pollenizer. Also, Merwad et al. (2015) studied the effect of pollenizers selected from different Egyptian governorates (Giza, Aswan, Rasheed, Noubaria,

150

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Table 6.11 Effect of different pollen sources on fruit set, fruit retention, and yield of cv. Sewi Pollenizer source

Fruit set%

Fruit retention%

Bunch weight (kg)

Yield/palm (kg)

1st season

2nd season

1st season

2nd season

1st season

2nd season

1st season

2nd season

Giza

61.3b

64.0d

49.33b

55.33b

14.83d

16.17e

118.7d

129.3e

Aswan

64.0b

67.3c

53.67a

60.57a

15.87c

17.23d

126.9c

137.9d

New Valley

57.0c

60.0e

50.00b

52.33bc

14.30e

18.70c

114.4e

149.6c

Noubaria

70.0a

71.0b

47.00c

50.67c

17.67b

21.10a

141.6b

168.8a

Rasheed

70.0a

74.0a

54.00a

59.00a

18.83a

19.63b

150.7a

157.1a

Source Sarrwy et al. (2014)

Table 6.12 Effect of different pollenizer sources on fruit set, fruit retention, bunch weight, and yield of cv. Amhat

Pollenizer

Fruit set%

Noubaria

Fruit retention %

Bunch weight (kg)

Yield/ palm(kg) 2012

2012

2013

2012

2013

2012

2013

a

a

a

a

18.9

a

20.3

a

e

15.3

e

86.0

c

85.3

57.0

d

d

62.0

d

2013 a

162.1a

e

109.9

122.3e

151.2

Aswan

69.2

70.3

50.0

52.3

13.7

Giza

80.3b

79.7b

55.7ab

60.3b

18.2b

19.5b

145.3b

155.7b

b

b

C

C

C

C

131.5

137.7C

C

Rasheed

80.0

80.3

52.7

57.0

16.4

17.3

New Valley

69.6C

73.0C

54.0bC

56.7C

15.4d

16.2d

123.5d

129.6d

Significant

S

S

S

S

S

S

S

S

Source Mustafa et al. (2014)

Table 6.13 Effect of different pollen sources on fruit set, fruit retention, and yield of cv. Hayany Pollen source

Fruit set%

Fruit retention%

Bunch weight (kg)

Yield/palm (kg)

1st season

2nd season

1st season

2nd season

1st season

2nd season

1st season

2nd season

Giza Aswan

75.3b

70.6b

34.0c

32.0c

14.6b

16.0a

117.3b

128.0a

70.3d

69.6b

29.6d

29.6d

10.6d

12.0b

85.3d

96.0b

New Valley

72.0c

70.3b

31.0d

32.3bc

12.0 cd

11.3b

96.0 cd

101.3b

Rasheed

78.6a

76.0a

42.3a

41.0a

16.3a

17.3a

130.6a

138.6a

Noubaria

75.6b

70.6b

35.6b

34.3b

13.3bc

15.6a

106.6bc

125.3a

Source Merwad et al. (2015)

New Valley) on fruit set and fruit retention of Hayany female cultivar (soft type). The results showed that Rasheed pollenizers were the most effective on fruit set and fruit retention and recorded the highest bunch weight and yield per palm, followed by the pollen from Giza governorate comparing with the other pollenizer sources (Table 6.13).

In the same line, Samy et al. (2015) used pollen from different Egyptian locations (Rasheed, El Noubaria, New Valley districts) as pollenizers for both Zaghloul and Samani female date palms (soft types). They found that the pollen from Rasheed district recorded the highest fruit set, fruit retention percentages, and yield of Zaghloul. The same pollenizer gave the highest

6

DNA Fingerprinting of Date Palm Pollen Sources …

151

Table 6.14 Fruit set, fruit retention, bunch weight, and yield of cv. Zaghloul as affected by different pollenizer sources during 2012 and 2013 seasons Pollenizer Source New Valley

Fruit set %

Fruit retention %

Bunch weight (kg)

Palm yield (kg)

1st season

1st season

1st season

2nd season

1st season

60.0

61.0

30.33

2nd season 32.00

14.83

2nd season 15.53

118.6

2nd season 124.26

Rasheed

63.0

62.0

34.66

36.00

17.60

17.93

140.8

143.46

El Noubaria

60.0

61.0

32.66

33.33

17.13

16.16

137.0

129.33

1.25

2.26

0.47

0.52

LSD 5%

2.17

2.97

8.22

4.20

Source Samy et al. (2015)

Table 6.15 Fruit set, fruit retention, bunch weight, and yield of Samani date palm as affected by different pollenizer sources during 2012 and 2013 seasons Pollenizer Source

Fruit retention %

Bunch weight (kg)

Palm yield (kg)

1st season

2nd season

1st season

2nd season

1st season

2nd season

1st season

2nd season

New Valley

79.66

37.50

39.00

70.66

16.0

14.33

128.0

114.66

Rasheed

72.00

39.00

40.00

69.33

17.7

14.70

141.6

117.60

El Noubaria

79.00

35.80

37.33

80.33

18.2

15.70

145.6

125.63

LSD 5%

Fruit set %

2.944

1.474

1.255

1.472

0.103

0.296

0.869

2.277

Source Samy et al. (2015)

value for fruit set, fruit retention of Samani female date palm, while the highest yield was recorded due to the pollen from El Noubaria district, compared with the other pollenizers from different districts (Tables 6.14 and 6.15). The genetic relationships recorded medium value (0.402 and 0.667) among Rasheed pollenizer and both Zaghloul and Samani date palms, respectively, while it recorded 0.282 between El Noubaria pollenizer and Samani female cultivar. On the other hand, Mostafa et al. (2016) mentioned that Sewi as a semidry and Amhat as a soft female type were pollinated with grains selected from male pollenizers brought from Giza, Aswan, and El-Wadi governorates. They found that the Aswan pollenizer gave the best effect on fruit set of cv. Amhat, while Giza pollen grains had a superior effect on cv. Sewi date palm compared with the other pollenizers (Fig. 6.12). Giza was the most suitable pollenizer for

pollinating Sewi female date palm concerning the bunch weight yield per palm. While the Aswan pollenizer had the greatest effect on bunch weight and palm yield of cv. Amhat (Figs. 6.13 and 6.14). The genetic relationship revealed low values (0.195 and 0.135) between Sewi female and both Giza and Aswan pollenizers, respectively, while the genetic relationship achieved high values between Amhat female and both Giza and Aswan pollenizers (1.000 and 0.979, respectively). Mostafa et al. (2018) studied the effect of pollen sources from five different Egyptian governorates (Noubaria, Rasheed, New valley, Aswan, and Giza) on four female cvs. (Sewi, Amhat, Samany, Zaghloul). The authors concluded that the Rasheed pollenizer followed by Noubaria one recorded the maximum bunch weight for cv. Sewi. In this concern, the genetic relationship was 0.571 and 0.143 among Sewi

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Fig. 6.12 Effect of different pollen sources on fruit set of cvs. Sewi and Amhat. Source Mostafa et al. (2016)

Fig. 6.13 Effect of different pollen sources on bunch weight of cv. Amhat. Source Mostafa et al. (2016)

Fig. 6.14 Effect of different pollen sources on yield per palm of cv. Amhat. Source Mostafa et al. (2016)

and both Rasheed and Noubaria, respectively. On the other hand, the Rasheed pollenizer followed by the Giza one gave the heaviest bunch weight of cv. Zaghloul and the genetic relationship recorded 0.762 and 0.810 among Zaghloul and both Rasheed and Giza pollens, respectively. As for cv. Samany, the Giza pollenizer followed by Noubaria and Rasheed pollenizers gave the highest bunch weight, the genetic relationship among cv. Samany and Giza, Noubaria and Rasheed pollenizers recorded 0.667, 0.095 and 0.143, respectively. Concerning cv. Amhat, it is

clear that Noubaria followed by Giza pollen grains recorded higher bunch weight values. The genetic relationship among cv. Amhat and both Noubaria and Giza recorded 0.524 and 0.429. This means that the differences between pollen grain sources have different effects on yield as measured by bunch weight or yield per palm (Fig. 6.15). Figure 6.16 shows the shape of various male strands that were selected from different Egyptian governorates as shown in an internal project report entitled selection, identification and

6

DNA Fingerprinting of Date Palm Pollen Sources …

153

Fig. 6.15 Effect of different pollenizer sources on bunch weight of cvs. Sewi, Amhat, Samany, and Zaghloul. Source Mostafa et al. (2018)

micropropagation of male date palms as a source of high xenic and metaxenic effects for fruit quality. Presented by Esam A.M. Mostafa (2010–2013), Pomology Department, NRC, Egypt. The effect of different pollen sources on fruit set and fruit traits of cvs. Sewi, Amhat, Samany, and Zaghloul are shown in Figs. 6.17, 6.18, 6.19 and 6.20 as presented in an internal project report entitled Selection, identification, and micro propagation of date palm pollinators as a source of high xenic and metaxenic effects for fruit quality. Presented by Esam A.M. Mostafa (2010–2013), Pomology Department, NRC, Egypt

6.6.2 Relevance of Pollen Sources to Fruit Traits The xenia effect influences the size and shape of the seeds, and the metaxenia effect influences fruit set, yield and fruit physical and chemical characteristics. However, different pollen grain sources show different effects on fruit physical and chemical traits. Sudhersan et al. (2010) recorded that Phoenix pusilla Gaertn. pollen, which closely resembles that of the date palm, was tested on date palm cvs. Barhi, Medjool and Sultana. The study revealed that P. pusilla pollen can fertilize date palm flowers, resembling date palm pollen in all three of the experimental cultivars. In the three cultivars studied, fruit production was similar up to the khalal stage. In the rutab and tamar stages, major variations in the

fruit and seed characteristics were noted. The maturity of fruits was delayed as compared to normal fruits and fruits were seedless or incompletely developed at maturity (Fig. 6.21). Sarrwy et al. (2014) pollinated cv. Sewi with pollen grains from five different regions in Egypt (Giza, Aswan, New Valley, Noubaria, Rasheed). They reported that the best fruit quality over two seasons was obtained from palms pollinated with both Rasheed and Noubaria as pollen sources. They added that genetic relationship reached high and medium values (0.792 and 0.618) between cv. Sewi cultivar and both Rasheed and Noubaria as pollen sources. Mustafa et al. (2014) studied the effect of pollen sources from five different districts [(Noubaria, Aswan, Giza, Rasheed, New Valley), in which their average yearly temperature ranged about 19.7, 26.7, 22.9, 20.5, and 25.2°C, respectively, Emad (2015)] on yield and fruit characteristics of cv. Amhat. They found that Noubaria gave the best fruit physical and chemical characteristics compared with the other pollen sources. The genetic relationship between the pollenizer from Noubaria and the commercial cultivar (Amhat) showed a high value (63.2%). In the same line, Samy et al. (2015) found that the pollen sources from El Noubaria district gave the best results concerning physical properties (weight, length, diameter, size), and also enhanced the chemical properties (TSS, and total, reducing and nonreducing sugars) for cv. Samani. The genetic relationship between the pollenizer and the female cultivar reached 0.282. The same trend was observed when the pollen

154 Fig. 6.16 Samples of male strands from different Egyptian governorates:- (a) Aswan, (b) Noubaria, (c) Rasheed, (d) New Valley, (e) Giza. (Photos by Mohamed M.S. Saleh)

M. M. S. Saleh et al.

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DNA Fingerprinting of Date Palm Pollen Sources …

155

Fig. 6.17 Effect of different pollen sources on fruit traits of cv. Sewi: - (1) Aswan, (2) Noubaria, (3) New Valley, (4) Rasheed, 5) Giza pollen sources. (Photo by Mohamed M.S. Saleh)

Fig. 6.18 Effect of different pollen sources on fruit traits of cv. Amhat: - (1) Aswan, (2) Noubaria, (3) New Valley, (4) Rasheed, (5) Giza pollen sources. (Photo by Mohamed M.S. Saleh)

Fig. 6.19 Effect of different pollen sources on fruit traits of cv. Samany: - (1) Aswan, (2) Noubaria, (3) New Valley, (4) Rasheed, (5) Giza pollen sources. (Photo by Mohamed M.S. Saleh)

from Rasheed district was used to pollinate cv. Zaghloul, where the value of genetic relationship was 0.402. The effect of pollen sources on fruit traits was observed by Merwad et al. (2015) who studied

the effect of pollen selected from different districts (governorates in Egypt) as pollen sources for cv. Hayany. They reported that Rasheed pollen recorded the highest fruit characteristics (fruit dimensions) and chemical composition of

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Fig. 6.20 Effect of different pollen sources on fruit traits of cv. Zaghloul. (1) Aswan, (2) Noubaria, (3) New Valley, (4) Rasheed, (5) Giza pollen sources. (Photo by Mohamed M.S. Saleh)

Fig. 6.21 Fruits of cv. Medjool. On the right, with P. pusilla as the pollen source, the seed appears very small and rudimentary, while on the left, with P. dactylifera as a pollen source, the seed is normal seed. Source Sudhersan et al. (2010)

Fig. 6.22 Average minimum and maximum temperatures in Alexandria, Egypt (Rasheed and Noubaria districts). Source (www.weather-and-climate.com)

6

DNA Fingerprinting of Date Palm Pollen Sources …

fruit (total soluble solids, reducing sugars, nonreducing sugars, total sugars). Additionally, Mostafa et al. (2016) noted that Sewi as a semidry date and Amhat as moist date were pollinated with male pollen sources from Giza, Aswan, and El-Wadi (New Valley) governorates. The results concerning physical and chemical fruit properties indicated that Giza and Aswan were the most suitable pollen sources for cv. Sewi. The genetic relationships among the two pollen sources and cv. Sewi were low (0.195 and 1.135 for Giza and Aswan, respectively). Mostafa et al. (2018) also studied the effect of pollen from Noubaria, Rasheed, New Valley, Aswan, and Giza, on four cvs. (Sewi, Amhat, Samany, Zaghloul). The results showed that Giza and Aswan pollens were the most suitable for pollinating cvs. Sewi and Amhat females, respectively, concerning physical and chemical fruit properties. The genetic relationship was high (0.985) among Giza pollenizer and cv. Sewi and recorded 0.190 between Aswan and Amhat pollen sources.

6.7

Environment and the Relation Between Pollen Source and Female Cultivar

From the abovementioned experiments, it could be concluded that no constant trend is detected for the genetic relationship between the pollen source and the commercial female cultivars, while there is a clear relationship between them due to ecogenetics for both the selected pollen source and the commercial female cultivar, since in most studies, the pollen imported from high humidity areas with moderate temperature such as Rasheed or Noubaria districts (Alexandria governorate, Figs. 6.22 and 6.23) were the most effective on fruit set, fruit retention, bunch weight, and yield per palm of the female cultivars grown under Giza governorate conditions (Figs. 6.24 and 6.25), these results could be explained by that reported by Swingle (1928)

157

who concluded that the direct effect of the male parent on the development of the date fruit is unique and definite, and varies with the male used for the fertilization of the female flowers, each male having approximately the same influence on the fruit of all varieties and having the same effect over different years. On the other hand, it is clear that the pollen sources selected from the areas characterized with low humidity and high temperature such as Aswan (Figs. 6.26 and 6.27) mostly recorded low fruit set, fruit retention, bunch weight and yield per palm. This may due to the negative effect of high temperature in Aswan on pollen grain viability. In this concern, Leonardo et al (2018) reported that quinoa pollen grain viability and pollen wall structure were affected by high temperatures at anthesis stage; they added that heat stress reduced the pollen viability by 30–70%.

6.8

Conclusion and Prospects

Generally, no constant trend for similarity relationship values was detected between the pollen sources and commercial female cultivars concerning the yield and fruit traits of date palms, so the presence of a high genetic relationship between the pollen source and the female cultivar is not indicated to induce high yield; sometimes the highest yield corresponds with low genetic relationship, also the same is true for fruit traits since the low traits may result from high similarity between the pollen source and the commercial female cultivar. The effect of pollen source on yield and fruit traits seems to correspond with the ecogenetics (gene-environment interactions). The direct physiological effect is related to the concentrations of free IAA among date fruits produced by pollinating the female flowers with various pollen sources that are likely responsible for some manifestation of the xenia and metaxenia phenomena. Additionally, work on metaxenia has shown that specific pollen sources influence cytokine and gibberellin

158

M. M. S. Saleh et al.

Fig. 6.23 Average relative humidity in Alexandria, Egypt (Rasheed and Noubaria districts). Source (www.weatherand-climate.com)

Fig. 6.24 Average minimum and maximum temperatures in Cairo, Egypt (Giza district). Source (www.weather-andclimate.com)

Fig. 6.25 Average relative humidity in Cairo, Egypt (Giza district). Source (www.weather-and-climate.com)

Fig. 6.26 Average minimum and maximum temperatures in Aswan, Egypt (Aswan district). Source (www.weatherand-climate.com)

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DNA Fingerprinting of Date Palm Pollen Sources …

159

Fig. 6.27 Average relative humidity in Aswan, Egypt (Aswan district). Source (www.weather-and-climate.com)

levels. Finally, the relationship between the pollen source and the female cultivars may need more study to detect it. On the other hand, the RAPD technique is useful to make comparisons among the date palm genotypes. DNA fingerprinting is considered as an indicator of the relationship between the pollen source and the economic female date palms.

References Abbas MF, Abdul-Wahid AH, Abass KI (2014) Metaxenic effect in date palm (Phoenix dactylifera L.) fruit in relation to level of endogenous auxins. AAB Bioflux 6:40–44 Al-Dous EK, George B, Al-Mahmoud ME et al (2011) De novo genome sequencing and comparative genomics of date palm (Phoenix dactylifera). Nat Biotech 29:521–527 Al-Mssallem IS, Hu S, Zhang X et al (2013) Genome sequence of the date palm (Phoenix dactylifera L.). Nat Com 4:2274 Billotte N, Marseillac N, Brottier P et al (2004) Nuclear microsatellite markers for the date palm (Phoenix dactylifera L.): characterization and utility across the genus Phoenix and in other palm genera. Molec Ecol Not 4:256–258 Denney JO (1992) Xenia includes metaxenia. Hort Sci 27:722–728 El-Hammady MM, Khalifa AS, El-Hammady AM (1977) The effect of date pollen on some physical and chemical characters of (Hayani variety). Res Bull 773 Fac Agric Ain Shams Univ, Cairo, Egypt El-Salhy AM, El-Bana AA, Abdel-Galil HA, Ahmed EF (2010) Effect of pollen grains suspensions spraying on yield and fruit quality of Saidy date palm cultivar. Acta Hort 882:329–336 El-Salhy AM, AbdAlla AY, Mostafa RAA (1997) Evaluation of some date palm male seedling in pollination of Zaghloul and Samani date palms under Assiut conditions. Assiut J Agric Sci 28:79–87

Emad FSA (2015) GIS-Mapping aridity and rainfall water deficit of Egypt. J Agric Env Sci Dam Univ, Egypt 14:17–40 Gros-Balthazard M (2013) Hybridization in the genus Phoenix: a review. Emir J Food Agric 25:831–842 Hamwieh A, Farah J, Moussally S et al (2010) Development of 1000 microsatellite markers across the date palm (Phoenix dactylifera L.) genome. Acta Hort 882:269–277 Hazzouri KM, Flowers JM, Visser HJ et al (2015) Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop. Nat Com 6:8824 Leonardo H, Janet BM, Kevin MM (2018) Effect of high temperature on pollen morphology, plant growth and seed yield in quinoa (Chenopodium quinoa Willd.). J Agr Crop Sci 205:33–45 Lynch M (1990) The similarity index and DNA fingerprinting. Mol Biol Evol 7:478–484 Merwad MA, Mostafa EAM, Ashour NE et al (2020) Evaluation, DNA fingerprinting and productivity of some superior seeded female Egyptian date palms. Inter J Latest Tech Eng Manag Appl Sci 9:68–75 Merwad MA, Mostafa EAM, Saleh MMS et al (2015) Yield and fruit quality of Hayany date palm as affected by different pollen grain sources. Inter J ChemTech Res 8:544–549 Moghaieb REA, Abdel-Hadi AA, Ahmed MRA, Hassan AGM (2010) Genetic diversity and sex determination in date palms (Phoenix dactylifera L.) based on DNA markers. Arab J Biotech 132:143–156 Mohamed A Awad (2010) Pollination of date palm (Phoenix dactylifera L.) cv. Khenazy by pollen grainwater suspension spray. J Food Agric Envir 8 (3&4):313–317 Mostafa EAM, Saleh MMS, Ashour NE et al (2016) The effect of metaxenia on fruit yield and the relation between some date palm pollinizers and two female cultivars using RAPD molecular markers. Res J Pharma Bio Chem Sci 7:971–984 Mostafa EAM, Saleh MMS, Ashour NE et al (2018) Productivity, fruit properties and genetic diversity by molecular markers of four Egyptian female date palm cultivars and five different pollinizers. Biosc Res 15:166–175 Mothew CA, Al-Rawi H, Al-Zubahidi A et al (1975) The effect of different type of pollination of individual

160 trees with different pollens. Proc 3rd Inter Palm and Date Conf Nov 30-Dec 4 1975 Baghdad, Iraq, pp 1– 17 Mustafa EAM, Heiba SAA, Saleh MMS et al (2014) Effect of different pollinizer sources on yield, fruit characteristics and phylogenetic relationships with Amhat cv. date palm (Phoenix dactylifera L.) in Egypt using RAPD markers. Inter J Agric Res 9:331– 343 Munir M, Alhajhoj MR, Sallam AAM et al (2020) Effects of indigenous and foreign pollinizers on the yield and fruit characteristics of date palm cultivar Khalas. Iraqi J Agric Sci 51:356–365 Nixon R (1928) Immediate influence of pollen in determining the size and time of ripening of the fruit of the date palm. J Hered 19:241–255 Nixon RW (1935) Metaxenia in dates. Proc Amer Soc Hort Sci 32:221–226 Olfati JA, Sheykhtaher Z, Qamgosar R et al (2010) Xenia and metaxenia on cucumber fruit and seed characteristics. Inter J Veget Sci 16:243–252 Osman AMA, Reuther W, Erickson LC (1974) Xenia and metaxenia studies in the date palm (Phoenix dactylifera L.). Date Grow Inst Rep 51:6–16 Price AL, Patterson NJ, Plenge RM et al (2006) Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38:904– 909 Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genet 155:945–959 Robbertse PJ, Coetzer LA, Johannsmeier MF, Swart DJ (1996) Hass yield and fruit size as influenced by pollination and pollen donor: a joint progress report. South Afr Avocado Grow Assoc Yearb 19:63–67 Sambrook J, Fritsch KF, Maniatis T (1989) Molecular cloning. 2nd ed. Cold Spring Harbor, New York Heiba Samy AA, Hoda Ali BM, Mostafa Esam AM et al (2015) The phylogenetic map between three pollinizers and their impact on fruit set, yield and fruit quality of Zaghloul and Samani date palms. Catarina 11:51– 58 Sarrwy SMA, Haiba AAA, Attia SAA et al (2014) Influence of different pollen grains sources on yield and fruit quality of Siwi date palm, and their phylogenetic relationships using RAPD markers. Mid East J Agric Res 3:330–337 Sedgley M, Griffin AR (1989) Sexual reproduction in tree crops. Academic Press, London, UK

M. M. S. Saleh et al. Sedra H, Lashermes Philippe, Trouslot Pierre et al (1998) Identification and genetic diversity analysis of date palm (Phoenix dactylifera L.) varieties from Morocco using RAPD markers. Euphy 103:75–82 Shaheen MA, Bacha MA, Nasr TA (1989a) Effect of male type on fruit chemical properties in some date palm cultivars. Ann Agric Sci 34:265–281 Shaheen MA, Bacha MA, Nasr TA (1989b) Effect of male type on fruit setting, yield and fruit physical properties in some date palm cultivars. Ann Agric 34:283–299 Soliman SS, Kassem HA, Al-Obeed RS et al (2020) Influence of pollen source and its mineral content on fruit retention and quality of Kadary date palm cultivar (Phoenix dactylifera L.) J Chem Bio Phys Sci 10:437– 446 Sudhersan C, Jibil Manuel S, Al-Sabah L (2010) Xenic and metaxenic effect of Phoenix pusilla pollen on certain date palm cultivars. Acta Hort 882:297–302 Swingle WT (1928) Metaxenia in the date palm: possibly a hormone action by the embryo or endosperm. J Hered 19:257–268 Tamura K, Stecher G, Peterson D et al (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729 Welsh J, McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucl Acid Res 18:7213–7218 Welsh J, Chada K, Dalal SS et al (1992) Arbitrarily primed PCR fingerprinting of RNA. Nucl Acids Res 20:4965–4970 Weingartner U, Kaeser O, Long M, Stamp P (2002) Combining cytoplasmic male sterility and xenia increases grain yield of maize hybrids. Crop Sci 42:1848–1856 White JW, Castillo JA (1990) Association between productivity, root growth and carbon isotope discrimination in Phaseolus vulgaris under water deficit. Austral J Plant Phys 17:189–198 Williams JGK, Kublick AR, Livak KJ et al (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl Acids Res 18:6531– 6535 Zaid A, De Wet PF (2002) Climatic requirements of date palm In: Zaid A (ed) Date palm cultivation, Plant Prod Prot Paper 156 Rev. 1, FAO, Rome, pp 57–72 Zhang H, Gao S, Lercher MJ et al (2012) EvolView, an online tool for visualizing, annotating and managing phylogenetic trees. Nucl Acids Res 40:W569–W572

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Gender Determination of Date Palm Summar Abbas Naqvi, Waqar Shafqat, Muhammad Salman Haider, Faisal Saeed Awan, Iqrar Ahmad Khan, and Muhammad Jafar Jaskani

Abstract

Date palm is dioecious, characterized by unisexual flowers located on separate male or female trees. Dioecy has always been an issue in date palm breeding because the sex of the plant cannot be known until it reaches its reproductive stage, which takes 5‒9 years to first flowering. Hence, early sex identification

S. A. Naqvi
I. A. Khan
M. J. Jaskani (&) Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan e-mail: [email protected] S. A. Naqvi e-mail: [email protected] I. A. Khan e-mail: [email protected] W. Shafqat Department of Horticultural Sciences, Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Fort Pierce, FL 34945, USA e-mail: [email protected] M. S. Haider Key Laboratory of Genetics and Fruit Development, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China e-mail: [email protected] F. S. Awan Center for Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad, Pakistan e-mail: [email protected]

can enhance breeding and genetic improvement success in date palm. The breeding programs help in the selection and identification of superior seedlings of important traits. However, the sex of dioecious plants is usually tedious to identify. Sex determination may not only enhance genetic resources but also gear up breeding programs. No doubt, substantial progress has been made in understanding mechanisms of sex identification in date palm using morphological, physiological, biochemical, and cytological procedures but the discrimination in male and female plants by these techniques is not reliable. Sexuality in plants is regulated by genetic, epigenetic, and physiological mechanisms, and the most specialized mechanism of sex determination is the sex chromosomes. Molecular markers have been applied accurately in date palm breeding programs to differentiate male and female seedlings, but the development of more reliable molecular markers is needed. Hence, this chapter is focused on an understanding of floral biology, genetics of sex, and methods applied for sex determination in date palm.

7.1

Introduction

The term dioecious refers to plant taxa with separate individual male and female plants; only 7% of the angiosperm genera (13,000) possess dioecious species and 6% of the angiosperm

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species (240,000) are exclusively dioecious. Dioecious species have evolved from their hermaphrodite ancestors, which have both male (androecium) and female (gynoecium) organs (Kumar et al. 2014). In dioecious plants, abortion occurs in the gynoecium of male flowers whereas the androecium of female flowers at different stages of organ development dividing plants into two groups Type I and Type II (Diggle et al. 2011). Type I species show considerable alterations in the androecium or gynoecium during different developmental phases (Phoenix dactylifera L., Vitis vinifera L. and Dioscorea tokoro Makino ex. Miyabo, and it is speculated that independent evolution must have occurred in these species. Whereas, androecium and gynoecium are entirely missing in the male and female flowers, respectively, of Type II species (Spinacea oleracea L. and Populus trichocarpa Torr. & A. Gray ex. Hook) and may have evolved independently (Mitchell and Diggle 2005). The growth and development of dioecious taxa may be slower than cosexual plant species for two reasons. First, dioecious plants are unable to self-pollinate, thus need efficient pollination for seed production (Oster and Erikson 2007; Schlessman et al. 2014; Wilson and Harder 2003). Pollinator direction in a dioecious plant is an important factor because the movement of pollen from male to female flower enables seed production (Vamosi et al. 2006). Pollination through insects favors dioecious species because it encourages the directional movement of pollinators from staminate (male) to pistillate (female) flowers (Beach 1981). Previous field experiments have demonstrated that pollinator (male) site and pollen density are the main factors that determine fruit set and seed production (Oster and Erikson 2007; Otero-Arnaiz and Oyama 2001; Xia et al. 2013). Second, dioecious plants are unable to colonize unpopulated sites because about half of individuals (50%) in a population participate in seed set. In date palm, fruit development varies with respect to the male plant used to fertilize female plants, which is known as the metaxenial effect. Therefore, it is of vital importance to select superior males for crossing and breeding purposes (Raza et al. 2020). Pollination in date

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palm is carried out naturally by means of insects and wind, while manual pollination is also practiced ensuring higher productivity in commercial orchards (Haider et al. 2014a, b; Maryam et al. 2015, 2017; Qasim and Naqvi 2012). Dioecy restricts inbreeding and therefore plays a vital role in evolution by exchanging the genetic material of both parents. Outcrossing can lead to the formation of new genotypes that have resilience and adaptability to a variety of environmental conditions. Plants crossbred with external pollen produce healthier offspring (Cannabis sativa L.) as compared to plants fertilized with their own pollen (Milewicz and Sawicki 2013). However, sex determination is a major problem for the offspring as it is hard to identify plant sex at a juvenile stage. In crops such as date palm, pistachio, papaya, cloudberry, and kiwi, sex can only be determined at the flowering phase, which is a lengthy process, taking about 5‒7 years in date palm (depending on cultivar type, climate, and growth conditions). Therefore, it is important to differentiate the dioecious plant gender at an early phase of juvenility. Many researchers have adopted the identification of chromosomes and the application of molecular markers to distinguish male and female plants (Maryam et al. 2016). Sexual (seed) propagation in date palm has a substantial disadvantage of producing a genetically mixed population that may lack desirable fruit characteristics and faces problems in gender discrimination at the seedling stage. About 50% of the seedlings produced by the sexual method are either male or female, and therefore it is a waste of time and resources to manage a large number of seedlings until the emergence of flowers. Instead, vegetative propagation (basal suckers) secures genetic integrity, yet does not meet the requirement of the commercial plantation as only 20‒30 suckers are produced by a date palm over its lifespan. Due to the long lifespan and late flowering habit, it is difficult for growers at the seedling stage to observe the date palm sex, so they choose a specific proportion of male and female trees for industrial plantations. One male can fertilize 50 female date palm trees, yet male to female ratio for commercial orchards

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is 1:20 (Maryam et al. 2015). Date palm breeding is an extended endeavor and requires a full description of morphology, physiology, genetics, and morphogenesis (Haider et al 2018; Naqvi et al. 2015; Zango et al. 2017; Zehdi-Azouzi et al. 2015). Hence, sex determination at an early phase of the life cycle is of utmost importance to hasten the male and female plants selection for further breeding programs and can save time and resources to evaluate the efficiency and potential of the hybrids. The sex determination in date palm is done with morphological and molecular markers. Various genes govern the phenotypic characteristics of the plant and can be divided into qualitative and quantitative traits. Previously, morphological characterizations (for example, plant height, trunk width, leaf and leaflet length, spathe length and width) have been employed to check the diversity in various crops; however, both qualitative and quantitative phenotypic traits are mostly sensitive to the environment and can only be used at sexual maturity (Gros-Balthazard et al. 2020; Haider et al. 2015; Naqvi et al. 2015). Whereas molecular markers are a valuable tool to discriminate gender at any phase of plant growth and development (Awan et al. 2017; Ballardini et al. 2013; Maryam et al. 2016). The most advanced molecular markers, including random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter simple sequence repeats (ISSR), and biochemical analysis have been successfully used for gender determination in many dioecious species. Moreover, secondary metabolites are also used for male and female plant identification, but this method is restricted to plants that produce an ample quantity of metabolites (Fu et al. 2020; Tuskan et al. 2012). In dioecious species, the presence of sex chromosomes (allosomes X/Y) is also implicated to determine sexual dimorphism (Honys and Twell 2004). In this technique, totipotent cells (pollen grains or root tips) are used, colchicine is used to cease their multiplication at the metaphase of meiosis or mitosis and

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observed under the microscope after staining with specific dye (Khosla and Kumari 2015). A karyogram is prepared based on the presence of X or Y chromosomes and ploidy numbers. Recently, high-throughput sequencing technology (microRNA) has been applied to identify the number of differentially expressed genes (DEGs) induced at sex differentiation stages of dioecious plants (Mohanty et al. 2019).

7.2

Floral Biology of Date Palm

The phenotype of either sex in date palm is important due to the dioecious behavior (Elmeer and Mattat 2012). Flowers having nonfunctional gynoecium in male and androecium in female flowers are borne on separate trees (Haider et al. 2013, 2018; Othmani et al. 2017). In date palm, flower spathes are produced on the previous growth in leaf axils. Rarely both sex type flowers, pistillate and staminate, are observed in the same spathe. Inflorescence of the male and female flowers are dissimilar, physically, and morphologically (Al-Juburi et al. 2001; Chao and Krueger 2007; Raza et al. 2020). A typical inflorescence of date palm generally consists of several unbranched rachillae arranged spirally that are attached with a single flattened rachis. Male and female flowers on the rachillae are sessile and are enclosed in a fibrous, hard sheath called a spathe that protects the flowers from any unstable environmental issues such as excessive sunlight and heat during the delicate stages of flower development (Chao and Krueger 2007). When the flower reaches the maturity stage, the spathe splits to expose the entire inflorescence (Zaid and DeWet 2002). In nature, the date palm progeny population can be half male and half female, which cannot be distinguished by morphological traits at an early stage before flowering and fruiting. The date palm originates from the base axis of the leaves, and then at anthesis, the inflorescence spikes exert pressure on sheaths, which crack open longitudinally (Fig. 7.1).

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Fig. 7.1 Flowers sex type in the date palm. a Male flower, b Female flower. Source Chao and Krueger (2007)

7.2.1 Sex Types In general, the sex of a particular plant is monoecious, bisexual, or dioecious. Monoecious means that female and male flowers are found on a single individual plant, but at different positions or both sex organs (gynoecium and androecium) present in one flower (bisexual or hermaphrodite flower). In contrast, dioecious means one sex type, i.e., one plant produces either male or female flowers. Differentiation of any date palm flower types cannot be discerned at an early flower development stage, because the stamen and carpel primordia arise in the unisexual flower (Fig. 7.2).

7.2.1.1 Monoecious and Bisexual Flowers The date palm tree can be either all male or female trees but occasionally some palms produce both types of flowers on the same tree (Sidky and Eldawyati 2012). The true date (Phoenix dactylifera) and the Canary Island date palm (P. canariensis Chab.) have one sex type, all male or female trees. Masmoudi-Allouche et al. (2009) reported the modification of the female sex in date palm through in vitro hermaphrodism induction. Date palm flowers in female trees can exhibit new characteristic and physiological dimensions which are quite rarely observed in the process of flower development. A stunted stamen type in pistillate date palm flowers shows a unique character to proliferate

under in vitro conditions, without carpel growth blockage, leading to hermaphrodite flower morphology (Masmoudi-Allouche et al. 2009).

7.2.1.2 Dioecious Flowers Dioecy is an infrequent sexual system, occurring in only some species (Guttman and Charlesworth 1998; Field et al. 2013). In date palm, no cultivar produces both megaspores and microspores; a single plant can be either female (producing megaspores) or male (producing microspores). However, Othmani et al. (2017) reported for the first time natural hermaphrodism in female date palm cv. Alligue. Floral hermaphroditism in date palm needs to be explored for the process of selffertilization to recognize sex-related genes and markers that regulate the development of sex organs. Figure 7.3 gives a better understanding of the dioecious behavior of date palm spathes and inflorescence. This distinguishes the flower structure, spathe length and width, and flower density of both date palm sex types. 7.2.1.3 Male Flower A mature male tree of date palm produces 5‒10 large spathes each year. Male spathes are wider and shorter than female spathe. Just prior to opening (cracking), the male spathe sheath softens slightly and changes color from green to brownish (Fig. 7.4). Flowers in the range of 10,000‒15,000 are found in male spathes (Bekheet and Hanafy 2011), which are crowded at the rachis end (Fig. 7.4). Male flowers are

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Fig. 7.2 Sexual characteristics of flowering plants (L-R). a Basic architecture of female, hermaphrodite, and male flowers, b Typical date palm female, hermaphrodite, and

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male flowers; scale bar 1 cm. Source Lebel-Hardenack and Grant (1997, Othmani et al. (2017)

Fig. 7.3 Variation in spathe size and flower density in date palm. a Female tree, b Male tree. Source Intha and Chaiprasart (2018)

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Fig. 7.4 Male inflorescence. a Male spathe, b Male spathe opening, c Male inflorescence. Source Islam (2017)

usually pleasantly scented and typically have 6 stamens, encircled by sepals and petals which have a waxy scale structure. Stamens are composed of 2 small yellowish pollen sacs.

7.2.1.4 Female Flower A mature female date palm tree produces 5‒20 spathes in spring. Female flowers are usually yellowish-green. The branchlets in female date

Fig. 7.5 Female inflorescence. a Female flower/inflorescence emerging from the spathe, b Slender and long branchlets in female tree inflorescence. (Photos by Summar Abbas Naqvi)

palm inflorescence are long and slender (Fig. 7.5). A total of 8000‒10000 female flowers are produced in 1 female spathe of date palm (Sidky and Eldawyati 2012). The female inflorescence is less dense at the rachis, having 3‒4 mm diameter, superior ovary (hypogynous), rudimentary stamens, and 3 carpels. The female flowers have 3 sepals and petals each, which are united together and show more yellow color on the opening.

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7.3

Genetics of Sex

Female date palms are of economic importance because they bear fruit and hence documentation of this sex type is highly desirable at an early growth stage of seedlings. Sex detection of the male or female plant by DNA-based markers is a very useful tool for early identification. A wide range of molecular markers like RAPD (George et al. 2007; Sharma et al. 2010; Shirkot et al. 2002), AFLPs (Danilova and Karlov 2006; Renganayaki et al. 2005; Wang et al. 2011), SSR markers (Cherif et al. 2013), ISSR and RAPD (Yunus et al. 2008), RAPD_SCAR (Dhawan et al. 2013; Xu et al. 2004) and ISSR-SCAR (Korpelainen et al. 2008) are used for this purpose. To identify male and female plants by RAPD primers, two special alleles of 600 bp and 750 bp for male and female, respectively, are tested. Another group of RAPDs for sex-specific (2 for male and 3 for female) markers of 3 different types were employed by Yunus et al. (2008). They also amplified the ISSR marker of 5 different types (340, 1010, 375, 590, 920 bp) for the male-specific identification. Similarly, Elmeer and Mattat (2012) used 14 different SSR primer pairs for date palm to screen out 34 cultivars based on the sex type. Date palm sex identification at the seedling stage can be observed by a male-specific sequence characterized amplified region (SCAR) marker (Kharb and Mitra 2017). Female and male date palm genomic DNA, isolated and amplified against SCAR primers, resulted in an amplicon of 406 bp in both samples (male and female) and a unique amplicon of 354 bp only in male samples. SCAR primer pair amplified a 406 bp amplicon (both male and female) and 354 bp fragment (only male) for assured sex differentiation in date palm (Al-Mahmoud et al. 2012). RFLP has many advantages in contributing to the development of complete genetic maps by isoenzymatic and morphological markers. Plant population allelic variation is larger in RFLP markers than morphological or iso-enzymatic markers. El-Khishin et al. (2003) made crosses of

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5 date palm cvs. (Damen, Bunarenga, Khalas, Fardh, Khenizi) with the male pollen (BN-96, DN-96, Fr-96, KL-96, KN-96) and studied a simple genetic feature according to Mendelian inheritance. AFLP fingerprints for the 2 parent clones were obtained from 32 basic combinations. The combination of 3 primers (E-AAG, MCAT; E-ACG, M-CAA, and E-AAC, M-CAG) showed smaller bands in males and few bands in female parents. The polymorphism between the 2 parents using the E-ACG and M-CAA combination was 4 and 55% based on the combination in male and female, respectively (El-Khishin et al. 2003).

7.4

Cytogenetics

Date palm sex can be discriminated from the cytological studies of root cells. Male plant cells have two fluorescent blocks of irregular intensity; in contrast, female cells have 2 identical blocks (Siljak-Yakovlev et al. 1996). Cytological observations on in vitro grown date palm (cv. Karama) revealed 36 chromosomes arranged as 18 bivalents of chromosomes in c-metaphase, 17 bivalents autosomal chromosomes, and 1 bivalent chromosome with XY in male or XX in female (AbdAlla and El-Kawy 2010). Date palm is a diploid plant species that uses genetically inherited XY heteromorphic sex chromosomes. DeMason and Tisserat (1980) reported for the first time that in some cases, some male palms produce showy bisexual flowers. Selbi et al. (2006) presented an equal number of chromosomes when analyzing chromosomal karyotypes in male and female, and 2n = 36. In several types of food, the genes that determine the sex of each group connect and form sex chromosomes. Male trees have generally heterogeneous and nonspecific chromosomes (XY) for male and female, and the female has homogeneous with two female chromosomes (XX). In polyploidy, a single Y chromosome is enough to determine male characters (SiljakYakovlev et al. 1996). When the ratio between the X-chromosomes and the set of autosomes is

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at least 1.0 (i.e., AA XX) in female plants; a ratio of 0.5 or less (similar to the male AA genotype, normal XYY) creates a male phenotype; and ratios of 0.5 and 1.0 (chromosomal structure of AAA, XXYY (Yamamoto 1938) with triploid lead to a phenotype that lies between the phenotype of hermaphrodite flowers. In karyotyping, chromomycin A3 is used to color the chromosomes of the roots of date palm species (Siljak-Yakovlev et al. 2002), thereby identifying the differences in heterochromatin on male and female chromosomes. The date palm genome size is approximately 700 Mb (Fakir

Fig. 7.6 Genome size in palms (Arecaceae). a DNA amounts range in each subfamily (= mean for subfamily). The number of species with genome size data is presented in brackets, b Distribution of genome sizes within each chromosome number (arrows point to the mean for each data set). Source Leitch et al. (2010)

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et al. 2018). Chromosome average length range is 0.99‒6.46 lm and complements in diploid date palm varieties consist of metacentric (m), submetacentric (sm), subtelocentric (st) and telocentric (t). In the Arecaceae family, genome size data of 5 subfamilies are presented in Fig. 7.6a (Dransfield et al. 2008). C-values ranged c. 33-fold from 0.9 pg in the diploid Phoenix canariensis (Coryphoideae) (2n = 36) (Suda et al. 2005) to 30.0 pg in the highly polyploid Voanioala gerardii J. Dransf. (Arecoideae) with 2n = c. 600 (Johnson et al. 1989). The large C-value for V. gerardii was clearly divergent

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(Fig. 7.6b) from the next largest genome size of diploid Pinanga subintegra Ridl. with 1C = 13.9 pg. The cytological data reflects that polyploidy is uncommon in palms and to date just 4 polyploid species are reported (Leitch et al. 2010); two tetraploids (Arenga caudata Lour. H. E. Moore), 2n = 64 and Rhapis humilis, Blume 2n = 72) and 2 rare, monotypic genera of high ploidy, c. 12 in Jubaeopsis caffra Becc. from South Africa (2n = 160–200) and c. 38 in V. gerardii from Madagascar (Roser 2000).

7.5

Methods of Gender Discriminations

7.5.1 Morphological Basis Plant identification through morphological features is comparatively the simplest method of selection. In the past, genetic variation in date palm was characterized through morphological descriptions such as plant form, shape, and structure, but results provided by these markers are ambiguous due to environmental and development stage effects (Haider et al. 2015; Naqvi et al. 2015; Raza et al. 2020). Large-scale

Fig. 7.7 Discrimination of date palm seedling sex at an early stage. a Seed morphology, b Seedling emergence pattern. Source Zango et al. (2016)

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morphological data are required for accurate plant identification that is only possible when a tree is grown and in a fully mature form. Cultivar and genotype identification through morphological data is a complicated exercise (Fatima et al. 2014). In date palm, Ageez and Madboly (2011) investigated whether there were a greater number and length of new leaves, leaf blade, and spine blade, in male than the female trees. Even, spine arrangement and size differences in male and female trees. Previously, it is also reported that the leaflet at the lower third of the leaf is strong and stiff in male as compared to female seedlings (Royal Botanic Gardens Kew 1914). Traditional date farmers in southeastern Niger customarily identify the date palm sex at two stages: seed and seedling. Seed having curved pointed tips and a smooth appearance can germinate a female seedling; straight with a smooth appearance would be male seedling (Fig. 7.7a). Secondly, for sex determination at the seedling stage, the seed is sown under the straw mat, and if the seedling pushes the straw mat and emerges out straight, it would be male (Fig. 7.7b). If a seedling bends under the straw mat, it would be female (Zango et al. 2016). No scientific studies support this means of gender identification.

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7.5.2 Biochemical Basis

7.5.3 Cytological Studies

Biochemical markers have been used by Bekheet et al. (2008) to differentiate the sex in date palms and reported that female adult plants and female offshoots showed an elevated level of peroxidase activities. The sex estimation of in vitro developed lines was identified by the activity of enzymes (glutamate oxaloacetate and acid phosphatase) that provide the strong differentiation in male and female plants. The biomarkers are also used for gender discrimination in date palm by analyzing leaf proteomics through mass spectrometry and twodimensional polyacrylamide gel electrophoresis (PAGE) (Sonia et al. 2013). Male and female comparison of the proteomic map identified one clear protein spot which is linked to gender in date palm and corresponds to the ABC superfamily ATP-binding cassette transporter. ABC protein is associated with pollen development and male fertility. Sex prediction in date palm is also possible by using chemical analysis that the female date palm trees have high ability of anabolism and the substances that are produced from this process. El-Yazal (2008) developed a leaf chemical composition method to discriminate gender in date palm and reported that female trees had a high content of total carotenoids, carbohydrates, anthocyanin, phenols, chlorophyll (a and b), indoles, free amino acids, nitrogen, reducing sugars, crude proteins, silicon, and dry matter while the male trees had high concentrations of proline and ash. Sugar is an essential element in the sap of date palms irrespective of sex (Rao et al. 2009). Plant sex may affect the sap quality in Phoenix and male date palms reveal higher levels of sugar and dry matter contents in comparison to female palms. Similarly, higher total phenolic content (TPC) is recorded in male date palm sap compared to female (2.04 vs.1.648 lg gallic acid equivalent mL−1, respectively) (Makhlouf-Gafsi et al. 2016).

It is important to create a genomic map of the date palm and to carry out cytogenetic analysis to detect the number and abnormalities in chromosomes. Expanding the Palm Gene Bank and managing biodiversity with molecular tools is also an important crop improvement objective. Techniques like in situ hybridization (ISH), fluorescent in situ hybridization (FISH), concomitant oncoprotein detection (CODFISH), and spectral karyotyping (SKY) are applied to identify chromosomes and chromosomal aberrations; hence identification of sex in seedlings may be worked out using these techniques (Al-Ani et al. 2010). Sex chromosomes were unidentified in date palm until Siljak-Yakovlev et al. (1996) stained root chromosomes with chromomycin A3 and identified clear variations in isolated male and female chromosome heterochromatin (Juarez and Banks 1998). Sexual discrimination of palm trees based on fluorescent in situ hybridization (FISH) was tested by Atia et al. (2017a), who hybridized complementary probe sequences to visualize the identified DNA sequences of cell preparations. Since then, in situ fluorescence hybridization (FISH) has been established as an effective and important method for specific genome DNA fragment detection (Fig. 7.8). Physical mapping of plant chromosomes with useful markers like ribosomal DNA genes (45S and 5S rDNA) is practiced in determining date palm gender and studying genomic organization (Atia et al. 2017a).

7.5.4 Molecular Markers DNA or the genetic fingerprinting method to identify individual organisms is based on the organization of an organism’s genetic material. The DNA fingerprinting technique has several advantages; the cell’s DNA content is not

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171

Fig. 7.8 Fluorescence in situ hybridization in date palm. a, b Green 45S rDNA probe in cv. Zagloul, c, d Green 45S rDNA and red 5S rDNA probes in cv. Siwy at

metaphase chromosome. Arrows in b and d indicate 45S rDNA sites present on a Y chromosome; scale bar= 5 µm. Source Atia et al. (2017a)

influenced by environmental factors, growth stages, or organ specificity. This technique starts with the DNA extraction that further divides into four different processes: polymerase chain reaction (PCR)-based, non-PCR-based, sequence analysis, and hybridization. Several DNA-based markers have been tested for genetic variation analysis, cultivar identification, gene mapping, and phylogenetic analysis (Zango et al. 2017; Zehdi-Azouzi et al. 2015). In date palm, restriction fragment length polymorphisms (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and simple sequence repeats (SSR) markers have been used to genetically differentiate cultivars, analyze genetic diversity and genetic relationships (Khanam et al. 2012; Maryam et al. 2016) (Fig. 7.9). Gender-specific PCR-based molecular markers have been used effectively for gender discrimination in date palm. Al-Mahmoud et al. (2012) used the polymorphic region in male and female date palms to design the DNA-based

assay that differentiates the male and female seedlings at an early growth stage. PCR-RFLP with the enzyme BclI restricts the male allele only and this site is absent in female allele, and HpaII has 3 restriction sites but the site at 180 bp out of 452 bp is only present in female date palm and absent in male date palm. They designed only PCR-based primers for gender-specific polymorphism and up to 90% accuracy these primers differentiated the gender in date palm, where the male samples amplified two bands while the female a single band. Williams et al. (1990) developed a RAPD technique for the first time which is widely applied up to now due to its simplicity. For plant sex determination, the RAPD method is a better option due to its simplicity, inexpensiveness, and is less timeconsuming. Moreover, the RAPD technique is more efficient, better studied, and uses smaller DNA than RFLP. AFLP markers have the efficiency to detect high polymorphism but are rarely used because they are expensive and laborious. ISSR is the simplest and most

172 Fig. 7.9 Amplification product of RAPD decamer primer GLB-6. M 1 kb DNA ladder, 1‒6 male plant sample, 7‒12 female plant DNA sample. Source Habib (2014)

S. A. Naqvi et al.

M

1 2 3 4 5

6

7 8 9 10 11

Selected fragment

reproducible technique, but for sex identification, it is rarely used. RAPD technology has comparatively low reproducibility and reliability and was improved by developing the sequence characterized amplified region (SCAR) markers from already known RAPD fragments. SCAR markers have codominance and are found to be more reliable, reproducible, and less sensitive to reaction conditions than RAPD markers. SCAR primers are longer (20‒30 bp) from the RAPD decamer and sequence-specific primers are used in many dioecious plants for gender discrimination. These primers can be designed from cloning and then sequencing of required polymorphic band amplified from the RAPD (PCR) amplification. Cherif et al. (2013) added that malespecific DNA markers can provide genetic evidence of an XY chromosome system. In the postgenomic era, identification of Date-SRY Gene and GWAS mapping of sex determination locus (Hazzouri et al. 2019) are some of the novel tools for sex identification in date palm (Mohi et al. 2019). Recently, Wang et al. (2020) developed the sex-linked SSR markers

and validated them in date palm. Markers mPdIRDP52 and DPM4 proved to be sex-linked with 100% accuracy. Mathew et al. (2014) constructed the first genetic map for date palm and identified the putative sex chromosome. They placed *4000 markers on the map using nearly 1200 framework markers spanning a total of 1293 cM. Approximately *1.9 cM/Mb were revealed on the map by this analysis. Date palm sexdetermination region analysis reported telomere repeats on linkage group 12 and displayed recombination in the full chromosome. Similarly, Atia et al. (2017b) identified and developed PCR-based molecular markers against gender discrimination in date palm genotypes. These markers were AFLP, conserved DNA-derived polymorphism (CDDP), start codon targeted polymorphism (SCoT), intron-targeted amplified polymorphism (ITAP), and RAPD (Fig. 7.10). They developed the procedure based on PCRbased sex-specific markers starting from genomic DNA purification to sequencing up to BLAST analysis (Fig. 7.11).

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Fig. 7.10 The amplicon pattern established from SCoT, CDDP, ITAP, and AFLP marker types discriminating male and female date palm varieties. Samples 1 M ¼ male-1, 2 M ¼ male-2, 3 M ¼ male-3, 4 M ¼ male-4, 5 M ¼ male-5, 6 M ¼ male-6, 1F ¼ female Zagloul-1, 2F

7.6

Conclusions and Prospects

Date palm being dioecious in nature, sex discrimination is very crucial at the early seedling stage. It is problematic for date palm experts to discriminate plant sex in the field based on morphological features. It takes more than five years after planting to reach the first flowering. There is no clear differentiation in sex-based chromosomes in date palm, although some cytological studies have suggested evidence of chromosome differentiation. Cross-pollination in nature has made date palm crop improvement difficult. Hence, the development of reliable molecular markers for sex determination is important for the success of date palm breeding

173

¼ female Zagloul-2, 3F ¼ female Hayany-1, 4F ¼ female Hayany-2, 5F ¼ female Samany-1 and 6F ¼ female Samany-2. BM ¼ bulk of males, and BF ¼ bulk of females. Source Atia et al. (2017b)

programs. Researchers have developed sexlinked DNA markers and identified the dateSRY gene for sex determination, which can gear up sex determination related studies in date palm. Gender-specific PCR-based markers can be further used as an efficient tool for molecular breeding of date palm. The in silico analysis of such fragments coupled with reverse genetic approaches will provide insights into the evolutionary origins of sex-linked loci and genome mapping of such traits in the date palm. Hence, the more detailed mapping of the Y chromosome will provide answers about the degeneration of the Y chromosome and future directions to manage the genetic diversity in this ancient, domesticated tree crop.

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S. A. Naqvi et al.

Fig. 7.11 The procedures used to develop sex-specific PCR-based marker in date palm starting from genome DNA purification to BLAST analysis. Source Atia et al. (2017b)

References AbdAlla MM, Abd El-Kawy AM (2010) Karyotype analysis for date palm (Phoenix dactylifera L) compared with tissue culture derived plants. New York Sci J 3(11):165–170 Ageez A, Madboly E (2011) Identification of male specific molecular markers in date palm Sewi cultivar. Egypt J Genet Cytol 40:201–214 Al-Ani B, Zaid A, Shabana H (2010) On the status of chromosomes of the date palm (Phoenix dactylifera L.). Acta Hort 882:253–268 Al-Juburi HJ, Al-Masry HH, Al-Banna M et al (2001) Effect of some growth regulators on some fruit characteristics and productivity of date palm trees (Phoenix dactylifera L.) II. Khaniezy cultivar. In: Zaid A, Hegarty V, Al Kaabi HHS (eds) Proceedings of the second international conference on date palms,

United Arab Emirates University, Al-Ain, UAE, pp 21–35 Al-Mahmoud ME, Al-Dous EK, Al-Azwani EK et al (2012) DNA-based assays to distinguish date palm (Arecaceae) gender. Amer J Bot 99(1):7–10 Atia MA, Adawy SS, El-Itriby HA (2017a) Date palm sex differentiation based on fluorescence in situ hybridization (FISH). In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date palm biotechnology protocols, vol 2. Humana Press, New York, pp 245–256 Atia MA, Sakr MM, Mokhtar MM et al (2017b) Development of sex-specific PCR-based, markers in date palm. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date palm biotechnology protocols, vol 2. Humana Press, New York, pp 227–244 Awan FS, Maryam Jaskani MJ et al (2017) Gender identification in date palm using molecular markers. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date

7

Gender Determination of Date Palm

palm biotechnology protocols, vol 2. Humana Press, New York, pp 209–225 Ballardini M, Mercuri A, Littardi C et al (2013) The chloroplast DNA locus psbZ-trnfM as a potential barcode marker in Phoenix L. (Arecaceae). ZooKeys 365:71–82 Beach JH (1981) Pollinator foraging and the evolution of dioecy. Amer Nat 118:572–577 Bekheet SA, Hanafy MS (2011) Towards sex determination of date palm. In: Jain SM, Al-Khayri JM, Johnson DV (eds) Date palm biotechnology. Springer, New York, pp 551–566 Bekheet SA, Taha HS, Hanafy MS et al (2008) Morphogenesis of sexual embryos of date palm cultured in vitro and early identification of sex type. J Appl Sci Res 4:345–352 Chao CT, Krueger RR (2007) The date palm (Phoenix dactylifera L.): overview of biology, uses, and cultivation. HortScience 42(5):1077–1082 Cherif E, Zehdi S, Castillo K et al (2013) Male-specific DNA markers provide genetic evidence of an XY chromosome system, a recombination arrest and allow the tracing of paternal lineages in date palm. New Phytol 197(2):409–415 Danilova TV, Karlov GI (2006) Application of inter simple sequence repeat (ISSR) polymorphism for detection of sex-specific molecular markers in hop (Humulus lupulus L.). Euphytica 151(1):15–21 DeMason DA, Tisserat B (1980) The occurrence and structure of apparently bisexual flowers in the date palm, Phoenix dactylifera L. (Arecaceae). Bot J Linn Soc 81(4):283–292 Dhawan C, Kharb P, Sharma R et al (2013). Development of male-specific SCAR marker in date palm (Phoenix dactylifera L.). Tree Genet Genomes 9(5):1143–1150 Diggle PK, Di-Stilio VS, Gschwend AR et al (2011) Multiple developmental processes underlie sex differentiation in angiosperms. Trends Genet 27(9):368– 376 Dransfield J, Uhl NW, Asmussen CB et al (2008) Genera palmarum: the evolution and classification of palms. Royal Botanic Gardens Kew, UK El-Khishin DA, Adawy SS, Hussein EH et al (2003) AFLP fingerprinting of some Egyptian date palm (Phoenix dactylifera L.) cultivars. Arab J Biotech 6 (2):223–234 Elmeer K, Mattat I (2012) Marker-assisted sex differentiation in date palm using simple sequence repeats. 3 Biotech 2(3):241–247 El-Yazal MA (2008) Sex determination of date palm (Phoenix dactylifera L.) through chemical composition of leaves. Fayoum J Agric Res Dev 22(2):76–87 Fakir N, Muhammad A, Ali GM et al (2018) Utility of morphological features, chemical composition of fruit and chloroplast genes in date palm (Phoenix dactylifera L.) characterization. Sarhad J Agric 32(2):378–388 Fatima G, Khan IA, Jaskani MJ et al (2014) Ecological significance of root anatomy in date palm (Phoenix dactylifera) cultivars from diverse origins. Int J Agric Biol 16:795–800

175 Fu L, Wang Q, Zhang M et al (2020) Electrochemical sex determination of dioecious plants using polydopamine-functionalized graphene sheets. Front Chem 8:1–8 George M, Nair SS, Malini KA et al (2007) Finite size effects on the electrical properties of sol–gel synthesized CoFe2O4 powders: deviation from MaxwellWagner theory and evidence of surface polarization effects. J Phys Appl Phys 40(6):1593–1602 Gros-Balthazard M, Battesti V, Ivorra S et al (2020) On the necessity of combining ethnobotany and genetics to assess agrobiodiversity and its evolution in crops: a case study on date palms (Phoenix dactylifera L.) in Siwa Oasis. Egypt. Evol Appl 00:1–23 Guttman DS, Charlesworth D (1998) An X-linked gene with a degenerate Y-linked homologue in a dioecious plant. Nature 393(6682):263–266 Habib M (2014) Development of sequence characterizes amplified revisions (SCAR) markers in date palm. Thesis Dissertation, University of Agriculture, Faisalabad Haider MS, Khan IA, Jaskani MJ et al (2014a) Biochemical attributes of dates at three maturation stages. Emir J Food Agric 26(11):953–962 Haider MS, Khan IA, Jaskani MJ et al (2015) Assessment of morphological attributes of date palm accessions of diverse agro-ecological origin. Pak J Bot 47(3):1143– 1151 Haider MS, Khan IA, Jaskani MJ et al (2018) Pomological and biochemical profiling of date fruits (Phoenix dactylifera L.) during different fruit maturation phases. Pak J Bot 50(3):1069–1076 Haider MS, Khan IA, Naqvi SA et al (2013) Fruit developmental stages effects on biochemical attributes in date palm. Pak J Agric Sci 50(4):577–583 Haider MS, Khan IA, Naqvi SA et al (2014b) Flesh and fiber characterization at three different edible stages of date fruit development. Int J Food Nutr Sci 3(4):16–22 Hazzouri KM, Gros-Balthazard M, Flowers JM et al (2019) Genome-wide association mapping of date palm fruit traits. Nat Comm 10(1):1–14 Honys D, Twell D (2004) Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol 5(11):1–13 Intha N, Chaiprasart P (2018) Sex determination in date palm (Phoenix dactylifera L.) by PCR based marker analysis. Sci Hortic 236:251–255 Islam SE (2017) Evaluation of date palm male types as pollinators for Zaghloul and Samany date palm cvs. grown in Qalyubia governorate. Mid East J Agric Res 6(4):1049–1056 Johnson MAT, Kenton AY, Bennett MD (1989) Voanioala gerardii has the highest known chromosome number in the monocotyledons. Genome 32:328–333 Juarez C, Banks JA (1998) Sex determination in plants. Curr Opin Plant Biol 1:68–72 Khanam S, Sham A, Bennetzen JL et al (2012) Analysis of molecular marker-based characterization and genetic variation in date palm (Phoenix dactylifera L.). Austr J Crop Sci 6(8):1236–1244

176 Kharb P, Mitra C (2017) Early sex identification in date palm by male-specific sequence characterized amplified region (SCAR) markers. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date palm biotechnology protocols, vol 2. Humana Press, New York, pp 199– 207 Khosla PK, Kumari A (2015) Methods of sex determination in dioecious angiospermous plants. Lakshya J Sci Manag 1:1–9 Korpelainen H, Virtanen V, Kostamo K et al (2008) Molecular evidence shows that the moss Rhytidiadelphus subpinnatus (Hylocomiaceae) is clearly distinct from R. squarrosus. Mol Phylogenetics Evol 48 (1):372–376 Kumar S, Kumari R, Sharma V (2014) Genetics of dioecy and casual sex chromosomes in plants. J Genet 93 (1):241–277 Lebel-Hardenack S, Grant SR (1997) Genetics of sex determination in flowering plants. Trends Plant Sci 2 (4):130–136 Leitch IJ, Beaulieu JM, Chase MW et al (2010) Genome size dynamics and evolution in monocots. J Bot 2010: Article ID 862516, https://doi.org/10.1155/2010/ 862516 Makhlouf-Gafsi I, Mokni-Ghribi A, Bchir B et al (2016) Physico-chemical properties and amino acid profiles of sap from Tunisian date palm. Sci Agric 73:85–90 Maryam, Jaskani MJ, Awan FS et al (2016) Development of molecular method for sex identification in date palm (Phoenix dactylifera L.) plantlets using novel sex-linked microsatellite markers. 3 Biotech 6:1–7 Maryam Jaskani MJ, Fatima B et al (2015) Evaluation of pollen viability in date palm cultivars under different storage temperatures. Pak J Bot 47(1):377–381 Maryam Jaskani MJ, Naqvi SA (2017) Date palm pollen storage and viability. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date palm biotechnology protocols, vol 2. Humana Press, New York, pp 3–14 Masmoudi-Allouche F, Châari-Rkhis A, Kriaa W et al (2009) In vitro hermaphrodism induction in date palm female flower. Plant Cell Rep 28(1):1–10 Mathew LS, Spannagl M, Al-Malki AGB et al (2014) A first genetic map of date palm (Phoenix dactylifera) reveals long-range genome structure conservation in the palms. BMC Genom 15(1):1–10 Milewicz M, Sawicki J (2013) Sex-linked markers in dioecious plants. Plant Omics J 6:144–149 Mitchell CH, Diggle PK (2005) The evolution of unisexual flowers: morphological and functional convergence results from diverse developmental transitions. Amer J Bot 92(7):1068–1076 Mohanty JN, Chand SK, Joshi RK (2019) Multiple microRNAs regulate the floral development and sex differentiation in the dioecious cucurbit Coccinia grandis (L.) Voigt. Plant Mol Biol Rep 37:111–128 Mohei EL, Mohasseb HAA, Al-Khateeb AA et al (2019) Identification and sequencing of Date-SRY gene: A novel tool for sex determination of date palm (Phoenix dactylifera L.). Saudi J Biol Sci 26(3):514–523

S. A. Naqvi et al. Naqvi SA, Khan IA, Pintaud JC et al (2015) Morphological characterization of Pakistani date palm (Phoenix dactylifera L.) genotypes. Pak J Agri Sci 52(3):645– 650 Oster M, Eriksson O (2007) Sex ratio mediated pollen limitation in the dioecious herb Antennaria dioica. Ecosci 14:387–398 Otero-Arnaiz A, Oyama K (2001) Reproductive phenology, seed set and pollination in Chamaedorea alternans, an understorey dioecious palm in a rain forest in Mexico. J Trop Ecol 17:745–754 Othmani A, Collin M, Sellemi A et al (2017) First reported case of spontaneous hermaphrodism in female date palm (Phoenix dactylifera L.), cv. Alligue. J Hort Sci Biotech 92(4):376–388 Qasim M, Naqvi SA (2012) Dates: a fruit from Heaven. In: Manickavasagan A, Essa M, Sukumar E (eds) Dates: production, processing, food and medicinal values. CRC Press, USA, pp 341–350 Rao PVKJ, Das M, Das SK (2009) Changes in physical and thermo-physical properties of sugarcane, palmyrapalm and date-palm juices at different concentration of sugar. J Food Eng 90:559–566 Raza MK, Jaskani MJ, Naqvi SA et al (2020) Exploitation of phenotypic diversity in male accessions of date palm (Phoenix dactylifera) and its use in germplasm conservation. Int J Agric Biol 24:133–144 Renganayaki K, Jessup RW, Burson BL et al (2005) Identification of male-specific AFLP markers in dioecious Texas bluegrass. Crop Sci 45(6):2529–2539 Roser M (2000) DNA amounts and qualitative properties of nuclear genomes in palms (Arecaceae). In: Wilson KL, Morrison DA (eds) Monocots: systematics and evolution. CSIRO, Melbourne, Australia, pp 538– 544 Royal Botanic Gardens Kew (1914) The sex of date palm seedlings. Bull Misc Info Kew 1914:159–162. https:// doi.org/10.2307/411539 Schlessman MA, Vary LB, Munzinger J et al (2014) Incidence, correlates, and origins of dioecy in the island flora of New Caledonia. Int J Plant Sci 175:271–286 Selbi W, Day AJ, Rugg MS et al (2006) Overexpression of hyaluronan synthase 2 alters hyaluronan distribution and function in proximal tubular epithelial cells. J Am Soc Nephrol 17(6):1553–1567 Sharma A, Zinta G, Rana S et al (2010) Molecular identification of sex in Hippophae rhamnoides L. using isozyme and RAPD markers. Forest Stud China 12(2):62–66 Shirkot P, Sharma DR, Mohapatra T (2002) Molecular identification of sex in Actinidia deliciosa var. deliciosa by RAPD markers. Sci Hortic 94(1–2):33– 39 Sidky RA, Eldawyati MM (2012) Proliferation of female inflorescence explants of date palm. Ann Agric Sci 57 (2):161–165 Siljak-Yakovlev S, Cerbah M, Coulaud J et al (2002) Nuclear DNA content, base composition,

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Gender Determination of Date Palm

heterochromatin and rDNA in Picea omorika and Picea abies. Theor Appl Genet 104(3):505–512 Siljak-Yakovlev S, Cerbah M, Sarr A et al (1996) Chromosomal sex determination and heterochromatin structure in date palm. Sex Plant Reprod 9(3):127–132 Sonia DDS, Laurent CL, Cosette P et al (2013) The date palm (Phoenix dactylifera L.) leaf proteome: identification of a gender biomarker to screen male parents. Pak Orthodontic J 6(1):18–23 Suda J, Kyncl T, Jarolimova V (2005) Genome size variation in Macaronesia angiosperms: forty percent of the Canarian endemic flora completed. Plant Syst Evol 252(3–4):215–238 Tuskan G, Di-Fazio SP, Faivre-Rampant P et al (2012) The obscure events contributing to the evolution of an incipient sex chromosome in Populus: a retrospective working hypothesis. Tree Genet Genomes 8(3):559–571 Vamosi JC, Vamosi SM, Barrett SCH (2006) Sex in advertising: dioecy alters the net benefits of attractiveness in Sagittaria latifolia (Alismataceae). Proc Royal Soc B: Biol Sci 273:2401–2407 Voigt FA, Jung S, Farwig N et al (2005) Low fruit set in a dioecious tree: pollination ecology of Commiphora harveyi in South Africa. J Trop Ecol 21(2):179–188 Wang F, Li F, Wang J et al (2011) Genetic diversity of the selected 64 potato germplasms revealed by AFLP markers. Mol Plant Breed 2(4):22–29 Williams JG, Kubelik AR, Livak KJ et al (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl Acid Res 18:6531– 6535 Wilson WG, Harder LD (2003) Reproductive uncertainty and the relative competitiveness of simultaneous

177 hermaphroditism versus dioecy. Amer Nat 162:220– 241 Xia J, Lu J, Wang ZX et al (2013) Pollen limitation and Allee effect related to population size and sex ratio in the endangered Ottelia acuminata (Hydrocharitaceae): Implications for conservation and reintroduction. Plant Biol 15:376–383 Xu WJ, Wang BW, Cui KM (2004) RAPD and SCAR markers linked to sex determination in Eucommia ulmoides Oliv. Euphytica 136(3):233–238 Yamamoto M (1938) Cultural conflicts and accommodations of the first and second generation Japanese. Social Process Hawaii 4:4–48 Yunus MF, Abd Aziz M, Kadir MA et al (2013) In vitro mutagenesis of Etlingera elatior (Jack) and early detection of mutation using RAPD markers. Turk J Biol 37(6):716–725 Zaid A, de-Wet PF (2002) Pollination and bunch management. Chapter 8 In: Zaid A (ed) Date palm cultivation. FAO, Rome Zango O, Cherif E, Chabrillange N et al (2017) Genetic diversity of Southeastern Nigerien date palms reveals a secondary structure within Western populations. Tree Genet Genomes 13(4):75 Zango O, Rey H, Bakasso Y et al (2016) Local practices and knowledge associated with date palm cultivation in Southeastern Niger. Agric Sci 7:586–603 Zehdi-Azouzi S, Cherif E, Moussouni S et al (2015) Genetic structure of the date palm (Phoenix dactylifera) in the old world reveals a strong differentiation between eastern and western populations. Ann Bot 116(1):101–112

Part III Genome Mapping and Bioinformatics

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Whole-Genome Mapping of Date Palm (Phoenix Dactylifera L.) Zafar Iqbal, Muhammad Naeem Sattar, and Jameel M. Al-Khayri

Abstract

A genome represents an organelle’s complete DNA repertoire and it is the starting point of genetic studies and genome mapping. Since the advent of genome mapping techniques, researchers have devoted concerted efforts to link phenotype to genotype. Initially, efforts were made to accomplish genotyping by isozymes, followed by the execution of various kinds of molecular markers such as simple sequence repeats (SSR), single nucleotide polymorphism (SNP), and random amplified polymorphic DNA (RAPD). Nonetheless, genome mapping was dramatically revolutionized as next-generation sequencing (NGS) technologies emerged. NGS technologies have produced a deluge of genome sequencing. The analysis of such sequencing data is

Z. Iqbal (&)
M. N. Sattar Central Laboratories, King Faisal University, Al-Ahsa, Saudi Arabia e-mail: [email protected]; [email protected] M. N. Sattar e-mail: [email protected]; naeem. [email protected] J. M. Al-Khayri Department of Agricultural Biotechnology, College of Agriculture and Food Sciences, King Faisal University, Al-Ahsa, Saudi Arabia e-mail: [email protected]; [email protected]

only possible through highly sophisticated computational tools. Presently, the low-cost sequencing technologies and highly sophisticated computational tools are of great significance in unraveling the complexity of plant genomes. Date palm (Phoenix dactylifera L.) is a socioeconomically important plant. Cultivar identification mostly relied on phenotypic traits rather than genotyping. Still, limited information is available on date palm genotyping and genome mapping. Nonetheless, advancements in whole-genome sequencing and genome assembly computational tools hold a substantial advantage to boost the identification of date palm cultivars, genome variants, and gene/genome annotation. In this chapter, advancements in date palm genome mapping are categorized into two main types: date palm genetic mapping in the pre-NGS and post-NGS eras. Various computational tools that hold an unparalleled advantage to conduct genome-wide association mapping, genome prediction, and selection, and with higher prediction accuracy of SNPs and other variations in date palm genomes are discussed. In addition, various databases related to date palm genomes are discussed, these databases encompass important information about the whole genome of certain date palm cultivars, molecular markers such as SNPs and SSR, and phylogenetic relationship of different date palm cultivars.

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Introduction

Genome mapping is a widely applicable technique to identify, map, and to locate a gene or intergenic gaps on a chromosome responsible for a particular trait. Genome mapping provides a critical foundation to link a phenotypic trait to a gene or genes. A genome represents an organelle’s complete DNA repertoire; the animal genome is localized at two parts: nucleus and mitochondrion, while plants have an additional component in the chloroplast. Although every type of genome is essential, the nuclear genome is by far the largest one of the three and contributes a vast majority of traits to an organism. A genome map can be interpreted as a roadmap, representing the relative proximity between various landmarks. While mapping genomes, chromosomes—the linear units of the nuclear genome— act as milepost guides to the traveler along a highway, and DNA markers include reference points marking unique positions along each chromosome. The foundation of genome mapping was laid down with Mendel’s Laws of Heredity in 1865. Later, researchers around the globe began untiring efforts to link phenotype(s) to the genotype(s) and this has become the pivotal component of molecular biology. Gregor Mendel carried out experiments on pea (Pisum sativum L.), family Fabaceae, and studied phenotypic traits such as color and surface texture of pea seeds and pods along with plant height; later in 1912, these traits were selected as the first genetic markers in biology when Vilmorin and Bateson carried out research on linkage maps in peas. Nevertheless, the idea of linkage groups remained unclear until 1948, when Lamprecht deciphered the first-ever pea genetic map with 37 markers spread over 7 linkage groups (Swiecicki et al. 2000). Higher plants presumptively have ca. 25000 or even more genes and for the vast majority of these genes, no function could be ascribed. Although significant progress has been achieved toward their characterization using model plant species, many questions remain. Nonetheless, the answers lie in genome mapping and genetic markers. Presently, wide arrays of

genetic markers are available for many crops including model plants such as Arabidopsis thaliana. Nonetheless, the first-ever developed genetic markers at the molecular level were isozymes, which are protein isoforms varying in amino acid composition and can be differentiated by electrophoretically resolving them in gel. The use of isoenzymes to classify populations and breeding lines of plants and to map their genetic variants has been the most favored approach, particularly in maize, wheat, and rice (Pham et al. 1990; Tang and Hart 1975; Zlokolica and Milošević 2001). Nonetheless, the use of isozymes became the least popular among researchers because of the small numbers of proteins in which isoforms occur, which are separable/distinguishable by electrophoresis. With the advancement of molecular biology, a significant number of techniques have been developed to determine the genetic variations at the DNA level, which later served as a genetic marker. The first-ever DNA-based genetic marker devised was the restriction fragment length polymorphism (RFLP) marker that could explicitly differentiate polymorphisms in the genomes (Botstein et al. 1980). The initial breakthrough advancement in genome mapping was accomplished with the advent of DNA sequencing techniques. The innovation of nextgeneration sequencing (NGS) technologies continued to make major contributions to genome mapping. In current genome mapping techniques, NGS has become a matchless alternative for genetic mapping at a global scale. The date palm (Phoenix dactylifera L.), family Arecaceae, is a globally grown plant species having a strong impact on worldwide agriculture and economics. The precise origin of the date palm is difficult to map and has been lost in antiquity. Nonetheless, the date palm cultivation records can be traced until 4000 B.C and were used to construct a moon god temple around Ur in southern Iraq-Mesopotamia (Popenoe 1973). This history has been supported by archaeological discoveries and corroborated by ancient historical remains of the Akadians, Babylonians, and Sumerians. Date palm tree is considered as a

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life symbol in the deserts as it can tolerate higher temperatures, water, and salinity stress. In addition, it has strong socioeconomic values due to its domesticated fruit, health benefits, and range of subsistence products. Date palm is cultivated in both the Old World and New World and the cultivation area has been stretched from East Indus Valley to the West Atlantic Ocean. Total world production of dates is ca. 8.2 million metric ton (MMT) and the area under date palm cultivation is 1.32 million hectares (ha) (FAOStat 2017). Egypt is the global leader in date production and cultivation, and produces ca. 1.2 MMT date per annum. Iran ranks as the secondlargest producer of the dates with nearly 1.05 MMT annual production. While the date’s thirdlargest producer is Saudi Arabia and produced 0.92 MMT date fruit. There are reportedly over 400 different date palm cultivars in Saudi Arabia alone (Pariona 2017). Further details on the history of the date palm can be found in Chap. 3 of this book entitled “A brief history of the origin of domesticated date palms”. Date palm is an ancient agricultural commodity and is widely distributed around the globe. Over the course of a long history, the date palm has undergone a long progression of domestication. During domestication, several factors such as migration, the effect of breeding, selection pressure, and environmental effects have led to phenotypically and genetically distinct populations (Doebley et al. 2006). In addition, notable human domestication traits linked to yield and fruit quality and history reveal that date palm sexual propagation has been going on since the Neolithic (Tengberg 2003). Presently, hundreds of cultivars have gained regional prominence, based on specific traits pertaining to physiology, morphology, and nutritional profiles. Nevertheless, cultivating regional date palm cultivars and early agricultural practices have facilitated monocropping, causing extreme genetic depression of the biodiversity and genetic erosion of many potential cultivars. Therefore, under the prevailing circumstances of global warming and genetic erosion, there exists a demand for the exploitation of the available germplasm at its full capacity to gain maximum

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yield potential. Luckily, the answer lies in genome mapping to identify and characterize the potential genes controlling specific traits. After identification, these genes can be incorporated into the new breeding programs for date palm. Besides the date palm’s socioeconomic values and global cultivation, it falls among those plants whose genomes are exploited least. The available date palm genetic resources can be genome mapped to execute or select the elite traits to integrate them into the desired cultivars. Several efforts have been successful to map certain genes of the date palm, and over time the molecular toolkit for date palm genetic mapping has been extended to include amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), RAPD, genome-wide association study (GWAS), single nucleotide polymorphism (SNP), simple sequence repeat (SSR), and expressed sequence tags (ESTs). Besides, the major groundbreaking advancement came after the NGS advent and until now the full-length genomes of many date palm cultivars have been sequenced (Al-Dous et al. 2011; Al-Mssallem et al. 2013; Hazzouri et al. 2015; Mohamoud et al. 2019). DNA markers offer insights into the diversity or similarity in the genomes, cultivars, varieties, or clones, which morphologically cannot be distinguished. In date palm, several DNA markers have been employed to determine the resemblance or variability of different varieties that are difficult to discern based on molecular characteristics. This chapter presents the date palm genetic mapping in the pre-NGS and post-NGS eras. Advances in whole-genome sequencing and genome assembly computational tools and their potential role in enhancing the identification of date palm cultivars, genome variants, and gene/genome annotation are also discussed. Other topics covered include various computational tools and databases related to the date palm genome, which have an unparalleled advantage of genome-wide mapping, genome prediction, and selection, and with a higher predictive accuracy of SNPs and other variations in date palm genomes.

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Types of Genomic Maps

Genome maps are of two major types: genetic and physical. Genetic maps are focused on recombination frequencies between genes and genetic markers, and linked markers/genes form linkage groups displaying their order. A physical map indicates the relative positions of genes and important DNA sequences, with distances usually calculated in base pairs (bp). Since actual bp distances are difficult to calculate directly, physical maps are essentially created by shattering the genome into hierarchically smaller pieces. Physical maps use real physical distances, so the graphical maps represent the genome map more accurately than a genetic map. Physical maps could be further categorized into three subtypes: cytogenetic or chromosomal maps, sequence maps, and radiation hybrid (RH) maps (Tiwari et al. 2016). The construction of genetic maps and linkage analysis techniques was developed by D.H. Morgan in 1911. In genetic maps, the distance between two consecutive markers or loci is measured to obtain recombination frequencies among them. It is actually a graphical representation of genes to position them as linkage groups on the basis of genetic distance and the length of linkage groups. In a genetic map, the linkage groups represent a specific chromosome and thus, the total number of linkage groups signifies the haploid number of chromosomes of a plant species. The linkage groups are characterized by morphological, molecular, or biochemical markers and the respective distance between any adjacent markers is measured in terms of centimorgans (cM). Many types of software (discussed in section Software for Genome mapping) are available with the varying capability to construct genetic maps and estimate genetic distances by calculating the recombination frequencies. A detailed genetic map is usually constructed by the following steps: (a) Selection of polymorphic parents, (b) Formation of mapping population,

(c) Application of selective primers to identify polymorphic DNA markers for mapping the population, (d) Segregationally scoring record of DNA markers used for mapping population, (e) Estimation of recombination frequencies through statistical analysis, (f) Selection and application of appropriate software to construct a linkage map, (g) Determination of linkage group/s, and (h) Accrediting the linkage groups to respective chromosomes. In the next section, a generalized approach is subdivided into two main sections: genetic mapping in the pre-NGS era and post-NGS era, to describe the main aspects of date palm genome mapping (Fig. 8.1).

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Genome Mapping in the PreNGS Era

During the early decades of genetic mapping, phenotypically proved genes on allelic variants were used as markers to build genetic maps of an organism. Nevertheless, the genetic measurements in the fully mapped organisms showed that the chromosomal recessions between the known genes contained huge gaps in genetic information. Such gaps could not be filled with simple linkage analysis due to the unavailability of any markers spanning those regions. Thus, the construction of high-resolution genetic maps necessitated the design of additional molecular markers. These molecular markers represent heterozygous sites, which are genetically silent and phenotypically unmeasurable. These heterologous genetic loci can be successfully employed for mapping analysis similar to a conventional heterozygous allelic pair. Moreover, these molecular markers are numerous alongside the genome and when linked to a map through linkage analysis, the missing voids are easily filled between the phenotypically known

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Fig. 8.1 A generalized schematic representation of date palm genome/genetic mapping. The genome/genetic mapping is divided into two eras: pre-NGS and post-NGS. All the techniques which have been employed or are being employed in both eras are mentioned. Finally, both approaches need computational analysis to achieve a meaningful objective of genome/genetic mapping in date palm. Abbreviations used are next-generation sequencing (NGS), genotyping by sequencing (GBS), restriction site associated DNA sequencing (RAD), reduced representation library (RRL), wholegenome re-sequencing (WGRS), simple sequence repeat (SSR), variable number of tandem repeats (VNTR), random amplified polymorphic DNA (RAPD), single nucleotide polymorphism (SNP), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), expressed sequence tag (EST), genome-wide association studies (GWAS), and quantitative trait loci (QTL). (Figure prepared by Z. Iqbal in Adobe Illustrator 2019)

genes. These molecular markers have no individual biological significance in genetic mapping; rather, they provide mere reference points along the entire chromosome. RAPD is based upon a PCR amplification technique, which does not necessarily require any prior sequence information of a particular DNA sample. This technique has been very valuable in the construction of genetic maps. Nucleotide sequence polymorphisms are the procedural outcome of RAPD through an arbitrary and highly polymorphic 10 bp PCR primer. This stochastic approach, however, necessarily requires very strictly optimized PCR reaction conditions in terms of reproducibility in results. These dominant molecular markers have been widely used for agronomic, disease resistance, and morphological traits in various crops. However, certain limitations such as poor reproducibility, fuzzy results, and difficult band scoring make them sometimes inappropriate for genetic mapping and linkage analysis. In date palm, RAPDs have been employed for varietal identification (Al-Khalifah and Shanavaskhan 2017) and somaclonal variations (Al-Khateeb et al. 2019; Al-Khateeb et al. 2020); however, they are not useful markers for the construction of genetic maps. Similarly, other PCR-based molecular markers, AFLPs, have been used only for germplasm characterization in date palm (ElAssar et al. 2005). Various restriction enzymes cut the DNA of different individuals in a population at the same position because the positions of target sites are the same on the homologous chromosomes. However, it is quite common that a silent mutation could take place and mutate a particular restriction site in a particular individual. Such

mutations can occur within the coding or noncoding regions (the region between two genes). Such sites can be found through Southern blot hybridization by probing that particular region, which will result in multiple fragments after restriction analysis. Thus, these multiple fragments constitutively give rise to restriction fragment length polymorphism (RFLP). Traditionally RFLPs were combined with Southern blot hybridization techniques, therefore, these are time-consuming and have been rarely applied in their standard form. RFLPs can be mapped with reference to a gene or another molecular marker on a chromosome similar to other chromosomal loci. However, in date palm, RFLPs have only been used for varietal identification (Corniquel and Mercier 1997) Simple sequence repeats (SSRs) are 1–6 bp long repeated sequences in the genome. Hybridization followed by sequencing are prerequisites to generate microsatellite markers for mapping and linkage analysis in plants. Once the sequencing of cloned microsatellite fragments is found and microsatellite markers are defined, the utility of these markers is very high. These unique sequences can be used to design polymorphic primer sequences specified for SSR loci. These molecular markers are codominant and multiallelic, which can detect DNApolymorphism more efficiently than other molecular marker systems. Moreover, SSR markers are universal markers for the genetic mapping of a genome and are quite efficient in characterizing plant germplasm collections. In pea, the first microsatellite-based genetic map was established in 2005 (Loridon et al. 2005) and subsequently, various linkage analyses were carried out using SSR markers. Another type of

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microsatellite markers is inter-SSR (ISSR) universal microsatellite markers. These markers are a combination of SSRs and RAPD in terms of their universality and polymorphism. These are mostly dominant; however, fewer have codominance in nature. These can be synthesized in many different combinations as tri- tetra and penta- nucleotides. In date palm, SSR and ISSR markers have been successfully used for DNA fingerprinting (Al-Hinai 2019), to investigate the population structure of different date palm cultivars (Shahzad et al. 2020) and characterization of intravarietal variability in cultivar Khalas from the GCC countries (Elmeer et al. 2020). Expressed sequence tags (ESTs) are valuable molecular markers obtained from partial cDNA sequencing. These are based upon the transcribed parts of the plant genome and provide an advantage over anonymous sequences as genetic markers. EST markers are usually useful in sequencing full genome and to identify the active genes associated with a trait of interest. These are highly conserved and have been used in mapping different plant systems. Various studies have unraveled the abiotic stress-responsive ESTs in date palm (El Rabey et al. 2015; Rekik et al. 2019; Safronov et al. 2017). In the present decade, the application of single nucleotide polymorphism (SNP) based markers along with the NGS techniques has brought about steady improvement in genetic mapping. Fine mapping using SNP-based DNA markers has been empirically studied in a limited number of research studies during the pre-NGS era (Wang et al. 2009). The SNP-based DNA markers are undoubtedly advantageous over the other DNA-based molecular markers; however, the absence of high-throughput SNPs availability and appropriate genotyping methods during the pre-NGS era limit their widespread applicability for genetic mapping. The unique SNP signatures have been used for varietal characterization in date palm (Faqir et al. 2019). Among plant species, the use of RFLP and QTL identification was reported for the first time in tomato (Solanum lycopersicum L.) cv. UC82B (Paterson et al. 1988). However, SSR markers kept their hierarchy in genetic and plant breeding

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studies due to high polymorphism, genome-wide association, and amenable automation (Gupta and Varshney 2000). The QTL regions associated with a specific trait of interest are identified by DNA markers. After QTL identification, the QTL region was refined by mapping the flanking DNA markers for their physical location on a physical map. This process was followed by subsequent cloning, sequencing, and developing of DNA markers inside the identified QTL region (Fig. 8.1). Comparing the molecular marker development, mapping, and QTL resolution shows that the QTL resolution was extremely low (10–30 cM) in the pre-NGS era as compared to the post-genomic era (0.5–10 cM) (Jaganathan et al. 2020). In addition, transposable elementbased molecular markers have also been employed to demonstrate somaclonal variations among date palm plants developed via tissue culture (Mirani et al. 2020). The molecular markers toolbox in date palm is very limited. Although there have been a few studies available regarding RAPD, AFLPs, SSR, and SNPs markers, these are not enough to assess the genetic diversity, construction of genetic linkage maps, and to facilitate marker-assisted breeding in date palm (Zhao et al. 2017). The gene-based genetic markers can be very useful for QTL-mapping, molecular breeding, and targeted cloning in date palm. Zhao et al. (2013) analyzed *28,000 ESTs of date palm genome and identified *5000 SSR primer pairs for ESTSSR markers (Zhao et al. 2013). Nearly 20 random primers were used to differentiate 12 date palm cultivars. Generally, the available molecular markers have been mostly used for varietal clustering in date palm into related groups. Moreover, these markers have been used for varietal identifications, germplasm characterization, identification of somaclones during in vitro micropropagation and/or marker-assisted breeding for biotic and abiotic stresses (Al-Khateeb et al. 2019). However, a date palm genetic linkage map using molecular markers is far from developed due to the longer time required to establish progenies and to conduct backcrosses and consecutive recurrent selections. Moreover, the classical breeding approach is not applicable

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as it requires almost 30 years of continuous breeding to perform 3 backcrosses in date palm. Thus, deployment of post-NGS approaches may be the only solution to map the date palm genome.

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Genome Mapping in the PostNGS Era

High-density linkage mapping has been strengthened by the large-scale adoption of NGSbased population genotyping (Hazzouri et al. 2020; Varshney et al. 2019). Since its invention, the sequencing cost of NGS technology has been reduced 100-fold, thus thousands to millions of DNA reads can be generated in a single run due to the parallelization of the sequencing process. The timeline of high-density genetic linkage mapping in the pre- and post-NGS eras has also been enormously shortened. The emerging NGSassociated protocols such as reduced representation libraries (RRLs), restriction site-associated DNA sequencing (RAD), genotyping by sequencing (GBS), whole-genome resequencing (WGRS), and skim GBS are quite efficient and able to determine and map an enormously high number of SNPs in a single-run (Varshney et al. 2019). Moreover, the dramatically lower cost for NGS has enabled researchers to gain a deeper insight into the genomic regions of whole mapping populations. Besides, new markers for a QTL region can be generated in comparatively reduced time without tiresome efforts, which could not be practically possible in the pre-NGS era (Fig. 8.2). A genetic linkage map with moderate density (*1000 loci) in the pre-NGS era required substantial time and effort and multiple researchers. Comparatively, a highly saturated genetic map with 100,000 loci can be constructed within a few months using the current post-NGS techniques, which are amenable to high technical efforts (Yang et al. 2015). In this post-NGS era, SNP arrays based upon resequencing facility are being used to generate high-density marker profiles in many crops (Roorkiwal et al. 2018). The high-quality SNP chips are now available and are preferable to

other high-density genotyping portfolios (Yuan et al. 2019). These SNP chip arrays have been successfully developed and deployed for linkage mapping in cotton (CottonSNP80K) and wheat (TaBW280K) (Rimbert et al. 2018). Reduced automation for SNP markers has broken the hierarchy of SSR markers in crop improvement breeding programs during the past two decades (Jaganathan et al. 2020). Thus, coupled with cost-effective whole-genome sequencing techniques, the latest SNP genotyping platforms are available to genotype thousands of samples in the shortest possible time. Quite recently, New York University, Abu Dhabi has identified *12 million SNPs from 62 date palm cultivars as a part of the Plant and Animal Whole Re-sequencing (WGRS) project (Masmoudi 2019). The development of such a high-density SNP genotyping platform may allow the identification of alleles at single genomic loci using SNP-based markers. The bulked segregant RNA seq (BSR-Seq) technique is based upon the sequencing of the whole transcriptome. BSR-Seq assays have particular significance for the larger and complex genomes like wheat, barley, and some tree plants. This technique is very important to generate a genetic mapping of those crops for which a reference genome is not available. It simplifies the identification of a particular region by developing markers near or inside the gene of interest. For instance, BSR-Seq was successfully deployed to map the grain protein content (GPC) region in wheat to the shortest distance of 0.4 cM as compared to previously reported 30 cM using conventional techniques (Trick et al. 2012). This approach has been extensively used for the quick discovery of candidate genes and genetic markers linked to the gene of interest. Similarly, the QTL-Seq technique has also been widely used for rapid gene discovery for the genetic traits in crops. This QTL-Seq has been successfully applied for rapid identification of QTLs related to blast resistance in rice (Takagi et al. 2013), early flowering in cucumber (Lu et al. 2014), seed and root weight traits in chickpea (Singh et al. 2016a), resistance to Fusarium wilt and sterility mosaic disease in pigeon pea (Singh et al. 2016b) and many other

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Fig. 8.2 Comparison of genetic mapping in the pre- and post-NGS eras. Addition of NGS-based molecular markers has reduced the time span of genetic mapping to less than one-half that of pre-NGS era. (Figure prepared by M.N. Sattar in Adobe Illustrator 2019)

genetic mapping studies. This technique has been adopted to map the QTLs related to both qualitative and quantitative traits in many other crops. Genome-wide association studies (GWAS) provide a comparable alternative to QTLmapping approaches in tree plants to map the important loci for different traits. In date palm, the application of the GWAS approach has been successfully deployed for high-resolution mapping. Genome mapping using the GWAS approach necessitates the availability of highquality genome assemblies and in this context,

two whole-genome draft assemblies are available in date palm cv. Khalas. Coupling high-density GBS and WGRS approaches in date palm enable the discovery of a plethora of novel SNP markers across the date palm genome in many studies. Nevertheless, these post-NGS approaches lead to the identification and characterization of novel sex determination loci (Mathew et al. 2014), fruit development and maturation pathways (Hazzouri et al. 2019), and genome-wide diversification studies in date palm (Hazzouri et al. 2015; Mathew et al. 2015).

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Genome Mapping Software

Release of the first date palm genome sequence led to a deluge of information spawning a vast array of periodic genome mapping and sequencing data. All standard genome mapping and NGS techniques have contributed significantly to date palm genome mapping. Nonetheless, both produced an enormous amount of data; the processing of which would not have been feasible without sophisticated bioinformatics tools. Many such tools have been developed which have streamlined the entire process of plant genome mapping, from initiation to publication. The knowledge gleaned from genome mapping has proven extremely invaluable in producing high-density genetic maps, GWAS, linkage analysis, variety identification, and improved assessment of date palm diversity to boost breeding and production programs. Some essential bioinformatics tools of prime importance that can be executed to perform date palm genome mapping are discussed in the following section. Assembly by short sequencing (ABySS) was designed to amass large data sets produced by sequencing large genomes. ABySS is extremely handy to recognize indels and novel sequences in large genomes and can accurately and swiftly assemble 3.5 billion short-sequence reads created on the Illumina platform. It is of particular use for the assembly of certain genomes where no reference genome is available. ABySS is based on the distributed representation of a De Bruijn graph which enables parallel computation of the assembly algorithm across a cluster of computers. This program conducts a two-step assembly in the initial step, the contigs are extended without paired-end information; in the subsequent step, paired-end information is used to merge the expanded contigs. ABySS is built in C ++ language and has successfully been executed to achieve spruce mitochondrial and plastid genomes sequenced through the shotgun sequencing platform. In addition, genomes of several other plant species have been assembled via ABySS (Jackman et al. 2015). Similarly,

variations between mitochondrial genomes of cotton (Gossypium hirsutum L. and G. barbadense L.) have been mapped through ABySS, while both genomes were sequenced through a Solexa paired-end, 90 bp read (Tang et al. 2015). All these studies pinpoint that ABySS is a wonderful computational tool that can be chosen to perform the date palm genome and/or organellar genome assembly even if they are sequenced using diverse NGS platforms. PyroBayes is an automated computational tool built-in Unix environment to analyze SNPs in genomes. It is an advanced and modified version of PolyBayes and built on the Bayesian inference engine (Marth et al. 1999). It measures the likelihood that differences at a given locus of multiple alignment positions are real-time sequence changes and not sequencing errors. PyroBayes show the results of each SNP with an SNP probability score. This tool was built to read data created from 454 sequencing platforms. Trait analysis by association, evolution, and linkage (TASSEL) is a versatile computational tool that provides unlimited utilities to evaluate evolutionary patterns, trait associations, and linkage disequilibrium. It is based on a general linear model and mixed linear model-based methodologies to control population and family structure of the target genes or traits. The mixed linear model reduces Type I error in mapping complex pedigrees, families, and population structure. TASSEL software interprets findings by allowing linkage statistics to be computed and visualized graphically, while some embedded middleware facilitates database searching and data import. Some other features include indels evaluation, estimation of diversity statistics, integration of genotypic and phenotypic data, computation of incomplete or missing data, and estimation of key/principal components. The interactive, user guide, sample data sets, and instructional guide are available and can be accessed at http://www.maizegenetics.net/tassel (Bradbury et al. 2007). Besides offering association tools, TASSEL allows evaluation of diversity and its analysis such as average pairwise divergence and segregating loci, data

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extraction, and its analysis including SNPs and indels extraction, sequence alignment, neighborjoining dendrogram, and a plethora of data graphing and data management functions. To import data files in flat formats, TASSEL is integrated with genomic diversity and phenotype connection (GDPC) to supplement an interface to relational databases. GDPC uses various data sources, enabling users to download filtered and customized data, and helps to export tabdelimited text files. The computational tools like TASSEL could accelerate the genome mapping of larger genomes like date palm. Genome association and prediction integration tool (GAPIT) is an R package based computational tool that is handy to compute genomewide association (GWAS) and gene prediction (or selection). This software runs in an R environment and uses limited coding. This software uses state-of-the-art statistical approaches for computational genetics, such as efficient mixed model association (EMMA), P3D, compressed linear mixed model (CMLM), and CMLM genomic simulation and collection. This software can accommodate large amounts of datasets of ca. 10000 individuals and 1 million SNPs with extremely short processing time, while offering user access and succinct tables and graphs to decipher results. Thus, GAPIT shares wonderful utilities of analyzing large datasets, reduced computational time, and delivers detailed findings with high-quality R objects and graphs. GAPIT accomplish the reduction in computational time by subdividing genotypic data into several formats. GAPIT will read either in HapMap format or in the numerical format needed for the EMMA R kit (Lipka et al. 2012). The R package based GAPIT is a modern tool that offers huge room to customize the genome analysis, nonetheless, this has not yet been employed in date palm genome mapping. However, it holds great potential to be executed in the date palm genome mapping. VarScan is an open-source program to identify gene variants in NGS data. VarScan holds a substantial advantage to deal with Roche/454, Illumina/Solexa, and other platforms generated short sequences and have an unparalleled

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performance of sequence alignment compared to other developed algorithms. It is a platformindependent program that can detect somatic mutations, SNPs, alteration in somatic copy numbers, germline variations, chromosome position indels, and read counts. In addition, it can analyze and detect variations in a single sample as well as in pooled samples with an ability to detect small (1%) frequency variations in pooled samples. In multiple sequence alignment, this program can discard the reads, which are present at multiple positions and demonstrates poor sequence homology when aligned with the reference sequence, and finally, aligned sequences are chosen to map the changes such as SNPs and indels. To predict variations, VarScan determines the maximum range, the total number of contigs and reading strands detected for each allele, and the average consistency of the nucleotide (Koboldt et al. 2009). VarScan has proven its supremacy over its competing computational tools in identifying mutations in somatic tumor cells to differentiate different subclones. This computational tool can cover the bottlenecks in the study of date palm genome, solve problems associated with date palm genome study, and can differentiate between actual SNPs data and sequencing errors. ALLPATHS assembler is a higher ranked computational software able to achieve de novo genome assembly on the basis of graph theory (Gnerre et al. 2011). A graph is created after using the spectrum, also referred to as the sequence graph, which implements a detailed and comprehensively simplified approach to minimize the graph to just a few or no branches. Finally, scaffolding is performed to combine all contigs or subgraphs to obtain the global assembly of the target genome. To minimize error, ALLPATH, first of all, detects low-quality reads and then boosts them with certain heuristics, if this works then the contig is retained or vice versa. This software has a very specialized method to retain, discard or modify the contigs and is based on very versatile algorithms. First, it generates a catalog of all k-mers from the reads then calculates their functions, then a simple graph is constructed to gain optimized k-mers,

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optimized k-mers are the ones that occur frequently and vice versa, weak k-mers are substituted with the new one. When the new read shared is ten times better than the former, the read is updated otherwise discarded. This k-mers is a coding scheme that is a unique integer assigned to a specific DNA. This simplification helps in building a database and achieving a multiple local alignment based on the k-mers which ultimately leads to the generation of a unipath by placing each read in the proper orientation and at a suitable place. Eventually, graphs are generated on the basis of unipaths and multiple sequence alignment that are subsequently linked and then cleaned to achieve the genome assembly. The resultant cumulative graph, after performing scaffolding and graph simplification, is referred to as the global assembly of the target genome. To achieve the assembly of larger genomes, local assembly is achieved first by identifying all possible seed unipaths. These seed unipaths are identified after filtering all unipaths, which can overlap on either side with other unipaths. Subsequently, these unipaths are expanded and their neighborhoods are constructed using unipath intervals. Assembly of all the neighborhoods together yields the graph whose edges are unipath. Such different local sequence graphs are joined together to create a global sequence graph. ALLPATH can be employed in a date palm genome assembly process that can be extended to all DNA sequence data types, not just quick read data, but also traditional sequence reads. QTL Ici Mapping (version 4.2) is free public software that can create high-density genetic maps and perform QTL mapping. This projectbased application has a user-friendly interface and can be assessed at http://www.isbreeding.net/ news/?type=detail HYPERLINK “http://www. isbreeding.net/news/?type=detail&id=123” and HYPERLINK “http://www.isbreeding.net/news/ ?type=detail&id=123”id=123” (Zhang et al. 2019). This computational tool is equipped with integrated functionalities including (A) Analysis of variance (AOV) for multi-environmental phenotype trials, AOV technology applied genotype through the study of environmental

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stability and unbalanced (or incomplete phenotypic) data may also be analyzed; (B) binning of redundant markers (BIN); (C) translating SNP genotype data into algorithm format (i.e., from AA, AT, CC, GG, TT, TC, TG, AC, AG, GT to A, B, H, X, nonetheless, SNP genotype data of two parents is required, of the two inbred parents); (D) mapping of biparental population and their genetic linkage map construction (MAP), in the MAP feature, the approximate recombination frequency, and LOD score are documented in a temporary external file to improve the capacity to manage large numbers of markers. The cumulative number of markers in the current version have exceeded up to 20,000; (E) consensus mapping of multiple genetic linkage maps having same markers (CMP); (F) mapping of digenic epistasis and additive genes in a bi-parental population (BIP); (G) mapping of segregation distortion loci in a biparental population (SDL); (H) mapping of digenic epistasis and additive genes with chromosome segment substitution (CSL); (I) QTL mapping of biparental population interacting through the environment (MET); and (J) QTL mapping of clustered association mapping population (NAM). In addition, this program also offers some supplemental analysis including the display of completed genetic linkage map (MapShow), estimation of recombination between 2 loci in 20 biparental populations (2pointREC), and analysis of variance of different studies (ANOVA). Genetic analysis for multiparental pure line (GAPL) is another free-ware to build QTL map of pure line populations derived from four-way or eight-way crosses and to produce high-density genetic linkage maps. The software is similar to QTL Ici mapping and is project-based with userfriendly interfaces. The latest version of GAPL (v1.2) can be accessed online http://www. isbreeding.net/news/?type=detail HYPERLINK “http://www.isbreeding.net/news/?type= detail&id=121” and HYPERLINK “http://www. isbreeding.net/news/?type=detail&id=121”id= 121”. This software performs four important functions including (A) conversion of SNP genotyping data to the compatible format (i.e. from AA, TT, CC, GG, AT, AC, AG, TC, TG,

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GT to A, B, H. The SNP genotypes of the four or eight inbred parents are needed to use this functionality); (B) construction of genetic linkage maps in multiparental pure-line populations (PLM); (C) binning of redundant markers (BIN); (D) detection of a gene in multiparental pure-line populations (PLQ). TetraploidMap is another computational program to perform linkage and QTL mapping in an experimental cross of an autotetraploid species through the use of dominant markers (AFLP) and codominant markers (SSR), Hackett et al. (2017). In this program, the major limitation is the size that is restricted to 800 markers, and QTL mapping is performed separately for each parent due to the difficulties in achieving a precise consensus map for both parents. Current genotyping techniques now yield datasets of thousands of SNPs, which can be scored as SNP dosages in autotetraploid species, identifying among AAAB, AABB, and ABBB heterozygotes, rather than merely using the presence or absence of an allele. Dosage data are more descriptive regarding recombination, leading to higher density graphs. The latest software, TetraploidSNPMap, makes effective usage of dosage data and has a new improved interface for displaying SNPs clustering, phase calling, and swiftly arranging large SNPs dataset by employing multidimensional scaling analysis. It also has new QTL mapping routines based on a specialized Markov model, which simultaneously predicts the impacts of alleles of both parents using dosage data. TetraploidMap is a Windows-based program that allows data exploration and data entry and can be downloaded freely from the GitHub repository. AutoSNP is a Perl-written computational tool to uncover indels and SNPs polymorphisms in EST files (Duran et al. 2009). This program is extremely handy in tackling sequencing errors, especially those related to the reverse transcription process. AutoSNPdb integrates SNP discovery tools and sequence annotation into a relational database to easily classify different genes or traits, associated SNP, and indel polymorphisms. The software has been successfully implemented to characterize a total of 14832

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candidate polymorphisms in the maize EST sequence data, additionally, it has also been employed to map SNP polymorphism in rice, barley, and Brassica. This program is categorized as one of the pioneer software for outstanding analysis of SNPs polymorphism.

8.6

Available Databases to Map Date Palm Genome

Several web-based date palm databases have been built. Most of those available are designed to help the researcher and breeder to identify a dominant majority of date palm cultivars by opting for highly specific polymorphic markers. In addition, these databases enable researchers to search genetic variations among different date palm cultivars, thus staging the next level of information for breeding and genetic studies. In the succeeding section, these databases are discussed.

8.6.1 Date Palm Genome Database (DRDB) This database is the first of its kind as an online, user-friendly web interface that is well-equipped with an incorporated visualization tool to observe date palm genomic features. The DRDB database was built with the key aim to assist the plant breeder/researcher to recognize the plethora of date palm varieties using excellently selected polymorphic SSRs and SNPs markers. It is comprised of several searching functionalities and can be accessed freely online—http://drdb. big.ac.cn/home (He et al. 2017). In the DRDB, 62 different date palm cultivars from Africa, Egypt, Sudan, Middle East and Asia have been sequenced thoroughly using the HiSeq nextgeneration sequencing platform. Then 246,445 SSRs and 6,375,806 SNPs were annotated in the genome assembly. In SSRs, the percentage of most abundant mononucleotide SSRs was 58.92%, followed by dinucleotide (29.92%), tri(8.14%), tetra- (2.47%), penta- (0.36%), and hexanucleotide SSRs (0.19%). For most

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(174,497; 70.81% of the total) SSRs, highquality PCR primers were built. For the collected 4,177,778 (65.53%) SNPs, high-quality PCR primers were also made. In the browser page of the DRDB, a phylogenetic tree of the recruited date palm cultivars based on their SNP data is depicted; the SNPs data vividly forms three subclades based on their geographic distribution including North Africa, Sudan, Egypt, and the Middle East. Further details of SSRs and SNPs data pertaining to a particular date palm cultivar can also be seen by clicking on the name on the phylogenetic tree. In addition, SSRs and SNPs data search can also be retrieved by customizing the parameters such as SNPs quality and product size in SSRs. At the marker selection page, SSRs and SNPs markers are categorized at region, cultivar and country level. This information can be retrieved either alone or in combination, and are equipped with the position of the markers, allele frequency, reference, and other essential details of SNPs markers or position, size, and type of SSR markers. Besides, the DRDB database supplemented some other information related to SNP annotation, again users can customize SNPs search and putative functional consequences of those SNPs including frameshift substitution, non-frameshift substitution, synonymous and nonsynonymous. Furthermore, this page is supplemented with information from external sources including UNIPROT. All the information on the DRDB database is freely available and can be downloaded.

8.6.2 Plant Genome and System Biology (PGSB) PGSB is a major online-available database containing the databases of many important plants. Nonetheless, the date palm genome database is one of its important integral parts, which is a joint venture of Weill Cornell Medical College, Qatar and the German Research Center for Environmental Health, Germany. The date palm genome database can be accessed directly at

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http://pgsb.helmholtz-muenchen.de/plant/pdact/ index.jsp or https://qatar-weill.cornell.edu/ research/research-highlights/date-palm-researchprogram. Presently, the work is ongoing and date draft sequence version 3.0 is available which encompasses date palm genome sequence and genome annotation. The available files are of date palm genome draft assembly created by shotgun next-generation DNA sequencing”. The key features of the available genome include: an estimated genome size of ca 650 Mb, ca, 57000 scaffolds, an N50 scaffold of about 30 kb with exceptionally reduced gap size, GC content 38%, ca. 3.5 million new high-quality SNPs distributed among 9 date palm genomes and the Khalas cv. as a reference genome, ca. 25000 predicted genes (excluding transposons), 381 Mb of assembled sequences comprising ca. 90% of genes and 60% of the genome sequences were comprised of highly repetitive sequences and the chloroplast genome. The available data can be viewed on a separate page which further splits into two major portions: contig tables and genetic elements. In the contig tables, the sequence of up to 12 kb contigs is presented, which were obtained after NGS reads, and quality and validity of contigs are inspected manually and based on mate-pair validity. The quality of these 12 Kb scaffolds is approximately identical to other plant draft sequences, such as papaya and rice. Larger scaffolds are likely to have errors, so researchers should be vigilant in designing PCR primers when spanning gaps in the sequences. In addition, genetic elements and contigs data can also be searched with a small scale of customization, researchers can access the data either by name/id or by free text methods. Again, all the information on this database is free and can be downloaded from http://qatar-weill.cornell.edu/ research/research-highlights/date-palm-researchprogram/date-palm-draft-sequence.

8.6.3 Date Palm Molecular Markers Database (DPMMD) DPMMD is a more comprehensive type of database than the two previously mentioned. It

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contains information useful for basic and applied research; for example, related to date palm genetics, genomics, and molecular breeding and can be accessed freely at http://dpmmd. easyomics.org/index.php. DPMMD has information of more than 3,611,400 DNA markers including SNPs, SSRs and SSR-SNPs. In addition, it provides information on genetic linkage maps, KEGG pathways, DNA barcodes, and date palm markers related to articles indexed in PubMed journals. In DPMMD, a list of SSR and SNPs primers is integrated from some previously published data (Al-Dous et al. 2011; Mokhtar et al. 2016) and compiled in a relational database using MySQL environment. All the information of SSR markers was subcategorized into 4 different subdatabases; Genic SSR includes 9650 markers, Genic SSR-SNP contains 21731 SSR-SNP markers, SNPs markers have 3,000,000 SNPs markers, and intergenic SSR-SNPs have 57251 markers which are situated within the SSR flanking sites. The two remaining subdatabases are related to R-genes SSR markers and DNA barcode. The R-genes subdatabase has 42 integrated SSR markers related to 16 separate genes of disease resistance. The DNA barcode was retrieved based on NCBI sequences of 138 date palm cultivars. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database is another component of DPMMD, KEGG is a valuable method for decoding genes and their biochemical interactions and here 896 SSR markers were mapped to 111 pathways; all these pathways are freely accessible to researchers. The last important subdatabase is related to date palm genetic maps, which describe correlation analysis, association studies, and physical mapping. DPMMD contains the first-ever constructed date palm genetic linkage maps spanning ca. 4000 DNA markers covering a total of 1293 cM distance. The location and reference sequence of different markers are also mapped on this linkage.

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8.7

Date Palm Genome Assemblies and Mapping in the ThirdGeneration Sequencing Era

Whole-genome sequencing and the generation of genome assemblies have been continuously evolving over time. Radically, new DNA sequencing tools and bioinformatics approaches are steadily refining existing NGS approaches and thus the quality of genome assemblies is improving. Ultimately, the high-throughput capacity of NGS has enabled researchers to access the whole-genome investigations of the complex genome for population genomics, disease mapping, improved yields and others. The third-generation genome sequencing approaches characteristically use long reads in kilobases (Kb) rather than base pairs (bp) which are cost-effective due to simplicity in preparation and sequencing methods. These approaches are quite promising in unraveling longer, complex, and repetitive genomes to help bridge the unexplored genomic regions. Currently, two types of technologies are included in the third-generation sequencing, i.e., long-reads sequencing platforms and long-range scaffolding technology (Jiao and Schneeberger 2017). The most widely adopted long-read sequencing technique is the singlemolecule real-time (SMRT) technology, which gives an average read length of up to 20 Kb. Using SMRT technology in date palm, researchers at New York University Abu Dhabi (NYUAD) generated a comprehensive genome assembly of male and female members of various date palm varieties located at two farms in the United Arab Emirates (Hazzouri et al. 2019). Currently, this presents the most complete whole-genome sequence of date palm available, which may help to identify the genes related to fruit color and sugar content. Recently, Chenopodium quinoa C.L. Willdenow (Willd.) genome has been assembled with average read length *12 Kb (Jarvis et al. 2017). Another most commonly used long-read technology is synthetic long-read (SLR) technology with an

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average read length encompassing 8–10 Kb. SLR can be exclusively adopted to resolve the haplotype of polyploidy plant genomes. Another promising technology is Nanopore (Oxford Nanopore Technologies) technology, which can generate long reads of more than 5 Kb, although *12 Kb median length was reported for wild tomato Solanum pennellii Correll genome sequencing (Schmidt et al. 2017). Until now, the whole genomes of wild tomato, Arabidopsis thaliana (L.) Hyenh. and Oryza coarctata Roxb. have been sequenced completely using Nanopore technology. Besides all the long-read technology progress, it is still not possible to assemble a whole diploid plant genome (such as date palm) with only sequencing reads from NGS data. Therefore, longrange scaffolding techniques are also equally essential. This necessitates the extension of obtained contigs into scaffolds and finally aligning them into complete chromosomes. Following the current NGS technologies in hand, additional genetic and physical mapping is necessary to translate the genetic information onto respective chromosomes. An alternate third-generation approach is Hi-C offered by Dovetail Genomics (https://dovetailgenomics.com), which is based on chromosome conformation capture sequencing. The long-range mate-pair data created by HiC can be used for chromosome phasing and scaffolding in a very user-friendly way (Sedlazeck et al. 2018). Additionally, the Hi-C and Oxford Nanopore technologies can be used to construct physical maps using very long DNA fragments. Another recent collaboration between Phase Genomics and Pacific Biosciences has released FALCON-Phase technology (Kronenberg et al. 2018). This recently announced tool is promising to overcome haplotyping problems during diploid plant genome assemblies, such as date palm. FALCON-Phase tool combines the SMRT long-read data and Hi-C data to construct the fully phased chromosome assemblies. Another recently announced technology is GemCode offered by 10X Genomics (www. 10xgenomics.com). GemCode is similar to the SLR approach of Illumina, however, it can handle even longer reads with comparatively less

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read depth. The median captured read length is more than 100 Kb. This approach has only been applied to the diploid pepper (Capsicum annuum L.) genome (Hulse-Kemp et al. 2018). Another promising approach is optical mapping, which facilitates the fingerprinting of larger genomic fragments assembling highly fragmented genomes. Optical mapping has promisingly improved the scaffolding and ultimately reduced the need for genetic and physical mapping (Jiao and Schneeberger 2017).

8.8

Conclusions and Prospects

Date palm genome mapping generally has remained limited to pre-NGS techniques including AFLP, RFLP, RAPD, GWAS, SNPs, SSR, and ESTs. The execution of these DNA markers has been limited to identifying different cultivars/varieties, population and sex determination. Although, a big breakthrough occurred with the execution of NGS after sequencing the complete genomes of different date palm cultivars (Al-Mssallem et al. 2013; Al-Dous et al. 2011; Hazzouri et al. 2015; Mohamoud et al. 2019); nonetheless, the date palm genome remained an ignored area. However, the first date palm genetic map was inferred by Mathew et al. (2014), which revealed long-range genome structure conservation in the palms and demonstrated the gender-segregating region in date palm to LG12 suggesting that this 5–13 Mb genetic region may contain gender-based genetic alteration. Later, several web-based databases and the Kyoto Encyclopedia of Genes and Genomes pathways for date palm were assembled to better underpin the functional and comparative genomics of the date palm. Until now, studies entailing the comparative and functional genomics of date palm have been very limited. The contemporary NGS-based sequencing techniques coupled with omics technologies have explored gene functions and their involvement in biochemical pathways such as circadian and diurnal pathways in date palm (Safronov et al. 2017). Subsequently, the prospects of genetic mapping as a complementary method for the identification

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Whole-Genome Mapping of Date Palm …

of QTLs related to abiotic stress have also been discussed (Hazzouri et al. 2020). Over time, improvements in whole-genome sequencing and assembly approaches for diploid date palm will fundamentally boost the genetics and plant breeding research in date palm. It will better help to understand the date palm genome by identification of genomic variants and ultimately relate them to physiological, morphological, agronomical, and economical traits such as improved yield, biotic and abiotic stress tolerance, sex determination, secondary metabolites, and others. Better date palm genome assemblies can also unravel the genotype-phenotypeenvironment interactions in date palm. Over 100 genomes of date palm and its wild relatives are currently available in GenBank (https://www. ncbi.nlm.nih.gov/genome/?term=txid42345[orgn ]), which can open new opportunities to investigate and answer the longstanding questions regarding date palm genomic origin and study the date palm genome in depth. Thus, wholegenome resequencing of economically important date palm cultivars has been necessarily required to have high-quality reference subgenomes to capture genetic variations in the genomes of these cultivars. Many unresolved questions include the time of date palm domestication, precise date palm origin, and mechanisms behind the genetic diversity in date palm cultivars. These and many other longstanding questions can be answered by constructing high-density genetic maps using phenotypic and genotypic traits of different date palm cultivars, worldwide.

References Al-Dous EK, George B, Al-Mahmoud ME et al (2011) De novo genome sequencing and comparative genomics of date palm (Phoenix dactylifera). Nat Biotech 29 (6):521–527. https://doi.org/10.1038/nbt.1860 Al-Hinai M (2019) Fingerprinting of some Omani date palm cultivars using SSR markers. Paper presented at the The Sixth International Date Palm Conference (SIDPC), Abu Dhabi, UAE Al-Khalifah NS, Shanavaskhan AE (2017) Molecular Identification of date palm cultivars using random amplified polymorphic DNA (RAPD) markers. In: AlKhayri JM, Jain SM, Johnson DV (eds) Date Palm

197 biotechnology protocols, Vol II, Germplasm conservation and molecular breeding. Springer New York, New York, NY, pp 185–196. https://doi.org/10.1007/ 978-1-4939-7159-6_16 Al-Khateeb SA, Al-Khateeb AA, Sattar MN, Mohmand AS (2020) Induced in vitro adaptation for salt tolerance in date palm (Phoenix dactylifera L.) cultivar Khalas. Biol Res 53(1):37 Al-Khateeb SA, Al-Khateeb AA, Sattar MN et al (2019) Assessment of somaclonal variation in salt-adapted and non-adapted regenerated date palm (Phoenix dactylifera L.). Fres Environ Bull 28(5):3686–3695 Al-Mssallem IS, Hu S, Zhang X et al (2013) Genome sequence of the date palm Phoenix dactylifera L. Nat Comm 4:2274. https://doi.org/10.1038/ncomms3274 Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Amer J Hum Genet 32(3):314–331 Bradbury PJ, Zhang Z, Kroon DE et al (2007) TASSEL: software for association mapping of complex traits in diverse samples. Bioinform 23(19):2633–2635. https://doi.org/10.1093/bioinformatics/btm308 Corniquel B, Mercier L (1997) Identification of date palm (Phoenix dactylifera L.) cultivars by RFLP: partial characterization of a cDNA probe that contains a sequence encoding a zinc finger motif. Int J Plant Sci 158(2):152–156. https://doi.org/10.1086/297425 Doebley JF, Gaut BS, Smith BD (2006) The molecular genetics of crop domestication. Cell 127. doi:10.1016/j.cell.2006.12.006 Duran C, Appleby N, Clark T et al (2009) AutoSNPdb: an annotated single nucleotide polymorphism database for crop plants. Nucl Acids Res 37:951–953. https:// doi.org/10.1093/nar/gkn650 El-Assar AM, Krueger RR, Devanand PS, Chao C-CT (2005) Genetic analysis of Egyptian date (Phoenix dactylifera L.) accessions using AFLP markers. Gen Res Crop Evol 52(5):601–607. https://doi.org/10. 1007/s10722-004-0583-z El Rabey HA, Al-Malki AL, Abulnaja KO, Rohde W (2015) Proteome analysis for understanding abiotic stress (salinity and drought) tolerance in date palm (Phoenix dactylifera L.). Int J Genom 2015:1–11. https://doi.org/10.1155/2015/407165 Elmeer K, Mattat I, Al Malki A et al (2020) Intra-cultivar variability at microsatellite loci in date palm cultivars across the GCC countries. QScience Connect 2020(1). doi:https://doi.org/10.5339/connect.2020.3 FAOStat (2017) Food and Agriculture Organization of the United Nations Statistics Division. http://faostat3.fao. org/home/E Faqir N, Muhammad A, Ali GM et al (2019) Development of a Single Nucleotide Polymorphism-based genetic identification key for date palm cultivars. Crop Sci 59(6):2509–2520. https://doi.org/10.2135/crop sci2019.01.0016 Gnerre S, Maccallum I, Przybylski D et al (2011) Highquality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Nat Acad

198 Sci USA 108(4):1513–1518. https://doi.org/10.1073/ pnas.1017351108 Gupta PK, Varshney RK (2000) The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphy 113 (3):163–185. https://doi.org/10.1023/a: 1003910819967 Hackett CA, Boskamp B, Vogogias A et al (2017) TetraploidSNPMap: software for linkage analysis and QTL mapping in autotetraploid populations using SNP dosage data. J Hered 108(4):438–442. https://doi.org/ 10.1093/jhered/esx022 Hazzouri KM, Flowers JM, Nelson D et al (2020) Prospects for the study and improvement of abiotic stress tolerance in date palms in the post-genomics era. Front Plant Sci 11(293). https://doi.org/10.3389/fpls. 2020.00293 Hazzouri KM, Flowers JM, Visser HJ et al (2015) Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop. Nat Commun 6:8824. https://doi.org/10.1038/ncomms9824 Hazzouri KM, Gros-Balthazard M, Flowers JM et al (2019) Genome-wide association mapping of date palm fruit traits. Nat Comm 10(1):4680. https://doi. org/10.1038/s41467-019-12604-9 He Z, Zhang C, Liu W et al (2017) DRDB: An online date palm genomic resource database. Front Plant Sci 8 (1889). https://doi.org/10.3389/fpls.2017.01889 Hulse-Kemp AM, Maheshwari S, Stoffel K et al (2018) Reference quality assembly of the 3.5-Gb genome of Capsicum annuum from a single linked-read library. Hort Res 5(1):4. https://doi.org/10.1038/s41438-0170011-0 Jackman SD, Warren RL, Gibb EA et al (2015) Organellar genomes of white spruce (Picea glauca): assembly and annotation. Gen Bio Evol 8(1):29–41. https://doi. org/10.1093/gbe/evv244 Jaganathan D, Bohra A, Thudi M, Varshney RK (2020) Fine mapping and gene cloning in the post-NGS era: advances and prospects. Theor Appl Genet. https:// doi.org/10.1007/s00122-020-03560-w Jarvis DE, Ho YS, Lightfoot DJ et al (2017) The genome of Chenopodium quinoa. Nature 542(7641):307–312. https://doi.org/10.1038/nature21370 Jiao W-B, Schneeberger K (2017) The impact of third generation genomic technologies on plant genome assembly. Curr Opin Plant Biol 36:64–70. https://doi. org/10.1016/j.pbi.2017.02.002 Koboldt DC, Chen K, Wylie T et al (2009) VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinform 25 (17):2283–2285. https://doi.org/10.1093/ bioinformatics/btp373 Kronenberg ZN, Hall RJ, Hiendleder S et al (2018) FALCON-Phase: Integrating PacBio and Hi-C data for phased diploid genomes. bioRxiv:327064. https:// doi.org/10.1101/327064 Lipka AE, Tian F, Wang Q et al (2012) GAPIT: genome association and prediction integrated tool. Bioinform

Z. Iqbal et al. 28(18):2397–2399. https://doi.org/10.1093/ bioinformatics/bts444 Loridon K, McPhee K, Morin J et al (2005) Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.). Theor Appl Genet 111(6):1022–1031. https://doi.org/10.1007/s00122-005-0014-3 Marth GT, Korf I, Yandell MD et al (1999) A general approach to single-nucleotide polymorphism discovery. Nat Genet 23(4):452–456. https://doi.org/10. 1038/70570 Masmoudi K (2019) Identify and characterize SNP markers for the candidate genes HKT1; 5 and SOS1 in 18 Date palm cultivars. Technical Report. Development of sustainable date palm production systems in the GCC countries of the Arabian Peninsula Mathew LS, Seidel MA, George B et al (2015) A genome-wide survey of date palm cultivars supports two major subpopulations in Phoenix dactylifera. G3: Genes|Genomes|Genetics 5(7):1429–1438. https://doi. org/10.1534/g3.115.018341 Mathew LS, Spannagl M, Al-Malki A et al (2014) A first genetic map of date palm (Phoenix dactylifera) reveals long-range genome structure conservation in the palms. BMC Gen 15(1):285. https://doi.org/10.1186/ 1471-2164-15-285 Mirani AA, Teo CH, Markhand GS et al (2020) Detection of somaclonal variations in tissue cultured date palm (Phoenix dactylifera L.) using transposable elementbased markers. Plant Cell Tiss Organ Cult 141(1):119– 130. https://doi.org/10.1007/s11240-020-01772-y Mohamoud YA, Mathew LS, Torres MF et al (2019) Novel subpopulations in date palm (Phoenix dactylifera) identified by population-wide organellar genome sequencing. BMC Gen 20(1):498. https://doi.org/10. 1186/s12864-019-5834-7 Mokhtar MM, Adawy SS, El-Assal SE-DS, Hussein EHA (2016) Genic and intergenic SSR database generation, SNPs determination and pathway annotations, in date palm (Phoenix dactylifera L.). PLoS ONE 11(7): e0159268. https://doi.org/10.1371/journal.pone. 0159268 Pariona A (2017) Leading countries growing dates (fresh date palm fruits). Retrieved from (https://www. worldatlas.com/articles/world-leading-countriesgrowing-fresh-dates.html) Paterson AH, Lander ES, Hewitt JD et al (1988) Resolution of quantitative traits into Mendelian factors by using a complete linkage map of restriction fragment length polymorphisms. Nature 335 (6192):721–726. https://doi.org/10.1038/335721a0 Pham JL, Glaszmann JC, Sano R et al (1990) Isozyme markers in rice: genetic analysis and linkage relationships. Genome 33(3):348–359. https://doi.org/10. 1139/g90-054 Popenoe PB (1973) The date palm. Field Research Projects, Coconut Grove, Miami, Florida, USA Rekik I, Chaâbene Z, Kriaa W et al (2019) Transcriptome assembly and abiotic related gene expression analysis of date palm reveal candidate genes involved in

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response to cadmium stress. Comp Biochem Physiol 225: https://doi.org/10.1016/j.cbpc.2019.108569 Rimbert H, Darrier B, Navarro J et al (2018) High throughput SNP discovery and genotyping in hexaploid wheat. PLoS ONE 13(1): https://doi.org/10. 1371/journal.pone.0186329 Roorkiwal M, Jain A, Kale SM et al (2018) Development and evaluation of high-density Axiom(®) CicerSNP Array for high-resolution genetic mapping and breeding applications in chickpea. Plant Biotech J 16 (4):890–901. https://doi.org/10.1111/pbi.12836 Safronov O, Kreuzwieser J, Haberer G et al (2017) Detecting early signs of heat and drought stress in Phoenix dactylifera (date palm). PLoS ONE 12(6): https://doi.org/10.1371/journal.pone.0177883 Schmidt MH-W, Vogel A, Denton AK et al (2017) De novo assembly of a new Solanum pennellii accession using nanopore sequencing. Plant Cell 29(10):2336– 2348. https://doi.org/10.1105/tpc.17.00521 Sedlazeck FJ, Lee H, Darby CA, Schatz MC (2018) Piercing the dark matter: bioinformatics of long-range sequencing and mapping. Nat Rev Genet 19(6):329– 346. https://doi.org/10.1038/s41576-018-0003-4 Shahzad R, Kanwal S, Yasmeen E et al (2020) DNA fingerprinting and population structure of date palm varieties grown in Punjab Pakistan using simple sequence repeat markers. Int J Agri Biol 23:943– 950. https://doi.org/10.17957/ijab/15.1373 Singh VK, Khan AW, Jaganathan D et al (2016a) QTLseq for rapid identification of candidate genes for 100seed weight and root/total plant dry weight ratio under rainfed conditions in chickpea. Plant Biotech J 14 (11):2110–2119. https://doi.org/10.1111/pbi.12567 Singh VK, Khan AW, Saxena RK et al (2016b) Nextgeneration sequencing for identification of candidate genes for Fusarium wilt and sterility mosaic disease in pigeonpea (Cajanus cajan). Plant Biotech J 14 (5):1183–1194 Swiecicki W, Wolko B, Weeden N (2000) Mendel’s genetics, the Pisum genome and pea breeding. In: 100 years of Genetics for Plant Breeding. Mendel Centenary Congress, Vorträge für Pflanzenzüchtung, 48:65-76 Takagi H, Abe A, Yoshida K et al (2013) QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J 74(1):174–183. https://doi.org/ 10.1111/tpj.12105 Tang KS, Hart GE (1975) Use of isozymes as chromosome markers in wheat-rye addition lines and in triticale. Genet Res 26(2):187–201. https://doi.org/10. 1017/s0016672300015986

199 Tang M, Chen Z, Grover CE et al (2015) Rapid evolutionary divergence of Gossypium barbadense and G. hirsutum mitochondrial genomes. BMC Gen 16(1):770. https://doi.org/10.1186/s12864-015-1988-0 Tengberg M (2003) The date palm: from traditional resource to green wealth. In: Al-Istirātījīyah, Markaz al-Imārāt lil-Dirāsāt wa-al-Buḥūth, UAE Tiwari VK, Faris JD, Friebe B, Gill BS (2016) Genome Mapping. In: Reference module in food science.. Elsevier, pp 2–11. https://doi.org/10.1016/B978-0-08100596-5.00220-1 Trick M, Adamski NM, Mugford SG et al (2012) Combining SNP discovery from next-generation sequencing data with bulked segregant analysis (BSA) to fine-map genes in polyploid wheat. BMC Plant Biol 12(1):14. https://doi.org/10.1186/14712229-12-14 Varshney RK, Pandey MK, Bohra A et al (2019) Toward the sequence-based breeding in legumes in the postgenome sequencing era. Theor Appl Genet 132 (3):797–816. https://doi.org/10.1007/s00122-0183252-x Yang H, Li C, Lam H-M et al (2015) Sequencing consolidates molecular markers with plant breeding practice. Theor Appl Genet 128(5):779–795. https:// doi.org/10.1007/s00122-015-2499-8 Yuan Y, Scheben A, Batley J, Edwards D (2019) Using genomics to adapt crops to climate change. In: Sustainable solutions for food security. Springer, pp 91–109 Zhang L, Meng L, Wang J (2019) Linkage analysis and integrated software GAPL for pure-line populations derived from four-way and eight-way crosses. The Crop J 7(3):283–293. https://doi.org/10.1016/j.cj. 2018.10.006 Zhao Y, Keremane M, Prakash CS, He G (2017) Characterization and amplification of gene-based simple sequence repeat (SSR) markers in date palm. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date Palm biotechnology protocols, Vol II, Germplasm conservation and molecular breeding. Springer New York, New York, pp 259–271. https://doi.org/10. 1007/978-1-4939-7159-6_21 Zhao Y, Williams R, Prakash CS, He G (2013) Identification and characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.). BMC Plant Biol 12(1):237. https://doi.org/10.1186/14712229-12-237 Zlokolica M, Milošević M (2001) Isozymes as genetic markers in maize breeding. Bio Plant 44(2):207–211

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Date Palm (Phoenix dactylifera L.) Chloroplast Genome M. Kamran Azim

Abstract

The chloroplast is a significant organelle of autotrophic cells which contains an autonomous, double-stranded, circular DNA molecule (cpDNA) known as the chloroplast genome. It harbors crucial genes for maintenance of the chloroplast. The chloroplast genome encodes several components of the photosystems and enzymes involved in biosynthetic pathways. Understanding the molecular genetics of the chloroplast is important owing to its small size, conserved nature, determined gene organization and the favorable capability for transgenic expression. Hence, chloroplast genome sequences have been contributory in molecular phylogenetics and classification of plants. Chloroplast genome sequences of date palm (Phoenix dactylifera L.) were among the first crop organelle genomes reported. Date palm chloroplast genome sequences of four cultivars: Khalas from Saudi Arabia, Aseel from Pakistan, and Naghal and Khanezi from Oman are currently available. Similar to other flowering plants, the chloroplast genome of date palm is a

M. K. Azim (&) Department of Biosciences, Mohammad Ali Jinnah University, 22-E, Block 6, PECHS, Karachi, Pakistan e-mail: [email protected]; [email protected]

double-stranded, circular DNA molecule exhibiting a quadripartite structure with a large and a small single-copy region (termed LSC and SSC, respectively) that are separated by two copies of inverted repeat regions (IR). With a size of * 158,000 bp, date palm cpDNA contains approximately 130 functional genes. Many single nucleotide variants (SNVs) as well as heteroplasmy, more than one type of organellar genome within an individual, have been observed in the date palm chloroplast genome. Chloroplast genome sequences from date palm cultivars have improved our insights into chloroplast biology, conservation, diversity and intracellular gene transfer.

9.1

Introduction

The chloroplast is an important cellular organelle of plant cells and photosynthetic algae (Sugiura 2003; Xiong 2009). From the standpoint of evolution, the chloroplast evolved by endosymbiosis between photosynthetic cyanobacteria and non-photosynthetic protists (Howe 2003; Raven 2003; Xiong 2009). Along with mitochondrial and nuclear DNA, photosynthetic organisms encompass self-replicating DNA in the chloroplast, designated as cpDNA or chloroplast genome. Mainly, in higher plants, the inheritance of cpDNA arises through the paternal or maternal parent; in some cases, biparental inheritance has

© Springer Nature Switzerland AG 2021 J. M. Al-Khayri et al. (eds.), The Date Palm Genome, Vol. 1, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-73746-7_9

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also been observed (Hansen 2007; McKinnon 2001). The chloroplast genome of angiosperms is a double-stranded, circular DNA with a range in size of 120,000–160,000 bps (Odintsova and Yurina 2006). Among higher plants, cpDNA is highly conserved in size, gene content and organization (Olmstead and Palmer 1994). Archetypal cpDNA comprised small and large single-copy regions, designated as SSC and LSC, respectively, separated by two duplicated inverted repeat regions, designated as IRA and IRB (Ravi 2008) (Fig. 9.1). With the regular size of 20,000–30,000 bps, the IR sequences of chloroplast genome of angiosperms are extremely conserved. The IR regions commonly vary in length among cpDNA sequences (Odintsova and Yurina 2006; Ogihara et al. 2002). Also, the boundaries of IR regions with LSC and SSC are vital for expansion and contraction in the cpDNA (Goulding et al. 1996; Plunkett and Downie 2000). Variation in size of cpDNA is typically due to the expansion or contraction of the inverted repeat (IR) regions (Downie and Jansen 2015). Analysis of cpDNA sequences from an array of plants species has revealed 60–200 genes (Leister 2003). Based on their functions, genes in the cpDNA are categorized into three sets: photosynthesis, photosynthetic metabolism and transcription/translation (Odintsova and Yurina 2006). Only 10% of the genes required for fully functional chloroplast is encoded by cpDNA, whereas the outstanding proteins in chloroplasts are encoded by the nuclear DNA (Jarvis and Robinson 2004). The date palm (Phoenix dactylifera, L.) is a key fruit crop of the Arecaceae family. It is generally grown in dry regions of the Middle East, South Asia and Africa (Al-Farsi and Lee 2008). As the chloroplast genome is matrilineal, insights into its evolution and sequence variation offer beneficial data for evolving propagation technologies. Complete sequences of hundreds of cpDNA from diverse species are available in the NCBI Organelle Genome Resources database (http:// www.ncbi.nlm.nih.gov/genomes/). It is notable that the majority of available cpDNA sequences have been submitted in the Gene bank during the

M. K. Azim

last 12 years. This is principally due to modernizations in (a) next-generation sequencing technologies and (b) bioinformatics tools for sequence data. In this chapter, current trends in chloroplast genome sequencing, data mining and analysis are discussed.

9.2

Chloroplast Genome Sequencing

Significant progress has been made in chloroplast genome sequencing strategies. As an initial step, cpDNA is purified from a chloroplast-rich fraction of leaves (Jansen 2005). Conventionally, the purified cpDNA is digested by restriction enzymes or random shearing followed by ligation of the cpDNA fragments in cloning vectors. The clones containing chloroplast DNA fragments are then processed for Sanger DNA sequencing. This methodology was utilized with some modifications for the sequence determination of several chloroplast genomes until the advent of nextgeneration DNA sequencing technologies in 2007. For instance, sequencing of complete chloroplast genomic DNA of tobacco (Nicotiana tobacum L.) (Shinozaki 1986), maize (Zea mays L.) (Maier et al. 1995), Chinese wheat (Triticum aestivum L.) (Ogihara et al. 2000), Korean ginseng (Panax schinseng Nees) (Ki-Joong, 2004), cucumber (Cucumis sativus L.) (Jin-Seog 2005) and potato (Solanum tuberosum L.) (Chung 2006) was carried out by Sanger sequencing. Moreover, a number of cpDNA sequences have been carried out with the rolling-circle-amplification (RCA) technique which amplifies the original DNA prior to sequencing (Jansen 2005). For the sequencing of cpDNA of cotton (Gossypium hirsutum L.) (Lee 2006) and Citrus x sinensis (L.) Osbeck (Baucher 2006), purified cpDNA was first exposed to RCA. The yields of RCA were then treated with restriction enzymes and cloned for sequencing. In the sequencing of cpDNA of shining clubmoss {Huperzia lucidula (Michaux) Trevisa} and welwitschia (Welwitschia mirabilis Hook f.), the RCA technique was utilized followed by shotgun sequencing (McCay 2008; Wolf 2005).

9

Date Palm (Phoenix dactylifera L.) Chloroplast Genome

203

Fig. 9.1 The Gene map of date palm cv. Aseel (Phoenix dactylifera) chloroplast genome. The thick line indicates the extent of two inverted repeat (IRA, IRB) regions. Genes inside the circle are transcribed counterclockwise,

whereas genes outside the circle are transcribed clockwise (figure constructed by M. Kamran Azim, Karachi, Pakistan)

After completion of the human genome project in 2000, high throughput and rapid DNA sequencing was among the main tasks in current genomic research (Straiton et al. 2019). While DNA sequencing by the Sanger method is considered the gold standard for whole genome sequencing projects, it necessitates a hefty arrangement and highly qualified human resources (Beck et al. 2016). The techniques involved

in genome-level DNA sequencing by the Sanger method are cloning of DNA into vectors, growth of host and purification of vectors followed by Sanger sequencing (Kozińska et al. 2019). However, during the past 14 years, the nextgeneration sequencing (NGS) technologies have been adopted for genome-level sequencing of microbes, animals, plants and humans (Kumar et al. 2019; Zhao and Struan 2011). The NGS

204

M. K. Azim

technologies are non-Sanger-based sequencing technologies for massively-paralleled highthroughput DNA sequencing at extraordinary speed (Tripathi et al. 2019). The chloroplast genome sequences of four date palm cultivars have been reported using two different NGSTs: pyrosequencing and Illumina sequencing technology (Khan et al. 2012, 2018; Yang et al. 2010). Pyrosequencing and Illumina sequencing are based on the principle of sequencing by synthesis. Pyrosequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. It relies on light detection based on a chain reaction when pyrophosphate is released (Harrington et al. 2013). Illumina sequencing technology is the widely adopted NGS technology. Illumina sequencing technology supports massively parallel sequencing using a method that detects single bases as they are incorporated into growing DNA strands (McCombie et al. 2019).

9.3

Date Palm Chloroplast Genome Assembly and Annotation

Next-generation sequencing (NGS) generates huge quantities of sequence data by massively paralleled sequencing of DNA fragments. The data generated necessitate bioinformatics proficiency to analyze and interpret the NGS data. The NGS datasets of date palm chloroplast DNA obtained from the pyrosequencing and Illumina systems were assembled using different assembling software such as CLC Genomics Workbench (CLCbio, Denmark) (Khan et al. 2010). Recently, the NOVOPlasty, a program for de novo assembly of organelle genomes from NGS data has been used for the assembly of chloroplast DNA (Dierckxsens et al. 2017; Rasheed et al. 2020). Annotation of chloroplast genomes was carried out by ORF Finder (http://www.ncbi. nlm.nih.gov/projects/gorf/), Dual Organellar Genome Annotator (the DOGMA server) (Wyman et al. 2004) and BLAST (Altschul et al. 1990). Besides, annotation of some tRNAs was accomplished by tRNAscan-SE (Lowe and Eddy

1997) and after sequence similarity searching with annotated plastomes. Characterization of repeat sequences was done using the REPuter program (Kurtz et al. 2001). The circular genome map of date palm chloroplast genome was built with a number of bioinformatics tools including the GenomeVx online tool (Conant 2008). For analysis of gene order, GeneOrder tool was utilized (Celamkoti et al. 2004). Multiple sequence alignments of chloroplast genomes were constructed by the mVISTA comparative genomics server (Frazer et al. 2004). The maximum parsimony-based phylogenetic tree construction of 25 open-reading frames was carried out by MEGA4 program (Tamura et al. 2007). Thus different bioinformatics software have been used to analyze and interpret the date palm chloroplast cpDNA sequence data.

9.4

Organization of Date Palm Chloroplast Genome

Thus far, the chloroplast genomes of four date palm cvs. Khalas, Aseel, Khanezi and Naghal have been sequenced (Khan et al. 2012, 2018; Yang et al. 2010). First, the chloroplast genome sequence of cv. Khalas was reported from Saudi Arabia (Yang et al. 2010), followed by cv. Aseel sequence from Pakistan (Khan et al. 2012). Recently, Khan et al. (2018) reported chloroplast genome sequences of two Omani date palm cvs. Khanezi and Naghal. Date palm chloroplast genomes are characteristic double-stranded circular DNA molecules, which share the quadripartite structure with other angiosperms (and Arecaceae family members) (Khan et al. 2012). Sequence analysis revealed the chloroplast genome sizes of cvs. Khalas, Aseel, Khanezi and Naghal as 158,462, 158,458, 158,211 and 158,210 bp, respectively (Table 9.1) (Khan et al. 2018). Analysis of date palm chloroplast genome sequences showed a close association with the broadleaf cattail (Typha latifolia L.). The incidence of few forward and inverted repeats in date palm chloroplast genome point to conserved sequence arrangement.

9

Date Palm (Phoenix dactylifera L.) Chloroplast Genome

205

Table 9.1 Statistics of complete chloroplast genomes for four date palm (Phoenix dactylifera) cultivars Total length (bp)

Khalas

Aseel

Naghal

Khanezi

158,462

158,458

158,210

158,211

GC%

37.2

37.2

37.3

37.3

Cumulative length of protein coding genes (bp)

83,904

81,408

82,153

82,144

GC%

37.9

37.8

37.9

37.9

Cumulative length of tRNA coding genes (bp)

9050

9050

9050

9050

Cumulative length of rRNA coding genes (bp)

3568

2735

2960

2960

Citation

Yang et al. (2010)

Khan et al. (2012)

Khan et al. (2018)

Genbank Accession No.

GU811709

FJ212316

MF197494, MF197495

9.4.1 The Chloroplast Genome of cv. Khalas Yang et al. (2010) reported complete sequence of cv. Khalas chloroplast genome-based on pyrosequencing technology. The length of cv. Khalas cpDNA is 158,462 bp and it has a quadripartite structure of SSC region of 17,712 bp and LSC region of 86,198 bp separated by the pair of IR regions, 27,276 bp. Analogous to most flowering plants, the Phoenix dactylifera chloroplast genome possesses 112 unique genes and 19 duplicated fragments in the IR regions (Daniell et al. 2016). The intersections between SSC/IRs and LSC/IRs demonstrate features of sequence extension in evolution. Yang et al. (2010) characterized 78 single nucleotide variants (SNVs) as key intravarietal polymorphisms, most of which were positioned in genes with important biochemical roles. RNA-seq data revealed 18 polycistronic transcription units and three highly expressed genes, i.e. rrn23, atpF and trnA-UGC (Yang et al. 2010).

9.4.2 The Chloroplast Genome of cv. Aseel Khan et al. (2012) reported the date palm cpDNA sequence of cv. Aseel from Pakistan, by means of Sanger-based and Illumina sequencing technologies. Similarly to cv. Khalas from Saudi

Arabia (Yang et al. 2010), the size of the cpDNA of cv. Aseel was 158,458 bp with the pair of IR sequences of 27,276 bp that were separated by a SSC region of 17,711 bp and LSC region of 86,195 bp (Fig. 9.1). Genome annotation identified 89 protein-coding, 38 tRNA and 8 rRNA genes (Table 9.1). The cpDNA contained 41% non-coding (including intergenic sequences, pseudogenes and introns) and 59% coding regions. Out of 89 protein-coding sequences, 16 genes contained intronic regions. Among these, the genes ycf3, clpP and rps12 contained two introns each. Date palm chloroplast genome contained 38 genes for transfer RNAs (30 distinct genes), and, of these, 8 genes contained intronic regions. Four rRNA genes were limited to and duplicated in the inverted repeat sequences. Altogether, 20 genes (including ycf15 pseudogene) and one 3′-exon of rps12 trans-splicing protein were present in the inverted repeats. These 20 genes included 7 protein-coding, 8 tRNA and 4 rRNA genes. The comparative analysis of chloroplast genome sequences of cvs. Khalas and Aseel showed variation in intervarietal sequences in the LSC region. These sequence polymorphisms included (i) 2 single nucleotide variants (SNVs) in intergenic sequences, (ii) one SNV in rpoc1 gene, and (iii) a 4 base pair insertion in intergenic region between genes accD and psaI (Fig. 9.2). Khan et al. (2012) also identified a polymorphic spot in

206

mononucleotide SSR situated at nucleotide 120,710. They further analyzed cv. Aseel cpDNA sequence with date palm cpDNA sequence entries deposited in nucleotide databases. These entries belong to partial genes and intergenic regions of the date palm chloroplast genome. Sequence analysis pointed out a number of possibly valuable sequence variants in date palm.

9.4.3 Chloroplast Genomes of cvs. Naghal and Khanezi Khan et al. (2018) reported on the chloroplast genome sequences of cvs. Naghal and Khanezi from Oman using next-generation DNA sequencing (NGS). The lengths cpDNA of Naghal and Khanezi cvs. were 158,210 bp and 158,211 bp, respectively. The assembly of NGS reads and annotation of chloroplast genome showed the archetypal quadripartite structure. Structurally, chloroplast genomes of these cultivars were encompassed by a pair of IR regions (Khanezi/Naghal = 27,273 bp/27,272 bp), the LSC region (Khanezi/Naghal = 86,090 bp/ 86,092 bp) and the SSC region (Khanezi/Naghal = 17,575 bp/17,574 bp). In the cvs. Khanezi and Naghal cpDNA sequences, 227 and 229 randomly distributed microsatellites were found, respectively. Whole chloroplast genome-based phylogenetic tree construction showed that cvs. Khalas and Aseel formed clades with cvs. Khanezi and Naghal. This finding pointed out intervarietal association among these cultivars with available chloroplast genomes (Yang et al., 2010).

Fig. 9.2 Alignment of cpDNA sequences from date palm cvs. Khalas and Aseel, Typha latifolia, Dioscorea elephantipes (L’Hér.) Engl. and Acornis calamus L.

M. K. Azim

Sequence analysis of cvs. Khanezi and Naghal revealed variations in inverted repeats (IRA and IRB) as well as in large single-copy (LSC) region and small single-copy (SSC) region. For example, two inverted repeats showed variation of one base pair in length (Naghal = 27,272 bp and Khanezi = 27,273 bp). In the case of LSC, the two chloroplast genomes varied in length by two base pairs (Khanezi = 86,090 bp and Naghal = 86,092 bp). Similarly, the SSC was 17,575 bp and 17,574 bp for Khanezi and Naghal, respectively. The coding sequences in both Khanezi and Naghal possess similar GC content and length relative to the chloroplast genome (Khan et al. 2018).

9.5

Comparison with Other Monocot Species

The date palm is a monocotyledonous species. Therefore, comparison of chloroplast genomes of date palm cvs. was carried out with 10 species from 6 monocot families [2 species from family Acoraceae (Goremykin et al. 2005); 4 species from family Poaceae (Maier et al. 1995; Masood et al. 2004; Ogihara et al. 2000; Wu et al. 2009] and 1 species each from families Dioscoreaceae (Hansen et al. 2007), Orchidaceae (Chang et al. 2006), Araceae (Mardanov et al. 2008) and Typhaceae (Guisinger et al. 2010)]. The analysis showed that length of date palm cpDNA was larger than the compared monocots, excluding Typha latifolia (Typhaceae) and Lemna minor (Araceae). The GC and AT contents of date palm cpDNA are in close range with monocots. A maximum parsimonyphylogenetic tree construction by mVISTA server

(position 61,464–61,499; date palm cv. Khalas numbering) indicating 4 bp insertion in cv. Khalas (figure constructed by M. Kamran Azim, Karachi, Pakistan)

9

Date Palm (Phoenix dactylifera L.) Chloroplast Genome

(Brudno et al. 2003; Frazer et al. 2004) of complete chloroplast genome sequences of 11 monocot species including date palm revealed a grouping of date palm cpDNA sequences with Typha latifolia, Dioscorea elephantipes and Phalaenopsis aphrodite Rchb. f.

9.6

Conclusion and Prospects

Currently, full-length chloroplast genome (cpDNA) sequences of four date palm cultivars are available in nucleotide databases. These are Khalas, Naghal and Khanezi grown in the Arabian Peninsula (Saudi Arabia and Oman) and cv. Aseel cultivated in South Asia (Sindh Province of Pakistan). Sequencing of date palm cpDNA was primarily carried out by massively paralleled next-generation DNA sequencing technology. Date palm cpDNAs are characteristic doublestranded circular DNA molecules, which share the quadripartite structure with other angiosperms and Arecaceae family members. Molecular phylogeny showed close association of date palm cpDNA sequences with broadleaf cattail (Typha latifolia L.). Comparative analysis of partial date palm cpDNA sequences, submitted to nucleotide databases from countries in northern Africa and the Middle East, showed several single nucleotide variants (SNVs). This has provided information related to diversity in chloroplast genome of date palm cultivars growing in Egypt, Tunisia, Morocco, Algeria, Saudi Arabia, Oman and Pakistan. Insights gained from complete chloroplast genome sequences have enhanced our understanding of plant biology and diversity. Chloroplast genomes have made noteworthy inputs to phylogenetic studies of plant lineage. Further, during past 15 years, research on genetic engineering of the chloroplast has focused in the overexpression of target genes to improve biotic stress tolerance. In future, the chloroplast genome would be utilized to express genes from different organisms, including plants, microbes, humans and other animals for application in agriculture, biomedicine and other areas of biotechnology.

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References Al-Farsi MA, Lee CY (2008) Nutritional and functional properties of dates: a review. Crit Rev Food Sci Nutr 48(10):877–887 Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410 Baucher MG (2006) The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var ‘Ridge Pineapple’: organization and phylogenetic relationships to other angiosperms. BMC Plant Biol 6:21 Beck TF, Mullikin JC, Comparative Sequencing Program NISC et al (2016) Systematic evaluation of Sanger validation of next-generation sequencing variants. Clin Chem 62(4):647–654 Brudno M, Do CB, Cooper GM et al (2003) LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA. Genome Res 13(4):721– 731 Celamkoti S (2004) GeneOrder 3.0: Software for comparing the order of genes in pairs of small bacterial genomes. BMC Bioinform 5:52 Chang C, Lin H, Lin I et al (2006) The chloroplast genome of Phalaenopsis aphrodite (Orchidaceae): comparative analysis of evolutionary rate with that of grasses and its phylogenetic implications. Mol Biol Evol 23:279–291 Chung HJ (2006) The complete chloroplast genome sequences of Solanum tuberosum and comparative analysis with Solanaceae specie identified the presence of a 241-bp deletion in cultivated potato chloroplast DNA sequence. Plant Cell Rep 25:1369–1379 Conant GC (2008) Genome Vx: simple web-based creation of editable circular chromosome maps. Bioinform 24:861–862 Daniell H, Lin CS, Yu M et al (2016) Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol 17(1):134 Dierckxsens N, Mardulyn P (2017) Smits G (2017) NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucl Acids Res 45(4): Downie SR, Jansen RK (2015) A comparative analysis of whole plastid genomes from the Apiales: expansion and contraction of the inverted repeat, mitochondrial to plastid transfer of DNA, and identification of highly divergent noncoding regions. Syst Bot 40(1):336–351 Frazer KA, Pachter L, Poliakov A et al (2004) VISTA: computational tools for comparative genomics. Nucl Acids Res 1:32 (Web Server issue):W273–279 Goulding S, Olmstead R, Morden C et al (1996) Ebb and flow of the chloroplast inverted repeat. Mol Gen Genet 252:195–206 Goremykin VV, Holland B, Hirsch-Ernst KI et al (2005) Analysis of Acorus calamus chloroplast genome and its phylogenetic implications. Mol Biol Evol 22:1813– 1822 Guisinger MM, Chumley TW, Kuehl JV et al (2010) Implications of the plastid genome sequence of Typha

208 (Typhaceae, Poales) for understanding genome evolution in Poaceae. J Mol Evol 70:149–166 Hansen AK (2007) Paternal, maternal and biparenatal inheritance of the chloroplast genome in Passiflora (Passifloraceae): implication for phylogenetic studies. Amer J Bot 94(1):42–46 Hansen DR, Dastidar SG, Cai Z et al (2007) Phylogenetic and evolutionary implications of complete chloroplast genome sequences of four early-diverging angiosperms: Buxus (Buxaceae), Chloranthus (Chloranthaceae), Dioscorea (Dioscoreaceae), and Illicium (Schisandraceae). Mol Phylogenet Evol 45:547–563 Harrington CT, Lin EI, Olson MT et al (2013) Fundamentals of pyrosequencing. Arch Pathol Lab Med 137 (9):1296–303 Howe CJ (2003) Evolution of the chloroplast genome. Phil Trans R Soc Lond 358:99–107 Jarvis P, Robinson C (2004) Mechanisms of protein import and routing in chloroplast. Curr Biol 14:1064– 1077 Jansen RK (2005) Methods for obtaining and analyzing whole chloroplast genome sequences. Meth Enzymol 395:348–384 Jin-Seog K (2005) Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome. Plant Cell Rep 25 (4):334–40 Kozińska A, Seweryn P, Sitkiewicz I (2019) A crash course in sequencing for a microbiologist. J Appl Genet 60(1):103–111 Khan A, Khan IA, Asif H et al (2010) Current trends in chloroplast genome research. Afr J Biotech 9 (24):3494–3500 Khan A, Khan IA, Heinze B et al (2012) The chloroplast genome sequence of date palm (Phoenix dactylifera L. cv. ‘Aseel’). Plant Mol Biol Rep 30:666–678 Khan AL, Asaf S, Lee I-J et al (2018) First chloroplast genomics study of Phoenix dactylifera (var. Naghal and Khanezi): a comparative analysis. PLoS ONE 13 (7):e0200104 Ki-Joong K (2004) Complete chloroplast genome sequences from Korean Ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants. DNA Res 11:247–261 Kumar KR, Cowley MJ, Davis RL (2019) Nextgeneration sequencing and emerging technologies. Semin Thromb Hemost 45(7):661–673 Kurtz S, Choudhuri JV, Ohlebusch E, Schleiermacher C, Stoye J, Giegerich R (2001) REPuter: the manifold applications of repeat analysis on a genomic scale. Nucl Acids Res 29:4633–4642 Lee SB (2006) Complete chloroplast genome sequencing of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms. BMC Genom 7:61 Leister D (2003) Chloroplast research in the genomic age. Trends Genet 19(1):47–56 Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucl Acids Res 25(5):955–964

M. K. Azim Maier RM, Neckermann K, Igloi GL et al (1995) Complete sequence of the maize chloroplast genome: gene content, hotspots of divergence and fine tuning of genetic information by transcript editing. J Mol Biol 251:614–628 Mardanov AV, Ravin NV, Kuznetsov BB et al (2008) Complete sequence of the duckweed (Lemna minor) chloroplast genome: structural organization and phylogenetic relationships to other angiosperms. J Mol Evol 66:555–564 Masood MS, Nishikawa T, Fukuoka S et al (2004) The complete nucleotide sequence of wild rice (Oryza nivara) chloroplast genome: first genome wide comparative sequence analysis of wild and cultivated rice. Gene 340:133–139 McCay SR (2008) The complete plastid genome sequence of Welwitschia mirabilis: an unusually compact plastome with accelerated divergence rates. BMC Evol Biol 8:130 McCombie WR, McPherson JD, Mardis ER (2019) Nextgeneration sequencing technologies. Cold Spring Harb Perspect Med 9(11):a036798 McKinnon GE (2001) Maternal inheritance of the chloroplast genome in Eucalyptus globulus and interspecific hybrids. Genome 44:831–835 Odintsova MS, Yurina NP (2006) Chloroplast genomics of land plants and algae. In: Maria Giardi T, Piletska EV (eds) Biotechnological applications of photosynthetic proteins: biochips, biosensors and biodevices. Springer Nature, Switzerland AG, pp 57–72 Ogihara Y, Isono K, Kojima T et al (2000) Chinese spring wheat (Triticum aestivum L.) chloroplast genome: complete sequence and contig clones. Plant Mol Biol Rep 18:243–253 Ogihara Y, Isono K, Kojima T (2002) Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA. Mol Genet Genom 266:740– 746 Olmstead RG, Palmer JD (1994) Chloroplast DNA systematic: a review of methods and data analysis. Amer J Bot 81(9):1205–1224 Plunkett G, Downie S (2000) Expansion and contraction of the cp inverted repeat in Apiaceae subfamily Apioideae. Syst Bot 25:648–667 Rasheed S, Zaidi S, Azim MK (2020) The chloroplast genome sequence of Momordica charantia L. (bitter gourd). Gene Reports 21:100963 Raven JA (2003) Genomics and chloroplast evolution: what did cyanobacteria do for plants? Genome Biol 4:209 Ravi V (2008) An update on chloroplast genomes. Plant Syst Evol 271:101–102 Shendure J, Mitra RD, Varma C et al (2004) Advanced sequencing technologies: methods and goals. Nat Rev Genet 5:335–344 Shinozaki K (1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5(9):2043–2049

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Date Palm (Phoenix dactylifera L.) Chloroplast Genome

Straiton J, Free T, Sawyer A et al (2019) From Sanger sequencing to genome databases and beyond. Biotechniq 66(2):60–63 Sugiura M (2003) History of chloroplast genomics. Photosyn Res 76:371–377 Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599 Tripathi P, Singh J, Lal JA et al (2019) Next-generation sequencing: an emerging tool for drug designing. Curr Pharm Des 25(31):3350–3357 Wolf PG (2005) The first complete chloroplast genome sequence of a lycophyte, Huperzia lucidula (Lycopodiaceae). Gene 350:117–128

209

Wu F, Kan D, Lee SB et al (2009) Complete nucleotide sequence of Dendrocalamus latiflorus and Bambusa oldhamii chloroplast genomes. Tree Physiol 29:847– 856 Wyman S (2004) Automatic annotation of organellar genomes with DOGMA. Bioinform 20(17):3252– 3255 Xiong AS (2009) Gene duplication, transfer and evolution in the chloroplast genome. Biotech Adv 27:340–347 Yang M, Zhang X, Liu G et al (2010) The complete chloroplast genome sequence of date palm (Phoenix dactylifera L.). PLoS ONE 5(9):e12762 Zhao J, Struan FAG (2011) Advances in whole genome sequencing technology. Curr Pharm Biotech 12 (2):293–305

Comparative Analysis of Date Palm (Phoenix dactylifera L.) Mitochondrial Genomics

10

Sajjad Asaf, Abdul Latif Khan, Ahmed Al-Harrasi, and Ahmed Al-Rawahi

Abstract

Date palm (Phoenix dactylifera L.) is a major fruit crop of arid regions, domesticated *7000 years ago in the Near and Middle East. With the advancement of next-generation sequencing technology, genomic data of date palm have increased rapidly and yielded new insights into this species and its origins. Four P. dactylifera mitochondrial genome assemblies are available in the National Center for Biotechnology Information (NCBI). These mitochondrial genomes are circular double-stranded DNA molecules ranging in size from 585,493 bp (P. dactylifera; MG257490) to 715,120 bp (in cv. Khanezi; MH176159) and their guanine-cytosine (GC) content range is 44.8–45.1%. Their protein-coding sequences are composed of 4.6–5.35% gene content. Repeat analysis of the P. dactylifera mt

S. Asaf
A. L. Khan (&)
A. Al-Harrasi (&)
A. Al-Rawahi Natural and Medical Science Research Center, University of Nizwa, Nizwa, Oman e-mail: [email protected] A. Al-Harrasi e-mail: [email protected] S. Asaf e-mail: [email protected] A. Al-Rawahi e-mail: [email protected]

genomes revealed 20–24 palindromic repeats, about 26–30 forward repeats and 35–49 tandem repeats, respectively. Similarly, a total of 233, 271, 242 and 271 simple sequence repeats (SSRs) were identified in P. dactylifera (unverified cultivars) and cvs. Khanezi, Khalas and Naghal, respectively. Pairwise mt genomic alignment among different P. dactylifera cultivar genomes showed a high degree of synteny. Phylogenetic analysis revealed that the mt genome of P. dactylifera forms one monophyletic clade. Evolutionary analysis based on the mitochondrial genome will help to understand the evolutionary changes of the various date palm cultivars.

10.1

Introduction

Date palm (Phoenix dactylifera L.) belongs to family Arecaceae and is an ecologically, culturally and economically important fruit tree in the arid and semiarid regions of North Africa and Southwest Asia. It is one of the oldest cultivated plants (Al-Shahib and Marshall 2003; Moussouni et al. 2017). Date palm is cultivated mainly in the Middle East and North Africa in an area of 1.2 million ha and produces about 7 million mt of fruit. Saudi Arabia, United Arab Emirates (UAE), Iran and Egypt are the main producing countries, together contributing 57% of the global production (Al-Dous et al. 2011). The date palm (2n = 36 chromosomes) is a dioecious,

© Springer Nature Switzerland AG 2021 J. M. Al-Khayri et al. (eds.), The Date Palm Genome, Vol. 1, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-73746-7_10

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212

monocotyledonous perennial tree that can be grown up to 1500 m elevation. In arid regions, dates fruits are an essential source of food and nutrients. Nutritionally, dates can provide 314 kcal energy per 100 g of fresh fruit (Baliga et al. 2011). Approximately 2000 named varieties of dates are known worldwide (Al-Khalifah and Askari 2003). Phoenix dactylifera is a strictly dioecious tree capable of living for over 100 productive years (Al-Mssallem et al. 2013). It is one of the oldest domesticated trees having socioeconomic importance (Mahmoudi et al. 2008). The earliest P. dactylifera cultivation dates back to 3700 BC (Al-Mssallem et al. 2013) in the area between the Euphrates and the Nile rivers (Gros-Balthazard et al. 2013). It is thought to be native to the Arabian Peninsula region, possibly originating from what is now southern Iraq (Al-Mssallem et al. 2013). Date palm tree was introduced to northern India, North Africa and southern Spain and it has a major economic role in arid zones (El-Juhany 2010). Saudi Arabia, a major producer of date fruit, has a high percentage (14%) of the world’s date palm trees, accounting for 340 varieties (Al-Maasllem 1996). Date palm fruit contains a high proportion of carbohydrates, salts, vitamins and minerals. The fats comprise 14 kinds of fatty acids, proteins that are composed of 23 different amino acids and a considerable quantity of dietary fiber in the fruit (Al-Shahib and Marshall 2003). Date palm is a monocotyledonous perennial, cross-pollinated dioecious tree (Barrow 1998; Cherif et al. 2013; Terral et al. 2012). Morphological differences in date palm are highly dependent on environmental factors and the genetic basis of the variety. These variations are also reflected in the genetic diversity, derived from nuclear, chloroplast (cp) and mitochondrial genomes. This chapter analyzes and compares the complete mitochondrial genomes of four P. dactylifera cultivars (P. dactylifera (unverified), and cvs. Khanezi, Khalas and Naghal. It also provides information for the further understanding of mt genome evolution, gene contents and repetitive sequences in these cultivars.

S. Asaf et al.

10.2

Date Palm Genomics

Only a few genome-wide studies on Phoenix dactylifera have been done (Al-Mssallem et al. 2013). Some recent studies on genomics (draft) used the Illumina GAII sequencing platform (AlDous et al. 2011) and estimated a genome size of 658 Mb, 58% genome assembly (382 Mb) and 25,059 predicted genes (Al-Mssallem et al. 2013). Bourgis et al. (2011) reported comparative transcriptomics of the mesocarp using pyrosequencing data from the Roche GS FLX Titanium platform (Bourgis et al. 2011). Transcriptomic profiles for P. dactylifera fruit development and full-genome assemblies of two P. dactylifera organelles {158,462, add new genomes cp for plastid (Yang et al. 2010) and 715,001 bp for mitochondrion (Fang et al. 2012), respectively} was reported by Yin et al. (2012). Mitochondrial genome research history is one of the groundbreaking and innovative discoveries with immense potential. With the improvement in Sanger sequencing facilities (Metzker 2005), it became more feasible to sequence the entire mtDNAs. New methods and sequencing technologies contributed to efficient and fast isolation, sequencing, assembly and annotation of mtDNAs (Burger et al. 2007; Cheng et al. 1994; DeSalle et al. 1993). The introduction of enormously parallel sequencing technologies is a game-changer in the mitochondrial genome, because they are cheaper, faster and can generate more data than the Sanger-based sequencing (Metzker 2010). Different types of nextgeneration sequencing (NGS) technologies such as Illumina and Ion Torrent have generated highquality mitochondrial genome assemblies from whole genomic data sets (King et al. 2014; Smith 2012). High-throughput sequencing has its own limitations (Hert et al. 2008). However, these limitations such as homopolymer errors, short read lengths and low coverage are not considered a major issue for mitochondrial genome assemblies because of their genome size (Seo et al. 2015). The excess of organelle-derived reads generation as compared to nuclear ones, and certainly is an annoyance for researchers during

10

Comparative Analysis of Date Palm …

nuclear DNA studies, but helpful to study mitochondrial genomics (Raz et al. 2011; Smith 2013). Organelle genomes, mainly mitochondrial (mt) and cp, are two extra chromosomal genomes present in plants and known for inheritance and evolutionary traits. Primarily, the inheritable mitochondrial organelle is the semi-autonomous part that produces adenosine triphosphate (ATP) to play a central role as the energy powerhouse (Gray 2012; Gray et al. 1999). The size of mtDNA in mammals is estimated at 15–17 kbp, whereas in plants, for example in angiosperms, it can vary from 200 to 700 kb and even be as large as 11 Mb in the striped corn catchfly (Silene conica L.) Gualberto and Newton (2017). The majority of mtDNA comprises the non-coding sequence that has an amply higher repeat number. Plant mitochondrial genomes are more vigorous than other eukaryotic organisms. However, a very few (140) mtgenomes of angiosperms are deposited in the NCBI organelle genome section, when compared to the 9208 for animals, to September 2019. The mitochondria play an important role in plant growth and development (Ogihara et al. 2005). Due to the recent genomic advancements in mitochondrial DNA, it is considered one of the most effective and significant genetic variants among species and subspecies. Compared to other eukaryotes, the mtDNA of plants have a large and complicated genomic structure (Li et al. 2009; Liu et al. 2011). Moreover, various angiosperm mt genomes are known for their foreign DNA uptake, due to their low mutation rate and horizontal gene transfer (Goremykin et al. 2008; Palmer and Herbon 1988).

10.3

General Features of the Mitochondrial Genome

Four Phoenix dactylifera mitochondrial genome assemblies are registered in the NCBI database. Among these two mt genomes are published and two unverified and unpublished. The first published genome was P. dactylifera cv. Khalas

213

from Saudi Arabia (Fang et al. 2012) while the second from cv. Khanezi was recently published by our group from Oman (Asaf et al. 2018). Subsequently, the mitochondrial genome of a parthenocarpic mutant variety was reported by Bhatt and Thaker (2019). Like other angiosperm, the mitochondrial genomes of date palm are circular double-stranded DNA molecules having sizes from 585,493 bp (P. dactylifera; MG257490) to 715,120 bp (in cv. Khanezi; MH176159) (Fig. 10.1; Table 10.1). The GC content range of these genomes is 44.8–45.1%. Like other angiosperm genomes such as Cucurbita pepo (982,833 bp) (Alverson et al. 2010 and Vitis vinifera (773,279 bp; Goremykin et al. 2008), these genomes are large in size.

10.4

Protein Coding, RRNA and TRNA Genes in the Mitochondrial Genome

The protein-coding sequence is composed of 5.35, 4.6 and 4.6% gene content in Phoenix dactylifera (NC016740), P. dactylifera (MH176159) and P. dactylifera cv. Naghal, respectively. On the other hand, in the unverified mt genome of P. dactylifera (MG257490), the non-coding region contains only 1.8%. Similarly, the gene content is similar among these mt genomes (Table 10.1; Fig. 10.1). There are about 43 protein-coding genes found in. P. dactylifera (NC016740) and 38 found in both P. dactylifera (MH176159) and P. dactylifera cv. Naghal mt genomes. The unverified mt genome contains only 16 protein-coding genes. The mt genomes of P. dactylifera does not have the genes encoding respiratory chain complex II, such as sdh3 and sdh4, which are only found in certain other angiosperms mt genomes (Lee et al. 2018). Similarly, these genomes contain one copy of RNA polymerase genes. Genes related to ribosomal small subunit such as rps1, rps2, rps3, rps4, rps7, rps11, rps12, rps13 and rps14 were detected in these genomes (Table 10.1; Fig. 10.1). However, rps15 was detected only in P. dactylifera; MG257490, while rps19 was

214

S. Asaf et al.

Fig. 10.1 Mitochondria genome map of Phoenix dactylifera. Features on the clockwise and counterclockwise transcribed strands are drawn on the inside

and outside of the circle, respectively. The figure represents the authors’ datasets of date palm genomes. Source Asaf et al. 2018

Table 10.1 Gene contents and total length of Phoenix dactylifera mitogenomes Features

P. dactylifera (NC016740)

P. dactylifera (MH176159)

P. dactylifera (Unpublished)

P. dactylifera (MG257490)

Genome size (bp)

715,001

715,120

715,094

585,493

G+C contents

45.1

45.1

45.1

44.8

Total gene contents

64

63

71

36

Protein-coding gene

43

38

38

16

rRNA

3

3

3

–

tRNA

18

22

30

20

Total protein-coding gene length

38,253

32,916

32,916

8133

The figure represents authors’ own datasets of date palm genomes. Source Asaf et al. 2018

10

Comparative Analysis of Date Palm …

found in cvs. Khanezi and Naghal. Similarly, genes related to complex I, such as nad1 and nad2, were only found in the Khalas cultivar. The Phoenix dactylifera mt genomes have a variable number of tRNA genes, ranging from 18 in cv. Khalas to 30 in cv. Naghal. However, the number of rRNA genes is the same in all mt genomes, except in the unverified one in which no rRNA gene annotated (Table 10.2). Among them, tRNA genes with five amino acids (A, W, L, G and V) have been noted to be not encoded. Twenty amino acids are regarded as essential for the synthesis of mitochondria protein (Table 10.2). The findings revealed that those missing tRNAs are provided via chloroplast or nuclear genomic DNA. Furthermore, it was found that four mt tRNA genes of higher plants are increasingly lost and exchanged by chloroplast-derived tRNA (Tian et al. 2007).

10.5

Repetitive Sequences in the Mitochondrial Genomes

Repeat sequences play a pivotal role in genome rearrangements and are considered essential to understand the taxonomy and phylogenetic analysis (Cole et al. 2018; Figueroa and Baco 2014). In addition, analyzing various mt genomes has shown that repeat sequences are essential to induce indels and substitutions (Christensen 2013). Repetition in sequence consists of a majority of disperse repeats and tandem repeats. These can be different in size in different plant mitochondrial genomes (Gualberto and Newton 2017). Dispersive repetitive sequences are segments of DNA occurring at multiple times and at more or less random positions of the genome, and tandem repeats are small segments of DNA repeated one after another (Gualberto and Newton 2017; Wang et al. 2015). REPuter was used to determine the repetitive sequences (direct, reverse and palindromic repeats) within these plastomes (Kurtz et al. 2001). For repeat identification via REPuter, the following settings were used: (1) a minimum repeat size of 30 bp, (2)  90% sequence identity and (3) Hamming distance of 1. Tandem Repeats Finder version

215

4.07 b was used to find tandem repeats by using default settings (Benson 1999). Repeat analysis of the Phoenix dactylifera mt genomes revealed 20–24 palindromic repeats, about 26–30 forward repeats and about 35–49 tandem repeats (Fig. 10.2). Among these repeats, 9 palindromic repeats are 30–44 bp in length found only in the unverified P. dactylifera mt genome. Similarly, among these genomes about 3, 10, 9 and 10 forward repeats of >90 bp length were detected in P. dactylifera (unverified), and cvs. Khanezi, Khalas and Naghal, respectively (Fig. 10.2).

10.6

Simple Sequence Repeats

Simple sequence repeats (SSRs), or microsatellites, are repeating sequences of typically 1–6 bp that are distributed throughout the genome (Asaf et al. 2016). To identify SSRs, Phobos version 3.3.12 was employed with search parameters set to  3 repeat units for pentanucleotide and hexanucleotide repeats,  4 repeat units for trinucleotide and tetranucleotide repeats,  8 repeat units for dinucleotide repeats and  10 repeat units for mononucleotide repeats (Mayer 2010). A total of 233, 271, 242 and 271 SSRs were identified in Phoenix dactylifera (unverified cultivars), and cvs. Khanezi, Khalas and cv. Naghal, respectively. The majority of SSRs in these mt genomes possess a mononucleotide repeat motif, varying in quantity from 61 in cv. Khalas to 106 in the unverified P. dactylifera. Dinucleotide SSRs, the second most common, range from 45 in unverified P. dactylifera to 56 in both Khalas and Khanezi cultivars. During bioinformatic analysis, 1, 3, 3 and 3 hexanucleotide SSR were detected in P. dactylifera (unverified cultivars), and cvs. Khanezi, Khalas and Naghal, respectively (Fig. 10.2e). In these mt genomes, mostly mono-nucleotide SSRs were A (adenine) and T (thymine) motifs that comprised the majority of dinucleotide SSRs as adenine guanine (AG) motif. Commonly, the mononucleotides and dinucleotides comprise A and T, which can also contribute to the bias in base-composition, and in conformity with other mt genomes (Asaf et al. 2016; Notsu et al. 2002).

216 Table 10.2 Gene contents comparison of Phoenix dactylifera mitochondrial genome with other species

S. Asaf et al. Product group

Gene

NC16740

N2

K2

MG257490

Complex I

nad1

+

–

–

–

nad2

+

–

–

–

nad3

+

+

+

+

nad4

+2

+2

+2

–

nad5

+

+

–

+

nad6

+

+

+

–

nad7

+

+

+

–

nad9

+

+

+

–

Complex III

cob

+

+

+

–

Complex IV

cox1

+

+

+

–

cox2

+

+

+

–

cox3

+

+

+

–

Complex V

atp1

+

+

+

+

Ribosomal large subunit

Atp4

+

+

+

+

atp6

+

+

+

–

Ribosomal small subunit

atp8

+

+

+

+

atp9

+

+

+

+

rpl2

+

+

+

–

rpl5

+

+

+

+

rpl16

+

+

+

–

rps1

+

+

+

+

rps2

+

+

+

–

rps3

+

+

+

–

rps4

+

+

+

–

rps7

+

+

+

+

rps11

+

+

+

+

rps12

+

+

+

+

rps13

+

+

+

–

rps14

+

+

+

–

rps15

+

–

–

–

rps19

–

+

+

–

rrn5

+

+

+

–

rrn18

+

+

+

–

rrn26

+

+

+

–

ccmB

+

+

+

–

ccmC

+

+

+

–

ccmFc

+

+

+

+

ccmFn

+

+

+

–

matR

+

+

+

– (continued)

rRNA rRNA genes

Cytochrome C

Intron maturase

10

Comparative Analysis of Date Palm …

Table 10.2 (continued)

Product group

217 Gene

NC16740

N2

K2

MG257490

mttB

+

+

+

–

Cysteine

trnC

+

+

+

+2

Asperatic

trnD

+

+

+

–

Glutamic

trnE

+

–

+

–

Phenylalanine

trnF

+

+2

+2

+2

Methionine

trnM

+4

+5

+4

+3

Histidine

trnH

0

–

–

+

Isoeucine

trnl

0

–

–

–

Lysine

trnK

+2

+2

+

+2

Leucine

trnL

–

–

–

–

Asparagine

trnN

–

–

–

+

Proline

trnP

+2

+3

+3

+2

Glutamine

trnQ

+

+2

–

–

tRNA

Arginine

trnR

+

–

–

–

Serine

trnS

+3

+4

+4

+3

Tyrosine

trnY

+

+

+

–

Threonine

trnT

+

+

+

+

Tryptophan

trnW

–

–

–

+

+, presence of the gene; −, absence of the gene, numbers, indicate the number of genes present in mt genome. The table represents authors’ datasets of date palm genomes. Source Asaf et al. 2018

10.7

Mitochondrial DNA Comparison and Phylogenetic Analysis

Pairwise mt genomic alignment among different date palm cultivar genomes revealed the highest level of syntenic relationship. Cultivar Khanezi mt genome-based annotations were utilized for reference mapping purposes and to plot overall sequencing identification with mt genomics of the four cultivars in mVISTA (Fig. 10.3). Results showed the highest sequence-homology with Phoenix dactylifera cvs. Khalas and Naghal. However, relatively lower identity was also

observed with P. dactylifera unverified in different comparative genomics parts, particularly the rps19, ccmFc, nad7, nad4, cox2 and ccmFn regions. Furthermore, the non-coding regions exhibited greater divergence than coding regions. Moreover, the phylogenetic relationship of P. dactylifera with 20 other related monocot species were conducted utilizing complete mt genome following the maximum likelihood method. Results revealed that the mt genome of P. dactylifera forms one monophyletic clade (Fig. 10.4). This study will help scientists understand the evolution of various date palm cultivars mitochondrial genome with related species.

218 Fig. 10.2 Analysis of repeated sequences in four Phoenix dactylifera varieties mt genomes. a Totals of three repeat types, b Frequency of palindromic repeat, c Frequency of tandem repeat, d Number of different SSR types detected in P. dactylifera varieties mt genomes, e Frequency of forward repeat. (Figure constructed by Sajjad Asaf)

S. Asaf et al.

10

Comparative Analysis of Date Palm …

Fig. 10.3 Alignment visualization of four Phoenix dactylifera verities with Cocos nucifera mitochondrial genome sequences. VISTA-based identity plot showing sequence identity among the four P. dactylifera mt genome using P. dactylifera (MH176159) as a reference.

10.8

Conclusions and Prospects

In this chapter, we have reported and analyzed the complete mitochondrial genomes of four Phoenix dactylifera cultivars. Their size ranges from 585,493 bp to 715,120 bp in these cultivars. Genome organization and gene content is typical of the other angiosperm mitochondrial genomes. Phylo-

219

Vertical scale indicates the percentage of identity, ranging from 50 to 100%. Horizontal axis indicates the coordinates within the chloroplast genome. The figure represents authors’ datasets of date palm genomes. Source Asaf et al. 2018

genetic analysis revealed that P. dactylifera forms a monophyletic clade on the basis of complete mt genomes. This study will improve our understanding of P. dactylifera and the evolution of mitochondrial genome within the family Arecaceae. The current dataset will help future researchers understand the relationships of ancient and degraded remains of date palm cultivars, ushering in new knowledge on the natural history of the tree.

220

S. Asaf et al.

Fig. 10.4 Phylogeny of the Phoenix dactylifera mitochondrial genome with 20 other related monocot species. The phylogenetic tree was inferred using the maximum likelihood (ML) method based on entire mitochondrial genomes of these species. Numbers above the branches are the bootstrap values for ML (Figure constructed by Sajjad Asaf)

References Al-Dous EK, George B, Al-Mahmoud ME et al (2011) De novo genome sequencing and comparative genomics of date palm (Phoenix dactylifera). Nat Biotech 29 (6):521 Al-Khalifah N, Askari E (2003) Molecular phylogeny of date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia by DNA fingerprinting. Theor Appl Genet 107(7):1266–1270 Al-Maasllem I (1996) Date Palm (Phoenix dactylifera L.) Vol. 7. Encyclopedia Works Publishing & Distribution, Qatar Al-Mssallem IS, Hu S, Zhang X et al (2013) Genome sequence of the date palm Phoenix dactylifera L. Nat Commun 4:2274 Al-Shahib W, Marshall RJ (2003) The fruit of the date palm: its possible use as the best food for the future? Int J Food Sci Nutr 54(4):247–259 Alverson AJ, Wei X, Rice DW et al (2010) Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae). Mol Biol Evol 27(6):1436– 1448

Asaf S, Khan AL, Al-Harrasi A, Al-Rawahi A (2018) First complete mitochondrial genome of Phoenix dactylifera var. Khanezi. Mitochond DNA Part B Res 3(2):778–779 Asaf S, Khan AL, Khan AR et al (2016) Mitochondrial genome analysis of wild rice (Oryza minuta) and its comparison with other related species. PLoS ONE 11 (4): Baliga MS, Baliga BRV, Kandathil SM et al (2011) A review of the chemistry and pharmacology of the date fruits (Phoenix dactylifera L.). Food Res Int 44 (7):1812–1822 Barrow SC (1998) A monograph of Phoenix L. (Palmae: Coryphoideae). Kew Bull 53(3):513–575 Benson G (1999) Tandem repeats finder: a program to analyze DNA sequences. Nucl Acids Res 27(2):573– 580 Bhatt PP, Thaker VS (2019) Extremely diverse structural organization in the complete mitochondrial genome of seedless Phoenix dactylifera L. Vegetos 32(1):92–97 Bourgis F, Kilaru A, Cao X et al (2011) Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning. Proc Nat Acad Sci 108(30):12527–12532

10

Comparative Analysis of Date Palm …

Burger G, Lavrov DV, Forget L (2007) Sequencing complete mitochondrial and plastid genomes. Nat Protoc 2(3):603 Cheng S, Higuchi R, Stoneking M (1994) Complete mitochondrial genome amplification. Nat Genet 7 (3):350 Cherif E, Zehdi S, Castillo K et al (2013) Male specific DNA markers provide genetic evidence of an XY chromosome system, a recombination arrest and allow the tracing of paternal lineages in date palm. New Phytol 197(2):409–415 Christensen AC (2013) Plant mitochondrial genome evolution can be explained by DNA repair mechanisms. Genome Biol Evol 5(6):1079–1086 Cole LW, Guo W, Mower JP et al (2018) High and variable rates of repeat-mediated mitochondrial genome rearrangement in a genus of plants. Mol Biol Evol 35(11):2773–2785 DeSalle R, Williams AK, George M (1993) Isolation and characterization of animal mitochondrial DNA. In: Elizabeth AZ, Thomas JW, Rebecca LC, Allan CW (eds) Methods in enzymology, vol 224. Elsevier, p 176–204 El-Juhany LI (2010) Degradation of date palm trees and date production in Arab countries: causes and potential rehabilitation. Austral J Basic Appl Sci 4(8):3998– 4010 Fang Y, Wu H, Zhang T et al (2012) A complete sequence and transcriptomic analyses of date palm (Phoenix dactylifera L.) mitochondrial genome. PloS one 7(5):e37164 Figueroa DF, Baco AR (2014) Octocoral mitochondrial genomes provide insights into the phylogenetic history of gene order rearrangements, order reversals, and cnidarian phylogenetics. Genome Biol Evol 7(1):391–409 Goremykin VV, Salamini F, Velasco R et al (2008) Mitochondrial DNA of Vitis vinifera and the issue of rampant horizontal gene transfer. Mol Biol Evol 26 (1):99–110 Gray MW (2012) Mitochondrial evolution. Cold Spring Harb Perspec Biol 4(9): Gray MW, Burger G, Lang BF (1999) Mitochondrial evolution. Sci 283(5407):1476–1481 Gros-Balthazard M, Newton C, Ivorra S et al (2013) Origines et domestication du palmier dattier (Phoenix dactylifera L.). État de l’art et perspectives d’étude. Rev d’Ecol 4:1–15 Gualberto JM, Newton KJ (2017) Plant mitochondrial genomes: dynamics and mechanisms of mutation. Ann Rev Plant Biol 68:225–252 Hert DG, Fredlake CP, Barron AE (2008) Advantages and limitations of next-generation sequencing technologies: a comparison of electrophoresis and non electrophoresis methods. Electrophor 29(23):4618–4626 King JL, LaRue BL, Novroski NM et al (2014) Highquality and high-throughput massively parallel sequencing of the human mitochondrial genome using the Illumina MiSeq. Foren Sci Int Genet 12:128–135

221 Kurtz S, Choudhuri JV, Ohlebusch E et al (2001) REPuter: the manifold applications of repeat analysis on a genomic scale. Nucl Acids Res 29(22):4633– 4642 Lee H-O, Choi J-W, Baek J-H et al (2018) Assembly of the mitochondrial genome in the campanulaceae family using Illumina low-coverage sequencing. Genes 9(8):383 Li L, Wang B, Liu Y et al (2009) The complete mitochondrial genome sequence of the hornwort Megaceros enigmaticus shows a mixed mode of conservative yet dynamic evolution in early land plant mitochondrial genomes. J Mol Evol 68(6):665–678 Liu Y, Xue J-Y, Wang B et al (2011) The mitochondrial genomes of the early land plants Treubia lacunosa and Anomodon rugelii: dynamic and conservative evolution. PLoS ONE 6(10): Mahmoudi H, Hosseininia G, Azadi H et al (2008) Enhancing date palm processing, marketing and pest control through organic culture. J Organ Syst 3(2):29– 39 Mayer C (2010) Phobos Version 3.3. 12. A tandem repeat search program Metzker ML (2005) Emerging technologies in DNA sequencing. Genome Res 15(12):1767–1776 Metzker ML (2010) Sequencing technologies the next generation. Nat Rev Genet 11(1):31 Moussouni S, Pintaud J-C, Vigouroux Y et al (2017) Diversity of Algerian oases date palm (Phoenix dactylifera L., Arecaceae): heterozygote excess and cryptic structure suggest farmer management had a major impact on diversity. PloS one 12(4):e0175232 Notsu Y, Masood S, Nishikawa T et al (2002) The complete sequence of the rice (Oryza sativa L.) mitochondrial genome: frequent DNA sequence acquisition and loss during the evolution of flowering plants. Mol Genet Genom 268(4):434–445 Ogihara Y, Yamazaki Y, Murai K et al (2005) Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome. Nucl Acids Res 33 (19):6235–6250 Palmer JD, Herbon LA (1988) Plant mitochondrial DNA evolved rapidly in structure, but slowly in sequence. J Mol Evol 28(1–2):87–97 Raz T, Kapranov P, Lipson D et al (2011) Protocol dependence of sequencing-based gene expression measurements. PLoS ONE 6(5): Seo SB, Zeng X, King JL et al (2015) Underlying data for sequencing the mitochondrial genome with the massively parallel sequencing platform Ion Torrent™ PGM™. BMC Genom 16(1):S4 Smith DR (2012) Not seeing the genomes for the DNA. Brief Funct Genom 11(4):289–290 Smith DR (2013) RNA-Seq data: a goldmine for organelle research. Brief Funct Genom 12(5):454–456 Terral JF, Newton C, Ivorra S et al (2012) Insights into the historical biogeography of the date palm (Phoenix

222 dactylifera L.) using geometric morphometry of modern and ancient seeds. J Biogeogr 39 (5):929–941 Tian X, Zheng J, Hu S et al (2007) The discriminatory transfer routes of tRNA genes among organellar and nuclear genomes in flowering plants: a genome-wide investigation of indica rice. J Mol Evol 64(3):299–307 Wang X, Liu N, Zhang H et al (2015) Extreme variation in patterns of tandem repeats in mitochondrial control region of yellow-browed tits (Sylviparus modestus, Paridae). Sci Rep 5:13227

S. Asaf et al. Yang M, Zhang X, Liu G et al (2010) The complete chloroplast genome sequence of date palm (Phoenix dactylifera L.). PloS one 5 (9):e12762 Yin Y, Zhang X, Fang Y et al (2012) High-throughput sequencing-based gene profiling on multi-staged fruit development of date palm (Phoenix dactylifera, L.). Plant Mol Biol 78(6):617–626

Date Palm Bioinformatics Vadivel Arunachalam

Abstract

The science of bioinformatics is emerging as a tool to understand genome sequences. In this chapter, compilation of the advances in various aspects of bioinformatics in date palm is attempted. Computational tools are useful in discovering new molecular markers, analyzing gene function, mutations, microRNAs and structural biology. In silico mined new molecular markers covering simple sequence repeat regions, single nucleotide polymorphism/insertiondeletion regions from date palm genome sequences are reviewed in detail. The in silico analysis of gene sequences and their promoter motifs of date palm genome is briefly described. The genes, promoters or genomic regions analyzed using bioinformatics approaches in date palm covered here include the traits of abiotic stress tolerance, sex determination, invertase enzyme activity and color of fruit peel, phytochelators and metal-responsive genes. MicroRNA molecules and their target genes in date palm predicted using computational approaches are discussed. Digital image analysis of fruits and seeds, structural biology, molecular modeling and docking studies attempted in date palm are

V. Arunachalam (&) ICAR-Central Coastal Agricultural Research Institute Ela, Old Goa, Goa 403402, India e-mail: [email protected]

11

also reviewed. The chapter gives a list of computational resources and tools useful for bioinformatics workers and date palm research. A road map is presented covering the role of future bioinformatics of date palm and comparative genomics of palms.

11.1

Introduction

The explosion of data in biological genetic sequences has necessitated the requirement for computer software and other tools. Bioinformatics, the science dealing with the information explosion, handles the biological data effectively to obtain meaningful inferences and help store them in searchable databases. Genome sequences, gene annotations and other data sets generated from the date palm crop offer opportunities to harness bioinformatics software and database tools. Date palm is cultivated in oases of deserts, semiarid and arid regions especially in the Near East and North African countries. Whole draft sequence assemblies of nuclear, plastid and mitochondrial genomes of date palm are being generated to understand the crop’s DNA. Transcriptomes, proteomes, phenomes and metabolomes of the date palm have been generated by several researchers to understand the RNA, protein, phenotype and chemotype, respectively. In silico tools are employed to understand the data generated by these omics approaches to draw inferences about the mechanisms of the biological processes.

© Springer Nature Switzerland AG 2021 J. M. Al-Khayri et al. (eds.), The Date Palm Genome, Vol. 1, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-73746-7_11

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Molecular markers are important in understanding the diversity and linkage mapping of genes and associated genomic regions. In silico analysis of DNA sequences aids in discovering new molecular markers especially covering repeats and point mutations. Simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP)/indels identified by software on genome sequences serve as new molecular markers. Date palm is a dioecious plant where male and female flowers are borne on separate individuals. Hence bioinformatics was used to map the genomic region harboring the sex determining region. Bioinformatics also helps to understand the genomic regions of date palm plant’s response to critical biotic and abiotic challenges such as drought, salinity, red palm weevil and Bayoud disease. Mechanisms of peel color and sucrose content of date fruit have also been deciphered using genomics. Comparative omics studies of the mesocarp of oil-free date palm with oil-rich oil palm (Elaeis guineensis Jacq.) plants revealed the genes governing the lipid biosynthesis process. Other applications of bioinformatics on date palm plant are image analysis techniques, annotation of genes governing response to heavy metals and microRNA molecules. This chapter attempts to update the information covered in earlier detailed reviews on the subject by two books (Arunachalam 2012; Jain et al. 2011) and two reviews (Gros-Balthazard et al. 2018; Meerow et al. 2012). The objective of this chapter is to review the current knowledge on bioinformatics of date palm crop in terms of the computational tools used and the inferences drawn to identify the gaps in knowledge. This chapter contains information of the in silico predicted molecular markers and microRNAs, annotated genes and their promoter motifs, and digital phenotyping and structural biology of date palm.

11.2

Discovery of New Molecular Markers

The date palm is a diploid (2n = 36) plant whose genome is estimated to be of 670 Mb (Al-Dous et al. 2011) to 899 Mb (Hazzouri et al. 2019) size. Several versions of nuclear (Al-Dous et al. 2011; AlMssallem et al. 2013; Gros-Balthazard et al. 2018;

He et al. 2017; Thareja et al. 2018) and organelle genomes (Chaluvadi et al. 2019; Fang et al. 2012; Khan et al. 2012; Mohamoud et al. 2019; Sabir et al. 2014) of date palm are available. The above-listed genomic resources can be harnessed to mine the new candidate markers (simple sequence repeats and single nucleotide polymorphisms) by using bioinformatics softwares. Before the advent of whole genomes, transcriptome sequences (Hamwieh et al. 2010) of date palm were mined and 1000 SSR loci were located for developing new markers. AberlencBertossi et al. (2014) located SSR markers in the genic region and designed primers for amplification of SSR loci in cultivated date palm and its seven other related species and three members of the Coryphoideae subfamily of palms. The date palm genome was found to contain 166,760 SSR regions and SSRs occurred at a density of one in every 2.2 Kb (Manju et al. 2016) and 371,629 SSRs at a density of one in every 1.36 Kb (Xiao et al. 2016). About 229 SSRs per Mb were located on the date palm genome where primers could be designed (Mokhtar et al. 2016). SSR regions covered about 1.94% (Al-Mssallem et al. 2013) to 1.75% (Ferreira Filho et al. 2017) of the date palm genome. The SSR primers designed based on these regions of date palm genome were also found to cross-amplify the DNA of oil palm, palmyra (Borassus flabellifer L.), areca (Areca catechu L.) and coconut (Cocos nucifera L.) palms (Manju et al. 2016). Resequencing the genomes of 62 cultivars of date palm, and comparing them with known reference genome assemblies, He et al. (2017) found 264,655 SSR loci which are available as an online database. A pathway annotation work was undertaken in date palm using SSR regions and BLAST2GO approaches (Mokhtar et al. 2016) where 23 enzymes of starch and sucrose metabolism and 16 enzymes of amino/nucleotide sugar metabolism were tested. Among the trinucleotide repeats, most abundant motifs were of TAA and GAA type (Hamweih et al. 2010), AGG, AAG and AGC motifs (Zhao et al. 2012) in the date palm transcriptomes, whereas in the coding regions of the whole genome (Mokhtar et al. 2016) AAG repeat was predominant.

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Fig. 11.1 Abundant trinucleotide simple sequence repeats in date palm genome

SNPs occurred at a density of 1 SNP in every 217 bases of date palm genome (Al-Dous et al. 2011). SNP peaks at linkage group (LG) 12 (Hazzouri et al. 2019; Mathew et al. 2014) were found associated with sex or gender determining loci. SNP at start codon of VIR (virescence) loci governed by R2R3-MYB transcription factor was found to change the color of fruit peel from purple to yellow.

A consensus of AAG repeat motif as abundant in the date palm genome (Fig. 11.1) is observed from the above three reports. A database was generated to display the SNP variation (He et al. 2017) in the resequenced genomes of several cultivars along with the known genomes of date palm. The genomes used, SSR mining approaches and other details are listed in Table 11.1.

Table 11.1 Software and in silico resources, databases and URL links for SSRs Source/version of date palm genome

Software/method used and useful links

References

EST sequences Version 1 at qatar-weill.cornell.edu site

Perl script

Hamwieh et al. (2010)

EST sequences at qatar-weill.cornell.edu site

–

Zhao et al. (2012)

Al-Dous et al. (2011)

Websat program http://wsmartins. net/websat/

Cherif et al. (2013)

Version 2: Al-Dous et al. (2011)

SSR pipeline 2.0 http://www.mpl.ird.fr/ bioinfo/ Poncet et al. (2006) combines Tandom Repeat Finder (TRF), Blast and Primer3 tools

AberlencBertossi et al. (2014)

Version 1: 142,304 whole genome shotgun sequences from National Center for Biotechnology Information (NCBI) ftp site

MISA Perl script Thiet al. (2003); https://webblast.ipk-gatersleben.de/ misa/ (Fig. 11.2)

Manju et al. (2016)

version 3, Al-Dous et al. (2011) and at qatarweill.cornell.edu site

MISA Perl script Thiel et al. (2003)

Mokhtar et al. (2016) (continued)

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Table 11.1 (continued) Source/version of date palm genome

Software/method used and useful links

References

NCBI website

Msatfinder Thurston and Field (2005) https://github.com/knirirr/Msatfinder

Xiao et al. (2016)

Resequenced data of 62 cultivars, He et al. (2017) and published reference genomes

MISA Perl script Thiel et al. (2003)

He et al. (2017)

Resequenced data of 62 cultivars, He et al. (2017) and published reference genomes

MISA Perl script (Thiel et al. 2003) & results as DRDB http://drdb.big.ac.cn/ home

He et al. (2017)

Chloroplast genomes of 27 date cultivars and 14 species, Chaluvadi et al. (2019)

–

Chaluvadi et al. (2019)

Fig. 11.2 Work flow in SSR (simple sequence repeats) prediction

motifs metal-response elements (MREs) with a core structure of 5’-TGCRCNCG-3’ in 5’ end of the metallothionein (MT) genes in model plants of rice and Arabidopsis (Dąbrowska, 2012) are listed, which need to be matched by in silico approaches in date palm MTs. Aquaporin gene PdPIP1; 2 regions of date palm studied by Patankar et al. (2019) indicate abundant abiotic (drought and salinity) stress-inducible motifs in its 2 Kb promoter region. Patankar et al. (2019) also found 40 members representing the four groups of aquaporins in date palm genome. Genomes/materials used, gene/SNP annotation/ promoter motif approaches and other details are listed in Table 11.3.

The genomes used, SNP mining approaches and other details are listed in Table 11.2.

11.3.2 Phytochelators and Metallothioneins

11.3

Annotation of Genes and Promoter Motifs

11.3.1 Abiotic Stress-Responsive Genes Wax biosynthesis genes were expressed two-fold in heat- and drought-stressed date palm (Safronov et al. 2017). The desaturases of date palm (Sham and Aly 2012) were analyzed and their role in stress response was established using bioinformatics approaches. Motif abundances in abiotic (heat, drought) stress-induced genes (Safronov et al. 2017) were worked by bioinformatics approaches (Table 11.3). Promoter

The date palm seed/pit is also known for its heavy metal absorption abilities and is highly useful in phytoremediation of polluted sites to remove toxic metals (reviews: Al-Najar et al. 2019; Sivarajasekar et al. 2019) especially cadmium, copper, lead, chromium and harmful dioxins (Hanano et al. 2016). Phytochelator synthase and metallothionein genes are cysteinerich proteins known to play a crucial role in detoxification of heavy metals due to their metalbinding properties (review: Cobbett and Goldsbrough 2002). Phytochelator synthases date palm phytochelatin synthase (PdPCS1) was studied in silico by Zayneb et al. (2017) which is found to

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Table 11.2 Software and in silico resources, databases and URL links for mining of SNPs Source/version of date palm genome

Software/method used and useful links

References

Version 2 ACYX02000000 (NCBI), Al-Dous et al. (2011)

BW, Li and Durbin (2009); SAMtools, Li et al. (2009) http://samtools.sourceforge.net/

Al-Dous et al. (2011)

Date palm genome ACYX00000000 (NCBI) and at qatar-weill.cornell.edu

SAMtools, Li et al. (2009) for SNP

Mathew et al. (2014)

Plastid and mitochondrial genomes of 9 cultivars

BW, Li and Durbin (2009); SAMtools, Li et al. (2009); VCF tools, Danecek et al. (2011); http://vcftools.sourceforge.net/

Sabir et al. (2014)

Version 3, Al-Dous et al. (2011) and at qatarweill.cornell.edu site

Perl script

Mokhtar et al. (2016)

Resequenced data of 62 cultivars, He et al. (2017) and published reference genomes

SAMtools, Li et al. (2009), VCF tools, Danecek et al. (2011) and results as DRDB http://drdb.big.ac.cn/ home

He et al. (2017)

Resequencing data of Thareja et al. (2018)

SAMtools, Li et al. (2009); VCF tools, Danecek et al. (2011)

Thareja et al. (2018)

DRDB Date palm genomic resource database

Table 11.3 Software used for promoter motifs, annotation of genes and mapping markers Material/purpose

Software/method used and useful links

References

SNPEF 2.0.5 Cingolani et al. (2012) for effect of SNPs

Hazzouri et al. (2015)

Circos software, Krzywinski et al. (2009) for mapping SNPs

Mathew et al. (2014)

Al-Mssallem et al. (2013), date palm genome for oleosins

–

Hanano et al. (2016)

Heat and drought stress genes of date palm

Orthologue finder, Emms and Kelly (2015)

Safronov et al. (2017)

Heat and drought stress genes of date palm

Promoter motif abundance by AGRIS, Davuluri et al. (2003); PLACE, Higo et al. (1999)

Safronov et al. (2017)

Aquaporin PdPIP1;2 generegulatory regions

PlantPan 2.0, Chow et al. (2015) - promoter motifs

Patankar et al. (2019)

Annotation of SNPs Resequenced genomes, Hazzouri et al. (2015) Mapping of genes and markers Date palm genome ACYX00000000 (NCBI) Annotation of genes

be 5.7 Kb long with 528 amino acids which is distributed in 8 exons intervened by 7 introns. But the sugarcane metal chelator gene (SoPCS) gene is only 508 amino acids long distributed in 6 exons and intervened by 5 introns (Kolahi et al. 2018). Transcriptome resources of cadmium

treated (Rekik et al. 2019), gene expression data of metallothioneins and phytochelators in copper, cadmium or chromium (Chaâbene et al. 2018a, b) treated date palm seedlings offer an opportunity to use bioinformatic tools to explore interactions of individual metals with the date palm

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plant. Heavy metal chelation abilities of different plants and bioinformatic studies of the respective phytochelator synthase gene(s), structure, arrangement and their binding pocket studies show promise in understanding the mechanisms of metal binding in plants.

11.4

Mapping Genes and TraitLinked Markers

11.4.1 Gender Determination (SRY) Genic Region The date palm is dioecious and the sex of seedling-derived plants can only be identified as male or female at the time of flowering, i.e. 5– 8 years after planting. Extensive research is carried out on date palm to identify the sex of seedling date palms using biochemical and DNA markers (reviews: Awan et al. 2017; Heikrujam et al. 2015). The sex of date palm is proposed to follow the XY chromosome pattern similar to humans. The sex-linked gene SRY region possesses recombination arrest mechanism and is specific to male plants. Bioinformatic analysis of transcriptomes (Hamweih et al. 2010) and whole genomes (AlDous et al. 2011) of date palm and other dioecious species of Phoenix (Torres et al. 2018) was carried out between the male and female palms to understand the gene, SSR and SNP variation. The important genes present either exclusively or as additional copies, or show variation in SSR or SNPs in the SRY region found by genomic studies are alcohol dehydrogenase adh (Hamweih et al. 2010; Rajendran and Al-Mssallem 2007), MYB86-like gene (Cherif et al. 2016), three single-copy genes Cytochrome P450 gene CYP703, glycerol-3 phosphate acyl transferase 6 like GPAT3 or the LONELY GUY family of genes (LOG) (Torres et al. 2018), TBL3 (Transducin beta like 3 protein-3) (Ali et al. 2018). A 355 bp genomic region was specific to male and absent in female (Mohei et al. 2019) in the SRY region of date palm which is similar to 3’ end of the sex determining region in papaya and human genomes. The sex determining region of the date

palm is present in the telomeric region of the long arm of linkage group (LG) 12 (Hazzouri et al. 2019; Mathew et al. 2014) with a size of approximately 6–13 Mb (Cherif et al. 2016; Mathew et al. 2014) corresponding to a genetic distance of 26 cM (Mathew et al. 2014). The region also harbors 112 (Mathew et al. 2014) to 155 (Hazzouri et al. 2019) SNPs in the 6 Mb region. The SSR associated with sex in date palm is a compound motif of (ATG)2(AT)3C(ATG)(AT)3 which is basically an (AT)n repeat, probably interspersed with SNPs at 158 bases from the adh gene (Hamweih et al. 2010). The SSR markers with (GA)n repeat motifs (mPdCIR series) originally developed by Billotte et al. (2004) were found useful in sex determination in date palm. Some of them were (mPdCIR 48,78, 93) found heterozygous only in male palms (Elmeer and Mattat 2012) and few loci were found associated with sex determining region, viz., mPdCIR80, 50 and 52 by Cherif et al. (2013) and mPdCIR48 and 25 by Maryam et al. (2016). Few male-specific DNA fragments amplified by random primers OPD10-375 bp by Younis et al. (2008), (OPA02354 bp) by Dhawan et al. (2013), (OPC06SCAR-186 bp) by Al-Qurainy et al. (2018) and IS_A71_ ISSR((CA)8RG)-380 bp by AlAmeri et al. (2016), and a SCAR markerderived 354 bp fragment by Kharb and Mitra (2017) are reported. Priming sites of these random amplified regions and mining of SSRs with GA and AT repeat offer the opportunity to use gender-linked genomic resources (Al-Dous et al. 2011; Hazzouri et al. 2019). A consensus of either GA or AT repeats associated with sex-linked (SRY) genomic region in date palm was observed from the above reports. However, GATA repeats were found predominant in human Y (male) chromosome (Subramanian et al. 2003). The role of GA or TA or GATA repeats in Y chromosomes across dioecious organisms may offer interesting insights in recombination arrest region of Y chromosome organization. The fine physical mapping of the above-listed genes (Fig. 11.3) and genomic regions on date palm LG 12 chromosome developed by Mathew et al. (2014) by in silico tools were used by Premkrishnan and Arunachalam (2012) and Thiel et al. (2003) for

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Fig. 11.3 Sex-linked simple sequence repeats and genes in date palm

random markers and SSR motifs, respectively, to infer the genomic arrangement and the evolution of sex in date palm.

11.4.2 Fruit Traits Date palm fruits colors are yellow, purple (Fig. 11.4) or brown (Ghnimi et al. 2018) based on the cultivar. The yellow peel color of date

Fig. 11.4 Purple and yellow-colored date palm fruits

fruit is governed by a dominant negative mutation suppressing the purple color anthocyanin pigment production (Hazzouri et al. 2019). R2R3 MYB transcription factor (VIR locus) virescence gene (Hazzouri et al. 2015), orthologous to oil palm (Singh et al. 2014) determines the color of peel/pericarp of the date fruit. Retrotransposon insertion of 397 bp size in the exon region leads to truncation of gene R2-R3-MYB gene by 62 amino acids (Hazzouri et al. 2019) changing the

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Fig. 11.5 Workflow of bioinformatics-assisted identification of microRNA

color from purple to yellow. The taste of dry date fruit varies due to the sucrose, fructose and glucose contents (Ghnimi et al. 2017). The high sucrose content in the fruits of the delicious date variety Deglet Noor is due to the deletion of two of the three copies of invertase gene (Malek et al. 2020). A large deletion of 40 Kb region in cell wall invertase gene on linkage group 14 was found to influence sucrose content of dried date fruits (Hazzouri et al. 2019).

content in heat and drought-stressed date palm (Safronov et al. 2017). Two important target genes of salt-stressed miRNAs were the potassium channel AKT2-like protein and vacuolar protein sorting associated protein (Yaish et al. 2015). The potassium channel AKT2-like gene later was also found to be differentially expressed in salt stress treatments (Yaish et al. 2017). The materials and software used to locate miRNA target genes are listed in Table 11.4.

11.5

11.6

Prediction of MicroRNAs and Their Targets

MicroRNA (miRNA) molecules negatively regulate the gene expression at the posttranscriptional stage. The miRNAs were predicted using bioinformatics tools using a flow of similar work (Fig. 11.5) by a few researchers in date palm. The miRNAs present in date palm, two species of oil palm and banana were compared by Da Silva et al. (2016). They found the target genes in most miRNAs as transcription factors and the role of miR160 in regulating the primary root growth by regulating ARF (auxin response factor) genes in palms (Da Silva et al. (2016). After studying the miRNAs at the stages of fruit development of date palm (Xin et al. 2015), pda-nov-miR110 and pda-nov-miR204 were found to negatively regulate pyruvate kinase and beta galactosidase genes, respectively. Pyruvate kinase enzyme gene express at endosperm and works at the last step in glycolysis of plastid (Xiao et al. 2019). Metabolome of date palm indicate an eight-fold increase in galactose

Image Analysis and Structural Biology

11.6.1 Image Analysis Software is also used in phenotyping of date palm varieties in various roles from identifying the varieties by leaflet anatomy captured by fluorescence microscopy (Arinkin et al. 2014) differences in the size and shape of the seed (Terral et al. 2012). Red palm weevil is a serious insect pest in date palm whose incidence, before damage, is captured by infrared/thermal camera and inferred by the crop water stress index (Golomb et al. 2015). The materials and software used for image analysis reports on date palm are listed in Table 11.5.

11.6.2 Structural Biology The structural biology of the date palm genes is a fascinating area where in silico approaches play a crucial role. The phytochelator synthase gene

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Table 11.4 Software/in silico resources and URL links for analysis of RNA sequences Material/purpose

Software/method used and useful links

References

RNA analysis microRNA targets in stages of fruit development

psRNATarget, Dai and Zhao (2011) http://plantgrn.noble. org/psRNATarget

Xin et al. (2015)

microRNA targets in salinity- stressed roots, leaves

psRNATarget, Dai and Zhao (2011)

Yaish et al. (2015)

qPCR data for stability of reference genes for salinity and droughtstressed dates

miRDeepFinder, Xie et al. (2012)

Patankar et al. (2016)

Prediction of microRNA targets in date palm genome at NCBI (000413155.1_DPV01)

psRNATarget, Dai and Zhao (2011)

Da Silva et al. (2016)

Table 11.5 Software used for image analysis of date palm Material/purpose

Software/method used

References

Seeds of date varieties varying shape

Elliptical Fourier transform by R package, Terral et al. (2010)

Terral et al. (2012)

Date varieties leaflet cross-sectional images

LabView

Arinkin et al. (2014)

Thermal images red weevil affected date

Watershed image processing algorithm, Cohen et al. (2012)

Golomb et al. (2015)

Date varieties varying fruit color

Tomato analyzer, Darrigues et al. (2008); ImageJ, Abràmoff et al. (2004)

Hazzouri et al. (2019)

structure of date palm was predicted using the available structure of a similar gene from the cyanobacteria genus Nostoc as a template (Zayneb et al. 2017). Silicon transport protein genes of date palm displayed the hourglass-like structure (Bokor et al. 2019). Oleosins of date palm seeds possess a hydrophobic domain of 12 amino acid long (proline knot) motif that offers stability to lipid droplets (Hanano et al. 2016). Mutations in the R2R3-MYB transcription factor gene determining the peel color of oil palm (Singh et al. 2014) and date palm (Hazzouri et al. 2015, 2019) can be further analyzed using the 3D structure of the orthologous gene WEREWOLF from Arabidopsis made available by Wang et al. (2020). Materials and the software used for structure prediction of date palm genes and other docking studies are listed in Table 11.6.

11.7

Roadmap for Future Date Palm Bioinformatics and Palm Comparative Omics

The availability of several genomic resources of date palm offers the possibility to harness bioinformatic approaches to understand the genomic variations among cultivars and species. The date palm genome also offers the opportunity as a model to understand several other biological processes unexplored in palms with unique features such as the role of a large (40 Kb) deletion in cell wall invertase gene (Hazzouri et al. 2019), start codon mutations and XY recombination arrest region. The knowledge is currently available on organelle genomes and trait-linked genomic variation for fruit peel color,

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Table 11.6 Software/in silico resources and URL links for structural biology of date palm Gene name

Software/method used and useful links

References

rpoA gene RNA polymerase subunit A-secondary structure prediction

Genomic synteny, http://binf.gmu.edu: 8080/ Ka/Ks values, http://services.cbu.uib. no/tools/kaks/index_htm

Dhieb et al. (2012)

PhSERK gene—secondary and tertiary structure and ligand-binding sites

Phyre2, http://www.sbg.bio.ic.ac.uk/phyre2

Rekik et al. (2013)

DnMRE11 double-stranded break repair proteinligand binding

http://consurf.tau.ac.il/ for each residue’s conservation score; Ligand binding by COACH software, Yang et al. (2013)

Rekik et al. (2015)

Larvicidial property by ADMT properties, Feixiong et al. (2012)

Hussein et al. (2018)

Genes of date palm

Docking studies Cholinesterase of mosquito - chromone 1,2 from date seed

sugar content and sex determination. Sex determination regions of date palm need to be studied across the dioecious palms such as palmyra and double coconut (Lodoicea maldivica (J.F. Gmelin) Persoon)) for conserved regions. Date palm seeds with high potential for bioremediation of heavy metals and toxins show promise in exploring the in silico approaches to compare with model plant species which are poor in metal-responsive genes to uncover the science of metal scavenging in plants. Genes and markers linked to iron content in fruit, resistance to Bayoud and brittle leaf diseases of palm and the dwarf trait in other species such as Phoenix pusilla Roxb. and P. roebelenii O’Brien, need to be explored in future using in silico tools.

11.8

Conclusions and Prospects

Date palm genomes and other omics resources are increasingly available and are subject to in silico analysis to infer the biological processes in date palm. The resources also pave the way to infer similar mechanism in other palms by using comparative omics resources of other palms. Sex determination is one major area where vital information is generated using genomics and inferred by the bioinformatics approaches. The genes and markers linked to the trait inferred by

the studies form a major breakthrough in understanding dioecious plants, especially with the XY chromosome pattern. SNP markers identified so far in nuclear and organelle genomes of date palm offer potential for developing SNP chips for molecular marker-assisted selection of date palm. Microsatellites and microRNAs, gene sequences and their promoter motifs of date palm cast light on diversity, abiotic stress tolerance and heavy metal-scavenging abilities. Molecular modeling and docking studies increase the understanding of the structure of genes and binding sites and crucial residues. Tabulated information on the status of research by material and software employed for in silico studies on date palm omics is furnished. The tables presented herein can guide researchers to identify gaps, to plan future experiments and to draw inferences on the nutritional value iron content of fruits and biotic stress tolerance.

References Aberlenc-Bertossi F, Castillo K, Tranchant-Dubreuil C et al (2014) In silico mining of microsatellites in coding sequences of the date palm (Arecaceae) genome, characterization, and transferability. App Plant Sci 2(1):1300058 Abràmoff MD, Magalhães PJ, Ram SJ (2004) Image processing with ImageJ. Biophoton Int 11(7):36–42

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Al-Ameri AA, Al-Qurainy F, Gaafar AZ et al (2016) Male specific gene expression in dioecious Phoenix dactylifera L. (date palm) tree at flowering stage. Pak J Bot 48 (1):131–135 Al-Dous EK, George B, Al-Mahmoud ME et al (2011) De novo genome sequencing and comparative genomics of date palm (Phoenix dactylifera L.). Nat Biotech (6):521 Ali HBM, Abubakari A, Wiehle M et al (2018) Genespecific sex-linked genetic markers in date palm (Phoenix dactylifera L.). Genet Res Crop Evol 65(1):1–10 Al-Mssallem IS, Hu S, Zhang X et al (2013) Genome sequence of the date palm Phoenix dactylifera L. Nat. Comm 4:2274 Al-Najar B, Bououdina M, Vijaya JJ et al (2019) Removal of toxins from the environment using Date Palm seeds. In: Naushad M, Lichtfouse E (eds) Sustainable agriculture reviews 34 Springer, Cham, pp 207–245 Al-Qurainy F, Al-Ameri AA, Khan S et al (2018) SCAR marker for gender identification in Date Palm (Phoenix dactylifera L.) at the seedling stage. Int J Genom 2018:3035406 Arinkin V, Digel I, Porst D et al (2014) Phenotyping date palm varieties via leaflet cross-sectional imaging and artificial neural network application. BMC Bioinform 15(1):55 Arunachalam V (2012) Genomics of cultivated palms. Elsevier Inc, UK Awan FS, Jaskani MJ, Sadia B (2017) Gender identification in date Palm using molecular markers. In: AlKhayri JM, Jain SM, Johnson DV (eds) Date palm biotechnology protocols, vol II. Humana Press, New York, pp 209–225 Billotte N, Marseillac N, Brottier P et al (2004) Nuclear microsatellite markers for the date palm (Phoenix dactylifera L.): characterization and utility across the genus Phoenix and in other palm genera. Mol Ecol Notes 4(2):256–258 Bokor B, Soukup M, Vaculík M et al (2019) Silicon uptake and localisation in date palm (Phoenix dactylifera) - a unique association with sclerenchyma. Front Plant Sci 10:988 Chaâbene Z, Hakim IR, Rorat A et al (2018a) Copper toxicity and date palm (Phoenix dactylifera) seedling tolerance: monitoring of related biomarkers. Environ Toxicol Chem 37(3):797–806 Chaâbene Z, Rorat A, Hakim IR et al (2018b) Insight into the expression variation of metal-responsive genes in the seedling of date palm (Phoenix dactylifera). Chemosph 197:123–134 Chaluvadi SR, Young P, Thompson K et al (2019) Phoenix phylogeny, and analysis of genetic variation in a diverse collection of date palm (Phoenix dactylifera) and related species. Plant Diver 41(5):330–339 Cherif E, Zehdi S, Castillo K et al (2013) Male-specific DNA markers provide genetic evidence of an XY chromosome system, a recombination arrest and allow the tracing of paternal lineages in date palm. New Phytol 197(2):409–415 Cherif E, Zehdi-Azouzi S, Crabos A et al (2016) Evolution of sex chromosomes prior to speciation in

233 the dioecious Phoenix species. J Evol Biol 29 (8):1513–1522 Chow CN, Zheng HQ, Wu NY et al (2015) PlantPAN 2.0: an update of plant promoter analysis navigator for reconstructing transcriptional regulatory networks in plants. Nucl Acids Res 44(1):1154–1160 Cingolani P, Platts A, Wang LL et al (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6(2):80–92 Cobbett C, Goldsbrough P (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Ann Rev Plant Biol 53(1):159–182 Cohen Y, Alchanatis V, Prigojin A et al (2012) Use of aerial thermal imaging to estimate water status of palm trees. Precis Agric 13(1):123–140 Da Silva AC, Grativol C, Thiebaut F et al (2016) Computational identification and comparative analysis of miRNA precursors in three palm species. Planta 243(5):1265–1277 Dąbrowska G (2012) Plant metallothioneins: putative functions identified by promoter analysis in silico. Acta Biol Cracov Bot 54(2):109–120 Dai X, Zhao PX (2011) psRNATarget: a plant small RNA target analysis server. Nucl Acids Res 39(suppl_2):155–159 Danecek P, Auton A, Abecasis G et al (2011) The variant call format and VCFtools. Bioinform 27(15):2156–2158 Darrigues A, Hall J, van der Knaap E et al (2008) Tomato analyzer-color test: a new tool for efficient digital phenotyping. J Amer Soc Hort Sci 133(4):579–586 Davuluri RV, Sun H, Palaniswamy SK et al (2003) AGRIS: Arabidopsis gene regulatory information server, an information resource of Arabidopsis cisregulatory elements and transcription factors. BMC Bioinformat 4(1):25 Dhawan C, Kharb P, Sharma R et al (2013) Development of male-specific SCAR marker in date palm (Phoenix dactylifera L.). Tree Genet Genom 9(5):1143–1150 Dhieb A, Elleuch A, Kriaa W et al (2012) Molecular characterization and in silico analysis of RNA polymerase alpha subunit gene (rpoA) in date palm (Phoenix dactylifera L.) cv. Deglet Nour. Genes Genom 34(6):599–608 Elmeer K, Mattat I (2012) Marker-assisted sex differentiation in date palm using simple sequence repeats. 3 Biotech 2(3):241–247 Emms DM, Kelly S (2015) OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol 16(1):157 Fang Y, Wu H, Zhang T et al (2012) A complete sequence and transcriptomic analyses of date palm (Phoenix dactylifera L.) mitochondrial genome. PloS one 7(5):37164 Feixiong C, Li Weihua, Yadi Z et al (2012) Admet SAR: a comprehensive source and free tool for evaluating chemical ADMET properties. J Chem Inf Model 52 (11):3099–3105

234 Ferreira Filho JA, de Brito LS, Leão AP et al (2017) In silico approach for characterization and comparison of repeats in the genomes of oil and date palms. Bioinform Biol Insights 11:1177932217702388 Ghnimi S, Al-Shibli M, Al-Yammahi HR et al (2018) Reducing sugars, organic acids, size, color, and texture of 21 Emirati date fruit varieties (Phoenix dactylifera L.). NFS J 12:1–10 Ghnimi S, Umer S, Karim A et al (2017) Date fruit (Phoenix dactylifera L.): an underutilized food seeking industrial valorization. NFS J 6:1–10 Golomb O, Alchanatis V, Cohen Y et al (2015) Detection of red palm weevil infected trees using thermal imaging. In: Stafford JV (ed) Precision agriculture ‘15. Wageningen Academic Publishers, Netherlands, pp 322–337 Gros-Balthazard M, Hazzouri KM, Flowers JM (2018) Genomic insights into date palm origins. Genes 9 (10):502 Hamwieh A, Farah J, Moussally S et al (2010) Development of 1000 microsatellite markers across the date palm (Phoenix dactylifera L.) genome. Acta Hort 882:269–277 Hanano A, Almousally I, Shaban M et al (2016) Biochemical, transcriptional, and bioinformatic analysis of lipid droplets from seeds of date Palm (Phoenix dactylifera L.) and their use as potent sequestration agents against the toxic pollutant, 2, 3, 7, 8Tetrachlorinated Dibenzo-p-Dioxin. Front Plant Sci 7:836 Hazzouri KM, Flowers JM, Visser HJ et al (2015) Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop. Nat Comm 6:8824 Hazzouri KM, Gros-Balthazard M, Flowers JM et al (2019) Genome-wide association mapping of date palm fruit traits. Nat Comm 10(1):1–14 He Z, Zhang C, Liu W et al (2017) DRDB: an online date palm genomic resource database. Front Plant Sci 8:1889 Heikrujam M, Sharma K, Prasad M et al (2015) Review on different mechanisms of sex determination and sexlinked molecular markers in dioecious crops: a current update. Euphy 201(2):161–194 Higo K, Ugawa Y, Iwamoto M et al (1999) Plant cisacting regulatory DNA elements (PLACE) database: Nucl Acids Res 27(1):297–300 Hussein MA, Zyaan OH, Abo-Bakr H et al (2018) Synthesis, molecular docking and insecticidal activity evaluation of chromones of date palm pits extract against Culex pipiens (Diptera: Culicidae). Int J Mosq Res 5(4):22–32 Jain SM, Al-Khayri JM, Johnson DV (eds) (2011) Date palm biotechnology. Springer Science & Business Media, Dordrecht Khan A, Khan IA, Heinze B et al (2012) The chloroplast genome sequence of date palm (Phoenix dactylifera L. cv.‘Aseel’). Plant Mol Biol Rep 30(3):666–678 Kharb P, Mitra C (2017) Early sex identification in date palm by male-specific sequence-characterized

V. Arunachalam amplified region (SCAR) markers. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date palm biotechnology protocols, vol II. Humana Press. New York, NY, pp 199–207 Kolahi M, Yazdi M, Goldson-Barnaby A et al (2018) In silico prediction, phylogenetic and bioinformatic analysis of SoPCS gene, survey of its protein characterization and gene expression in response to cadmium in Saccharum officinarum. Ecotoxicol Environ Saf 163:7–18 Krzywinski M, Schein J, Birol I et al (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19(9):1639–1645 Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinform 25(14):1754–1760 Li H, Handsaker B, Wysoker A et al (2009) The sequence alignment/map format and SAMtools. Bioinform 25 (16):2078–2079 Malek JA, Mathew S, Mathew LS et al (2020) Deletion of beta-fructofuranosidase (invertase) genes is associated with sucrose content in date palm fruit. Plant Direct 4(5): Manju KP, Manimekalai R, Naganeeswaran SA et al (2016) Microsatellites mining in date palm (Phoenix dactylifera L.) and their cross transferability across Arecaceae family. Plant Omics 9(3):191–197 Maryam, Muhammad JJ, Awan FS, et al (2016) Development of molecular method for sex identification in date palm (Phoenix dactylifera L.) plantlets using novel sex-linked microsatellite markers. 3 Biotech 6 (1):22 Mathew LS, Spannagl M, Al-Malki A et al (2014) A first genetic map of date palm (Phoenix dactylifera) reveals long-range genome structure conservation in the palms. BMC Genom 15(1):285 Meerow AW, Krueger RR, Singh R et al (2012) Coconut, date, and oil palm genomics. In: Schnell R, Priyadarshan P (eds) Genomics of tree crops. Springer, New York, NY, pp 299–351 Mohamoud YA, Mathew LS, Torres MF et al (2019) Novel subpopulations in date palm (Phoenix dactylifera) identified by population-wide organellar genome sequencing. BMC Genom 20(1):498 Mohei EL, Mohasseb HAA, Al-Khateeb AA et al (2019) Identification and sequencing of date-SRY gene: a novel tool for sex determination of date palm (Phoenix dactylifera L.). Saudi J Biol Sci 26(3):514–523 Mokhtar MM, Adawy SS, El-Assal SEDS et al (2016) Genic and intergenic SSR database generation, SNPs determination and pathway annotations, in date palm (Phoenix dactylifera L.). PLoS one 11(7):0159268 Patankar HV, Al-Harrasi I, Al-Yahyai R et al (2019) Functional characterization of date palm aquaporin gene PdPIP1; 2 confers drought and salinity tolerance to yeast and Arabidopsis. Genes-Basel 10(5):390 Patankar HV, Assaha DV, Al-Yahyai R et al (2016) Identification of reference genes for quantitative realtime PCR in date palm (Phoenix dactylifera L.) subjected to drought and salinity. PloS one 11 (11):0166216

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Poncet V, Rondeau M, Tranchant C et al (2006) SSR mining in coffee tree EST databases: potential use of EST–SSRs as markers for the Coffea genus. Mol Genet Genom 276(5):436–449 Premkrishnan BV, Arunachalam V (2012) In silico RAPD priming sites in expressed sequences and iSCAR markers for oil palm. Int J Genom 2012: Rajendran P, Al-Mssallem IS (2007) Genetic basis for sex determination in date palm (Phoenix dactylifera L.). In: Keshavachandran R, Nazeem PA, Girija D (eds) Recent trends in horticultural biotechnology. New India Publishing Agency, New Delhi, India, pp 641–647 Rekik I, Chaabene Z, Grubb et al (2015) In silico characterization and molecular modeling of doublestrand break repair protein MRE11 from Phoenix dactylifera v degletnour. Theor Biol Med Model 12(1):23 Rekik I, Chaâbene Z, Kriaa W et al (2019) Transcriptome assembly and abiotic related gene expression analysis of date palm reveal candidate genes involved in response to cadmium stress. Comp Biochem Phys C 225: Rekik I, Elleuch A, Kriaa W et al (2013) Molecular cloning and in silico analysis of three somatic embryogenesis receptor kinase mRNA from date palm. Genetika 45:837–853 Sabir JS, Arasappan D, Bahieldin A et al (2014) Whole mitochondrial and plastid genome SNP analysis of nine date palm cultivars reveals plastid heteroplasmy and close phylogenetic relationships among cultivars. PLoS ONE 9(4):94158 Safronov O, Kreuzwieser J, Haberer G et al (2017) Detecting early signs of heat and drought stress in Phoenix dactylifera (date palm). PLoS ONE 12 (6):0177883 Sham A, Aly MA (2012) Bioinformatics based comparative analysis of omega-3 fatty acids in desert plants and their role in stress resistance and tolerance. Int J Plant Res 2:1 Singh R, Low ETL, Ooi LCL et al (2014) The oil palm VIRESCENS gene controls fruit colour and encodes a R2R3-MYB. Nat Comm 5:4106 Sivarajasekar N, Prakashmaran J, Naushad M et al (2019) Recent updates on heavy metal remediation using date stones (Phoenix dactylifera L.) - date fruit processing industry waste. In: Naushad M, Lichtfouse E (eds) Sustainable agriculture reviews 34 Springer, Cham, pp 193–206 Subramanian S, Mishra RK, Singh L (2003) Genomewide analysis of Bkm sequences (GATA repeats): predominant association with sex chromosomes and potential role in higher order chromatin organization and function. Bioinform 19(6):681–685 Terral JF, Newton C, Ivorra S et al (2012) Insights into the historical biogeography of the date palm (Phoenix dactylifera L.) using geometric morphometry of modern and ancient seeds. J Biogeogr 39(5):929–941 Terral JF, Tabard E, Bouby L et al (2010) Evolution and history of grapevine (Vitis vinifera) under domestication: new morphometric perspectives to understand seed domestication syndrome and reveal origins of ancient European cultivars. Ann Bot 105(3):443–455

235 Thareja G, Mathew S, Mathew LS et al (2018) Genotyping-by-sequencing identifies date palm clone preference in agronomics of the state of Qatar. PLoS ONE 13(12):0207299 Thiel T, Michalek W, Varshney R et al (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet 106(3):411–422 Thurston MI, Field D (2005) Msatfinder: detection and characterisation of microsatellites. https://github.com/ knirirr/Msatfinder Accessed 06 Jan 2020 Torres MF, Mathew LS, Ahmed I et al (2018) Genuswide sequencing supports a two-locus model for sexdetermination in Phoenix. Nat Comm 9(1):3969 Wang B, Luo Q, Li Y et al (2020) Structural insights into target DNA recognition by R2R3-MYB transcription factors. Nucl Acids Res 48(1):460–471 Xiao Y, Xia W, Ma J et al (2016) Genome-wide identification and transferability of microsatellite markers between Palmae species. Front Plant Sci 7:1578 Xiao Y, Xia W, Mason AS et al (2019) Genetic control of fatty acid composition in coconut (Cocos nucifera), African oil palm (Elaeis guineensis), and date palm (Phoenix dactylifera). Planta 249(2):333–350 Xie F, Xiao P, Chen D et al (2012) miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Mol Biol 80(1):75–84 Xin C, Liu W, Lin Q et al (2015) Profiling microRNA expression during multi-staged date palm (Phoenix dactylifera L.) fruit development. Genom 105(4):242– 251 Yaish MW, Patankar H V, Assaha DV et al (2017) Genome-wide expression profiling in leaves and roots of date palm (Phoenix dactylifera L.) exposed to salinity. BMC Genom 18(1):246 Yaish MW, Sunkar R, Zheng Y et al (2015) A genomewide identification of the miRNAome in response to salinity stress in date palm (Phoenix dactylifera L.). Front Plant Sci 6:946 Yang J, Roy A, Zhang Y (2013) Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment. Bioinform 29(20):2588–2595 Younis RA, Ismail OM, Soliman SS (2008) Identification of sex-specific DNA markers for date palm (Phoenix dactylifera L.) using RAPD and ISSR techniques. Res J Agric Biol Sci 4(4):278–284 Zayneb C, Imen RH, Walid K et al (2017) The phytochelatin synthase gene in date palm (Phoenix dactylifera L.): phylogeny, evolution and expression. Ecotoxicol Environ Saf 140:7–17 Zhao Y, Williams R, Prakash CS et al (2012) Identification and characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.). BMC Plant Biol 12(1):237

Index

A Agrobacterium tumefaciens, 121 Agrobiodiversity, 109, 122 Alcohol dehydrogenase, 228 Allosomes, 163 Amplified Fragment Length Polymorphism (AFLP), 88, 90, 103, 105, 106, 110–112, 114, 116, 163, 167, 171–173, 183, 186, 187, 193, 196 Anatomy, 33, 41, 230 Ancient remains, 182, 219 Androecium, 162–164 Aneuploidy, 84 Angiosperm, 37, 161, 202, 204, 207, 213, 219 Anthesis, 10, 157, 163 Archaeobotany, 55 Arecaceae, 3, 4, 29, 56, 111, 123, 168, 182, 202, 204, 207, 211, 219 Array-based markers, 114, 123 Assembly by Short Sequencing (ABySS), 190 Association mapping, 116, 117, 181, 192 Autosomes, 167

B Bacteria, 19, 20 Bioinformatics, 38, 120, 190, 195, 202, 204, 215, 223, 224, 226–228, 230–232 Biology, 4, 9, 29, 34, 37, 38, 44, 55, 103, 107, 161, 163, 182, 201, 207, 223, 224, 230, 232 Biotic stress, 78, 80, 83, 84, 118–121, 187, 197, 207, 232 Biparental inheritance, 201 Breeding, 34, 60, 77, 83, 101, 106, 112, 114, 116–120, 123, 124, 138, 144, 161–163, 173, 182, 183, 187, 188, 190, 193, 195, 197 Bunch weight, 149–153, 157

C Chemical mutagen, 77 Chemical traits, 153 Chloroplast, 46, 47, 103, 108, 114, 182, 201, 202, 207, 212, 215 Chloroplast DNA (cpDNA), 47, 64, 65, 119, 122, 123, 201, 202, 204–207

Chloroplast genome, 119, 194, 201, 202, 204–207, 219, 226 Chromosome, 38, 39, 64, 77, 78, 81, 82, 84, 92–94, 101, 102, 111, 112, 114, 116, 137, 161–163, 167, 168, 170–173, 182, 184, 186, 191, 192, 196, 211, 228, 232 Chromosome breakage, 82 Climate, 3, 7, 12, 14, 17, 19, 23, 25, 59, 162 Cloudberry, 162 Colchicine, 163 Commercial female cultivars, 135, 136, 149, 157 Comparative genomics, 39, 196, 204, 217, 223 Computational genomics, 181, 183 Concomitant Oncoprotein Detection (COD FISH), 170 Conserved DNA-Derived Polymorphism (CDDP), 112, 172, 173 Control influences, 84 Conventional breeding, 111 CRISPR/Cas9, 121 Cryoprotectant, 83, 91 Cultivars, 10, 12, 16–22, 33, 38, 55, 63, 66–70, 79, 81, 84, 88–93, 101, 102, 107–115, 118, 119, 121–123, 136–151, 153, 157, 159, 162, 164, 167, 169, 171, 181, 183, 187, 188, 193–197, 201, 204–207, 211, 212, 215, 217, 219, 224–227, 229, 231 Cultivation, 4, 6, 14–17, 19, 25, 34, 38, 55–58, 60, 62, 64, 66, 67, 70, 80, 108, 182, 183, 212 Culture cycle, 83 Cytogenetics, 38, 88, 167, 170, 184 Cytological abnormalities, 84 Cytological marker, 101, 102, 111

D Database, 33, 181, 183, 190–196, 213, 223–225, 227 Date, 3, 4, 11–15, 16, 18, 21–23, 25, 34, 55, 56, 61, 64, 67, 68, 70, 84, 102, 119, 183, 212, 231 Date fruits, 3, 11, 12, 22, 23, 56, 137, 157, 183, 212, 224, 229, 230 Date palm (Phoenix dactylifera), 3, 10, 11, 29, 55–58, 69, 78, 107, 135, 136, 181, 182, 201, 202, 205, 211 Date palm genome, 39, 77, 101, 102, 111, 116, 118, 121, 123, 168, 181, 183, 184, 186–197, 214, 217, 219, 223–227, 231, 232

© Springer Nature Switzerland AG 2021 J. M. Al-Khayri et al. (eds.), The Date Palm Genome, Vol. 1, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-73746-7

237

238 Date Palm Genome Database (DRDB), 193, 194, 226, 227 Date palm genomics, 167, 193, 197, 212, 227 Date palm genotypes, 80, 111, 141–143, 146, 147, 159, 172 Date Palm Molecular Marker Database (DPMMD), 194, 195 Date-SRY Gene, 172, 173 Dendrogram, 145–148, 191 De novo, 62, 83, 112, 191, 204 Diffusion, 58–60 Digital phenotyping, 224 Dioecious, 3, 9, 37, 38, 67, 78, 91, 107, 111, 135, 161–164, 172, 173, 211, 212, 224, 228, 232 Diploid, 38, 39, 167–169, 196, 197, 224 Dispersal, 34, 36, 57, 64 Distribution range, 34, 36, 45, 59 Diversity Array Technology (DArT), 107 DNA bank, 109 DNA barcode, 107, 113, 121, 122, 195 DNA damage, 77, 93, 94 DNA fingerprinting, 136, 137, 159, 170, 187 DNA marker, 86, 88, 102, 110, 111, 122, 136, 172, 173, 182–184, 187, 195, 196, 228 DNA polymorphisms, 106 DNA repair, 78, 94 DNA storage, 33 DNA taxonomy, 102, 114, 121, 123 Docking, 223, 231, 232 Domestication, 3, 4, 23, 25, 37, 55–57, 59–62, 64, 66–70, 88, 108, 117, 183, 197 Domestication model, 62 Domestication syndrome, 56–58, 67, 68, 70

E Eco-genetics, 135, 136, 157 Endosymbiosis, 201 Evolution, 6, 29, 34, 44, 48, 50, 56–58, 66, 68, 92, 107, 108, 116, 118, 122, 123, 162, 201, 202, 205, 212, 217, 219, 229 Evolutionary history, 30, 34, 55, 56 Evolution of fruit trait, 57, 66, 68, 136 Expressed Sequence Tag (EST), 103, 183, 186, 187, 193, 196, 225 Ex situ conservation, 109 Ex vitro, 82

F Fertilization, 17, 19, 136, 137, 157 Flowers, 3, 9–11, 13, 14, 16, 18, 34, 36–38, 42, 44, 45, 47, 48, 55, 56, 60–67, 82, 108, 117, 135, 137, 153, 157, 162–168, 224 Fluorescent In Situ Hybridization (FISH), 170 Forward repeat, 211, 215, 218

Index Fossils, 32, 36–38 Fruit, 3, 4, 6, 7, 9, 11–18, 21–23, 25, 30, 34, 37, 38, 44, 45, 55, 56, 58, 62, 66–70, 84, 86, 88, 108, 111, 115–118, 120, 135–139, 144, 145, 147, 149, 153, 155–157, 162, 167, 183, 189, 202, 211, 212, 223, 225, 229–232 Fruit color, 12, 69, 117, 118, 195, 231 Fruit peel color, 231 Fruit retention, 149–151, 157 Fruit set, 13, 16, 18, 80, 86, 90, 135, 149–153, 157, 162 Fruit traits, 55, 57, 66, 68–70, 136, 153, 155–157, 229 Fungi, 19

G Gametoclone, 82 GC contents, 194, 206, 213 Gene annotation, 223 Gene contents, 202, 211–214, 216, 219 Gene pyramiding, 118 Genetic changes, 79, 80, 83, 84, 90–93 Genetic diversity, 30, 34, 48, 50, 60, 62, 78, 101, 102, 105, 106, 109–114, 116, 122, 123, 138, 139, 144, 147, 149, 171, 173, 187, 197, 212 Genetic engineering, 207 Genetic fidelity, 78, 84, 91–94 Genetic mapping, 106, 114, 116, 117, 119, 124, 181–184, 186–189, 196 Genetic relationship, 108, 114, 135, 136, 144–146, 149, 151–153, 155, 157, 171 Genetic resources, 91, 94, 107, 109, 110, 114, 116, 118, 120, 123, 161, 183 Genetics, 7, 18, 19, 30, 34, 40–42, 44–46, 48, 55–57, 59, 61–66, 68, 70, 71, 77–84, 86, 89–94, 101, 102, 104, 105, 107–112, 114, 116–124, 135–137, 143–145, 152, 161–163, 167, 169, 170, 172, 173, 181–184, 186–188, 190–197, 201, 212, 213, 223, 228 Genetic similarity, 145 Genome Association and Prediction Integration Tool (GAPIT), 191 Genome editing, 77, 94, 121 Genome instability, 94 Genome mapping, 173, 181–184, 188–191 Genome mapping software, 190 Genome size, 38–40, 78, 116, 168, 169, 194, 204, 212, 214 Genome Wide Association Study (GWAS), 69, 106, 108, 114, 116–118, 172, 183, 186, 189–191, 196 Genomics, 4, 36, 38, 39, 47–50, 55, 56, 61–63, 65, 66, 70, 77, 78, 80, 88, 89, 93, 103–105, 111, 119–121, 137, 142, 143, 170, 172, 184, 188, 191, 195–197, 202–204, 211–213, 215, 217, 223, 224, 228, 229, 231, 232 Genomic selection, 118, 119

Index Genotype, 69, 70, 80–82, 90–93, 101, 109, 112, 136, 143, 145, 146, 148, 162, 168, 169, 181, 182, 188, 192, 193, 197 Genotyping by Sequencing (GBS), 106, 107, 112, 116, 118, 186, 188, 189 Gulf, 56, 60, 61, 64, 118 Gynoecium, 162–164

H Habitat, 7, 8, 12, 30, 34–36, 44, 60, 109 Harvest, 3, 21, 22, 68, 135 Hermaphrodite, 162, 164, 165, 168 High-throughput genotyping, 107, 114 High-Throughput Phenotyping Platform (HTPP), 108, 114, 118, 119 Hybridization, 4, 7, 34, 38, 47, 48, 50, 56, 57, 60, 62–66, 70, 103, 106, 107, 122, 171, 186 Hypogynous, 166

I Illumina sequencing, 204 Image analysis, 223, 224, 230, 231 Inflorescence, 3, 9–11, 13, 18, 38, 67, 82, 84, 87, 135, 163, 164, 166 Insertion-deletion, 223 In silico, 223–228, 230–232 In situ conservation, 109 In Situ Hybridization (ISH), 170, 171 Inter Simple Sequence Repeats (ISSR), 88–90, 92, 104, 105, 109, 112, 114, 136, 163, 167, 171, 187 Introgression, 62–66, 71, 108, 114 Intron-Targeted Amplified Polymorphism (ITAP), 112, 172, 173 Invertase, 69, 118, 223, 230, 231 Inverted repeat region, 201, 202 In vitro, 38, 78–84, 86, 88–94, 109, 120, 121, 164, 167, 170, 187 Irrigation, 3, 13, 14, 17, 19, 25, 67, 135 ITS regions, 108, 114

K Kompetitive Allele Specific PCR (KASPTM), 120

L Leaves, 3, 9, 10, 13, 17, 18, 23, 30, 33, 36, 68, 84, 87, 108, 118, 163, 169, 202, 231 Linkage Disequilibrium (LD), 63, 112, 117–119, 190 Locus barcode, 121

M Mapping genes, 228 Marker, 46, 48, 78, 87–92, 101–107, 109–112, 114, 116–119, 121–124, 136–139, 142, 143, 145, 146,

239 148, 161, 164, 167, 169–173, 182, 184, 186–189, 192–195, 224, 227–229, 232 Marker-assisted backcross, 118 Marker-assisted recurrent selection, 111, 118 Marker-Assisted Selection (MAS), 107, 114, 116, 118, 119, 232 Maximum Likelihood (ML), 47, 217, 220 Megaspores, 164 Meiosis, 84, 163 Meristematic explants, 93 Metabolism, 78, 88, 111, 113, 202, 224 Metal absorbing ability, 226 Metaphase, 163, 171 Metaxenia, 12, 135–137, 139, 149, 153, 157 Methylation-Sensitive Amplified Polymorphism (MSAP), 90, 92 MicroRNA, 163, 223, 224, 230–232 Microspores, 164 Mitochondrial genome, 47, 48, 190, 211–213, 215–217, 219, 220, 223, 227 Mitosis, 82, 92, 163 Molecular markers, 78–80, 84, 86, 90, 93–95, 101–103, 105, 107, 109, 111–114, 116, 118, 122–124, 139, 144–147, 161–163, 167, 170–173, 181, 184, 186, 187, 189, 223, 224 Molecular modeling, 223 Molecular studies, 30, 49, 59, 60, 108, 137, 138 Monocotyledonous, 3, 25, 119, 135, 206, 212 Monoecious, 164 Monograph, 4, 31–33, 39, 40, 44 Mononucleotide repeat, 215 Morphology, 7, 33, 41, 44, 46, 84, 85, 163, 164, 169, 183 Mosquito, 232 Multiplication cycle, 83, 84 Mutagenesis, 77, 79, 93 Mutagenic agent, 84

N National Center for Biotechnology Information (NCBI), 195, 211, 213, 225–227, 231 NCBI Organelle Genome Resources database, 202 Next Generation Sequencing (NGS), 106, 119, 137, 181–183, 186–188, 190, 191, 193–196, 202–204, 206, 211, 212 North Africa, 3, 4, 14–17, 19, 20, 22, 23, 25, 29, 30, 55, 56, 58, 59, 62–66, 68, 118, 138, 194, 211, 212 Nuclear genome, 47, 122, 136, 148, 182 Nucleolar heterochromatin, 82 Nucleotide databases, 206, 207

O Offshoot, 5, 6, 9, 18, 19, 34, 67, 70, 84, 90, 92, 170 Oil palm, 30, 39, 69, 85, 90, 91, 93, 112, 116–118, 120, 121, 224, 229–231 Organelle, 47, 48, 181, 182, 201, 202, 204, 212, 213, 224, 231, 232 Organized cultures, 79

240 Organogenesis, 18, 80, 93 Origin, 3, 4, 12, 13, 21, 23, 25, 34, 36, 37, 49, 55–70, 82, 84, 101, 107, 108, 122, 173, 182, 183, 197, 211 Overexpression, 117, 207

P Palindromic repeat, 101, 211, 215, 218 Palm, 5, 6, 13, 18, 19, 23, 25, 29, 30, 34, 36–39, 59, 67, 68, 90, 91, 107, 112–114, 118, 120, 121, 149–152, 157, 170, 224 PCR-based marker, 103, 111, 173, 174 Perennial, 14, 15, 17, 21, 55–58, 62, 66, 70, 212 Pests, 13, 19, 21, 22, 25, 230 Phenology, 45, 68, 109 Phoenix, 3–6, 9, 11, 12, 19, 20, 29–42, 44–50, 55–60, 62–64, 66, 69, 70, 81, 88, 94, 101, 107, 112–114, 116, 118, 122, 123, 136, 170, 228 Phoenix acaulis, 5, 7, 30, 31, 36, 39, 45, 47 Phoenix andamanensis, 5, 7, 31, 34, 35, 40, 42, 44, 46 Phoenix atlantica, 5, 7, 30, 31, 39, 40, 42, 44–47, 65, 108, 114 Phoenix caespitosa, 5, 7, 30, 31, 34, 35, 40, 42, 44, 46, 47 Phoenix canariensis, 5, 7, 20, 30, 31, 34, 38–40, 42, 44–47, 57, 69, 108, 114, 122, 164, 168 Phoenix chevalieri, 32, 40, 43–45 Phoenix dactylifera, 3, 8, 10, 11, 29, 41, 47, 55–57, 59–61, 66, 69, 71, 78, 113, 122, 135, 136, 164, 181, 182, 202, 203, 205, 211–220 Phoenix iberica, 32, 40, 43–45, 108 Phoenix loureiroi, 30, 31, 34–36, 38–40, 42, 44, 45, 47, 69 Phoenix paludosa, 5, 7, 30, 31, 34, 35, 39, 40, 42, 44, 46, 47 Phoenix pusilla, 5, 7, 31, 36, 39, 42, 44, 45, 47, 153, 156, 232 Phoenix reclinata, 5, 7, 30, 31, 33–35, 38–40, 43–47, 69, 122 Phoenix roebelenii, 7, 30, 31, 34, 35, 39, 40, 43–47, 69, 232 Phoenix rupicola, 6, 8, 30, 31, 34–36, 39, 40, 43–47 Phoenix sylvestris, 6, 8, 30, 31, 33, 34, 36–40, 42, 44–49, 57, 69, 107, 108, 114, 123 Phoenix theophrasti, 5, 6, 8, 30, 31, 34, 35, 37, 39, 40, 42, 44–48, 58, 60, 63–66, 69, 107, 108, 114 Photosynthetic, 9, 201, 202 Photosystems, 201 Phylogenetic analysis, 48, 148, 171, 211, 215, 217, 219 Phylogenetics, 46, 48–50, 56, 102, 107, 110, 122, 123, 135, 137, 138, 145, 181, 201, 207, 217 Phylogenetic tree, 45, 46, 108, 123, 137, 138, 145–147, 149, 194, 204, 206, 220 Phylogenomics, 39, 47, 48 Phylogeny, 29, 34, 46–48, 102, 107, 108, 122, 123, 207, 220 Physical traits, 153 Physiology, 163, 183 Phytochelator, 223, 226, 228, 230 Phytoremediation, 226

Index Pistillate flower, 10 Plant Genome and System Biology (PGSB), 194 Plant systematic, 122 Plastid genome, 48, 121, 190 Ploidy, 79–82, 120, 163, 169 Pollen, 12, 14, 17, 36–38, 59, 135–139, 145–153, 155–157, 159, 162, 163, 166, 167, 170 Pollenizer, 136, 144, 146, 148–153, 157 Pollination, 3, 10, 13–18, 38, 45, 67, 68, 84, 102, 135–138, 149, 162, 173 Pollinator, 38, 45, 137, 153, 162 Polyacrylamide Gel Electrophoresis (PAGE), 88, 104, 106, 170 Polymerase Chain Reaction (PCR), 86, 89, 90, 101, 103–107, 111, 112, 135, 137, 139, 140, 142, 143, 145, 171–173, 186, 194 Polymorphism, 88, 90, 92, 102–107, 109, 110, 114, 119, 138–144, 163, 167, 171, 172, 181–183, 186, 187, 193, 223 Polyploidy, 82, 84, 102, 167, 169, 196 Polysomy, 82 Post-NGS, 181, 183, 184, 186, 188, 189 Pre-NGS, 181, 183, 184, 186–189, 196 Primordia, 164 Processing, 22, 106, 190, 191, 231 Promoter motif, 223, 224, 226, 227, 232 Propagation, 18, 38, 66, 67, 70, 78, 91, 93, 102, 121, 153, 162, 183, 202 Protein coding genes, 205, 213, 214 Proximity matrix, 145–147 Pyrosequencing, 106, 119, 204, 205, 212

Q QTL mapping, 114, 116, 187, 189, 192, 193 Quadripartite, 201, 204–207 Quantitative Trait Loci (QTL), 69, 114, 116–119, 186–189, 192, 197

R Rachillae, 9, 10, 163 Rachis, 9, 30, 33, 163, 164, 166 Random Amplified Polymorphic DNA (RAPD), 88–90, 92, 103, 104, 106, 110–114, 135–139, 141–146, 148, 159, 163, 167, 171, 172, 181, 183, 186, 187, 196 RAPD–PCR markers, 138 Reduced Representation Library (RRL), 186, 188 Regenerated plants, 81, 93 Regeneration, 77, 79–81, 83, 86, 92 Relative humidity, 12, 13, 158, 159 Re-sequencing, 117, 188, 197, 224, 227 Restriction Fragment Length Polymorphism (RFLP), 88, 90, 92, 103, 107, 110, 114, 116, 167, 171, 182, 183, 186, 187, 196 Restriction site associated DNA sequencing (RAD), 186, 188 Ribosomal RNA, 213

Index Root, 5, 6, 9, 12, 17, 18, 20, 23, 34, 80, 82, 104, 111, 118, 119, 163, 167, 168, 170, 188, 230, 231

S Secondary metabolites, 68, 163, 197 Seed morphometrics, 58 Seeds, 3, 11, 12, 18, 23, 33, 34, 36, 37, 45, 46, 56–59, 67, 68, 70, 91, 102, 107–109, 112, 115, 135–137, 149, 153, 156, 162, 169, 182, 188, 192, 223, 226, 230–232 Seed shape, 58 Seed size, 58, 68 Sequence-based markers, 106 Sequence Characterized Amplified Region (SCAR), 106, 112, 145, 167, 172, 228 Sequence of Gene Recognition (sgRNA), 121 Sex determination, 18, 38, 111, 116, 161–163, 169, 171–173, 189, 196, 197, 223, 228, 232 Sex-specific PCR based markers, 112 Simple Sequence Repeat (SSR), 88, 103–105, 110–112, 114, 116, 117, 137, 138, 167, 171, 172, 181, 183, 186–188, 194–196, 206, 211, 215, 218, 223–225, 228, 229 Single-copy region, 201, 202 Single Nucleotide Polymorphism (SNP), 46, 47, 78, 79, 86, 88, 106, 107, 111, 112, 114, 116–121, 137, 138, 149, 181, 183, 186–196, 223–228, 232 Single Nucleotide Variants (SNVs), 201, 205, 207 Software, 143, 184, 190–193, 204, 223–225, 227, 230–232 Somaclonal variation, 18, 77–82, 84, 86, 90, 92–94, 186, 187 Somaclone, 78, 80, 82–84, 86, 87, 187 Somatic embryogenesis, 67, 78, 80, 81, 83, 85, 88, 89, 93, 94, 120, 121 Species distribution, 36, 37, 60 Spectral Karyotyping (SKY), 170 Staminate flower, 162, 163 Start codon targeted polymorphism (SCoT), 88, 91, 110–112, 172, 173 Storage, 21, 22, 91, 108, 109 Sugar content, 12, 195, 232 Sympatry, 34 Synteny, 211, 232 Systematics, 29, 30, 34, 47, 48, 50, 109, 122

T Tandem repeats, 103, 211, 215, 218

241 Targeting Induced Local Lesions in Genome (TILLING), 101, 120, 124 Taxonomy, 4, 7, 19, 20, 29, 31, 34, 39, 50, 102, 114, 121–123, 215 Temperature, 9, 11–14, 17, 22, 25, 82, 90, 91, 104, 143, 153, 156–158, 183 Thinning, 17 Tissue culture, 18, 67, 77–84, 87, 88, 90, 92–94, 116, 120 Totipotent cells, 163 Trait analysis by association, evolution and linkage (TASSEL), 190, 191 Transcription factor, 69, 78, 88, 90, 108, 113, 118, 121, 225, 229–231 Transcriptome, 78, 112, 188, 223, 224, 227, 228 Transfer RNA, 205 Transposable element, 39, 104, 187 Transposon, 86, 88, 104, 105, 194 True-to-type, 70, 79, 81, 89, 91 Trunk, 3, 5–9, 18, 23, 108, 163

U Unisexual flower, 161, 164

V Variable Number of Tandem Repeats (VNTR), 186 Vegetative propagation, 67, 79, 162 Virescens, 69, 117, 118, 225, 229

W Weevils, 19, 25, 38, 224, 230, 231 West Asia, 55, 56, 62–64, 66, 68, 211 Whole Genome Resequencing (WGRS), 63, 186, 188, 189 Wild ancestor, 57, 58 Wild relative, 30, 57, 58, 60, 62–64, 68–70, 107, 123, 197

X Xenia, 12, 135–137, 149, 153, 157

Y Yield, 9, 14–18, 23, 24, 67, 68, 93, 112, 117, 118, 121, 135–139, 145, 149–153, 157, 183, 192, 193, 195, 197, 202