Genomic Designing for Abiotic Stress Resistant Fruit Crops 3031098749, 9783031098741

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Chittaranjan Kole   Editor

Genomic Designing for Abiotic Stress Resistant Fruit Crops

Genomic Designing for Abiotic Stress Resistant Fruit Crops

Chittaranjan Kole Editor

Genomic Designing for Abiotic Stress Resistant Fruit Crops

Editor Chittaranjan Kole Raja Ramanna Fellow Department of Atomic Energy Government of India ICAR—National Institute for Plant Biotechnology New Delhi, India

ISBN 978-3-031-09874-1 ISBN 978-3-031-09875-8 (eBook) https://doi.org/10.1007/978-3-031-09875-8 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 This work is subject to copyright. All rights are solely and exclusively licensed 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

Dedicated to

Dr. K. L. Chadha Padma Shri Awardee, former Deputy Director General (Horticulture), Indian Council of Agricultural Research and Founder President of the Indian Academy of Horticultural Sciences With regards and gratitude for his generous appreciations of my scientific contributions and service to the global academic community, and his constant support and encouragement during my professional journey!

Preface

Crop production is drastically affected due to external or environmental stresses. The biotic stresses cause significant yield losses in the range of 31–42% together with 6–20% loss during the post-harvest stage. The abiotic stresses also aggravate the situation with crop damage in the range of 6–20%. Understanding the mechanisms of interaction of plants with the biotic stresses caused by insects, bacteria, fungi, viruses, and oomycetes, etc., and abiotic stresses due to heat, cold, drought, flooding, submergence, salinity, acidity, etc., is critical to develop resilient crop varieties. Global warming and climate change are also causing emergence of new diseases and insects together with newer biotypes, and physiological races of the causal agents in one hand and aggravating the abiotic stress problems with additional extremes and unpredictability. Development of crop varieties resistant and/or adaptive to these stresses is highly important. The future mission of crop improvement should, therefore, lay emphasis on the development of crop varieties with optimum genome plasticity by possessing resistance or tolerance to multiple biotic and abiotic stresses simultaneously. A moderate estimation of world population by 2050 is about 9.3 billion that would necessitate an increase of crop production by about 70%. On the other hand, the additional losses due to climate change and global warming somewhere in the range of 10 to 15% should be minimized. Therefore, increase in the crop yield as well as minimization of its loss should be practiced simultaneously focusing both on ‘adaptation’ and ‘mitigation’. Traditional plant breeding practiced in the last century contributed a lot to the science of crop genetic improvement. Classical plant breeding methods including selection, hybridization, polyploidy, and mutation effectively catered to the basic F5 needs—food, feed, fiber, fuel and furniture. The advent of molecular breeding and genetic engineering in the latter part of that century complimented classical breeding that addressed the increasing needs of the world. The twenty-first century came with a gift to the geneticists and plant breeders with the strategy of genome sequencing in Arabidopsis and rice followed by the tools of genomics-aided breeding. More recently, another revolutionary technique, genome or gene editing, became available for genetic correction of crop genomes! The travel from ‘plant breeding’ based on visual or perceivable selection to ‘molecular breeding’ assisted by linked markers to vii

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‘transgenic breeding’ using genetic transformation with alien genes to ‘genomicsaided breeding’ facilitated by known gene sequences has now arrived at the age of ‘genetic rectification’ employing genome or gene editing. Knowledge on the advanced genetic and genomic crop improvement strategies including molecular breeding, transgenics, genomic-assisted breeding and the recently emerged genome editing for developing resistant, tolerant and/or adaptive crop varieties is useful to students, faculties and scientists in the public and private universities and organizations. Whole genome sequencing of most of the major crop plants followed by genotyping-by-sequencing has facilitated identification of exactly the genes conferring resistance, tolerance or adaptability leading to gene discovery, allele mining and shuttle breeding which is turn opened up the scope for ‘designing’ or ‘tailoring’ crop genomes with resistance/tolerance to biotic and abiotic stresses. To my mind, the mission of agriculture in this century is FHNEE security meaning food, health, nutrition, energy and environment security. Hence, genome designing of crops should focus on breeding of varieties with higher yields and improved qualities of the five basic F5 utilities; nutritional and neutraceutical compounds; and other industrially and aesthetically important products, and possibility of multiple utilities. For this purpose of ‘precise’ breeding employment of the genetic and genomic techniques individually or in combination as and when required, will play a crucial role. The chapters of the 12 volumes of this twin book series entitled, “Genomic Designing for Biotic Stress Resistant Crops” and “Genomic Designing for Abiotic Stress Resistant Crops”, will deliberate on different types of biotic and abiotic stresses and their effects on and interaction with crop plants; will enumerate the available genetic diversity with regard to biotic or abiotic stress resistance among cultivars; illuminate on the potential gene pools for utilization in interspecific gene transfer; will brief on the classical genetics of stress resistance and traditional breeding for transferring them to their cultivated counterparts; will discuss on molecular mapping of genes and QTLs underlying stress resistance and their marker-assisted introgression into elite crop varieties; will enunciate different emerging genomics-aided techniques including genomic selection, allele mining, gene discovery and gene pyramiding for developing smart crop varieties with genetic potential to produce F5 of higher quantity and quality; and also will elaborate the case studies on genome editing focusing on specific genes. Most of these chapters will discuss on the success stories of genetic engineering in the relevant crops specifically for generating crops with resistance and/or adaptability to diseases, insects and abiotic stresses. There are obviously a number of reviews and books on the individual aspects of plant molecular breeding, genetic engineering and genomics-aided breeding on crops or on agro-economic traits which includes the 100-plus books edited by me. However, there is no comprehensive reviews or books available that has coverage on crop commodity groups including cereals and millets, oilseeds, pulses, fruits and nuts, vegetables and technical or industrial crops, and modern strategies in single volumes with precise focuses on biotic and abiotic stresses. The present volumes will fill this gap with deliberations on about 120 important crops or their groups.

Preface

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This volume on “Genomic Designing for Abiotic Stress Resistant Fruit Crops” includes seven chapters focused on apple, banana, citrus, grapevine, almond, cherries, and berries contributed by 36 scientists from 9 countries including India, Iran, Italy, Lithuania, Morocco, Spain, Tunisia, UK, and USA. I remain immensely thankful for their highly useful contributions. I am indebted to my wife Phullara who as always has assisted me directly in editing these books and indirectly through maintaining an academic ambience to pursue my efforts for science and society pleasantly and peacefully. New Delhi, India

Chittaranjan Kole

Contents

1 Genomic Approaches to Improve Abiotic Stress Tolerance in Apple (Malus × domestica) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Madhushree Dutta, Rajesh Kumar Singh, and Gaurav Zinta

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2 Genomic Designing of Abiotic Stress Tolerance in Banana . . . . . . . . . . I. Ravi, M. Mayil Vaganan, T. Anithasree, K. Stellamary, and S. Uma

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3 Genomic Design for Abiotic Stress Resistant Citrus . . . . . . . . . . . . . . . . Angelo Sicilia, Supratim Basu, and Angela Roberta Lo Piero

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4 Development of Abiotic Stress Resistant Grapevine Varieties . . . . . . . Sanjay Kumar Singh, Satyabrata Pradhan, Hare Krishna, M. Alizadeh, Chavlesh Kumar, Nripendra Vikram Singh, Amol K. Jadhav, D. Ramajayam, Rahul Dev, and Rakesh Singh

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5 Genomic Designing for Drought Tolerant Almond Varieties . . . . . . . . 161 Pedro J. Martínez-García, Ossama Kodad, Hassouna Gouta, Sama Rahimi Devin, Angela S. Prudencio, Manuel Rubio, and Pedro Martínez-Gómez 6 Applications of Biotechnological Tools for Developing Abiotic Stress Tolerant Cherries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Shiv Lal and Mahendra Kumar Verma 7 Genomic Design of Abiotic Stress-Resistant Berries . . . . . . . . . . . . . . . . 197 Rytis Rugienius, Jurgita Vinskien˙e, Elena Andri¯unait˙e, Šar¯un˙e Mork¯unait˙e-Haimi, Perttu Juhani-Haimi, and Julie Graham

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Contributors

Alizadeh M. Horticulture Department, Gorgan University of Agricultural Sciences & Natural Resources, Gorgan, Iran Andriunait˙ ¯ e Elena Lithuanian Research Centre for Agriculture and Forestry, Institute of Horticulture, Babtai LT-54333, Lithuania Anithasree T. ICAR-National Research Center for Banana, Trichy, TN, India Basu Supratim Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR, USA Dev Rahul ICAR-Vivekananda Parvatiya Dugalkhola, Almora, Uttarakhand, India

Krishi

Anusandhan

Sansthan,

Devin Sama Rahimi Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran Dutta Madhushree Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology (CSIR-IHBT), Palampur, Himachal Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Gouta Hassouna Institut de l’Olivier, Sfax, Tunisia Graham Julie James Hutton Institute, Dundee DD2 5DA, Scotland, UK Jadhav Amol K. Division of Fruits and Horticultural Technology, ICAR-Indian Agricultural Research Institute, New Delhi, India Juhani-Haimi Perttu Lithuanian Research Centre for Agriculture and Forestry, Institute of Horticulture, Babtai LT-54333, Lithuania Kodad Ossama Departement d’Arboriculture Et Viticulture, Ecole Nationale d’Agriculture de Mekne’s, Mekne, Maroc Krishna Hare ICAR-Indian Institute of Vegetable Research, PO Jakhini (Shahanshahpur), Varanasi, Uttar Pradesh, India xiii

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Contributors

Kumar Chavlesh Division of Fruits and Horticultural Technology, ICAR-Indian Agricultural Research Institute, New Delhi, India Lal Shiv ICAR-National Research Center On Seed Spices, Ajmer, Rajasthan, India Lo Piero Angela Roberta Dipartimento di Agricoltura Alimentazione e Ambiente, Università degli Studi Catania, Catania, Italy Martínez-García Pedro J. Departamento de Mejora Vegetal Grupo de Mejora Genética de Frutales, CEBAS-CSIC, Espinardo, Murcia, Spain Martínez-Gómez Pedro Departamento de Mejora Vegetal Grupo de Mejora Genética de Frutales, CEBAS-CSIC, Espinardo, Murcia, Spain Morkunait˙ ¯ e-Haimi Šarun˙ ¯ e Lithuanian Research Centre for Agriculture and Forestry, Institute of Horticulture, Babtai LT-54333, Lithuania Pradhan Satyabrata Division of Fruits and Horticultural Technology, ICARIndian Agricultural Research Institute, New Delhi, India Prudencio Angela S. Departamento de Mejora Vegetal Grupo de Mejora Genética de Frutales, CEBAS-CSIC, Espinardo, Murcia, Spain Ramajayam D. ICAR-National Research Centre for Banana, Tiruchirapalli, India Ravi I. ICAR-National Research Center for Banana, Trichy, TN, India Rubio Manuel Departamento de Mejora Vegetal Grupo de Mejora Genética de Frutales, CEBAS-CSIC, Espinardo, Murcia, Spain Rugienius Rytis Lithuanian Research Centre for Agriculture and Forestry, Institute of Horticulture, Babtai LT-54333, Lithuania Sicilia Angelo Dipartimento di Agricoltura Alimentazione e Ambiente, Università degli Studi Catania, Catania, Italy Singh Nripendra Vikram ICAR-National Research Centre On Pomegranate, Solapur - Pune Highway, India Singh Rajesh Kumar Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology (CSIR-IHBT), Palampur, Himachal Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Singh Rakesh ICAR-National Bureau of Plant Genetic Resources, New Delhi, India Singh Sanjay Kumar Division of Fruits and Horticultural Technology, ICARIndian Agricultural Research Institute, New Delhi, India Stellamary K. ICAR-National Research Center for Banana, Trichy, TN, India Uma S. ICAR-National Research Center for Banana, Trichy, TN, India

Contributors

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Vaganan M. Mayil ICAR-National Research Center for Banana, Trichy, TN, India Verma Mahendra Kumar ICAR- Indian Agricultural Research Institute, New Delhi, India Vinskien˙e Jurgita Lithuanian Research Centre for Agriculture and Forestry, Institute of Horticulture, Babtai LT-54333, Lithuania Zinta Gaurav Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology (CSIR-IHBT), Palampur, Himachal Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India

Abbreviations

2-DE 6mA ABA ABF ABRE ADH AF AFLP AFP AGPase ALA AMFs AP AP2/EREBP APEDA APX AQP ARF ASR ATAF ATG8f BAC BSA bZIP CAGR CAPS Cas9 CAT CBD CBF

Two-dimensional electrophoresis N6-methadenine Abscisic acid ABRE binding factor ABA-responsive element Alcohol dehydrogenase Annual-fruiting Amplified fragment length polymorphism Antifreeze protein (from Antarctic fish) ADP-glucose pyrophosphorylase 5-aminolevulinic acid Arbuscular mycorrhizal fungi Apetala gene AP2/ethylene-responsive Agricultural and Processed Food Products Export Development Authority Ascorbate peroxidase Aquaporin Auxin response factor Abscisic acid-stress and ripening-inducer Arabidopsis transcription activation factor Autophagy-related protein 8f Bacterial artificial chromosome Bulk segregant analyisis Basic leucine zipper Compound annual growth rate Cleaved amplified polymorphic sequences CRISPR-associated protein 9 Catalase Convention on Biological Diversity C-repeat binding factor xvii

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CCS cDNA CDPK CEBAS CG CIB CIRAD CMT COR CQD CR CRISPR CRT CS CSIC CUC DAM DEG DHAR DHN DI DM DNMT DRE DRE(B) DREB DRM DSB EBI ECe ECs ECw EMS EPA ERD ERF ESP EST ET F1 F3H FAO FAOSTAT FDA

Abbreviations

Copper chaperone for superoxide dismutase Complementary DNA Calcium-dependent protein kinase Centro Edafología y Biología Aplicada del Segura Candidate gene Cold-induced gene Centre de cooperation International en Recherché Agronomique pour le Développement Chromomethylase Cold responsive Carbon quantum dot Chilling requirement Clustered regularly interspaced short palindromic repeats C-repeat Cabernet sauvignon Consejo Superior de Investigaciones Científicas Cup-shaped cotyledon Dormancy associated MADS-box Differentially expressed gene Dehydroascorbate reductase Dehydrin Deficit irrigation Downey mildew DNA methyltransferase Drought-responsive element Drought-responsive element binding Dehydration responsive element binding protein Domain-rearranged methyltransferase Double-strand break European Bioinformatics Institute Electrical conductivity of equivalent Electrical conductivity for soil Electrical conductivity for water Ethylmethyl sulphonate Environmental Protection Agency Early response to dehydration Ethylene response factor Exchangeable sodium percentage Expressed sequence tag Evapotranspiration First filial generation Flavanone 3-hydroxylase Food and Agriculture Organization FAO Corporate Statistical Database Food and Drug Administration

Abbreviations

FLS FPP GA GBS GES GM GMO GolS GP GPCR GS GTPases GWA GWAS HK HR HR HS Hsf HSF HsfA2 HSP HT ICE1 ICE1 IGG InDel INIBAP IPCC IPGRI ISSR ITC JA K+/Na+ KASP Lb LD LEA LG LOD LT LT LTR(E/B) Ma MABCB

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Flavonol synthase Farnesyl diphosphate Gibberellic acid Genotyping by sequencing Geraniol synthase Genetically modified Genetically modified organism Galactinol synthase1 Guaiacol peroxidase G protein-coupled receptors Genomic selection Guanosine triphosphatase Genome-wide association Genome-wide association study/ studies Histidine kinase Heat requirement Homologous recombination Heat stress Heat shock factor Heat shock transcription factor Heat stress factor A2 Heat shock protein High tolerance Inducer of CBF expression Little elongation complex subunit 1 Intergovernmental Group Insertion/deletion International Network for the Improvement of Banana and Plantain Intergovernmental Panel on Climate Change International Plant Genetic Resources Institute Inter-simple-sequence repeat Indian Tobacco Company Jasmonic acid Potassium/sodium ratio Kompetitive allele amplification Late blooming Linkage disequilibrium Late embryogenesis abundant protein Linkage group Logarithm of odds Low temperature Low tolerance Low-temperature-responsive (element/binding) Musa acuminata Marker-assisted backcross breeding

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MANT MAPK MAPKK MAPKKK MAS MASS Mb MDA miRNA MSAP MT MTP MYB MYC NAC042 NAC68 NAM NCBI NGS NHB NHEJ NIP NIR NMU NOS NP NPBT NPC NR NRCB NSP O·−2 OE OIV PA PAC PCD PDS PGIP PIP PIP1; 1 PIP1; 2 PIR PM PN

Abbreviations

Methyl anthranilate Mitogen-activated protein kinase Mitogen-activated protein kinase kinase Mitogen-activated protein kinase kinase kinase Marker-assisted selection Marker-assisted seedling selection Musa balbisiana Malondialdehyde MicroRNA Methylation sensitive amplified polymorphism Medium tolerance Metal tolerance protein Myeloblastosis Myelocytomatosis NAC domain-containing protein 42 NAC domain-containing protein 68 No apical meristem National Center for Biotechnology Information Next generation sequencing National Horticultural Board Non-homologous end-joining Nodulin-like plasma membrane intrinsic protein Near-infrared spectrometry N-nitroso-methyl urethane Nitric oxide synthase Nanoparticle New plant breeding techniques Nuclear pore complex Nitrate reductase National Research Center for Banana Nanoscale particle Superoxide radical Overexpression International Organization of Vine and Wine Proanthocyanidin P1-derived artificial chromosome Programmed cell death Phytoene desaturase Polygalacturonase-inhibiting protein Plasma membrane intrinsic protein Aquaporin PIP1-1 Aquaporin PIP1-2 Protein information resource Powdery mildew Photosynthetic rate

Abbreviations

POD PP2C PRD PRP PS I/II PSY PTM Put QD QD qPCR QTL QTLs RAD RAPD RdDM RDI REMAP RFLP RIP RLK RNAi RNA-Seq RNP ROP ROP ROS RT-PCR SAP SAR SCAR sgRNA sHSF sHSP SIB SiNP SIP siRNA SLAF-seq SNP SnRK2 SOD SPL SRAP SSN

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Peroxidase C-type protein phosphatase partial root drying Plant proline-rich protein Photosystem I and II Phytoene synthase Post-tranlational modification Putrescine Quarentadias (Name of a banana variety in Portuguese language) Quantum dot Quantitative PCR Quantitative trait locus Quantitative trait loci Restriction site associated DNA Random amplified polymorphic DNA RNA-directed DNA methylation Regulated deficit irrigation Retrotransposon-microsatellite amplified polymorphism Restriction fragment length polymorphism Ribosome inactivating protein Receptor-like kinass RNA interference RNA Sequencing Ribonucleoproteins Repressor of primer protein Repressor of primer protein Reactive oxygen species Real time–PCR Switch-activating protein Sodium absorption ratio Sequence-characterized amplified region Single guide RNA Small heat stress transcription factor Small heat shock protein Swiss Institute of Bioinformatics Silicon nanoparticle Small intrinsic protein Small interfering RNA Specific length amplified fragment sequencing Single nucleotide polymorphism SNF1-related protein kinases2 Superoxide dismutase Squamosa promoter binding protein-like Sequence-related amplification polymorphism Site-specific nuclease

xxii

SSR SWEETs TA TAC TALE TALEN TE TF TGE TIP TK TSS UNDESA UniProtKB UPOV USDA VLT VNTRS Vv WBF WGS WL WT YAC ZFN ZIP

Abbreviations

Simple sequence repeat Sugars will eventually be exported transporters Tartaric acid Transformation-competent artificial chromosome Transcription activator-like effector Transcription activator-like effector nuclease Transposable element Transcription factor Targeted genome editing Tonoplast intrinsic protein Traditional knowledge Total soluble sugars United Nations Department of Economic and Social Affairs UniProt Knowledgebase International Union for the Protection of New Varieties of Plants United States Department of Agriculture Very low tolerance Variable number of tandem repeats Vitis vinifera World Banana Forum Whole-genome shotgun Waterlogging Wild type Yeast artificial chromosomes Zinc-finger nuclease Zipper protein

Chapter 1

Genomic Approaches to Improve Abiotic Stress Tolerance in Apple (Malus × domestica) Madhushree Dutta, Rajesh Kumar Singh, and Gaurav Zinta

Abstract Apple ranks third in global fruit consumption owing to its high nutritional properties, specifically antioxidant and mineral constituents. Apples are widely grown in temperate regions of the world. Apple is a perennial woody fruit tree with high commercial value. In recent years, the cultivation and production of apple is declined due to abiotic stresses associated with climate change. Heat, cold, salinity and drought are the major stresses which affect apple productivity. Apple genetic resources available can be exploited to breed varieties resistant against diverse abiotic stresses, which can help expand the cultivation area of apples. Also, the mechanisms underlying abiotic stress tolerance need to be clarified. Although, molecular markers and modern plant breeding techniques have helped in the identification and characterization of genes involved in stress resistance. However, genetic manipulation and molecular breeding approaches can pave ways for the development of stress resistant apple cultivars. Keywords Abiotic stress · Breeding · Genome editing · Molecular markers · QTLs

1.1 Introduction Fruits are an essential source of vitamins, minerals, antioxidants, and fibers, which form an integral part of a healthy human diet (Amao 2018). Fruit is a fleshy and mature part of the plant ovary that can be sweet (apples, oranges and strawberries) or non-sweet in its raw state (Mintah et al. 2012). Apple is one of the most favored fruits with economic and cultural significance that is widely grown over the temperate regions of globe (Spengler 2019). Over the last decades, many efforts have been made to improve apple yield and productivity, which has led to cheaper and year-round availability of apple (Sharma et al. 2014). The global annual production of apples M. Dutta · R. K. Singh · G. Zinta (B) Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology (CSIR-IHBT), Palampur, Himachal Pradesh 176061, India e-mail: [email protected]; [email protected] Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh 201002, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 C. Kole (ed.), Genomic Designing for Abiotic Stress Resistant Fruit Crops, https://doi.org/10.1007/978-3-031-09875-8_1

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has doubled from 41 million tons in 1990 to 86 million tons in 2018 (FAO 2020). Apple is the third largest produced fruit after bananas and watermelon (FAO 2020). Interestingly, whole fruit is edible (except the seeds), which is a key source of market consumables like jam, jelly, tea and wine (Spengler 2019). Apart from nutritional value, they contain excellent immune boosters including active phytochemicals that are beneficial for humans (Boyer et al. 2004). Such active bio-ingredients are mostly found in the pulp and peel of apples. The list of such bioactive substances comprises polyphenols, polysaccharides, plant sterols, pentacyclic triterpenes, and organic acids with a significant difference in concentration in pulp and peel (Patocka et al. 2020). In the current scenario, there is still a great prospect for developing and utilizing bioactive substances in apples. The consumption of apple and its extracts rich in phytochemicals has been linked to reduced risk of cancer, cardiovascular disease, diabetes, and many other chronic diseases, including asthma. Mainly polyphenols govern such health benefits through antioxidant and anti-inflammatory activities and by modifying biomarkers in different cell signalling pathways (Patocka et al. 2020). The adverse climatic factors negatively affect apple productivity and quality. For instance, late spring frosts cause more severe crop loss than those of low winter temperatures. The flower bud and young fruit stages exposed to late spring frosts are vulnerable to low temperature (Aygün and San ¸ 2005; Tomasz et al. 2008). Environmental conditions also promote the development of apple scab and powdery mildew, which are some of the well-known fungal diseases affecting commercially important cultivars (Sansavini et al. 2004). Such infections cause significant fruit yield and quality losses (Blagov 2011). Additionally, climate plays an essential role in determining the overall crop production at a global scale by influencing the yield and quality traits. The global and regional scale climate change factors jointly pose a severe threat to sustainable apple crop production (Tharaga et al. 2021). The predictions for an increase in temperature are made from 1 to 6 °C by the year 2100, while CO2 concentration might increase to 850 ppm (Collins et al. 2013; IPCC 2009). Such conditions will inevitably affect apple production along with associated changes such as more frequent drought episodes and decline in the net chilling hours. Presently, the traditional apple farming is under stress owing to these climatic aberrations (Basannagari and Kala 2013). It has been observed that insufficient winter chilling units have caused a dramatic decline in apple production (Singh et al. 2016). The indicators of climate change at mid-hills and low hills are apple scab and pest attack, respectively (Singh et al. 2016). The traditional apple breeding practices takes years for commercial variety release. The development of new cultivars by conventional breeding is also constrained by a narrow gene pool, self-incompatibility, inbreeding depression, longer juvenile period and larger plant size (Brown and Maloney 2003). Together, such limitations of breeding techniques combined with biotic and abiotic stresses have limited apple production and the situation has become alarming for farmers and plant scientists. Thus, improving stress resilience and nutritional value of apple is of high importance in the face of global climate change.

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1.2 Abiotic Stresses Plants are exposed to different abiotic and biotic stresses in their natural habitat, and to endure stressful conditions various signaling pathways are initiated. Here we discuss the impact of abiotic stresses and underlying molecular regulators in apple (Fig. 1.1).

1.2.1 Heat Stress Heat stress is one of the most ominous abiotic factors which limit productivity and quality, thereby incurring huge economic losses. It has been reported earlier how high temperature affects the morphological, anatomical, physiological and biochemical changes in the plant system. The success in overcoming heat stress is limited owing to poor knowledge of heat stress effects during critical stages of fruit development in crops. There is an urgent need to improve heat stress tolerance by using breeding and biotechnological approaches (Sharma et al. 2020). Generally, heat stress alters the several biological processes in the plant system. Photosynthetic apparatus is vulnerable to damage at high temperatures leading to a reduced photosynthetic rate (Wang et al. 2018). However, plants on encountering such adverse conditions undergo a series of damaging events like protein misfolding, denaturation, and production of radicals like reactive oxygen species (ROS; Li et al. 2018). Such misfolded or truncated proteins are highly toxic for the cellular processes. On encountering such misfolded proteins, cells trigger cytoplasmic protein response or unfolded protein

Fig. 1.1 Molecular regulators involved in different abiotic stress responses in apple

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response in the endoplasmic reticulum, which stimulates the occurrence of autophagy (Deng et al. 2011). Autophagy is an evolutionarily conserved pathway that degrades unwanted cytoplasmic constituents and facilitates the circulation of cellular proteins. Its role in nutrient cycling in plants is well documented, which underpins plant tolerance to various biotic and abiotic stresses (Wittenberg et al. 2018). Studies show that autophagy plays a crucial role in basal thermotolerance in apples. It was found that MdATG18a improved thermotolerance by enhancing autophagic activity, protecting chloroplasts, maintaining higher levels of photosynthesis, scavenging toxic ROS, and inducing heat shock protein (HSP) expression in apple (Huo et al. 2020). In addition to this, global warming has also affected apple production by causing them to flower earlier, making them susceptible to freezing injury (Zhang et al. 2021). Nuclear pore complexes (NPCs) are central channels controlling nucleocytoplasmic transport by regulating plant development and stress responses. Recently, the components of NPC were studied, and it was found that they are a well-characterized nucleoporins group, MdNup62, interacts with MdNup54, forming the central NPC channel (Zhang et al. 2021). This interaction of nucleoporins is further associated with MdHSFs to regulate flowering and heat resistance in apples (Zhang et al. 2021). Additional analyses in heat stress demonstrated that plant proline-rich proteins (PRPs) are characterized cell wall proteins activated during stress events. Nine PRP genes were studied amongst which, MdPRP6 positively regulated heat stress tolerance in transgenic plants (Zhang et al. 2021). The MdPRP6 overexpressing plants showed comparatively lesser oxidative damage and higher photosynthetic capacity suggesting the role of PRP proteins in the apple in abiotic stress. Such findings will aid the future characterization and mechanism of PRP proteins in apples on accounting adverse environments.

1.2.2 Drought Stress Drought is another major limiting factor for crop productivity (Cabello and Chan 2012). Since climates are warming and water is limited, drought has become a global concern threatening future crop production (Zhao and Running 2010). Naturally, plants have developed several molecular and physiological mechanisms to cope with stress. Drought triggers key regulatory genes that in turn regulate physiological processes such as stomatal closure (Taiz and Zeiger 2002) and the detoxification of reactive oxygen species (ROS) (Sun et al. 2018; Chen et al. 2019). Likewise, heat and drought conditions also generate ROS leading to membrane damage and oxidative stress (Tsugane et al. 1999). The role of autophagy in combatting such stress conditions is also reported. It has been found that overexpression of MdATG18a in apple plants enhances their tolerance to drought stress, probably because of greater autophagosome production. Those processes are known to help degrade aggregated protein and limit oxidation damage (Sun et al. 2018). Another important agricultural technique to improve drought resistance is to inoculate plants with arbuscular mycorrhizal fungi (AMFs; Chitarra et al. 2016). AMFs are well-known symbionts with most terrestrial plants and play a crucial role in adaptation to various stresses

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(Huang et al. 2020). The role of AMFs on the drought resistance of plants is very complex, which involves many metabolic pathways (Wu and Xia 2006). It facilitates drought adaptation by uptake of plant nutrients and water uptake and transport, increased plant osmotic regulation, induced hormone signalling responses, improved gas exchange capacity and water use efficiency, and enhanced antioxidant capacity (Yang et al. 2014). Furthermore, MAPK signalling genes were upregulated during drought, suggesting their important role in facilitating the interaction between AMF and apple trees, leading to drought tolerance in apples (Huang et al. 2020). Plant transcription factors (TFs) also play a key role in regulating stress responses (Century et al. 2008). Amongst such, the homeodomain–leucine zipper (HD-Zip) family inevitably regulates drought responses (Yang et al. 2018). The apple HD-Zip gene MdHB7 led to significant endogenous abscisic acid (ABA) accumulation which further caused ROS detoxification and stomatal closure in response to drought, whereas RNA interference (RNAi) lines of this gene had the opposite effect (Zhao et al. 2020). Furthermore, ethylene response factors (ERFs) affect anthocyanin biosynthesis. A well-characterized ERF protein MdERF38 is involved in drought stress-induced anthocyanin biosynthesis. Molecular experiments showed the interaction between two partners ERF protein (MdERF38) and a positive modulator of anthocyanin (MdMYB1), promoting drought resistance (Jian-Ping An et al. 2020). MdMYB88 and MdMYB124 are the positive regulators of drought tolerance (Li et al. 2020). In apple, 42 apple specific miRNAs are present, out of which miR156, miRn249, miR408, miR395 are the positive regulators of drought stress tolerance (Li et al. 2020).

1.2.3 Cold Stress Premature fruit drop is one of the major concerns during cold stress. During the early development, apple fruits are exposed to abnormal cold conditions. Studies indicate apple trees with early developing fruits are subjected to abscission owing to ABA production. Abscission induction causes upregulation of ABA biosynthesis (MdNCED1) and metabolism (MdCYP707A) genes, and ethylene biosynthesis (MdACS1) and receptor (MdETR2) genes in the pedicel (Lee et al. 2021). Once the ABA in the pedicel spreads to adjacent organs, increasing ABA orchestrates cold response (Lee et al. 2021). Cold tolerance in apples is mediated by the ethylene biosynthesis gene MdERF1B, which upregulates the expression of the coldresponsive gene MdCBF1 in apple seedlings (Wang et al. 2021). Moreover, another positive regulator, MdCIbHLH1, functions upstream of CBF-dependent pathways facilitating the binding of MdERF1B to target gene promoter and increasing transcription rate (Wang et al. 2021). In a nutshell, upregulation of such key genes resulted in enhanced ethylene biosynthesis production leading to cold tolerance in apple cultivars (Wang et al. 2021).

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1.2.4 Salinity Stress Salt stress has the ability to hamper plant growth and development (Yang and Guo 2018). Approximately one-fourth of the global cultivated land area is salinized, and such conditions are going to worsen more in the upcoming years due to rapid climatic changes (Zhu 2016). The perennial woody apple trees are non-halophyte (Flowers et al. 2010). However, salt damages in apple-producing zones are contributed mainly by improper fertilization and irrigation that has adversely affected the overall stature of the plant. Due to such secondary salt damage, considerable economic losses are caused. Hence, developing salt-tolerant varieties is the need of the hour. The role of microRNAs in salt resistance is reported (Kumar et al. 2018). These small noncoding RNAs function by inhibiting or degrading the complementary mRNAs (Catalanotto et al. 2016). The first identified miRNA in plants is miR156 that targets and regulates the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors (Cardon et al. 1997). The miR156/SPL regulatory modulate the MdWRKY100 transcription factor in salt-tolerant pathways (Jiang et al. 2017; Ma et al. 2020).

1.2.5 Nutrient Stress The apple farming system is more sustainable owing to lower nutrient removal in the yield, high nutrient recycling and retention in the system (Wu et al. 2008). The nutrient uptake and removal by apple trees (through harvest and pruning) is comparatively lower. Apple trees have lower rooting densities making them inefficient in using nitrogen (Neilsen and Neilsen 2002). The growth of apple production is reliant on nutrient accumulation over multiple seasons (Wünsche and Lakso 2000). Annually only 50% of total uptake N is retained leading to yield of 90 t ha–1 (Neilsen et al. 2001). On the other hand, water loss beneath the root zone during the coolest months were greater than scheduled irrigation application (Neilsen and Neilsen 2002). Thus, in the irrigation systems if water is added to meet plant demand, then water and N movement through root zone can be reduced.

1.2.6 Role of MdCAX Proteins in Abiotic Stress Tolerance Calcium plays an important role in maintaining homeostasis for plant survival and growth. Besides, studies on many fruits have shown their role in regulating fruit development, quality, ripening, and preventing it from adverse climatic alterations (Michailidis et al. 2020). CaCA (Ca 2+ /cation antiporter) superfamily comprises several subgroups of exchangers, including Ca2+ /H + exchangers (CAXs) (Taneja et al. 2016). The role of CAX proteins is widely explored in preventing excessive

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accumulation of Ca2 + in the cytosol by promoting its efflux into vacuoles (Pittman et al. 2016). Interestingly, such CAX proteins are recently characterized in apple cultivars playing a significant role in building acquired tolerance against various abiotic stresses. Based on protein studies conducted by Yeast 2Hybrid, it was revealed CAX proteins interact with multiple stress key regulators like SOS2, CXIP1, MHX, NRAMP3, and MTP8 (Mao et al. 2021).

1.3 Traditional Breeding Methods Traditional breeding is one of the main strategies used to improve agronomic traits. Furthermore, such breeding techniques are usually a long-term and expensive process requiring many resources to meet the end. To complicate such events more, sexual breeding involved in the conventional breeding process is not always feasible because cultivars involved at times are incompatible, sterile, or polyembryonic (Talon and Gmitter 2008). However, after successful breeding, even repeated backcrosses are required to recover elite characters of improved cultivars, causing further delay in the breeding program. Such processes are even longer in the case of rootstock breeding (25 years and more). New plant breeding techniques (NPBTs) can overcome such limitations in traditional breeding to obtain improved organoleptic traits and resistance to biotic and abiotic stress. For carrying out such activities, thorough knowledge about the target gene is essential to imply techniques such as genome editing and cisgenesis (Salonia et al. 2020).

1.4 Molecular Breeding 1.4.1 Molecular Markers It is essential to have proper genetic analysis and marker-assisted selection (MAS) for efficient plant breeding. MAS is used to fish out specific traits or quantitative trait loci (QTLs) by use of genetic markers, usually named as named marker-trait association and marker-locus-trait association, respectively (Ru et al. 2015). Studies on genetic markers like isozymes, restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPD), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs) are conducted to provide insights into modern breeding ways (Pereira-Lorenzo et al. 2009). On the other hand, marker-assisted seedling selection (MASS), uses DNA markers to produce genetically improved cultivars from biparental crosses (Ru et al. 2015). MASS can also have additional advantages, e.g. identifying individuals with multiple disease resistance alleles, marking out phenotypic variance caused by environmental factors, and further revealing unexpected outcomes in context to genotype and environmental

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interactions (Ru et al. 2015). Although such DNA markers proved to be promising in generating improved seedling selection, especially for traits with low heritability, still less work is reported for rosaceous fruit trees. Recently, single nucleotide polymorphisms (SNPs) have been preferred for their ability to genotype at a large-scale concomitant with release 8 K, 20 K, and 450 K SNP arrays for apple using the Infinium II or Axiom system (Chagné et al. 2012a; Bianco et al. 2014, 2016a, b). Furthermore, whole-genome sequences are reported for ‘Golden Delicious’ (Velasco et al. 2010) and the doubled haploid ‘GDDH13’ (Daccord et al. 2017). Thus, such improvised tools have made the breeding process efficient by screening out target genes involved in the development of valuable traits.

1.4.2 Quantitative Trait Loci (QTLs) QTL mapping is usually conducted in a biparental population to uncover the markers associated with the trait of interest. QTLs for various traits of interest have been identified using standard protocols. Such traits include assessment of malic acid content, flesh firmness and softening, harvest time, and polyphenol and sugar components (Liebhard et al. 2003; Kenis et al. 2008; Costa et al. 2010; Longhi et al. 2012; Chagné et al. 2012b; Kunihisa et al. 2014). The genetic variations determining the flesh firmness and crispness was identified using genetic markers based on QTL. Such developmental studies depicted the role of ethylene response factors, MdERF2 and MdERF3 binding to the MdACS1 promoter and oppositely regulate its transcription during post-harvest ripening in apple (Li et al. 2016). Additionally, two potential QTLs identified as Ma and Ma3 on linkage group (LG) 16 and LG8 are key players in regulating acidity in apples. The research was done using pedigree-based QTL mapping software, FlexQTL (Verma et al. 2019). However, such QTL studies on apple acidity put forth few loopholes owing to the bi-allelic nature of the QTL models used in QTL mapping. Surprisingly, multiple Q alleles are found, each with a different effect contributing to software inability to uncover QTL genotype estimates for parents with correct designated alleles (Verma et al. 2019). Other challenges with QTLs include limited crossover of markers, need for a larger population of related individuals and a lower rate of recombination events (Costa 2015; Soto-Cerda and Cloutier, 2012).

1.5 Genome-Wide Association Studies (GWAS) The genome of cultivated apples is the base for generating many tools to navigate the genetic analysis to its best (Velasco et al. 2010). Domesticated apple has thousands of genes as compared to other crops to be used for genome-wide functional studies. Many of such genes are capable of providing resistance to abiotic stress, enhancing flavor and agronomical traits (Pereira-Lorenzo et al. 2009). The genomic selection

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process overcomes the limitations of MAS and marker-assisted recurrent selection (MARS). Alternatively, a genome-wide association study (GWAS) is based on using diverse populations of unrelated individuals to explore genotypic-phenotypic association studies. Numerous genetic markers are used covering the entire genome with at least one in linkage disequilibrium (Flint-Garcia et al. 2005). Amongst fruit trees, GWAS was initially applied for studies in pears and apples (Cao et al. 2012). However, GWAS exercised for apples was a challenging issue due to the heterozygous nature of apples together with whole-genome duplication and the rapid rate of linkage disequilibrium (Bianco et al. 2016a, b). Nonetheless, GWAS was still able to explore six useful traits in apples (e.g. fruit ripening, browning, firming) (Kumar et al. 2013). SNP-trait associations are also studied by GWAS to sight the differentiation between cider and dessert apples (Leforestier et al. 2015). Thus, genomic selection has recently emerged as another tool to accelerate apple breeding.

1.6 Modern Plant Breeding Techniques (NPBT) NPBTs are an alternative measure for advancing biotic and abiotic stress tolerance, nutritional quality and crop performance (Cao et al. 2016). To achieve the goal of genetically improved tree crops, cisgenesis and genome editing are the two promising methods. Genome editing produces specific, stable and inheritable mutations at a distinct location of the genome, whereas cisgenesis revolves around the transfer of genes across cross-compatible species (Salonia et al. 2020). Unlike traditional breeding, NPBT makes only minimal modifications in targeted genotypes, thereby preserving the genetic background of selected cultivars (Salonia et al. 2020). The above two methods are broadly discussed underneath the umbrella NPBT.

1.6.1 Cisgenesis The term cisgenesis was first coined by Schouten (2006) and Malabarba et al. (2021). It is defined as “the genetic modification of plants using genes that originate only from the species itself or from a species that can be crossed conventionally with this species”. Broadly such breeding methods involve the transfer of genes, including introns and controlling sequences from one genotype to another of the same or sexually compatible species (Schouten et al. 2006; Lusser and Davies 2013). The major advantage of cisgenesis is the ability to overcome “linkage drag” (unwanted gene transfer along with the gene of interest) (Jacobsen and Schouten 2007). However, cisgenesis has several other drawbacks that make it to take a backseat at many instances. Particularly, cisgene insertion in the host genome could either induce a negative effect on the gene or could potentially modify its function (Vanblaere et al. 2014). Secondly, the lack of efficient promoters and selectable markers remains the major bottleneck in its broader application (Limera et al. 2017). Mostly cisgenesis

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Table 1.1 Genetic modifications obtained by using new plant breeding technology to combat abiotic and biotic stress events Target Gene

Trait

References

1. MdATG18a

Heat and Drought tolerance by enhancing autophagic activity

Huo et al. (2020), Sun et al. (2018)

2. MdNup62, MdNup54, MdHSFs

Heat tolerance

Zhang et al. (2021)

3. MdPRP6

Heat tolerance

Zhang et al. (2021)

4. MdHB-7

Drought tolerance

Zhao et al. (2020)

5. MdERF38

Drought tolerance by anthocyanin Jian-Ping An et al. (2020) biosynthesis

6. MdMYB88 & MdMYB124

Drought tolerance

Li et al. (2020)

7. MdBES1 8. miRn249, miR408, miR156

Cold tolerance & Drought Sensitivity Drought tolerance

Liu et al. (2021) Li et al. (2020)

9. MdERF1B

Cold tolerance

Wang et al. (2021)

10.MdHcrvf2

Resistance to apple scab

Joshi et al (2011)

11.MdRvi6

Resistance to apple scab

Krens et al. (2015

12.MdFB_MR5

Resistance to fire blight

Kost et al. (2015)

13.MdDIPM-1, MdDMP2

Resistance to fire blight

Malnoy et al. (2016)

14.MdPDS

Albino phenotypes

Nishitani et al. (2019)

technology is worked out for apples and grapes with an aim to induce scab and fire blight resistance (Joshi et al. 2011; Vanblaere et al. 2011, 2014; Gessler et al. 2014; Krens et al. 2015; Würdig et al. 2015) (Table 1.1).

1.6.2 Genome Editing Genome editing involves modification of the selected sequences in the genome for crop improvement (Limera et al. 2017). These tools aim to edit, delete or replace specific sequences at the target genome site. CRISPR/cas9 gene editing technology rely on repairing double-stranded breaks through non-homologous end joining and homology-directed repair (Gaj et al. 2013). Apart from these, TALEs which are a recognized class of zinc finger transcription factors, are used for mutagenesis events. These nucleases disrupt the DNA adjacent to recognition sites (Gaj et al. 2013). CRISPR/Cas9 is a revolutionary molecular method that generates site directed mutagenesis. It works through guide RNA and Cas9, producing double-stranded break adjacent to gRNA annealing location (Bortesi et al. 2015). However, CRISPR/cpf1 is more efficient as compared to Cas9 (Ledford et al. 2015). CRISPR/Cas knockout targeted mutagenesis is implemented in several fruit tree species such as apples, pear and strawberry (Nishitani et al. 2019; Zhou et al. 2018; Charrier et al. 2019).

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Furthermore, CRISPR-Cas9 knock-out lines led to improved crop cultivars with fungal and bacterial resistance (Pompili et al. 2019). CRISPR/Cas system is successfully used to induce mutation in apple phytoene desaturase, producing the albino phenotype (Nishitani et al. 2019). Apple plants with visible albino phenotypes have slower growth rates with long life span and genomic heterozygosity (Nishitani et al. 2019). Despite recent advances, CRISPR/Cas9 system in perennial plants has still a few obstacles to overcome. Recently, base editing is being used in apple to create precise genomic edits (Malabarba et al. 2021).

1.6.3 Trans-Grafting Trans-grafting refers to grafting a genetically modified (GM) part with a non-GM part. In this cultivation process, GM roots improve the performance of commercially approved scion varieties’ performance and produce non-GM products. It is a cultivation method to exploit the interactive association between partner plants possessing different genomes (Mudge et al. 2009). Hence, trans-grafted plants stand more acceptable in the public’s eyes (Igarashi et al. 2016). In apple cultivation, it has been used mainly for maintenance and propagation of clone strains and for altering plant vigor, architecture, and precocity. Apple rootstocks have deep interaction with soil that support the growth of scion through water and mineral uptake.

1.6.4 Germplasm Conservation By In Vitro Methods The plant genetic resources with economic importance are the core source for conservation (Benelli et al. 2013). Cryopreservation in liquid nitrogen is one of the effective ways to preserve germplasm (Engelmann 2012). Apple is a woody perennial plant whose dormant bud are amenable of cryopreservation. For the cryopreservation of dormant buds, scions or branches are exposed to a cooler temperature. The dehydration is usually performed at –5 °C for days to reduce the moisture content by up to 25%. Such a step is crucial to obtain higher recovery after cryopreservation (Benelli et al. 2013). The cryopreservation methods in apple involve encapsulation-dehydration, vitrification, encapsulation-vitrification and droplet vitrification (Pereira-Lorenzo et al. 2009). In encapsulation-dehydration, shoot tips are coated with alginate or silica gel before dipping into liquid nitrogen. The vitrification step involves treating the shoot tips with vitrification solutions like cryoprotectants, while encapsulation-vitrification is a joint process of the above two steps followed one by one (Pereira-Lorenzo et al. 2009).

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1.7 Conclusion and Future Prospects Apple is an ancient worldwide crop with many nutritional benefits. Technologies such as CRISPR/Cas9 can help plant scientists to bring high yielding and disease-resistant apple cultivars in the market. Such techniques can help overcome traditional breeding limitations, including a long juvenile period, gametophytic self-compatibility, high heterozygosity and the time invested in screening out phenotypic variance. Also, tools like GWAS can enable us to use the wide genetic diversity provided by nature to make apples desirable in global markets.

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Wu FQ, Liu HB, Sun BS, Wang J, Gale WJ (2008) Net primary production and nutrient cycling in an apple orchard-annual crop system in the Loess Plateau, China: a comparison of Qinguan apple, Fuji apple, corn and millet production subsystems. Nutr Cycl Agroecosyst 81:95–105 Wu QS, Xia RX (2006) Arbuscular mycorrhizal fungi influence growth, osmotic adjustment and photosynthesis of citrus under well-watered and water stress conditions. J Plant Physiol 163:417– 425 Wünsche JN, Lakso AN (2000) Apple tree physiology: implications for orchard and tree management. Compact Fruit Tree 33:82–88 Würdig J, Flachowsky H, Saß A, Peil A, Hanke M-V (2015) Improving resistance of different apple cultivars using the Rvi6 scab resistance gene in a cisgenic approach based on the Flp/FRT recombinase system. Mol Breed 35:95 Yang PM, Huang QC, Qin GY, Zhao SP, Zhou JG (2014) Different drought-stress responses in photosynthesis and reactive oxygen metabolism between autotetraploid and diploid rice. Photosynthetica 52:193–202 Yang Y, Guo Y (2018) Elucidating the molecular mechanisms mediating plant salt-stress responses. New Phytol 217:523–539 Yang Y, Luang S, Harris J, Riboni M, Li Y, Bazanova N, Hrmova M, Haefele S, Kovalchuk N, Lopato S (2018) Overexpression of the class I homeodomain transcription factor TaHDZipI-5 increases drought and frost tolerance in transgenic wheat. Plant Biotechnol J 16:1227–1240 Zhang XW, An JP, Zhang XW, Bi SQ, You CX, Wang XF, Hao YJ (2020) The ERF transcription factor MdERF38 promotes drought stress-induced anthocyanin biosynthesis in apple. Plant J 101(3):573–89 Zhang X, Gong X, Li D, Yue H, Qin Y, Liu Z, Li M, Ma F (2021) Genome-wide identification of PRP genes in apple genome and the role of MdPRP6 in response to heat stress. Int J Mol Sci 22:5942 Zhang C, An N, Jia P, Zhang W, Liang J, Zhou H, Dong Z, Ma J, Caiping Z, Han M, Ren X, Xing J (2019) MdNup62 interactions with MdHSFs involved in flowering and heat-stress tolerance in apple Zhao M, Running SW (2010) Drought-induced reduction in global terrestrial net primary production from 2000 through 2009. Science 329:940–943 Zhao S, Gao H, Jia X, Wang H, Ke M, Ma F (2020) The HD-Zip I transcription factor MdHB-7 regulates drought tolerance in transgenic apple (Malus domestica). Environ Exp Bot 180:104246 Zhou J, Wang G, Liu Z (2018) Efficient genome editing of wild strawberry genes, vector development and validation. Plant Biotechnol J 16:1868–1877 Zhu JK (2016) Abiotic stress signaling and responses in plants. Cell 167:313–324

Chapter 2

Genomic Designing of Abiotic Stress Tolerance in Banana I. Ravi, M. Mayil Vaganan, T. Anithasree, K. Stellamary, and S. Uma

Abstract Banana is a tropical and subtropical fruit that is grown around the world and its fruits are valued for their flavor, nutritional value, and stable food for many African and Asian countries. Besides, its fruits are available throughout the year. This crop has been intensively cultivated in many parts of the world to meet the demand. During earlier days, the banana research has been done mostly on its fruit development ripening, and transport. Later on, more focus has been given to biotic stresses, especially diseases. However, there was not enough research on abiotic stresses in bananas compared to other economically important cereal and fruit crops, despite its huge economic value. Climate change is visible and felt by all the banana stakeholders. In recent past years, bananas are grown in non-traditional areas due to favorable climates. However, there is a negative impact of climate change on traditional banana growing areas, by the frequent occurrence of drought stress, high temperature, flash flood, etc. The traditional banana irrigated lands are becoming saline due to secondary salinization. The effect of all these abiotic stresses on banana has been discussed and the roles of modern biotechnological tools in banana improvement programs are also highlighted. The concerted efforts of all the banana stakeholders are required to address the negative impact of abiotic stresses through an exchange of existing technologies and investment in developing new technologies to sustain banana production.

2.1 Introduction Banana and plantains (Musa spp referred to as bananas) are grown in 135 countries, with a total production of 155 million tonnes (75% bananas and 25% plantains) on 11 million hectares. Banana contributes 37% of total fruit production in the world (FAOSTAT 2018). India made a significant jump in banana production after the 1990s with the integration of intensive area expansion and the adoption of scientific cultivation of bananas. Other than India, the leading producers are China, Brazil, I. Ravi (B) · M. M. Vaganan · T. Anithasree · K. Stellamary · S. Uma ICAR-National Research Center for Banana, Thogamalai road, Thayanur Post, Trichy, TN, India e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 C. Kole (ed.), Genomic Designing for Abiotic Stress Resistant Fruit Crops, https://doi.org/10.1007/978-3-031-09875-8_2

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Philippines, Ecuador, Indonesia, Mexico, Guatemala, and Colombia in terms of area and production. India and China alone contribute 28% of global banana production (FAOSTAT 2018). The highest average productivity was recorded in Indonesia and Guatemala nearing 50 tons per hectare. Smallholder farmers grow bananas primarily for local or regional markets, with only about 16% of production reaching international markets (FAOSTAT 2018). Around 1000 different banana varieties are produced and consumed around the world, but the Cavendish-type banana is the most popular, accounting for more than 47% of total production. It’s difficult to get precise data on global banana output since bananas are often grown on tiny farms and traded informally, making them untraceable. Local bananas, for example 70–80% of production in Africa has been grown for over 1000 years. The majority of these are cooking bananas, which are a common and significant staple item (www.fao.org). Despite the fact that bananas are only grown in tropical and subtropical areas of the world, they are one of the most frequently consumed fruits on the planet. It is valued for its flavor, nutritional content and year-round availability. The most typical way to eat the Cavendish group of dessert bananas is as ripe fresh fruits. It’s a high-value cash crop with a lot of potential for supplying raw materials to the growing agro-industry, and it’s also a crucial crop for food security. It may be grown in a variety of situations and yields fruit all year if the weather is favorable. The majority of bananas harvested in India are for local use. Although Cavendish bananas account for more than half of all bananas produced in India, the country is home to a diverse range of bananas. Bananas are grown both in family gardens and in large-scale commercial operations. Commercial production is mostly reliant on irrigation, whilst subsistence farming is done in rainy weather. Consumers’ focus has shifted to hygienic and healthy food products as food and beverage advancements and health awareness have increased. Bananas are high in vitamin B6, C, potassium, manganese, and other nutrients. It aids digestion and contributes to the proper functioning of the nervous system and metabolism. Bananas has long been regarded as a staple, low-cost, and widely available fruit food. This has resulted in continuous product development to meet the consumers’ economic, environmental, and social well-being 2 consciousness. Banana demand will rise further in the future as people become more health conscious (Mordor Intelligence 2018). In terms of world export trade banana ranks 5th most important commodity after coffee, cereal, sugar, and cocoa. With the international banana trade of more than US$45 billion, banana has a huge impact on the economy of many countries (FAO 2019). Banana contributes of 37% of total fruit production in the world. Banana farming generates approximately $7 billion in revenue in India and provides a living for millions of farmers. India contributes 20.48% of world banana production with the production of 31.75 million tons in an area of 8.98 lakh hectares (NHB 2020). Banana alone contributes 2.3% to the Agricultural GDP of India. Banana exports to different countries totalled US$13.7 billion with a volume of 22.3 million tons in 2018 (exports represent less than 16% of global production). Presently, India shares 0.34% of the total export of bananas with a value of over 600 crores and an export volume of 146.7 thousand metric tons (APEDA 2020). Following good agricultural practices in India opens up a new vista of export to

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far-flung countries, given the Philippines banana industry’s declining production, increased production costs, and disease spread. This will increase export volume and share from 0.3 to 10–15% of international trade with a value of $2–3 billion. In the world, bananas are among the most traded fruits. Bananas were traded for approximately 22.7 million tonnes in 2017, accounting for nearly 20% of global production. The banana industry generated USD 11 billion in revenue, far exceeding the value of any other exported fruit (Hays 2009; ITC 2018; UNDES 2019; FAO 2019). Asia is the world’s largest banana producer, while Latin America and the Caribbean countries are the world’s largest banana exporters, accounting for roughly 80% of global exports (UNDES 2019). Banana farming is a major source of income and employment for more than 70 million people in Africa. In 2016, the banana industry’s retail value was estimated to be between $20 and $25 billion, and it supported millions of banana farmers and workers in the world (InAfrica24 2016). Looking at country-level production and trade figures. In 2017, Ecuador was the largest producer and exporter, with USD 3 billion, followed by Costa Rica with USD 1.1 billion and the Philippines with USD 1.3 billion. Meanwhile, the top importing countries that year were the Belgium (USD 1.4 billion), United States (USD 2.5 billion), and Russia (USD 1.1 billion) (UNDES 2019). Due to these high figures, the overall banana trade balance (export–import) fluctuated significantly between 2015 and 2018. This variation is primarily due to three seasons in banana demand as well as climate-related production challenges such as cooler temperatures, severe flooding, and mudslides (Craymer 2018; FAO 2019; CIRAD 2019). The production, purchase, transportation, and marketing of bananas are all handled by five major multinational trading companies. Fresh Del Monte, Dole, Fyffes, and Noboa are among them, as is Chiquita, which recently relocated its headquarters to Geneva, Switzerland. Fyffes, based in Ireland, is the primary banana supplier in Europe. Tesco, Sainsbury’s and Asda account for roughly 60% of retail banana sales in the UK. They are in a strong position to influence import prices because they sell 80% of the bananas available to consumers. In the U.S., major retailers are increasing their bargaining power in global trade by buying directly from grower (Bananalink 2014; www.fao.org). Mordor Intelligence predicts that the banana sector will grow at a CAGR of 1.21% from 2019 to 2024, reaching a global consumption volume of 136 million tonnes by 2025. Rising demand in producing countries, particularly the Asia–Pacific region, which currently accounts for 61% of global consumption, is expected to drive this anticipated growth (FAO 2019).

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2.2 Classification and Description of Banana Simmonds and Shepherd created the nomenclature system used to classify banana cultivars in 1955. It divides cultured bananas into genome groups based on their ancestors’ wild species’ relative role, and then into subgroups, which are groups of strongly associated cultivars. The problems and inconsistencies associated with a taxonomy based on Musa paradisiaca and Musa sapientum are removed with this system. The ones related to Musa acuminata and Musa balbisiana are classified in Simmonds and Shepherd’s Genome-based System (1955) based on their contribution, denoted by the letters A for acuminata and B for balbisiana. The genome group to which a cultivar is assigned is determined by the no. of chromosome sets their genome (ploidy) and the species that donated them. Triploid cultivars can belong to one of three genome groups: AAB, AAA and ABB. Diploid cultivars can belong to either AA or AB genome groups. BBB genome group is recognised by some taxonomists, but its existence has yet to be proven. Breeders create the majority of tetraploid cultivars. Genomes are further divided into subgroups, a collection of cultivars derived from one another through somatic mutations. Cultivar names are separated by inverted commas and preceded by the genus name, and if known, group and subgroup name. For instance, AAA Cavendish group (Musa) and Subgroup ‘Robusta’. Based on the 15 characters, this system was chosen, they are different in. Musa acuminata and Musa balbisiana are two species of Musa. On a scale of one (typical Musa acuminata) to five (exceptional Musa acuminata), Total scores can range from 15 to 75. The expected range for AA and AAA are 15, and, 55 for ABB, 45 for AB 75 for BB and AAB is 35. Seedless, parthenocarpic, and vegetative propagated triploids (ABB, AAA, AAB, BBB genome) hybrids between M. acuminata subspecies (AA genome) or M. balbisiana and M. acuminata in all cultivated bananas (Mc Key et al. 2010). Each of the four wild Eumusa species has its own genome: Musa acuminata (AA genome), Musa balbisiana (BB genome), Australimusa species (TT genome) and Musa schizocarpa (SS genome) (Davey et al. 2013). Bananas that have been cultivated, including hybrids within or between the two Musa species, can have diploid or tetraploid genomes. Hundreds of banana cultivars are developed around the world. Till now, many farmers choose AAA genome (Cavendish subgroup) to commercialize bananas in both domestic and global markets (Tripathi et al. 2020).

2.2.1 Banana Plant Description Banana is the largest herbaceous tree-like perennial plant. It is a perennial herb, because, after fruiting by a single plant, many followers (suckers) arise from the sides of the rhizome. Each sucker bears fruits bearing bunch and this cycle continues. The pseudostem of banana plants is mainly composed of a dense smattering of spirally arranged and overlapping of leaf sheaths. The ‘true’ stem situatin of the rhizome, an

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inner core stem (encircled by leaf sheaths), which is an extension of the rhizome’s apical meristem, and the peduncle that bears fruits and terminates in the male bud. The rhizome clump formed by many banana suckers is referred to as a ‘banana mat’ in horticulture. In botanical nomenclature, the suckers/stools are called genet. The above-ground shoots are called ramets (www.promusa.org). Generally, all the cultivated crops propagated vegetatively and some of the wild bananas produce seeds in addition to the production of genets. Mostly banana flowers are sterile and produce non-pollinated parthenocarpy fruits. The roots are produced in the subterranean rhizome. The primary roots emerge from the rhizome’s central cylinder’s surface. Primary roots give rise to secondary and tertiary roots. Although the rhizome is usually a corm, it is scientifically a rhizome (IPGRI-INIBAP and CIRAD 1996). In the vegetative phase, the apical meristem of the rhizome appears as a flattened dome, and it becomes convex when the vegetative is transformed into the floral stage. Flower bracts replace the place of leaves during the transition from the vegetative phase into the reproductive stage. The protuberances separate into female flowers and then follow male flowers. The banana leaf is one of the largest leaves in the plant kingdom. The leaf emerges as a rolled cylinder (cigar leaf) through the center of the pseudostem. The distal end of the leaf forms leaf sheath and forms part of pseudostem. The petiole of the leaf develops into the midrib, which divides the blade into two lamina halves. The lower surface is referred to as abaxial and the upper surface of the leaf is adaxial. Growing sucker produces the first rudimentary leaves it referred as scale leaves. Foliage leaves are mature leaves that have a petiole, sheath, blade, and midrib. The veins of the lamina from the midrib to the margin, grow parallel to each other in S shape. There are no branches in veins. It was causing leaves to tear easily. Cigar leaf is an unopened newly appeared leaf that is rolled as a cylinder and has a precursory appendage at the tip of the leaf that withers and falls off after the entire leaf has unfurled. Every week, if conditions are favorable, a new leaf emerges from a plant. The poor climate and management conditions take more than two weeks. A sucker is a lateral shoot that emerges from a rhizome that is buried underground. A peeper is a sucker that has just emerged from the soil surface, whereas a maiden sucker is a fully grown sucker with leaf tissue. Suckers are classified into two types based on their morphology: 1. Sword suckers, characterized by narrow leaves like the sword and attached with mother plants. 2. Water suckers, which have broad leaves and have an independent growth with a weak connection to the parent plant and it is not suitable for planting material for commercial cultivation. The sucker that replaces the parent plant after fruiting is known as a ratoon or follower. A complex structure that contains flowers that will mature into fruits is called an inflorescence. The scientifical name for banana inflorescence is thyrsi. A thyrse is a flowering plant with lateral branche a main axis (Endress 2010). The central axis of the thyrse of banana is the peduncle with a transitional, female and male section. All nodes of the peduncle have a bract, in that female and male sections subtends an axillary cushion of tissue from which the flower initials arise. Fahn (1953) and Moncur (1988) observed a sequence of flower initials within each hand and flower initials represented a very compressed cincinnus. Cincinni have a false main axis from which individual flowers arise alternately in sequence (Buys and Hilger 2003).

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Fahn (1953) recorded cincinni in the seeded diploids Musa acuminata and Musa balbisiana as well as in the cultivated triploid ‘Dwarf Cavendish’ (Cavendish subgroup, AAA). Moncur (1988) examined the Cavendish sub-group and observed that the sequence of flower formation in the cincinni moved from right to leave (when viewed the thyrsi from the side with the apex pointing upwards) and alternated between the double rows of flowers. In a hanging bunch in the field, the sequence moves from left to right and can best be seen in the transitional hand that precedes the male phase of the inflorescence. The cincinnus in this hand has a mixture of fruitforming (female) and male flowers. The male flowers are observed at the extremity of the cincinnus and mostly as male buds or bells. Female flowers (pistillate) that develop into fruits and male flowers (staminate) that produce pollen are the two types of flowers in bananas. They are sterile in the majority of cultivated bananas. The ovary of a pistillate flower produces a seedless fruit (without the need for pollination). The bract (altered leaf) exposes a group of female flowers that grow into a fruit hand and are usually arranged in two rows. Fingers in the palm of one’s hand. The number of hands in a bunch varies with genotype, environmental conditions, and management. The distal part of the thyrsi develops pollen-producing staminate flowers. The female flowers develop into a fruit. The amount of pollen in most cultivated bananas is reduced or absent. The bunch is a descriptive term for all the fruits that have their hands attached to the peduncle.

2.3 Abiotic Stresses on Banana Bananas are sensitive to a wide range of abiotic stresses such as soil moisture deficit, salinity, extreme temperatures, wind and water stagnation, or flood inundation. These stresses may harm plant growth, fruit development, and yield.

2.3.1 Drought Stress Tolerance in Banana One of the major constraints to plant productivity is water accessibility (Boyer 1969) and it’s one of most important determinants of plant distribution. Around 35% of a world’s land surface is arid or semiarid, with insufficient precipitation, and even regions with adequate precipitation can be water-limiting environments requiring supplementary irrigation for agriculture. Invariably most of the agricultural regions face limited water availability of variable duration. The banana grows well in the tropics and subtropics, and it prefers hot, humid weather. Bananas are grown everywhere in the world, from the tropical areas to the humid subtropics and semi-arid tropics, distance from the ground up to 2000 m (Simmonds 1966). The banana grows well in the tropics and subtropics, and it prefers hot, humid weather. In agriculture, drought mostly refers to soil moisture deficit stress which affects crop growth and yield. Drought is defined as “a condition in which the amount of water available

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from rainfall and/or irrigation is insufficient to meet the crop’s transpiration needs” (Tuberosa 2012). When yearly rainfall begins to fall below 1,100 mm/year, drought can reduce banana yield by up to 65% (van Asten et al. 2011). As a result, varieties of bananas that can generate a logical yield while using a lesser amount of water are the most optimistic option for preserving banana production from the devastating impacts of drought. A cultivar is regarded as drought tolerant when it possesses the traits of high water-use efficiency with negligible or non-significant growth and yield reduction. Drought effects can be seen in bananas at various stages of development, including early stage, floral initiation, flowering, bunch/finger development (Robinson and Alberts 1986; NRC for Banana 2008). The drought stress effect manifestation depends on the growth stage and time under stress (Ravi et al. 2013). Droughtresistant banana genotypes show a lot less reduction in leaf area, gas exchange and higher net photosynthetic rate, as well as a leaf water retention capacity (Bananuka et al. 1999). Overheating and dehydration of cells cause wilting and also drying of leaves, which are the most visible phenotypic symptoms, as well as a possible reduction in bunch yield (Ravi and Vaganan 2016; van Asten et al. 2011). For example, when banana plants were deprived of water for four weeks during flowering, bunch weight (ranging from 18.83% to 42.07%), Fruit length and radius were reduced during harvest in the cultivars’ ‘Karpuravalli’ (ABB), ‘Robusta’ (AAA) and ‘Rasthali (AAB)’ (NRCB 2008). A considerable lowers in crop yield can be caused by an insufficient in the plant’s photosynthetic rate, which is heavily influenced by the chlorophyll content of the leaves, as well as stomatal closure (Flexas and Medrano 2002). Furthermore, a relationship between yield and leaf area suggests that chlorophyll content and leaf area are important determinants of harvestable yield (Surendar et al. 2013). Other major symptoms are, the pseudostem, girth may get reduced and weaken, and finally, it snaps or collapses. Other drought susceptibility symptoms are expressed as stunted plant growth, choking of bunch throw, and rosetting appearance of leaves.

2.3.1.1

Molecular Biology of Drought Stress Tolerance in Banana

Both apoplastic and symplastic pathways control water movement in plants (Aharon et al. 2003). The aquaporin (AQP) family of proteins regulates the symplastic pathway (Amodeo 1999). When plants are under abiotic stress, water can be transported across membranes using the symplastic pathway (Suga et al. 2002; Lian et al. 2004). Several genes encoding AQP proteins have been discovered in various plant species. Four groups of orthologs have been identified. Among the group, the plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), small intrinsic proteins (SIPs) and nodulin-like plasma membrane intrinsic proteins (NIPs) are distinguished by amino acid sequences that are highly conserved and intron positions that are stereotypical (Maurel et al. 2008). Sreedharan et al. (2013) identified the MusaPIP1;2 aquaporin gene as a positive factor in abiotic stress tolerance in bananas. Transgenic banana plants overexpressing MusaPIP1;2 showed better abiotic stress survival

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characteristics, including reduce malondialdehyde content, increase relative water content and high proline content compared to controls. Xu et al. (2014) isolated the MaPIP1;1 over expressed in Arabidopsis and found that it improved salt by preventing membrane damage and maintaining a elevated cellular potassium/sodium ratio. Besides, over expression of this gene has enhanced drought stress through decreased membrane injury and improved osmotic balance. Many genes have been isolated and characterized for their function. The details are in Table 2.1.

2.3.1.2

Water Use Efficiency in Banana

Bhattacharyya and Madhav Rao (1985) studied soil cover in the ‘Robusta’ banana plantation, which significantly reduced water consumption and the number of irrigations required for the crop’s life cycle. At 60% depletion of available soil moisture, consumption ranged from 1024 mm with an 800-gauge thick black polyethylene cover to 1560 mm with no cover at 20% depletion. At low available soil moisture depletion levels, they reported a noticeable increase in water-use efficiency under soil covers. When compared to bare soil, soil covers reduced the periodic crop coefficient values, indicating that banana plants under soil covers have a lower water requirement (23–33%). Goenaga and Irizarry (2000) conducted a three-year field study of three ratoon ‘Grande Naine’ banana, four irrigation levels an Ultisol to find out the need of water, yield, and fruit quality traits. Irrigation were determined using Class A pan factors ranging from 0.0 (rainfed) to 1.0 (irrigated) in 0.25 increments. Drip irrigation was provided three times a week on alternate days when needed. Irrigation treatment and crop effects on yield, bunch weight, weight and fruit diameter of the third and last hands, bunch mean hand weight, and length of fruits of the third hand were all significant (p < 0.01). The R2 crop produced the highest marketable yield (47.9 t ha–1 ) when a pan factor of 1.0 was used to apply water the purchase of a drip irrigation system for a mountain farm was explained after it was determined that irrigating the crop with a pan factor of 1.0 was sufficient. In another field experiment, Pramanik and Patra (2016) found that drip irrigation improved yield, growth, fruit quality, leaf nutrient uptake and irrigation water use efficiency when compared to surface irrigation in the Gangetic plain of West Bengal. All of these characteristics were consistently and significantly improved as drip fertigation levels increased. When compared to conventional surface irrigation and soil fertilizer, fertigation at 80% RDF with drip irrigation at 60% cumulative pan evaporation produced maximum, yield, growth and fruit quality attributes, as well as elevated irrigation water use effectiveness and water savings of 40.4% in the ratoon crop and 41.7% in the plant crop of a banana.

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Table 2.1 Banana candidate genes used in research and their application Candidate Gene

Applications

Authors

AhSIPR10

In the presence of NaCl and mannitol, photosynthetic efficiency improves, and plasma membrane damage is reduced

Rustagi et al. (2014)

MaAGPase

Regulates biotic and abiotic stress signalling pathways, as well as fruit development and maturation

Miao et al. (2017)

MaAQP

Encourages early fruit development, as well as plant resistance to saline and osmotic stress, and accelerates post-harvest banana maturation processes

Hu et al. (2015)

MaARFs

Promotes early fruit development, as well as plant resistance to saline and osmotic stress, and accelerates post-harvest banana maturation processes

Hu et al. (2015)

MabZIP

Organ development, fruit maturation, and Hu et al. (2016) abiotic stress responses such as dry, cold, and salt, are all influenced by this protein

MusaNAC042

Modulates bananas’ response to abiotic stress Tak et al. (2017) by retaining high levels of total chlorophyll while decreasing MDA levels. (malondialdehyde)

MaHsfs

Fruit maturation, as well as biotic and abiotic stress are all involved in the development of various tissues or stages

Wei et al. (2016)

MaPIP1;2

Maintaining low levels of malondialdehyde while increasing proline concentrations, relative water content, and photosynthetic efficiency improves survival characteristics under abiotic stress

Sreedharan et al. (2013)

MaSODs

It is important for the removal of ROS caused Feng et al. (2015) by hormonal and abiotic stresses

MaPIP1;1

It improves saline and water stress tolerance while also reducing membrane damage, improving ionic distribution (K + /Na + ratio), and preserving osmotic balance

MusaDHN-1

This is caused in leaves by drought, alkalinity, Shekhawat et al. (2011) cold, oxidative stress, toxic substances, and treatment with signalling molecules such as abscisic acid, ethylene, and methyl jasmonate

MaCCS

It is produced as a result of light, heat, drought Feng et al. (2016) stress, abscisic acid, and indole-3-acetic acid

Xu et al. (2014)

(continued)

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Table 2.1 (continued) Candidate Gene

Applications

Authors

MusaSAP1

Reduces malondialdehyde levels and regulates polyphenol oxidases (PPOs), which play a key role in a variety of defence pathways

Sreedharan et al. (2012)

MusaWRKY71

It is important component for transcriptional Shekhawat et al. (2013) reprogramming is involved in several stress responses, including improved photosynthetic efficiency and reduced leaf membrane damage

MusaNAC68

Stress tolerance and root development are regulated by NaCl and mannitol

Negi et al. (2015)

WRKY

Regulated under a number of different of stresses, and involved in the development, growth, as well as ripening process of fruits

Goel et al. (2016)

PYL-PP2C-SnRK2

Regulates banana fruit maturation and tolerance to abiotic stresss

Hu et al. (2017)

Non-redundant DEGs Protein modifications, glycan metabolism alkaloid biosynthesis„ lipid metabolism, cofactor, hormone, terpenoids, amino acid biosynthesis, sugar-nucleotide, carbohydrate degradation and other secondary metabolites were among the processes it was involved in

Muthusamy et al. (2016)

MpASR

F. oxysporum f. sp. cubense and cold stress, low water, ABA, and high saline concentration show positive activity

Liu et al. (2010)

MaATG8f

Modulates abscisic acid biosynthesis, ROS metabolism, autophagic activity to positively regulate plant drought stress resistance

Li et al. (2019)

MaSWEETs

Elevate the sugar transport during the early stages of fruit development, as well as under biotic and abiotic stress

Miao et al. (2017)

Source Santos AS, Amorim EP, Ferreira CF, Pirovani CP (2018) Water stress in Musa spp.: A systematic review. PLoS ONE 13(12): e0208052. https://doi.org/10.1371/journal.pone.0208052

2.3.2 Salt Stress Tolerance in Banana Bananas are generally grown in places free from salt stress problems. Unlike other cereal crops, literature on the effect of salt stress on bananas is limited. Secondary salinization, caused by regular flood irrigation and poor drainage, is slowly making traditional banana-growing areas, particularly in command areas, more saline. Bananas are sensitive to salt stress in general, but Nendran and Robusta are particularly sensitive. In bananas, the cause of salinity problem is due to irrigation of underground water in Jordan Valley in Israel was recorded by Dunlap and McGregor (1932) and subsequently, the problem was solved through subsurface drainage to some extent. However, the problem resurfaced when surface irrigation was restored

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utilizing drip irrigation (Israeli et al. 1986). The effect of salinity in bananas was recorded by Wardlaw (1961) as there was a failure of the banana plantation and mentioned that 500 ppm of TSS in the soil water extract was toxic to the crop. In the banana plant, salinity toxicity symptoms and disorders have been documented in the Canary Islands (Garcia 1977), Colombia (Colmet-Daage and Gaytheyrou 1968) Ecuador (Charpentier and Martin-Prevel 1965), and Israel (Shapira et al. 2013). The quality of irrigation water significantly affects banana growth and yield. The banana growth and yield declined at ECw = 3.6 dS/m and ECs = 3.0 dS/m which are harmful to banana production. Under salinity conditions, the sodium level in the roots is six-fold more than the petiole, and the leaf has lesser sodium and chloride than the petiole (Israeli et al. 1986). Sodium partition is taking place in the ascending order of leaf < petiole < pseudostem < root to protect the productive photosynthetic leaf. Under K deficiency conditions, the Na was recorded in the conductive tissues, and this suggests for higher application of K under salinity conditions. Generally, salinity in the soil increases the osmotic pressure of soil water and results in developing osmotic stress, and in banana plants, the poor absorption of water leads to increased concentration of sap suggesting frequent and more irrigation to alleviate the effect of soil salinity. There is a significant linear correlation was found between EC of soil and water with chlorides content of roots and 3rd leaf. And also, a correlation was observed with Sodium Absorption Ratio (SAR) with sodium concentrations in the irrigation water, soil, and roots (Israeli et al. 1986). The sodium and chloride contents in roots are indicators of salinity stress. It is worthy to note that out of 100% Na+ and Cl− contents in a banana plant, 20% of chloride and 40% of sodium contents are present in underground (banana corm and roots) parts of the plant (Turner et al. 1982). Bananas are grown on above 30% of the land in India, despite adverse soil conditions such as pH > 8.5, ECe 4dSm−1 , and ESP > 15. Banana suffers from a salt injury in such saline-sodic soils, with external symptoms of marginal chlorosis of leaves and reduced photosynthetic activities, resulting in yield loss and lower finger weight. Salt has an effect on fruit development in susceptible banana varieties, particularly in the Cavendish group. Salt-related issues crop up in a dehydrated climate on alkali soils and/or when low-quality irrigation water is used. Excessive salinity raises sodium levels in roots, resulting in a significant decrease in potassium uptake, slowing growth, delaying flowering, and lowering yield. The optimal soil potassium and sodium ratio is 2.5, and when sodium makes up more than 8% of the total exchangeable cations the banana yields are reduced. Although the salt effect cannot be eliminated, a dual-purpose banana variety called ‘Kaveri Saba’ can be grown in such soils with frequent irrigation and adequate drainage.

2.3.2.1

Molecular Biology of Salt Stress in Banana

Plant (ROPs)—Rho-like GTPases is plant-particular molecular switches that are required for plant survival in the face of abiotic stress (Miao et al. 2017). Using genomic techniques, 17 novel ROP proteins from Musa acuminata (MaROPs) were

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identified and characterized. The two banana genotypes tested had similar responses to abiotic stress, according to their transcriptomic analysis. In response to salt stress, MaROP5g was the most extremely expressed of these. Under salt stress, over expression of MaROP5g in transgenic Arabidopsis thaliana resulted in longer primary roots and higher survival rates than wild-type Arabidopsis thaliana. MaROP5g confers increased salt tolerance by protecting membrane damage, increasing the Calcium concentration in transgenic plants and raising the cytosolic potassium and sodium ratio when compared to wild-types. Sreedharan et al. (2015) MusaPIP2;6 was identified as an aquaporin gene, and it was characterised by overexpression in transgenic banana plants to investigate its potential functions in bananas. It was discovered that using a constitutive or inducible promoter (pMusaDHN-1) to overexpress MusaPIP2;6 in transgenic banana plants results in higher salt tolerance than equivalent untransformed control plants. Under salt-stressed conditions, Musa PIP2;6-overexpressing transgenic banana plants had less membrane damage (MDA equivalents) and higher photosynthetic efficiency (Fv/Fm ratios). MicroRNAs (miRNA) play major roles in development, growth, stress responses, physiology, and RNA interference pathways, including siRNA and miRNA biogenesis, in plants, largely through negative regulation of transcription factors. Plants’ responses to abiotic stress, such as salinity, have been shown to be modulated by several miRNA families. Lee et al. (2015) miRNAs were discovered in response to salinity stress. MiRNAtargets have been predicted for some theoretical or unspecified proteins and unannotated genes, and their corresponding salt stress-responsive miRNAs showed inverse expression patterns. CL2012Contig (macmiR156 and mac-miR49 targets), C111260 (mac-miR157 target), CL1Contig6497 (miR528), CL7009Contig1 (miR529), CL7639 (target of mac-miR38) (target of mac-miR62) and C102056 (mac-miR38 target) (Lee et al. 2015). Mazumdar et al. (2020) through genome-wide analysis, members of the MYB gene family linked to stress in the banana (Musa acuminata Colla). MaMYB is a non-redundant set of 154 MYB genes that encode proteins with variable lengths that are mostly found in the nucleus. MaMYB is divided into three subfamilies based on conserved repeats: R1-MYB, R2R3–14 MYB, and R1R2R3-MYB, with the R2R3-MYB subfamily accounting for the majority (80.5%). MaMYB is involved in the regulation of metabolism, plant development and various stress responses, according to ontology predictions. The genes were found in abundance among the 11 banana chromosomes, with the maximum density in chromosome 9 (11.04%). The chromosomal distribution pattern strongly suggested that dispersed duplication was the primary cause of the MaMYB gene family’s expansion in the banana. MaMYB63, MaMYB14 and MaMYB110 had significantly increased expression in the roots of banana plants exposed to salt stress, according to quantitative RT-PCR analysis (Mazumdar et al. 2020).

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2.3.3 Temperature Stress Tolerance in Bananas 2.3.3.1

High Temperature Stress

High air temperatures (>38 °C) with bright sunshine cause scorching on the exposed leaves and sunburned fruit, particularly the top hands of the bunch. Elevated temperatures in the subtropical regions are extremely high, resulting in pale and short plants and scorching sun (Ziv 1963; Simmonds 1966). The correlation between the rate of emergence of banana leaves and temperature has been established in the tropics and subtropics based on field studies (Barker 1969; Ganry 1973). The optimal temperature for the emergence of fresh leaves, according to the findings, is around 28–30 °C (mean day-night temperature). Stoler (1962) discovered a link into bunch size, pseudostem and Tm as well as a lower optimum temperature of 22–24 °C. The optimum temperature for crop growth, according to Champion (1963) and Simmonds (1966), is around 25–27 °C. Higher temperatures result in more horizontal leaves, but the lamina fold more easily to compensate. The unfurled cigar leaves may lose turgor under high temperature and resulting in curvature and sun scorch taking place. Santos et al. (2015) discovered Musa acuminata ssp. burmannicoides var. Calcutta 4 (AA) leaves had genes that responded to extreme temperatures. To create a COLD and a HOT cDNA library, the plants were subjected to temperature stress (cold and higher) ranging from 5 °C to 25 °C and 25 °C to 45 °C, respectively. After quality analysis and vector trimming, they assembled 2,286 sequences from both libraries into 1,019 recognized transcripts, exists of 217 clusters and 802 singletons, which they dubbed Musa acuminata assembled expressed sequence tagged (EST) sequences (MaAES). Only 22.87% of these MaAES are unique because that don’t match any consisting sequences in the public databases. According to a global analysis of the MaAES data set, 10% of sequenced cDNAs were found in both cDNA libraries, while 42% and 48% were found in only the COLD or HOT libraries, respectively. 715 (31.28%) of the 2,286 high-quality sequences came from full-length cDNA clones, yielding a set of 149 genes. HSPs are coded for by a set of 27 cDNAs derived from these. Cytoplasmic proteins are produced among thirteen sHSPs genes. HSPs were discovered from twelve full-length cDNAs, nine of them are HSP family and are present in chloroplast and cytoplasm. Genes involved in the photosynthesis apparatus and those related to temperature stress, such as ribulose 1, 5 -biphosphate carboxylase/oxygenase (10.72%), HSP (1.71%), sHSP (8.50%) metallothionein-like protein (6.91%) and PS I/II (9.75%), were found among the 2,286 sequenced transcripts.

2.3.3.2

Low Temperature Stress

In subtropical environments, cooler growing conditions reduce banana fruit yield and quality. Crop cycling takes longer, low temperatures can harm plants and fruits, and bunches and fruits develop abnormally. Some cultivars are more susceptible to pests and diseases as a result of the cooler temperatures. Consumers should avoid

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the dull gray/yellow fruit with under-peel discoloration from chilling. The typical estimate measures of black spotting of leaves, chlorophyll photo-oxidation, vigor ratings and petiole blackening, are supported by a simple prototype sap flow test that shows a practical, objective measure of cold tolerance. After harvesting the peduncle was removed from the top hand from the bunch, and the number of sap drops on the cut surface of the hand was counted in the first minute. During the winter there was no sap flow in any of the ten Cavendish types (AAA) tested, and only two had sap flow in mid-November, well into the Australian spring. Sap flow was observed in all four tetraploid cultivars, one ABB cultivar and two AAB cultivars, throughout the winter months. Weinert et al. (2020) proposed a simple method for determining which banana genotypes are cold tolerant. Zhang et al. (2011) the biochemical response of a tolerant cv. Cachaco (Musa paradisiaca ABB cv. Dajiao) and a susceptible cv. Williams (Musa acuminata AAA cv. Williams) and banana under low temperature (LT) of 7 °C was investigated. In cv. Williams, the Low Temprature treatment resulted in higher levels of higher levels of malondialdehyde (MDA), hydrogen peroxide (H2 O2 ) and, lower photochemical efficiency (Fv/Fm) and net photosynthetic rate (P N), superoxide radical (O2). However in cv. Cachaco was more tolerant to LT than cv. Williams. Total scavenging capability (DPPH scavenging capability) in Williams decreased significantly after 120 h of LT treatment, but there were no significant changes in Cachaco. After 120 h of LT treatment, Cachaco showed a gradual increase in peroxidase (POD), ascorbate peroxidase (APX) but no significant changes in superoxide dismutase (SOD) and a significant decrease in catalase (CAT). After 120 h of LT treatment, Williams showed a significant decrease in all four antioxidant enzymes. They proposed that higher APX, POD, SOD, and DPPHscavenging capability can be used to explain to some extent the plantain’s elevated cold tolerance, providing hypothetical guidance for banana production and cold-resistant variety screening. Musa spp. ‘Dajiao’; ABB Group; has a higher frozen tolerance than Cavendish Banana (Musa sp. Cavendish; AAA). To better identify with the regulatory mechanisms of cold signaling pathways in this coldtolerant banana, the functions of MaMAPK3 and MaICE1 were investigated in these cultivar. Many abiotic factors have been shown to activate MAPK (mitogen-activated protein kinase) cascades (Lee et al. 2016; Yi et al. 2016). Plant plasma membrane receptors detect signals and activate the mitogen-activated protein kinase kinase kinase (MAPKKK). MAPKKK then phosphorylates mitogen-activated protein kinase kinase (MAPKK), and the activated MAPKK then phosphorylates mitogen-activated protein kinase (MAPK) (Zhang et al. 2018). It has been confirmed that the MAPKKK1-MAPKK2-APK4/ 6 cascade is involved in the positive regulation of cold treatment (Furuya 2013; Teige 2004). The wildtype (WT) plants, on the other hand, remained normal after being exposed to cold. Cold-responsive genes changes the expression in WT plants, RNAi of MaMAPK3 and altered oxidoreductase activity, but there was no changes in transgenic plants. In MaMAPK3 RNAi transgenic plants, MaICE1 interaction with MaMAPK3 was reduced, as was MaICE1 expression. MaICE1 overexpression conferred cold resistance in transgenic

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Cavendish banana (Musa spp. AAA group) plants, compared to WT plants. In the MaICE1-overexpressing transgenic plants, the POD P7 gene was upregulated and interacted with MaICE.

2.3.4 Flooding and Inundation in Banana There are not many studies on flooding tolerance in bananas. In banana growing areas, flooding occurs mostly through the cyclone and tropical storms. Bananas can relatively tolerate flooding with flowing water for 48 h compared to stagnation. The stagnant water kills the plant in 24 h. Banana roots are sensitive to low oxygen (O2) content, a consequence of water logging (WL). Aguilar et al. (2008) observed that root characters that favor reduced physical resistance to gas diffusion (R rho, s cm−3 ) may improve tolerance to hypoxic stresses of banana roots caused by an inundation of water. Root length under WL was significantly reduced by about 48% compared with well-drained control. Regardless of root type, treatment, and sampling time, a trend in root thickness was the same, in the order of Saba (ABB) > Tindok (ABB) > = Latundan (AAB) > Lakatan (AA) > Quarentadias (QD) (AA). Root diameter was highly associated with the B genome and ploidy. All of the five cultivars had > 10% porosity even in well-drained conditions. When waterlogged, QD (AA) had significantly reduced R rho along the root length whereas, the thicker roots of Saba and Latundan reduced R rho only at the base.

2.4 Modern Biotechnological Tools in Banana Improvement Modern biotechnological tools and advancements paved the way for faster genetic improvement of bananas. The conventional breeding in bananas is very slow and unable to produce many desirable hybrids, owing to its limitations of parthenocarpy, male sterility, poor seed set, and poor seed germination. In the meantime, the development of genetically modified (GM) bananas has been more successful and handier in bananas. Now, genome editing has come as a potential tool to manipulate any popular banana cultivars with any desirable traits. Especially it is very handy for conferring biotic and abiotic stress resistance. Bananas with genetically modified genomes are already available (Dale 2020). Three bananas have been edited with the CRISPR/Cas9 gene. Cavendish had the phytoene desaturase (PDS) gene knocked out, and tri-allelic knockouts had an analbino phenotype, as expected. In Plantain, the PDS gene was knocked out, and banana streak virus sequences were deleted using genome editing (Dale 2020). According to these findings, genome editing in bananas will not be genotype specific. Because they have an integrated cassette containing the Cas9 gene, the guide RNA gene, and selectable marker gene, all of these edited

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plants are transgenic. In seed propagated crops, it is possible to ‘breed away’ from the integrated CRISPR/Cas9 cassette. To make an edited ‘non-GM’ banana, only the guide RNA and Cas9 protein will need to be expressed transiently, perhaps as a ribonucleoprotein. It becomes a possibility to develop a protoplast generation and regeneration protocol to make sure that edited plants can be regenerated from a single edited cell (Dale 2020). This will be difficult. Abiotic stress tolerance has been more difficult to develop. Though the modified banana plants indicated positive at the glasshouse level, they could not be progressed to field trial. As of now, no banana cultivars with different genomes AA, AAA, AAB, and ABB have been amenable for genetic modification and not highly genotype-specific, unlike many other crops.

2.5 Future Perspectives and Challenges Bananas are one of the most widely produced and consumed fruits on the planet. Bananas are grown on a large scale, and it is common to see indiscriminate use of agrochemicals, pesticides. And other abrasive production methods for scheming irrigation and plant diseases, as well as the environmental impacts of banana production and resource waste. As a result, the cost of producing bananas continues to rise. High levels of competition among international traders and leading retail chains, which are exerting a strong downward influence on prices, continue to exert pressure on labourers’ earnings and are already economically deprived small scale farmers. There is no standard price for bananas and many years the price of bananas is not increasing, despite an increase in input cost. Because of low prices for production, unable to pay decent wages to the workers in this sector. The government and larger companies around the world are not investing enough to keep banana production going. Banana industry created a multi-stakeholder platform called the World Banana Forum (WBF) to address the industry’s challenges. This forum provides a venue for the major players in the global banana supply chain to come together and agree on best practises for long-term production. The WBF’s mission is to foster stakeholder association that results in practical outcomes for the betterment of the banana industry, as well as to achieve consensus on best practises in the areas of gender equity, workplace issues, sustainable production, and environmental impact. Producers, retailers, exporters, importers, consumer associations, research institutions, governments, trade unions, and other civil society organizations are all represented at the WBF (www.fao.org). Developing good cultivation practices, regulate diseases in plant and improving producer organizations, and maintain both domestic and global marketing strategies are all things the FAO is working on with governments to build a viable banana sector. The Intergovernmental Group on Bananas and Tropical Fruits (IGG) of the FAO serves as an intergovernmental forum for consultation and exchange on banana trends in banana and tropical fruit production, consumption, trade, and prices, as well as a regular assessment of the global short-term outlook and market situation.

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A study conducted by Varma and Bebber (2019) on Climate change has had a positive impact on banana cultivation, with 27 countries accounting for 86% of global dessert banana production increasing crop yield since 1961 as a result of improved growing conditions in some of the world’s most important producing and exporting countries. Furthermore, ten countries, including the world’s largest producer and consumer of bananas, India, and the fourth-largest producer, Brazil, are expected to see crop yields decline. However, this climate change favors countries like Ecuador (the largest exporter) and Honduras, and many African countries may get overall benefits in crop yields. The important point is that the vulnerable countries must prepare to combat the negative impact of climate change through investment in technologies like irrigation. Practical solutions already exist, but these are scattered across banana-producing countries to counteract predicted yield losses due to climate change. An open exchange of knowledge and ideas will be critical (Varma and Bebber 2019).

2.6 Conclusion Internationally, the banana is one of the important traded fruits and provides staple food for most of the African countries and nutritional security for many Asian countries. Being an important world traded fruit crop, it is facing challenges on production in the traditional areas due to changing climate. There are reports on climate change that, there may be a shift in banana production regions in the world by 2050. A lot of research and focus has been given to biotic stresses in bananas and a similar effort is required in abiotic research in coming years to sustain banana production. The major hurdle in the improvement of bananas is breeding for desirable traits in desired cultivars. Modern tools like genetic modification through transgenic and CRISPR may hasten the process of the banana improvement program. The banana researchers from all over the world and from all the disciplines need to give more attention to tackling the negative impact of abiotic stress in bananas. It is very important to know the fact that, the abiotic stresses cause irreversible damage to the banana plantation if it is not addressed at the earliest possible time. All banana stakeholders must come forward and cooperate to exchange their knowledge, exchange technologies, and investment to combat the negative impact of climate change-induced abiotic stresses in bananas.

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Teige M, Scheikl, E, Eulgem T, Dóczi R, Ichimura K, Shinozaki K, Dangl JL, Hirt H et al (2004) The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol Cell 15(1):141–152 Tripathi L, Ntui VO, Tripathi JN (2020) CRISPR/Cas9 based genome editing of banana for disease resistance. CurrOpin Plant Biol 56:118–126 Tuberosa R (2012) Phenotyping for drought tolerance of crops in the genomics era. Front Physiol 3 Turner DW (1970) Daily variation in banana leaf growth. Aust J Exp Agric Anim Husb 10(23):1–4 Turner DW (1971) Effects of climate on rate of banana leaf production. Trop Agri (trinidad) 48:283– 287 Turner DW, Lahav E, Short CC (1982) The growth and chemical composition of the Williams banana in relation to temperature. Banana Nutr Newsletter 5:15–16 UNDES (United Nations Department of Economic and Social Affairs) (2019) UN Varma V, Bebber DP (2019) Climate change impacts on banana yields around the world. Nat Clim Change 9:752–757 Van Asten PJA, Fermont AM, Taulya G (2011) Drought is a major yield loss factor for rainfed East African highland banana. Agric Water Manag 98:541–552 Wardlaw CW (1961) Banana diseases: Including plantains and abaca. London: Longmans, Green and Co Ltd 62–62 Wei Y, Hu W, Xia F, Zeng H, Li X, Yan Y et al (2016) Heat shock transcription factors in banana: genome-wide characterization and expression profile analysis during development and stress response. Sci Rep 6:1–11 Weinert MP, Peasley DL, Smith MK, Drenth A (2020) A simple cold tolerance tests for banana cultivars. Acta Hort 1272: 33–38. www.fao.org.http://www.fao.org/economic/est/est-commodities/ bananas/bananafacts/en/#.YFp6Cq8zZPY Xu Y, Hu W, Liu J, Zhang J, Jia C, Hu H et al (2014a) A banana aquaporin gene, MaPIP1;1, is involved in tolerance to drought and salt stresses. BMC Plant Biol 14:1–14 Xu Y, Hu W, Liu J (2014b) A banana aquaporin gene, MaPIP1; 1 is involved in tolerance to drought and salt stresses. BMC Plant Biol 14:59 Yi J, Lee Y, Lee D, Cho M, Jeon J, An G et al (2016) OsMPK6 plays a critical role in cell differentiation during early embryogenesis in Oryza sativa. J Exp Bot 67(8):2425–2437 Zhang M, Su J, Zhang Y, Xu J, Zhang S et al (2018) Conveying endogenous and exogenous signals: MAPK cascades in plant growth and defense. Curr Opin Plant Biol 45:1–10 Zhang Q, Zhang JZ, Chow WS, Sun LL (2011) The influence of low temperature on photosynthesis and antioxidant enzymes in sensitive banana and tolerant plantain (Musa sp.) cultivars. Photosynthetica 49:201–208 Ziv D (1963) High soil temperature damage to bananas. Hassadeh 44:298–302 (In Hebrew.)

Chapter 3

Genomic Design for Abiotic Stress Resistant Citrus Angelo Sicilia, Supratim Basu, and Angela Roberta Lo Piero

Abstract Citruses are native to subtropical and tropical regions of Asia and the Malay Archipelago, but are present in the Mediterranean basin for centuries. The subtropical semiarid regions in which Citrus species grow are characterized by water deficit representing not only a serious environmental threat itself but also imposing the use of low quality water for supplemental irrigation raising the salt concentration in the soils to critical levels. These adverse environmental conditions, drought and salinity, as well as the occurrence of extreme temperatures, could be particularly severe on woody crop survival such as citruses that are part of the backbone of traditional Mediterranean agriculture. The Citrus species have been traditionally selected for traits such as improved fruit yield and quality or alteration in harvesting periods, leaving out traits related to plant field performance. Conventional breeding strategies played an crucial role in citrus cultivar advancement, particularly by taking advantage of natural mutations, or by exploring the acquired tolerance of naturally occurring, or induced polyploids. However, cross hybridization of citrus, which could be a powerful tool for getting heterozygosity in yearly crops, has come to restricted progressions due to characteristic reproductive habits, such as polyembryony, incompatibility, male and/or female sterility and long juvenile period. In the last decades, the availability of high-throughput sequencing techniques and computational analysis have added valuable information in genomic data also in the Citrus genus. In this chapter, the analysis of the main abiotic stresses affecting citriculture worldwide is followed by the description of how the available genetic resources might help citrus breeding by the application of biotechnological tools such as the identification of quantitative trait locus (QTL) regions, marker assisted selection, genome wide association study and genomic selection. In addition, the transition from genetic A. Sicilia · A. R. Lo Piero (B) Dipartimento di Agricoltura Alimentazione e Ambiente, Università degli Studi Catania, via Santa Sofia 98, 95123 Catania, Italy e-mail: [email protected] A. Sicilia e-mail: [email protected] S. Basu Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR, USA e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 C. Kole (ed.), Genomic Designing for Abiotic Stress Resistant Fruit Crops, https://doi.org/10.1007/978-3-031-09875-8_3

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engineering to genome editing is also shown, highlighting the enormous potentiality of these novel tools but not hiding the remaining difficulties to put them into pratice. Keywords Citrus · Abiotic stress · MAS · GWAS · GS · Transgenesis · Genome editing · NPBT

3.1 Main Abiotic Stresses Affecting Citriculture: Drought, Salinity and Extreme Temperatures Citrus are part of the Rutaceae family, Aurantioideae subfamily. Most of the cultivated citrus species belong to the Citrus genus (Tanaka 1961; Swingle and Reece 1967). Several studies (Nicolosi et al. 2000; Barkley et al. 2006; Garcia-Lor et al. 2013; Curk et al. 2015, 2016; Oueslati et al. 2017; Wu et al. 2018) uncovered the presence of four originating taxa: Citrus maxima (Burm.) Merr. (the pummelos), Citrus medica L. (the citrons), Citrus reticulata Blanco (the mandarins) and Citrus micrantha Wester (wild papeda species). The other developed species Citrus aurantium L. (sour orange), Citrus sinensis (L.) Osbeck (sweet orange), Citrus paradisi Macf. (grapefruit), Citrus limon (L.) Burm. F. (lemon) and Citrus aurantifolia (Christm.) Swingle (lime) come about from the recombination of the ancestral taxa. A few genera (Poncirus, Fortunella, Eremocitrus, Microcitrus, and Clymenia) are interfertile consistent with Citrus species and constitute the complete citrus group (Swingle and Reece 1967). Water shortage periods contrarily influence citrus plant efficiency in numerous aspects, counting decrease in growth and metabolism, which leads to a diminish in both fruit quality and yield (Pérez-Pérez et al. 2008). As citrus culture happens primarily in dryland, citrus breeding is centered on the choice and utilize of scion-rootstock combinations with distinctive reactions to dry season. Long time of field perceptions revelead that Rangpur lime (Citrus limon (L) Osbeck) is one of the foremost drought-tolerant rootstocks (Davies and Albrigo 1994), whereas a few of the broadly utilized commercial rootstocks, such as trifoliate orange (Poncirus trifoliata) and sour orange (Citrus aurantium) are not tolerant to dry season challenges. In 2006, a study showed that the Cleopatra mandarin tree (Citrus reshni) can endure direct water stretch and is more productive in soil water use than Carrizo citrange [(Citrus sinensis (L.) Osb. X Poncirus trifoliata (L.) Raf.], in water shortfall conditions. The influence of recurrent water deficit on the physiological, molecular and hormonal changes in Valencia (VO) scion variety (C. sinesis Osbeck) grafted on two rootstocks with different soil water extraction capacities: (Citrus limonia Osbeck, “Rangpur Lime”, RL) and (C. sunki hort. Ex Tanaka, Sunki Maravilha, SM) was also investigated (Neves et al. 2017). The results show that epigenetic modifications including DNA methylation are involved in dry condition resistance in Citrus. Correspondling, frequencies of methylated polymorphic segments recognized by the methylation sensitive amplified polymorphism (MSAP) method are particularly diverse between VO/RL and VO/SM plants indicating that repetitive dry seasons trigger memory to stress more clearly in VO plants grafted onto SM

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than in VO plants grafted onto RL. Citrus has been classified as a salt-sensitive crop (Maas 1993) since of saline water irrigation diminishes tree growth and fruit yield (Grieve et al. 2007). Elevated salt levels within the soil influence all plant physiological processes by inducing nutritional imbalances, osmotic stress and ion toxicity. In Citrus, salinity leads to leaf nutrient deficiencies (Grattan and Grieve 1992), which might cause significant growth loss. The presence of salts within the nutrient solution applyes an osmotic force that diminishes the accessibility to free water through physical mechanisms that disable water extraction from soil by roots. In Citrus, growth reductions can be also related to an accumulation of toxic levels of Cl− , Na+ or boron (B) in leaves (Levy and Syvertsen 2004). It is well known that Cl− and Na+ harmfulness decreases the CO2 absorption in citrus trees causing a sharp slowdown of the photosynthetic process and inducing a conceivable disability of the electron flow. Cl− is a major osmotically active solute within the vacuole fulfilling crucial role in both turgor maintaince and osmotic regulation (Marschner 1995; Colmenero-Flores et al. 2007). Woody perennial plants display toxicity symptoms to chloride (Cl− ), rather than to sodium (Na+ ) accumulation (Munns and Tester 2008). In citrus, the prove of the harmuful impact of chloride is mostly affirmed by the relationship between genetic differences in the rate of Cl− accumulation and the salinity resilience (Moya et al. 2002). It is widely believed that the citrus resistance to salt stress is related to its ability to exclude Cl− , or to the plant ability to limit Cl− uptake and the upward transport, this last being subordinated upon the ability of the rootstock. Carrizo citrange [(Citrus sinensis L. Osb. X (Poncirus trifoliate) L. Raf.] and rough lemon (Citrus jambhiri Lush.) are examples of Cl− includers considered their restricted capacity to avoid Cl− , thus causing their affectability to salt stress (Bañuls and Primo-Millo 1995; Levy and Syvertsen 2004; Alvarez-Gerding et al. 2015). Cleopatra mandarin (Citrus reshni Hort. Ex Tan.) and Rangpur lime (Citrus limonia) are both as chloride excluder rootstocks, and for this reason considered salt-tolerant. Sour orange (Citrus aurantium) is classified as a good Na+ and Cl− excluder (Gimeno et al. 2009) and is commonly used in in areas with high pH and calcareous soils. In any case, it is exceedingly vulnerable to Citrus tristeza virus (CTV) infections (Moreno et al. 2008). Other trifoliate rootstocks such as trifoliate hybrids, C22 and C146 (Citrus sunki Hort. Ex Tan. × Poncirus trifoliata L. Raf ‘Swingle’) are tolerant to CTV and, likely due to the capacity of trifoliate orange parent to restrain toxic ion upward translocation (Walker 1986), they are more tolerant to saline irrigation water than the sour orange rootstocks. Low temperature is one of the foremost natural adversity a plant has to cope and it can possibly cause serious losses particularly to crops developing in subtropical and tropical areas such as Citrus varieties, which are consequently considered vulnarable to cold. Specifically, cold stress, comprising chilling (40

Partial or total drying of leaves and grape berries; Disruption of cell membranes and irreversible protein degradation

>35

Damage to the photosynthetic apparatus; Anthocyanin degradation in the grape berries

>30

Decrease on anthocyanin synthesis and increase in its degradation; Decoupling between anthocyanins and sugars and acidity of grapes

>25

Decrease on volatile compounds synthesis and increase in its volatilization in grapes from red grapevine varieties

>20

Decrease on volatile compounds synthesis and increase in its volatilization in grapes from white grapevine varieties

Adapted from Gutiérrez-Gamboa et al. (2021)

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4.2.3 Cold Tolerance Low temperature is amongst the detrimental abiotic stresses, which affects grapevine survival in temperate climate. Understanding the physiology of grapevine cold resistance have relevance from the perspective of agricultural production and breeding of highly productive cold tolerant grapevine genotypes (Ollat et al. 2017). Yield in grapes are restricted by the influence of winter frosts, especially, after long warm periods in summer. The most pertinent traits characterizing the resistance of the grape varieties to stressors of winter periods are water content of shoots, free and bound water contents and their ratio, proline, protein, sugars, amino acid and the content of ascorbic acid etc. The resistance of the lipid phase of cell membranes to destruction is considered as one of the important elements of resistance of grape varieties to low temperatures. An important role in maintaining the integrity of the lipid phase is imparted by the water-soluble antioxidant-ascorbic acid, which is able to restore membranebound tocopherol. Winter-hardy varieties are characterized by high content of bound water and great resistance of plant cells to dehydration, imparted by osmoprotectors like proline and sucrose (Jie et al. 2008). Nenko et al. (2019) estimated the various mechanisms of adaptation of grape plant to winter period stress factors. Correlation coefficients between the content of the bound water, proline and sucrose showed that in the more winter-hardy cv. ‘Kristall’, there was higher dependence of bound water on proline content, than for the cvs. ‘Dostoyniy’ and ‘Krasnostop AZOS’. Proline accumulation in plants is accompanied by preclusion of denaturation of various proteins and preservation of activity of enzymes, which also imparts winter hardiness (Jie et al. 2008). Rooy et al. (2017) reported an increase in sugars in response to low temperature stress, as sugars are able to improve the stabilisation of the biomembrane by reducing the freezing temperature of intercellular water. Under cold stress, of abscisic acid contentsincreased almost fivefold in ‘Krasnostop AZOS’; thereby, activating the cold resistance genes. The higher resistance of ‘Kristall’ variety to desiccation and formation of proteins is associated with increased proline content and strengthening the cell walls in response to stress factors (Davey et al. 2000).

4.2.4 Drought Tolerance Water unavailability is one of the main limiting factors for yield and quality of grapevines (Marguerit et al. 2012). Grapevine responses to drought stress involve various components related to developmental, physiological, hydraulic, and metabolic processes (Chaves et al. 2010; Serra et al. 2014; Ollat et al. 2017). Water stress results in a lower yield mediated by restrained photosynthesis (Marguerit et al. 2012).

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Various genes such as F3H, F305H, LDOX and DFR are up-regulate during the water-stress conditions. These are also involved in the biosynthesis of antioxidants and colouring imparting compounds like proanthocyanidins, anthocyanins and flavonols. Furthermore, upon prevalence of drought stress during berry ripening, expression of genes related to anthocyanin accumulation (UFGT ) and transport (GST ) takes place, leading to higher berry coloration (Deluc et al. 2009). Up regulation of various differentially expressed genes (DEGs) related to the proline biosynthesis was observed in drought treatment compared to control (Haider et al. 2017). Under drought stress, accumulation of compatible osmolyte like proline leads to reduction of the hydric water potential of grapevine along with maintaining turgor pressure during drought (Liang et al. 2013; Canoura et al. 2018).

4.2.5 Flooding and Submergence Tolerance The frequency of floodings increased about 65% over the last 25 years, thereby, causing globally more climate-related disasters than any other extreme climatic factors, (FAO et al. 2018). Flooding radically lowers O2 availability for root respiration and plant survival. Anoxia condition in the soil leads to the formation of toxic compounds such as H2 S, N2 , MnC2 , FeC2 and ROS, and impacts the biosynthesis of stress related hormones like, ethylene and abscisic acid in roots (Carvalho et al. 2015; Loreti et al. 2016). Morpho-physiological aspects of grapevine adaptation to flood stress indicate towards an overall reduction in photosynthetic rate, stomatal conductance and plant height, along with premature senescence and strifling of yield components (de Herralde et al. 2005; Mugnai et al. 2011). Studies have shown various degrees of tolerance to flooding for some grapevine rootstock genotypes. Genotypes 420A, K5BB, 1616C and 3309C have been noted to be moderate flood tolerant as compared to 41B, 110 R, 140Ru, and 1103 P (Striegler et al. 1993; de Herralde et al. 2005; Mugnai et al. 2011). V. riparia’s higher tolerance is attributed to its better ability to maintain ion homeostasis (sustaining KC uptake) during hypoxia (Mugnai et al. 2011). Zhu et al. (2018a, b) investigated the response mechanism to waterlogging through the study of the transcriptomic regulation networks of grapevine leaves. They noted 12,634 genes in both control and waterlogged grapevine plants, of which 6837 genes differentially expressed. Ruperti et al. (2019) studied the transcriptional and metabolic responses in K5BB rootstock under flooding. There was down regulation of the various genes involved in the biosynthesis of plant growth regulators like auxin, brassinosteroid and gibberellin.

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4.2.6 Nutrient—Use Efficiency Analysis of rootstocks grown under various nitrogen (N) supply conditions showed that they differed in their ability to distribute minerals between roots and shoot parts. Gautier et al. (2018) demonstrated variability in P acquisition and utilization efficiencies among genotypes. A hypothesis that rootstocks may differ in N satiety was proposed by Ollat et al. (2017) and the same was confirmed in a transcriptomic study through split-root experiment (Cochetel et al. 2017). The role of strigolactones in response to N availability was investigated further by Cochetel et al. (2018). As rootstocks mainly are interspecific hybrids, it is relevant to quantify the extent to which the species contain specific alleles that define and differentiate their nutrient efficiencies. Those particular alleles could be introgressed to breed highly efficient rootstocks. The assessment of variability amongexisting rootstocks from diverse genetic backgrounds can generate interesting data for the requisite purpose.

4.2.7 Water—Use Efficiency Water shortage can be problematic in areas characterized by temperate, Tropical (arid and semiarid) or Mediterranean climates (Garcia-Tejero et al. 2014) where table grapes are grown usually under irrigated conditions (Williams et al. 2010). Compared to wine grapes and other types, table grapes are very high annual yielders and are considerably of higher water productivity (Teixeira et al. 2009); thus requiring high water inputs to reduce the risks of loss of quality and yield of grape berry. Deficit irrigation strategies may differ for table—grape production from those used in wine—grape production as their desired berry quality attributes are different (Williams et al. 2010; Conesa et al. 2015). In classic deficit irrigation (DI) strategy water is supplied below full crop evapotranspiration (ETc) in the growing period. Among the entire set of yield and biophysical quality traits clusters, number of commercial clusters, berry weight, berry size/diameter, color and firmness are severely affected by deficit irrigation. The timing of stress imposition through deficit irrigation influences on yield, WUE yield and berry quality (Myburgh 2003). The majority of the studies on table grapes indicate internal berry traits (TSS, TA and pH) mostly influenced by deficit irrigation (El-Ansary and Okamoto 2008; Du et al. 2008). The effectson the berry traits are not linear, and depend on the genotype, climate, and growing conditions. The two other strategies are regulated deficit irrigation (RDI) and partial root drying (PRD) (English 1990; Fereres and Soriano 2007). The RDI during specific periods may benefit WUE, increase water saving and improve berry quality (McCarthy et al. 2002; Loveys et al. 2004). In table grapes, RDI is generally used in post-veraison phase, i.e. at the onset maturation, as reduced watering before this stage can drastically reduce berry size and yield (Conesa et al. 2015). PRD approach works on root exposure to alternate cycles of drying and wetting. This results in plants growing with reduced stomatal conductance to water vapour

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and without or with minimal signs of drought stress (Zhang et al. 1987; Dodd et al. 2006). PRD also decreases vegetative growth and increases WUE. It also promotes root growth at deeper soil layers in grapevine (Santos et al. 2005). Imposed water deficit changes berry growth and accelerates ripening via up-regulation of genes controlling flavonoids pathway leading to increased flavour and accumulation of anthocyanins in berries.

4.2.8 Other Abiotic Stresses 4.2.8.1

Solar Radiations

Amongst the various parts of the solar spectrum, only a small proportion of the UV-B forms the most energetic component of day-light spectrum used by plants for growth and development. The light is also considered limiting factors affecting growth and development in plants (Caldwell et al. 2003; Isah 2019). On grapevine plants ultraviolet UV-B radiation strongly influences growth and normal fruit development. A higher proportion of UV-B leads to reduction of shoot length and leaf area of grapevine, but increase in leaf thickness (Berli et al. 2013). Higher accumulation of terpenes also reported in response to UV-B (Gil et al. 2012). Higher adaptation of grapevines to solar radiations can be attributed towards its multifaceted physiological responses. Higher antioxidant enzyme activities and higher secondary metabolites also plays a major role in this adaptation. Higher accumulation of various phenolic compounds, flavonoids, cinnamate esters, coloring pigments in vacuoles of epidermal cells upon exposure to UV-B radiations takes place. These responses triggered by UV-B perception and signaling pathway, have recently been identified and characterized in grapes (Liu et al. 2015). In grapevine, in leaf epidermis and fruit berry skin, anthocyanins and flavonols increase due to UV-B (Berli et al. 2011; Kolb et al. 2001; Pollastrini et al. 2011). The quality of grape berries for wine-making is correlated with higher accumulation of phenolic compounds. Therefore, understanding the underlaying mechanism of perception, signaling and response of the grapevine to UV-B to improve productivity and fruit quality by genetic modification is important for wine industry.

4.2.8.2

Light Stress

It includes various phenomenon like photosynthetic activity variations, photoxidation and photoinhibition. In leaves, both the deficiency and excess of light induce the stomatal and metabolic responses such as accumulation of few to an array of reactive oxygen species and activities of various antioxidant enzymes. In V. vinifera leaves, light increased photoprotective compound concentration (Kolb et al. 2001). Leaf carotenoids increased with UV-B treatment; synthesis of carotenoids is from vioxanthin cycle and lutein epoxide cycle (Young et al. 2012). Downey et al. (2006) did

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not notice any significant difference in the accumulation of anthocyanin in berries of Syrah grape under shade and light exposed condition. However, increase in the berry flavonol concentration with high exposure to sunlight before veraison period was observed (Matus et al. 2009). Light enhanced the expression VvFLS and VvMYBF1 gene, which are involved in the flavonoid synthesis (Koyama et al. 2010).

4.2.8.3

Elevated CO2

Elevated temperature accelerated the vegetative growth and grape development, but reduced grape production (Arrizabalaga-Arriazu et al. 2020). In general, combined elevated temperature and CO2 atmospheric concentration hastened grape development and stimulated vegetative growth on the contrary, yield and yield-related parameters were affected negatively, mainly due to high frequency of heat waves in high temperature treatments, regardless of CO2 levels. Although elevated CO2 enhances photosynthesis in grapevine leaves, this is temporal; net photosynthesis usually slows or stabilizes at a lower level, particularly under relatively long-term elevated CO2 exposure (Arrizabalaga-Arriazu et al. 2020).

4.2.8.4

Salt Stress

Salinity is one of the major abiotic stress of edaphic origin that significantly affects the grapevine growth and development resulting in yield reduction (Ismail et al. 2013). Cultivated grape (V. vinifera) is considered as moderately susceptible to the salinity stress. The damage in the plant system is majorly by the chloride (Cl− ) anions as compared to the sodium (Na+ ) cation. The reduction in growth and development of the grapevine under the salinity stress can be attributed to the reduced stomatal conductance, modified electron transport rate, reduced leaf water potential, chlorophyll fluorescence, osmotic potential, leaf ion concentrations etc (Cramer et al. 2007a, b). Along with these physiological changes, salinity also causes molecular changes which results in the biochemical modification leading to formation of ROS, disorganization of cellular membrane, metabolic toxicity, reduced nutrient acquisition and hormones changes (e.g. abscisic acid and jasmonates) etc (Cramer et al. 2007a, b; Ismail et al. 2012). Various rootstocks and wild Vitis species differ broadly in the Cl¯ exclusion property (in reducing order; V. rupestris, V. cinerea, V. champini and V. berlandieri). This imparts the variation in the degree of salinity tolerance (Fisarakis et al. 2001).

4.2.8.5

Iron Chlorosis

It results from iron deficiency with high levels of soil bicarbonate in sensitive grapevine genotypes. In most of the cases, Fe deficiency results in reduction of

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Table 4.2 Grapevine rootstocks and their response to abiotic stresses (Corso and Bonghi 2014). High tolerance (HT), medium tolerance (MT), low tolerance (LT) and very low tolerance (VLT) Rootstock

Parents

Drought

Salinity

Iron chlorosis

101–14

V. riparia × V. rupestris

LT

MT

LT

3309C

LT

LT

MT

Schwarzmann

MT

MT

VLT

V. rupestris × V. berlandieri

HT

MT

VLT

1103 Paulsen

HT

HT

VLT

140 Ruggeri

HT

HT

MT

110 Richter

196.17 castel

V. riparia × V. rupestris

HT

HT

VLT

M2

V. riparia × V. vinifera

VLT

MT

HT

41B

V. berlandieri × V. vinifera

MT

LT

VLT

HT

HT

VLT

420A

V. riparia × V. berlandieri

LT

LT

VLT

5BB Kober

V. riparia × V. berlandieri

MT

LT

VLT VLT

M4

5C

V. riparia × V. berlandieri

LT

LT

M3

V. riparia × V. berlandieri

LT

LT

VLT

SO4

V. riparia × V. berlandieri

LT

LT

MT

M1

V. cordifolia × (V. riparia × V. rupestris)

VLT

MT

HT

longevity and productivity of grapevine (Covarrubias and Rombola 2013). Iron deficiency stress enhances activity of Fe-reductase enzyme and increases release of protons and organic compounds in roots. This results in lower pH and higher solubility of Fe (III); known as strategy I (Jiménez et al. 2007; Covarrubias and Rombola 2013). Bavaresco and Lovisolo (2000) observed different responses to iron chlorosis in various scion/rootstock combinations which is correlated with chlorophyll content and vegetative growth. Bavaresco et al. (1993) reported that 140 Ruggeri as an ironefficient rootstock which did not induce chlorosis even on calcareous soil while, 101–14 as an iron-inefficient rootstock. This Fe-chlorosis tolerance character of 140 Ruggeri to could be attributed to its higher root Fe (III)-reductase activity and its ability to release phenolic compounds in the medium (Ksouri et al. 2006). Various rootstocks and their responses to abiotic stresses are given in Table 4.2.

4.2.9 Use of Morphological Markers Morphological markers include various traits such as plant height, disease response, photoperiod, sensitivity, shape or color of flowers, fruits or seeds etc. These markers are vulnerable to environmental contentions, which limits their application in fruit crops. Longer generation time and large size of fruit trees are also major hindrances of its application. In grapes, an urgent need is felt to establish morphological markers,

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which would speed up the traditional breeding and also assist molecular breeding. But molecular markers are pivotal to study and preserve diversity in any germplasm.

4.2.10 Limitations and Prospect of Genomic Designing In fruit crops like grape, where targeted trait(s) are expressed later in plant development, like fruit and flower characteristics, owing to longer juvenile phase, genome designing using molecular method of breeding can be effective. Various aspects of molecular breeding like marker assisted selection (MAS), marker assisted backcross breeding (MABCB), QTLs, transgenics, cisgenics, genome editing etc. can be used. These methods can be exploited to target specific trait or character(s) to enhance higher quality, tolerance to biotic or abiotic stress(s) etc. Furthermore, enhancement of genetic diversity for useful traits targeting abiotic stresses may help the grape breeders for allele mining or QTL identification through association mapping. These methods also have some inherent limitations. Higher cost and skilled manpower are some fundamental drawbacks. Limited investment in fruit -tree biotechnology adds up to limitations. MABCB is also useful when the trait is of low heritability or highly affected by environment. Difficulties in efficient regeneration and transformations using various plant parts is a major challenge. Lack of complete genome sequence is a major bottleneck in grape genome design. Along with, the variations in regulatory requirements for transgenics in various nations around the world need to be addressed. Public acceptance of the transgenic plants and the ethical issues concerning with these need to be addressed. Besides regulatory approaches for cisgenics and genome edited grape plants are at a nascent stage in most of the nations.

4.3 Genetic Resource of Resistant Genes The family Vitaceae has rich genetic diversity comprising 16 genera and 950 species. Amongthese, the cultivated species Vitis vinifera genotypes are quite unique (even sister seedlings have distinct traits owing to their unique genetic makeup). The scion and the rootstock improvement of Vitis for abiotic stress tolerance depends mainly on thewild relatives, species and landraces. In the past, a few Vitis species were characterized for their abiotic stress tolerance/resistance in various parts of the world, for example V. acerifolia, V. arizonica, V. × champinii Planch., V. monticola Buckley, V. berlandieri Pl., V. rupestris Scheele, V. vinifera L. are for drought; and V. acerifolia, V. berlandieri Pl., V. riparia Michx, V. candicans Engelm. and V. × champinii Planch. are for salinity etc. (Table 4.3). Similarly, the indigenous Himalayan Vitis species, viz. V. parviflora has been reported for its drought tolerance. The countries like China, United States and Germany are exploiting different species—V. amurensis, V. quinquangularis, and V. davidii—as these are good source of biotic and abiotic stresses tolerance/resistance as well as

Rupestris 101–41, Riparia 420 A Salt Creek, Solonis 1616

V. candicans Engelm., V. lincecumii Buckley, V. rotundifolia Michx.

V. acerifolia, V. arizonica, V. × champinii Planch., V. monticola Buckley, V. berlandieri Pl., V. rupestris Scheele, V. vinifera L.

V. acerifolia, V. berlandieri Pl., V. riparia Michx, V. candicans Engelm., V. × champinii Planch.

V. berlandieri Pl., V. vinifera L.

V. monticola Buckley

Hot

Drought

Salinity

Iron

Lime

Source Ray (2002), Bose et al. (2001)

V. adstricta Hance, V. amurensis Rupr., V. acerifolia, V. labrusca L., V. riparia Michx., V. vinifera L. ssp. Italian Riesling, Viktorisan, sylvestris var. tipica, var. balcanica, var. aberrans, V. vulpine L., V. yenshanensis J.

Resistant varieties/rootstocks

Cold

Abiotic stress Resistance/tolerance Vitis species

Table 4.3 Sources of tolerance to various abiotic stresses

74 S. K. Singh et al.

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possessing drought and cold tolerance ability; and also give good growth on the poor soils. Furthermore, a member of NAC transcription factor family, VaNAC26 was upregulated in V. amurensis during cold, drought and salinity treatments and transgenic line of Arabidopsis with VaNAC26 has shown drought and salt tolerance. Adaptation to calcareous soils is an important characteristic required in grape rootstock as these soils are prevalent in many European viticultural regions. V. berlandieri being tolerant to calcareous soil, has been used extensively in rootstock breeding to impart this trait (Ray 2002).

4.3.1 Primary Gene Pool The gene pool which leads to production of fertile hybrids is known as primary gene pool. It includes plants of the same species or of closely related species. The species of Vitis easily cross and produce fertile interspecific F1 hybrids (Ray 2002). In such gene pool, genes can be exchanged between lines simply by making conventional crossing and backcross-assisted breeding. Grape species having the same chromosome number are inter-fertile. In grape many cultivars are identified for biotic stress resistance belonging to this gene pool, but only a few have been identified for abiotic stress tolerance. These can be used as primary gene pool for production of new hybrids for abiotic stress tolerance. Cultivar Italian Riesling has been identified for its cold tolerance.

4.3.2 Secondary Gene Pool The genetic material that leads to partial fertility on crossing with primary gene pool is referred to as secondary gene pool. Species of this group are closely related, and can cross and produce at least some fertile hybrids. As would be expected by members of different species, there are some reproductive barriers among members of the primary and secondary gene pools. A genetic barrier operates between Vitis and Muscadine grape genotypes due to chromosome number and genomic difference. Experimentally, Muscadinia rotundifolia can be hybridized with V. vinifera to improve disease resistance of vinifera and fruit quality of rotundifolia (Ray 2002).

4.3.3 Tertiary Gene Pool Members of this gene pool are more distantly related to the members of the primary gene pool. The primary and tertiary gene pools cannot be intermated, and gene transfer between them is impossible without the use of special nonconventional tools and techniques. Transfer of gene from such material to primary gene pool is

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possible with special techniques such as embryo rescue (or embryo culture), induced polyploidy (chromosome doubling), somatic hybridization, genetic engineering, and bridging crosses (e.g., with members of the secondary gene pool). In grapes there is a need to explore the related genera, viz. Ampelocissus, Cayratia, Cissus, Tetrastigma, Leea and Parthenocissus for their abiotic stress tolerance.

4.3.4 Artificially Induced/Incorporated Traits/Genes Genetic engineering has facilitated transfer of desired genes or traits, which seems difficult to be transferred otherwise to the target genotypes. Rojas et al. (1996) used the SOD gene from Arabidopsis to induce cold tolerance in V. vinifera ‘Cabernet Franc’. Similarly, DREB1b, a grapevine dehydration response element binding gene and VvCBF4, a C-repeat binding factor gene were observed to impart cold tolerance (Jin et al. 2009; Tillet et al. 2012). A stress associated protein from Chinese wild grape (V. amurensis), VaSAP15, was reported to enhance cold tolerance of transgenic grapes (Shu et al. 2021). The transcription factors from grapevine also have been used to enhance abiotic stress tolerance in other crops. A novel stress-responsive grapevine NAC transcription factor, VvNAC17, increased sensitivity to abscisic acid and enhanced salinity, freezing, and drought tolerance in transgenic Arabidopsis (Ju et al. 2020). Similarly, a HD-Zip transcription factor, VvHDZ4 enhanced drought tolerance in transgenic tomato (Li et al. 2021a, b). Genome editing is also being used to incorporate desirable change(s) in grape genome to have designed targeted phenotype.

4.3.5 Vitis Germplasm for Abiotic Stress Tolerance The European grapes have originated from Vitis vinifera type. Thus, Vitis vinifera spp. sylvestris is considered as a progenitor species existing in the central Asia, western Africa and Asia minor, while Vitis vinifera ssp. Caucasia is predominant species scattered in the Russian Union, Iran, Turkey, Kazakistan and Kashmir. This diversity has allowed grape cultivation to spread in almost all the six continents of the world. In India there are a large number of species scattered in Himalayan region and Western Ghats, where related species are found. In India, it is believed that there are over 25 wild species spread in Himalayan region. The different wild species in this region are V. parviflora and V. lanata. Native Indian species resembling Vitis lanata and V. palmata grow wild in the northwestern Himalayan foothills. Indigenous varieties, namely, ‘Rangspay’, ‘Sholtu White’ and ‘Sholtu Red’, in cultivation still in some pockets, are drought tolerant. These need to be conserved before they become extinct. Grapevine plants are influenced by many environmental stresses. The most important ones are: extreme temperatures or too high (or too low) irradiation, water

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logging, drought, lack of minerals in soil (their deficiency) and too high salinity of soil (Koyro et al. 2012).

4.3.5.1

Rootstocks

With increasing edaphic problems and other abiotic stresses, rootstocks are now being adopted by growers all over the world. Though, most of the farmers still continue with self-rooted grapes. At present, rootstocks are being adopted to overcome adverse effects of salinity and drought and also to modify scion physiology, including bunches and berry traits. Of the available rootstocks, Dog Ridge was recommended for its drought and salt tolerance ability. It has compatibility with promising varieties like Thompson Seedless and its clones. In the last decade, it has been noticed that Dog Ridge induced problems like uneven bud burst after pruning, less fruitfulness, increased deadwood in the cordons, and inverted bottle- neck symptoms. V. riparia Michx., V. berlandieri Planch, V. aestivalis Michx and V. rupestris Scheele were used as rootstocks for most of the cross-breeding and breeding. V. aestivalis, is a rootstock of higher vigor, disease resistance and environmental stress tolerance (Mortensen et al. 1990). V. berlandieri is originated from the mountains of central Texas and is recognized for adaptation to calcareous soils (Cousins 2005). Presently, rootstocks of the group like 110 R, 99 R, and 1103 P (V. berlandierii × V. rupestris) etc. having drought and salt tolerance and rootstock 110 R are being adopted to replace Dog Ridge as they inducebetter fruitfulness, moderate vigor, restricted uptake of chlorides and increased water use efficiency. In extreme weather situations, these rootstocks are being used for in-situ grafting, chip budding and green grafting. Almost all of the private wineries have introduced bench-grafting machines and are using grafted vines. Some of the rootstocks identified by different agencies for various abiotic stresses are listed in Tables 4.4, 4.5 and 4.6. German rootstock Börner and the Czech hybrid Bruci [(Vitis berlandieri × Vitis rupestris) × Vitis cinerea] are being used by the rootstock breeders of Czech Republic as donors for development of genotypes having resistance to phyloxera. Hybrid combinations involving V. rupestris and V. amurensis showed a moderate tolerance to chlorosis (Pavloušek 2009). The evaluation and assessment of drought tolerance is an important component of selection of suitable grape rootstocks and for further breeding work. The classification of grape rootstocks into five groups according to their tolerance to drought is presented in Table 4.7. Hofäcker (2004) presented a general evaluation of the drought and chlorosis resistance of rootstocks, most commonly grown in Europe; results of this analysis are presented in Table 4.8. Rootstocks 333 EM and 41B (Vitis berlandieri × Vitis vinifera), Frecal were selected for their higher degree of calcium tolerance (Pouget and Ottenwaeter 1978). Rootstock 140 Ru (V. berlandieri × V. rupestris), known for tolerance to lime chlorosis was selected by A. Ruggeri, an Italian breeder.

V. berlandieri and V. riparia

V. champini

V. berlandieri and V. vinifera

SO4

Kober 5 BB

99 Richter

140 Ruggeri

110 Richter

1103 Paulsen

41 B

Dogridge

Fercal

Riparia Gloire de Montpelier V. berlandieri and V. vinifera

Rupestris Saint George

1

2

3

4

5

6

7

8

9

10

11

Drought tolerant, adapted to acidic soils, and resistant to salinity and phylloxera

Drought tolerant, highly resistance to phylloxera and root knot nematodes; moderate resistance to dagger and lesion nematodes

Vigorous, moderate nematode resistance, higher resistant to phylloxera

Vigorous, nematode and phylloxera tolerance

Salient features

V. berlandieri and V. vinifera

V. berlandieri and V. vinifera

Resistance to drought and phylloxera. Sensitive to root knot and dagger nematode, and moderate resistant to root lesion nematodes

Not suitable for calcareous soils and dry sites. Higher resistance to phylloxera

Suitable for high lime containing European soils

Extremely vigorous, highly resistance to nematodes, moderate tolerance to phylloxera and high-lime. Higher level of suckering is a commercial drawback

Moderate vigorous, resistance to high-lime soils

V. berlandieri and V. rupestris Vigorous, salt tolerant, adaptable to clay-lime condition

V. berlandieri and V. rupestris Vigorous, tends to delay maturity, drought tolerant to drought and lime (up to 17%)

Berlandieri and Rupestris

Berlandieri and Rupestris

V. berlandieri and V. riparia

Parentage

Sl. no Rootstock

Table 4.4 Description of grapevine rootstocks of commercial importance

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Table 4.5 Rootstocks suggested based on global data for different situations Situation/problem

Rootstock

Drought/low moisture condition

1103 P, 140 RU, 110 R, 420 A, SO4 , 99 R, St. George, Dog ridge

Soil EC more than 2 m mhos/cm and water EC more than 1 m mhos/cm

Ramsey, Dog Ridge, 140 RU, 99 R, 110 R

Soil ESP more than 15% and/or water SAR more than 8

140 RU, 1613, Ramsey, Dog Ridge

Free calcium content of soil is more than 12%

140 RU, SO4 , 420 A

Chloride content of water is more than 4 meq/l

Ramsey, Dog Ridge B, 140 RU. Teleki 5-C

Poor vigor of the variety without any soil/water Dog Ridge, St. George, SO4 , 140 RU problem For increased nitrogen and potassium uptake

Dog Ridge, St. George, 34 EM, Ramsey

For increased bud break

1613, B2-56

Table 4.6 Tolerance of rootstocks to chlorosis (Cousins 2005; Chauvet and Reynier 1979)

Rootstock

Tolerance to chlorosis

References

SO4

Medium

Cousins (2005)

Börner

Low

420 A

Good

Kober 5BB, SO4

Medium

140 Ruggeri

Very good

1103 Paulsen, 110 Richter

Medium

Fercal

Very good

Chauvet and Reynier (1979)

Table 4.7 Evaluation of drought tolerance of individual rootstock varieties (Carbonneau 1985) Degree of resistance

Rootstock genotype(s)

Highly resistant

R 110, R 140, 44–53

Resistant

P 1103, 196–17, P 1447, SO4 , R 99, 7383

Less resistant

3309, 7405, 7903, 420 A, Fercal, RSB1, 7921, 5 BB, 161–49, 41 B, Rupestris du Lot, 101–14

Susceptible

Rupestris du Lot, 101–14, EM 333, 7924, Yuga,

Highly susceptible

7542, Vialla

4.3.5.2

Cold Resistance

China is one of the centres of origin of grapes, and a Chinese wild grapevine species, Vitis pseudoreticulata, shows good resistance to several abiotic and biotic stresses. Among the eighteen wild Vitis species native to China, the cold-resistant (high to

+ + + (+) ++

+ ++

Austria Hungary Hungary France France France Italy Italy Italy France France France Czech Republic France Italy Italy France

V. berlandieri × V. riparia

V. berlandieri × V. riparia

V. berlandieri × V. riparia

V. berlandieri × V. riparia

V. berlandieri × V. riparia

V. berlandieri

V. berlandieri × V. rupestris

V. berlandieri × V. rupestris

V. berlandieri × V. rupestris

V. berlandieri × V. rupestris

V. berlandieri × V. rupestris

V. riparia × V. rupestris

V. riparia × V. rupestris

V. riparia × V. rupestris

V. berlandieri × V. riparia

V. berlandieri × V. riparia

125 AA

5C

Teleki 8B

420A

161–49 Couderc

R.S.B.1

140 Ruggeri

1103 Paulsen

775 Paulsen

Richter 110

Richter 99

3309 Couderc

Schwarzmann

101–14 Millardet de Grasset

Cosmo 2

Cosmo 10

Rupestris du Lot

V. rupestris

France

+++

+++

Germany

V. berlandieri × V. riparia

Riparia Glorie de Montpellier V. riparia

++++

++++

Germany

V. berlandieri × V. riparia

Binova

+++

+++

++ + (+)

+ (+)

+ + (+)

+++ + (+)

+++

++++

++++

(continued)

+++

++++

+++++ +++++

+

++++

+ + (+)

++++

+ + + + (+)

++++

++++

++/+++

+ ++(+)

+ + + + (+)

+ + + (+)

+(+)

++++

++++

+ +(+)

+ + +(+)

+ + +(+ )

+++

SO4

+++

Austria

V. berlandieri × V. riparia

5 BB

Country of origin *Drought resistance *Chlorosis resistance

Parentage

Rootstock

Table 4.8 Drought and chlorosis tolerance of the most common European rootstocks (Hofäcker 2004)

80 S. K. Singh et al.

Italy Hungary

Castel 156–12 × V. berlandieri

Kober 5 BB × V. vinifera

Golia

Georgikon 28

Note: + = Very low, ++ = Low, +++ = Medium, ++++ = High, +++++ = Very high * signifies the common degree of ratings for the two stress factors i.e. drought and chlorosis resistance

France

Cabernet Sauvignon × V. berlandieri

333 E.M

++++

+++

Germany

Sylvaner × 1616 C

Sorisil Germany

(V. berlandieri × Colombard) × V. berlandieri × (V. riparia France × V. rupestris × V. candicans)

Fercal

France

France

161–49 C × 3309 C

Gravesac

Trolinger × V. riparia

France

V. solonis × V. riparia

Chasselas blanc × V. berlandieri

Germany

V. solonis × V. riparia

Sori

1616 Couderc

26 G

++++

Germany

(V. berlandieri × V. riparia) × V. cinerea

41B Millardet de Grasset

+ + + (+)

++

Germany

V. riparia × V. cinerea

Cina

++++

+ + + (+)

++++

++++

++++

+++

+++

+ + + (+)

+ + + (+)

++++

+++

++++

+++

+++

+++

++

++

+ + (+)

+ + (+)

+ + (+)

Rici

+ + + (+)

Germany

V. riparia × V. cinerea

Börner

Country of origin *Drought resistance *Chlorosis resistance

Parentage

Rootstock

Table 4.8 (continued)

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low) are: V. amurensis, V. yeshanensis, V. adstricta, V. pseudoreticulata, V. quinquangularis, V. piasezkii, V. hancockii, V. ficifolia, V. romanetii, V. davidii, V. piasezkii var. pagnucii, V. bashantica, V. liubaensis, V. qinlingensis, V. davidii var. cyanocarpa, V. wilsonae, V. baihensis, and V. davidii var. ninqiangensis. Among the seven wild Vitis species native to America, cold-resistant are V. riparia, V. arizonica, V. rupestris, V. rotundifolia and V. californica (all high resistance) and V. labrusca and V. cinerea (medium resistance). The most cold-resistant accession has been V. riparia Mcadams. These results are being used for selection of wild grape species for cold resistance breeding (Zhang et al. 2012a, b). Further, the cold hardiness of the European grape cultivar ‘Cabernet Sauvignon’ ranks ‘low’, while that of V. pseudoreticulata ranks ‘medium/high’ and V. amurensis Zuoshan-1 ranks ‘high’ (Zhang et al. 2015). Being a perennial ligneous vine growing in the cool continental climates, V. amurensis can withstand cold for a long time under natural winter. Hence, this can be used in studies related to cold tolerance mechanism to overcome winter-associated low temperature-related stress.

4.4 Glimpses on Classical Genetics and Traditional Breeding Irrespective of the several drawbacks of the traditional grape breeding, most of the breeding achievements in grape are from this section only. The traditional grape breeding started with the introduction of various grape genotypes around the word. Grape is basically an introduced crop to India, which was first introduced in 1300 AD by invaders from Iran and Afghanistan. Systematic collection, conservation and characterization of the indigenous as well as the introduced exotic genotypes are the fundamentals of traditional grape breeding. Apart from the introductions, selection of various somatic variants and clonal variants has been attempted successfully in grape. Hybridization to produce elite grape recombinants is the major traditional breeding followed in Indian and around the world. Polyploidy breeding has been used to exploit the presence of different chromosome number in various close relatives of the cultivated grape genotypes. Amongst the various methods of traditional breeding, mutation and in vitro mutagenesis have also been attempted as these methods are having advantage over others in terms of short time requirement.

4.4.1 Classical Breeding Efforts Classical or traditional grape breeding programs have produced several improved rootstock and scion genotypes having resistance or tolerance to various pest and disease. However, this method is highly time consuming and can even take decades to fully evaluate and release new improved cultivars. As mentioned above, perennial

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species pose specific biological problems in the development of mapping populations. The generation time is long (3–5 years from seed to seed), growing many individuals is cost and labour demanding, species are heterozygous, and several of them, grapevine included, are sensitive to inbreeding depression. Various genetic linkage maps for grape have been published (Lodhi et al. 1995; Dalbo et al. 2000; Doligez et al. 2002; Grando et al. 2003; Riaz et al. 2004). These maps have been utilized to map horticultural traits such as seedlessness and berry weight, and diseaseresistance traits such as powdery mildew resistance. However, there is no attempt to develop cytological maps in grape vine.

4.4.2 Limitations of Classical Endeavors and Utility of Molecular Mapping and Breeding Genotype–phenotype associations using linkage mapping have traditionally been used in grape breeding programs. Further establishment of linkage-mapping populations is time-consuming owing to the grape’s long pre-bearing period. Therefore, genomic selection (GS), association mapping, genome-wide association (GWA) (McCarthy et al. 2008) etc. using the molecular markers (Heffner et al. 2009) explores the new vistas of alternative breeding instruments. A brief account of molecular mapping in grape for various aspects has been discussed in later part of this chapter.

4.4.3 Breeding Objectives At present, Viticulture industry worldwide is confronting several challenges of biotic and abiotic nature, which adversely affects the production, productivity and berry quality. The variations in temperature considerably impact the grapevine growth and development. The temperature fluctuations influence the berry development and its composition. The effect of climate change manifested as occurrence of abrupt rains during the berry maturity causes berry cracking and subsequent rotting, which significantly reduces the grape production; thereby, causing huge economic losses to the growers. In addition, various biotic and abiotic stresses also contribute to production losses and deterioration quality. Keeping in mind the challenges of viticulture industry, grape breeding programme can be directed to achieve the following objectives: • To develop early maturing, seedless and high soluble solids containing cultivars for table purpose. • To develop varieties resistant to anthracnose, Phylloxera and chaffer beetle. • To develop varieties with medium vigour and productive basal bud, amenable for training on head or pandal system.

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For the tropical regions, the objectives of breeding should be: • To develop high yielding and high-quality varieties with increased fruitfulness of basal buds coupled with the less degree of apical dominance and suitable for variety of purposes such as table, raisin, wine and juice and resistance to diseases. To develop rootstocks resistant to salinity, nematodes and drought.

4.4.4 Classical Breeding Achievements Globally there are over 10,000 known grapevine varieties, of which 13 occupy more than one-third of the world’s vineyard area, while 33 varieties cover half of the global area under grape production. Some of the varieties are grown in various countries and thus are hailed as “international varieties”. The varieties include those being used for table use, wine making, raisin production, processing and rootstock (Fig. 4.1). The Vitis international variety catalogue identifies 21,045 names of varieties including 12,250 of V. vinifera; however, this consists of a substantial chunk of synonyms and homonyms (Anonymous 2017). Grape is basically an introduced crop to India, which was first introduced in 1300 AD by invaders from Iran and Afghanistan. Later intensive introduction taken place between 1930 and 1960s. As a result, many varieties introduced from different countries were recommended for commercial cultivation. Among introduced varieties, Thompson Seedless, occupy 80% of the area along with its clones. Variety Bangalore Blue occupies approximately 3% of the total area under grapes, while cvs. Anab-e-Shahi and Dilkhush have 7%, Sharad Seedless 5%, Perlette 3% and

Fig. 4.1 Vine-growing areas and production for different purposes

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Gulabi and Bhokri together occupy 2% area. About a decade back, many promising introduction such as Crimson Seedless and Victoria from South Africa and Autumn Seedless, Blush Seedless, Marquis and Autumn Royal from Davis University, California etc. were made in Maharashtra. Most of the grape varieties are now under IP protection; hence, Govt. of India through the Ministry of Agriculture initiated support for introduction of IP protected varieties of grape for testing and commercial production in India.

4.4.4.1

Somatic Variations and Clonal Selections

On identification of superior genotypes, a vegetative propagation especially hard wood cutting is used to maintain and multiply a highly desirable genotype. One or few cuttings might eventually create slight variation in the existing genotype as a result of somatic variation, which may also produce a different phenotype. It may result from mutation or epimutation events that initiated in a single cell. Such variable genotypes in the clonally propagated (cutting) populations are termed as clonal variation. It seems to have been an important means of variation in grape and have played an important role in the evolutionary history of grape as spontaneous mutations. Spontaneous mutations are frequently observed, especially in old cultivars and provide a valuable source of variation in grapes. These appear swiftly over one cycle of vegetative propagation. When mutation occurs only in one cell layer of the plant, it produces unstable chimera plants but when colonise a cell layer either L-1 or L-2 or both layers (periclinal) the mutation may be transmitted from one generation to another both by asexual or sexual propagation (if present in layer L2). The mutations reported involve all kinds of characteristic, including yield, earliness, colour of berries, hardiness, resistance to pest and disease. Some of the important somatic variants identified/developed over last four decades time are described in Table 4.9.

4.4.4.2

Mutation and in vitro Mutagenesis

Different mutagens have also been used to get mutants. Physical mutagens such as 60Co-gamma rays, beta rays, X-rays or thermal neutrons and with chemical ethyl methyl sulphonate (EMS), N-nitroso-methyl urethane (NMU) and Diazomethane are most commonly used. Seedless mutants of seeded cultivars such as Concord, Emperor, Habshi and Muscat of Alexandria may be of interest as table grapes. Examples New Perlette evolved with X-ray (2.5 kR) treatment, while Red Niagara from Niagara, Robin Cardinal from Cardinal are other examples. ARI-302 is a Seedless Mutant of Anab-e-Shahi developed at ARI, Pune, out of 280 sprouted cuttings of Anab-e-Shahi treated with ethyleimine. It has attractive, mid-season ripening and even maturity of bunches, which have a medium size and cylindrical shape. Berries are golden yellow in colour, ellipsoidal in shape, medium in size with a strong adherence. Juice is sweet with high TSS (22°Brix.) and 0.4–0.6% acidity. Mutant has

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Table 4.9 Promising somatic variants identified/developed worldwide over last four decades Parent

Mutants

Important character

Bequignol red

Bequignol variant

Variegated white/red berries

Cabernet Sauvignon

Bronze CS

Bronze berries

Malian/Shalistin

Bronze/white berries

RRM

Ramified bunch, extreme cluster proliferation and delayed anthesis

Carignan Chardonnay

Red Chardonnay

Red berries

Flame seedless

Early Flame

Shorter cycle

Gamay

Freaux, De Chaudenay

Red flesh

MPW

Multiple perianth whorls

Gravesac

53XX lines

Vigour variations

Italia

Rubi Italia

Light pink berries

Benitaka

Pink berries

Brazil

Black berries

Bicchieri

Red berries

La Notte

Longer berries

Dipinto

Shorter cycle

Mutated Italia

Cluster/berry size Short cycle

Ruby Okuyama

Red berries

CLS

Carpel-less

Mouvèdre Muscat of Alexandria

Flame Muscat

Red berries

Niagara red

Rosinha Seedless

Seedless

Niagara Branca

Niagara Rosada

Attractive rosy colour

Patricia

Patricia Branca

White berries

Pinot Noir

Pinot Meunier

Hairy epidermis

Pinot Gris

Pink berries

Pinot Blanc

Green/white berries

Pink/green variants

Colour variations

Sangiovese SO4

Binova

Hermaphroditism

Sultana

Pink Sultana

Pink berries

Red sultana

Red berries

Syrah

Star flowers

Calyptra anomalies

Ugni Blanc

Fleshless berry

No pulp

Source Ray (2002), Bose et al. (2001)

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best keeping quality and high yield but is susceptible to both the mildews. It is now under commercial cultivation for table purpose and also used for raisin making in Maharashtra. Gamma irradiation (5–100 Gy) has been found to increase the tetraploid plant formation frequency of primary (7%) and embryogenic calluses (7.6%); besides, the development ofsome aneuploid plants. Colchicine treatment was not found effective for the induction of tetraploid plants. In vitro mutagenesis cv. in Pusa Seedless (Vitis vinifera L.) by subjecting in vitro proliferated cultures to different gamma irradiation doses using 60 Co. Likewise, in vitro irradiated (20 Gy gamma rays) explants (var. Black Matrouh) exhibited significant increase in the drought tolerance.

4.4.4.3

Hybridization

Hybridization efforts have yielded several prominent varieties in India. Seedlessness is one of the prime characters for table and dried grape production in Asian countries. The seedlessness in most of the vinifera is due to ‘stenospermocarpy’ (Stout 1936), a process in which fertilisation occur but seed development failed soon after leaving small size or un-detectable traces of seed. Seedlessness in grape is also due to ‘parthenocarpy’ such as Black Corinth and no seed is produced. All the traits are governed by one or more factors. Seedlessness is governed by a recessive factor (Pandey and Pandey 1996). Different workers proposed different hypothesis for seedlessness in grape such as it is governed by one recessive gene, two recessive genes, many recessive genes, five dominant genes, three dominant genes and one dominant gene. Of late, it has been accepted that three independently inherited recessive genes is controlled by a dominant regulator gene (Bouquet and Danglot 1996) having an inhibitor locus, SdI (Seed Development Inhibitor). Gene pairs with epistatic action namely, B, a dominant gene for black fruit, and R, a dominant gene for red fruit are responsible, while both recessive genes contributes to white colour in grape. Likewise, two two genes namely, G for diglucosides or g for monoglucosides, and O for triphenols or o for diphenols controls the composition of fruit anthocyanins, have been identified. For fruit aroma, muscat flavor is controlled by five dominant complementary genes, methyl anthranilate is controlled by three dominant complementary genes, while volatile ester levels are determined by two genes. Systematic grape improvement programme started by ICAR-Indian Agricultural Research Institute, New Delhi way back in 1950s with introduction and evaluation of large number of varieties from different countries and their evaluation for different purposes. Over 350 grape varieties including some most promising ones from California, U.S.S.R., Yugoslavia, Australia, etc. had been collected at the Indian Agricultural Research Institute, New Delhi along with other centres located in country. Hybridization programme was initiated in late 1980s in the country using the parents having useful traits such as earliness, seedless, resistance to diseases etc. The varieties developed from breeding programme in India are given in Table 4.10.

Table and raisins Table and wine purpose

1996 2014

2013

2014

1980

Banqui Abyad × Perlette

(‘Hur’ × ‘Bharat Early’) × ‘Beauty Seedless

Hur × Cardinal

Black Champa × Thompson 1980 Seedless 1980

Hur × Beauty Seedless

Anab-e-Shahi × Queen of the Vineyards

Bangalore Blue × Black Champa

Pusa Urvashi

Pusa Aditi

Pusa Trishar

Pusa Swarnika

Arkavati

Arka Kanchan

Arka Shyam

Principal use(s)

Table and wine purpose

Table purpose, juice making and Munnakka preparation

Table purpose, juice making

Table purpose, juice making

Table purpose and raisin making

Juice and wine making

Year of release 1996

Parentage

Madelein Angevine × Rubi Red

Hybrid/variety developed

Pusa Navrang

Table 4.10 Indian grape improvement program involving diverse parentages/through hybridisation Major traits

Big sized round berries, moderate to heavy yielder

Golden yellow berries, seeded and muscat flavour

Yellowish green, seedless

Early with large berry, natural loose bunches tolerant to anthracnose and powdery mildew

Early maturing, suitable for sub-tropical conditions moderate tolerance to anthracnose, powdery mildew and termite

Early maturing, seedless having large round berry (2.7 g), tolerant to anthracnose and powdery mildew

Bunch is loose, seedless greenish-yellow berries

Earliness, uniform ripening, teinturier, resistant to anthracnose disease

Institution/centre

(continued)

ICAR-IIHR, Bangalore

ICAR-IIHR, Bangalore

ICAR-IIHR, Bangalore

ICAR-IARI, New Delhi

ICAR-IARI, New Delhi

ICAR-IARI, New Delhi

ICAR-IARI, New Delhi

ICAR-IARI, New Delhi

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Table purpose Table purpose

Wine making Juice making

Juice and wine making

1994

Black Champa × Thompson 1992 Seedless 1994 1994 1994

Angur Kalan × Black Champa

Angur Kalan × Anab-e-Shahi

Anab-e-Shahi × Queen of Vineyards

Bangalore Blue × Convent Large Black

Black Champa × Thompson 1994 Seedless

Diamond Jubilee × Rubi Red

Arka Majestic

Arka Neelamani

Arka Chitra

Arka Soma

Arka Trishna

Arka Krishna

ARI-27

2008

Table purpose

1994

Anab-e-Shahi × Thompson Seedless

Arka Sweta

Principal use(s)

Wine making

Table and raisin

Wine making

Year of release 1980

Parentage

Anab-e-Shahi × Bangalore Blue

Hybrid/variety developed

Arka Hans

Table 4.10 (continued) Major traits

Institution/centre

ICAR-IIHR, Bangalore

ICAR-IIHR, Bangalore

ICAR-IIHR, Bangalore

ICAR-IIHR, Bangalore

ICAR-IIHR, Bangalore

(continued)

ICAR-IIHR, Bangalore

ICAR-IIHR, Bangalore

Mid-season maturity, conical ARI, Pune shape bunches, tolerant to the mildews and anthracnose

Suitable for beverage industry, black berries, seedless and sweet

Deep tan coloured, male sterile, very sweet pulp

White berries, muscat flavour ICAR-IIHR, Bangalore

Seeded, berries-golden yellow with pink blush

Black coloured berries, all buds on a cane are fruitful

Bold and seeded, ideal for export

Prolific bearer, greenish yellow coloured, uniform seedless berries

Prolific bearer, bunches medium in size, yellowish green berries

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Juice making

Black Champa × Thompson 2008 Seedless

Pusa Navrang × Flame Seedless

A-18/3

Manjari Medika

2017

Table grape and raisin purpose

2008

V. labrusca var. Catawba × V. vinifera var. Beauty Seedless

ARI-516 (Punjab MACS Purple)

Principal use(s)

Table purpose, juice, munakka, and making of rose wine

Table purposes and making rose wine

Year of release 2008

Parentage

Cheema Sahebi × V. labrusca var. Catawba

Hybrid/variety developed

ARI-144

Table 4.10 (continued) Major traits

Institution/centre

Teinturier nature, juice of this variety is very dark in colour and contains anthocyanins

Coloured variety with rudimentary seed

Early ripening, bluish black berries, musky flavoured, tolerant to fungal diseases

ICAR-NRC for Grapes, Pune

ICAR-National Research Centre for Grapes, Pune

Developed by ARI, Pune Released by PAU, Ludhiana

Mid-season ripening, ARI, Pune cylindrical bunches, bluish black colour berries, tolerant to fungal diseases

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Polyploidy Breeding

Polyploidy has also been proved significance in commercial viticulture. Changes in ploidy level such as number of sets of genomes, has many practical applications as it affects crossability, fertility, cell size and heterozygosity. The polyploidy in grape arise spontaneously in the nature or can be induced by chemical agents. The spontaneously polyploidy in grape is seen as chance sports of normal diploid grape cultivar, or the chromosomal doubling take place at initial cells of the shoot meristem or from the latent buds near the pruning wood of grape. Several antimitotic agents like colchicine, trifluralin, and oryzalin have been able to induced polyploids. Polyploids progenies were also unexpectedly produced in the crosses of diploid and tetraploid grape cultivars. Among the polyploidy grapes, tetraploid grape varieties have great importance due to its large berry size. The tetraploid grape cultivars derived through budsport of the normal diploid cultivars are the source of important genetic material in tetraploid grape breeding programmes. For examples the grape genotypes Ishiharawase, Red Pearl, and Centennial are important tetraploid derived from the budsports of Campbell Early, Delaware and Rosaki, respectively (Yamane et al. 1978). Kyoho a tetraploid grape genotype derived from cross combination of Ishiharawase and Centennial, released for commercial cultivation in Japan (Yamane 1996) and cover the 32% grape area MAFF statistics (2015). Another tetraploid grape derived from the Kyoho have similar attributes like large berries and moderate disease resistance, of which Pione and Fujiminori are important one (Yamada and Sato 2016). Takao a hypo-tetraploid (aneuploid) with 2n = 75 (Yamane et al. 1978) obtained from the open-pollinated seedlings of Kyoho, which bear fruits in cluster with seedless berries and average berry weight of 4 to 5 g. This cultivar is cover around the 84 hectares area in Japan during the 2012 (Yamada and Sato 2016). Some other important tetraploids identified and registered are Sunny Rouge for early ripening; Suiho for very large seedless berries (Matsumoto et al. 1995); Honey Venus for high sugar content and firm flesh (Sato et al. 2004). Thus, the identification and development of the polyploids can add new gene pool in Vitis, which can directly explore in commercial viticulture or can be used as parent in varietal improvement programme.

4.4.5 Limitations of Traditional Breeding and Rationale for Molecular Breeding Keeping in view of the exponential rise in world population, there is a need for breeding new fruit genotypes having higher quality and productivity with novel traits to sustain under the fluctuating climatic situations However, for the genetic improvement of the fruit crops including grape through the conventional breeding methods are highly tedious owing to their perennial nature, inherent long juvenile phase, highly heterozygous nature, linkage drag, seedlessness etc. (Petri and Burgos

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2005; Rai and Shekhawat 2014; Limera et al. 2017). Genome designing using the biotechnological tools offers several ways to solve the problem of conventional grape breeding with greater extent. Designing the grape genome to confer resistance or tolerance to an array of abiotic stresses along with higher berry qualities for both table and processing purpose can make the grape breeders achieve the target within a short time period. This chapter entails the current understanding, applications, achievements and future prospects of various biotechnological tools like markerassisted gene introgression, molecular mapping, association mapping, QTLs, mapbased cloning, genetic engineering, gene editing nanotechnology etc. to design the grape genome particularly for abiotic stress tolerance or resistance.

4.5 Diversity Analysis In order to overcome the challenges posed by the prevailing biotic and abiotic factors, plants have evolved various characteristics that confer survival and ensure adaptation to certain unfavorable conditions, consequently, leading to diversity (Nwosisi et al. 2018). Genetic diversity can be defined as the differences in the genetic composition among individuals of a population, a species, an assemblage, or a community. These variations are evolved as a consequence of processes such as mutation, and physical or behavioral isolation of populations (Charlotte 2010). Genetic diversity analysis provides a powerful tool that enhances a comprehensive understanding of genetic variation and improves conservation strategies. Evaluation of genetic diversity and population structure generates important information for plant breeding-related research like genome-wide association studies (GWAS) and genomic selection (GS) (Naybom and Lacis 2021). Though genetic diversity is not always identifiable, it is quite valuable as a prerequisite to evolutionary adaptation to a changing environment. The greater the variation among individuals within a species, the greater will be the chances for adaptability to varying environmental conditions, which in turn is likely to improve the survival of a species under challenging scenarios (Charlotte 2010). The knowledge about the presence of genetic variation in germplasm is critical for effective conservation and utilization of Vitis germplasm in various breeding programme. V. vinifera is diploid with genome size about 475 Mb (Myles et al. 2010). One of the bases of plant gradation is to have access and knowledge of diversity extent in genetic collections and in different phases of gradation projects. It is also of great importance to get information about genetic distance in the population of breeding program (This et al. 2007). Precise breeding by selecting favorable genomic variants will help to improve plant productivity and efficiency. This, however, depends on a detailed understanding of the relationship between genotype and phenotype (Furbank and Tester 2011). Traditionally, ampelography, which deals with the differentiation and identification of grapevine varieties and hybrids, has been in use for cultivar identification in grapevines. It is generally done by experts who distinguish the vineyard species based on apparent characteristics of the leaves (Babellahi and Jafari

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2016). Besides morphological attributes, biochemical and molecular markers have been used to distinguish relationship of different grapes varieties (This et al. 2007). Therefore, the grapevine diversity analysis is discussed briefly hereafter.

4.5.1 Phenotype-Based Diversity Analysis Phenotype is the term used in genetics for the observable characteristics or traits of an organism (Fasoula et al. 2020). Phenotyping provides a strong foundation for any breeding selection process (Franco et al. 2005). Morphological characterization is the legally accepted methodology for patenting and registration of varieties (Chitwood 2021). Morphological diversity of some wild grapes belong to north of Iran has been depicted in Fig. 4.2. In the last years, morphological data have been used to resolve the complex problem of the definition and classification of crop accessions using multivariate statistical analyses (Manjunatha et al. 2007; Aghaei et al. 2008). Ampelography is the science of phenotypically distinguishing grapevines. For identification of grapevine cultivars, the International Organization of Vine and Wine (OIV) has developed the ampelographic scheme based on OIV descriptors (OIV, 2007). Leaf morphology observation is one of the most important methods in ampelography for cultivar determination. Grapevine leaves of different cultivars vary in chemical composition and morphology such as shape, dimension, colour and serration/ edge shape. These differences in morphometric characteristics have been acquired as evolutionary traits corresponding to specific gene expressions and their interaction with the environment

Fig. 4.2 Morphological diversity of fruit bunches of some wild grapevines collected from six different geographical areas, Golestan province, north of Iran (Photo: M. Alizadeh)

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to which each cultivar has been adapted to (Nicotra et al. 2011; Vlad et al. 2014). Morphological diversity of grape leaves of some wild and cultivated genotypes are depicted in Fig. 4.3. Phenotype-based analysis was already reported in numerous studies with different wild and cultivated grape genotypes and during the last decade a lot of energy was spent in order to investigate the genetic diversity of grape germplasms based on their morphological characteristics (Table 4.11). Most of the studies used grape leaf morphology for analysis. Some examples of such studies were summarized in Table 4.11. Chitwood et al. (2014) provided a morphometric analysis of more than 1200 grape accessions. The diversity of leaf morphs was be represented by the simple morphometric measures of circularity and aspect ratio (AR; Fig. 4.4).

Fig. 4.3 Morphological diversity of grape leaves of some Iranian wild and cultivated genotypes. The 1–5 samples are wild genotypes collected from northern forests of Iran. The 6, 7 and 8 samples are the cultivated varieties, i.e. Rashe, Perlette, Yaghoti, respectively (Photo: M. Alizadeh)

Table 4.11 Some examples of phenotype-based analysis of grapevine genotypes Research objective(s)

Study area

References

Morphological characterization of varieties

Turkey

Kara (1990)

525 cultivars were described by 151 morphological descriptors

Germany

Weihl and Dettweiler (2000)

Ampelographical description of new varieties

Czech

Pavloušek (2003)

Evaluation of diversity by morphological and AFLP markers

Argentina

Martinez et al. (2003)

Analysis of quantitative and qualitative traits in 90 genotypes

Iran

Fatahi et al. (2004) (continued)

4 Development of Abiotic Stress Resistant Grape Vine Varieties Table 4.11 (continued) Research objective(s)

Study area

References

Comparison of wild populations and old cultivars

Portugal

Coelho et al. (2004)

Morphological characterization of three wild populations

Portugal

Cunha et al. (2007)

Molecular and ampelographic characterization of 3 varieties

Spain

Santiago et al. (2007)

Discrimination of Fox grape genotypes through OVI descriptors

Turkey

Celik et al. (2008)

Characterization of wild collections

Mexico

Franco Mora et al. (2008)

Morphological relationships of wild and cultivated cultivars

Portugal

Cunha et al. (2009)

Ampelometry in Tunisian Vitis sylvestris

Tunisia

Harbi-BenSlimane et al. (2010)

Morphological variation and relationships of different populations

Georgia

Ekhvaia and Akhalkatsi (2010)

Ampelographic characterization

Turkey

Ates et al. (2011)

Ampelography of minor varieties

Spain

Garcia-Munoz et al. (2011)

Clustering of Tunisian cultivars

Tunisia

Lamine et al. (2014)

Morphological characterization of 136 table Brazil genotypes

Leão et al. (2011)

Ampelographic characterization of some accessions

Italy

Alba et al. (2011)

Ampelographic characterization of 20 varieties

Iran

Zeinali et al. (2012)

Screening of a gene pool

Russia

Troshin and Maghradze (2013)

Identification of minority red genotypes

Spain

Balda et al. (2014)

Morphological characterization of endangered wild genotypes

Turkey

Karatas et al. (2014a, b)

Ampelographic characterization of different Croatia varieties

Maleti´c et al. (2015)

Morphological diversity of 63 wild genotypes

Doulati Baneh et al. (2015)

Iran

Morphological Characterization of different Herzegoina varieties

Knezovi´c et al. (2017)

Ampelographic characterization of 21 varieties

Greece

Stavrakaki and Biniari (2017)

Identification of local varieties by OIV descriptors

Azerbaijan

Salimov et al. (2017)

Ampelography of germplasm

Romania

Popescu and Crespan (2018)

Morphological diversity of Russian genotypes grown in Iran

Iran

Tajalifar et al. (2020)

Identification of red cultivars in ancient vineyards

Spain

Jiménez-Cantizano et al. (2020)

Characterization of white varieties

Spain

Sancho-Galán et al. (2020)

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Fig. 4.4 AR and circularity among grape accessions. Averaged AR (major/minor axis of a fitted ellipse) and circularity (the ratio of area to perimeter squared times 4p) values of 1,213 accessions in the USDA germplasm collection are shown. In this population, high AR values indicate leaves with low length-to-width ratios, and leaves with low circularity have increased lobing and serration. Leaves from accessions exhibiting extreme AR and circularity values are shown. In this figure, the common name (boldface), place of origin (italics), and accession number (roman) of leaves is provided below the leaf photographs (Photo: Chitwood et al. (2014); Plant Physiology)

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Fig. 4.5 Example of scanned leaves of the 16 grape cultivars for the classification study (left) and corresponding binary images for automatic shape and edge recognition (right) to obtain morphocolorimetric parameters from each leaf (Photo: Fuentes et al. (2018); Computers and Electronics in Agriculture)

Automated image analysis for morphological and colour features extraction of scanned leaves rendered rapid, accurate and inexpensive methods to be used for ampelography/cultivar classification (Fuentes et al. 2018). The Computer algorithms and chemometric fingerprinting using near-infrared spectrometry (NIR) of plant leaves have been applied in ampelography to enhance the accuracy (Fuentes et al. 2018). An example of scanned grape leaves is shown is Fig. 4.5. This is a noticeable example of application of computer algorithms in ampelography. Flower sex phenotype (Fig. 4.6) and seed morphology are the important criteria which are generally used to distinguish subsp. sylvestris (dioecious, seeds with short beaks) from cultivated sativa forms (predominantly hermaphrodite, seeds with larger beaks). The morphological descriptors may be time saving but less reliable as these are influenced by environmental factors and subjective approach of the ampelographer (Vignani et al. 1996). However, until a few years ago, ampelography was the main method used for describing and identifying vine varieties (Schneider et al. 2008). Recently, molecular genetic markers, have become an essential tool for the identification of grape varieties (Leko et al. 2012; Žulj Mihaljevi´c et al. 2020).

4.5.2 Genotype-Based Diversity Analysis Traditionally, ampelography, ampelometry and chemical traits analysis has been used for biotype identification in grape (Imazio et al. 2002). However, these nongenetic marker tools have resulted in several false attributions, in particular when used at the clonal level. Furthermore, ampelography is environmentally sensitive and

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Fig. 4.6 Flower morphology of two grape subspecies. The dioecious flowers of V. vinifera subsp. sylvestris (Top). The hermaphrodite flowers of V. vinifera subsp. sativa (Bottom) (Photo: M. Alizadeh)

can lead to erroneous identification, especially in artificial conditions, and closely related cultivars (Vignani et al. 1996; This et al. 2004). Molecular analysis and DNA fingerprinting offers an alternative method that is not influenced by the environment and is relatively easy to perform (Van Heerden et al. 2018). However, the genotype based analysis of grapevine genomes have become highly useful techniques in recent years. Some genetic markers such as simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) have been used to solve cases of ambiguity, to fingerprint varieties and to search for the parents of prominent grapevines varieties (Imazio et al. 2002; Naybom and Lacis 2021). Application of some molecular markers to grapevine genetic analysis was summarized in Table 4.12.

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Table 4.12 Genotype-based analysis of grapevine genotypes and application of some molecular markers Marker name

Research objective(s)

Geographical area

References

RFLP

Identification of rootstock genotypes

France

Bourquin et al. (1992)

SSR

Genetic characterization of germplasm

Iran

Fatahi et al. (2003)

SSR

Molecular characterization of 9 varieties

Spain

Santiago et al. (2005)

SSR

Molecular and ampelographic characterization of 3 varieties

Spain

Santiago et al. (2007)

RAPD

Characterization of 46 local varieties

Turkey

Karatas et al. (2008)

SSR

Genetic diversity of 42 genotypes

India

Papanna et al. (2009)

ISSR

Ampelographic and molecular diversity among 44 varieties

Turkey

Sabir et al. (2009)

SSR

Molecular analysis of 1005 accessions and exploring parentages

Italy

Cipriani et al. (2010)

SSR

Genetic diversity in different genotypes

Korea

Cho et al. (2011)

ISSR; DAMD

Characterization of 21 local varieties

Iran

Seyedimoradi et al. (2012)

ISSR

Molecular and ampelographic characterization of 20 varieties

Iran

Zeinali et al. (2012)

SCoT

Genetic relationships within 64 varieties

China

Guo et al. (2012)

SSR; SNP

Genetic diversity of a large germplasm collection (2273 accessions)

Italy

Emanuelli et al. (2013)

SSR

Characterization of 317 India accessions

Upadhyay et al. (2013)

SSR

Genetic diversity and relationships amongst the Georgian germplasm

Imazio et al. (2013)

Georgia

(continued)

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Table 4.12 (continued) Marker name

Research objective(s)

Geographical area

References

SSR

Relationships among wild and cultivated accessions

Turkey

Karatas et al. (2014a, b)

SSR

Ampelographic, Croatia genetic characterization

Maleti´c et al. (2015)

SSR

Genetic diversity of 63 wild genotypes

Iran

Doulati Baneh et al. (2015)

SSR; SCoT

Genetic diversity, variety identification

Egypt

Ibrahim et al. (2016)

AFLP

Molecular and ampelographic characterization of 21 varieties belonging to “Mavroudia” group

Greece

Stavrakaki and Biniari (2017)

SSR

Varietal identification

South Africa

Van Heerden et al. (2018)

ISSR

Genetic diversity of 36 varieties

Palestine

Basheer-Salimia et al. (2019)

RAPD

Discrimination of 49 varieties

Greece

Biniari and Stavrakaki (2019)

SSR

Genetic characterization of Iranian genotypes and their relationships with Italian ecotypes

Iran

Khadivi et al. (2019)

RAPD; ISSR; SCoT

Genetic diversity of three grape varieties

Saudi Arabia

Abdel-Hameed et al. (2020)

SSR

Relationships among wild and cultivated of 243 accessions

Balkan region and Central Europe

Zdunic et al. (2020)

SSR; SNP

Genetic diversity of 212 accessions and parentage analysis

Croatia

Žulj Mihaljevi´c et al. (2020)

4.5.3 Relationship with Other Cultivated Species and Wild Relatives Grapes belong to the genus Vitis, which includes over 60 inter-fertile species spread broadly across the northern hemisphere (This et al. 2006). The phylogenetic relationships among these species are of keen interest for the conservation and use of the grape germplasm. V. vinifera or its hybrids cover the majority of commercial grape cultivation area across the world (Reisch et al. 2012). Various wild relative species have been used in breeding programme, to impart biotic and abiotic stress resistance

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in the progeny (Migicovsky et al. 2016). Cultivated grapes shows hermaphrodites sex form while the wild relatives are mostly dioecious in nature (Ocete et al. 2019). During the process of domestication, the wild ancestor of grapevine has probably changed from the dioecious to hermaphrodite sex form (Ramos et al. 2017). However, various wild Vitis species are being used in Vitis improvement programme to incorporate special traits of interest. In the due course of domestication of grapevine, a large diversity of characters was preferred. This resulted in the greater extent of diversity and heterozygosity in the cultivated form as compared to the wild form (Riaz et al. 2018; Cunha et al. 2020; D’Onofrio 2020). Various molecular studies have thrown lights regarding the relationship of V. vinifera or the cultivated genotypes with its wild relatives (This et al. 2006; Arroyo-García et al. 2006; Emanuelli et al. 2010).

4.5.4 Relationship with Geographical Distribution It was the between the Black Sea and Iran, where the domestication and cultivation of grapevine started between the 7th and 4th millennia BC (Zohary 1996; Zohary and Hopf 2000). Later on, the present days cultivated forms spread from this area in the Near East, Middle East and Central Europe (Arroyo-García et al. 2006). The genus Vitis contains about 100 species, most of which are inter-fertile. Most of the species are occurs in the Northern Hemisphere primarily in temperate zones. V. vinifera L. ssp. sativa (or vinifera) is covering the highest area under cultivation. The wild ancestor V. vinifera L. ssp. sylvestris represents the only Vitis taxon naturally found in Europe. Contrastingly, several species of genus Vitis have originated from the geographical area of North America and East Asia. Although not majorly in cultivation for human consumption but serves as important source of resistance to various biotic and abiotic stresses apart from direct use as rootstock (This et al. 2006). The genotyping of wild and cultivated forms from two important European and American germplasm repositories has provided a significant dataset capable of elucidating relationships within and between the two sub-species at the global level (Bacilieri et al. 2013; Riaz et al. 2018).

4.5.5 Extent of Genetic Diversity Various aspects of genetic variation are of interest, including the extent, nature, pattern, causes and relationship between the variation and the consequent phenotypic variation (Ewens 2013). The description of the extent genetic diversity in a species, and of the way in which it is structured, is prerequisite to determining what to conserve, and where and how to conserve it (Rao and Hodgkin 2002). Despite the importance of grapevine cultivation in human history and the economic values of cultivar improvement, largescale genomic variation data for grapevines are still lacking. Besides the widely used 10–20 K genotyping arrays (Myles et al. 2010;

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Laucou et al. 2018), the whole-genome resequencing data of various qualities were only reported very recently for 36 grapevine accessions in total (Zhou et al. 2017). Liang et al. (2019) in an extensive research work reported whole-genome genetic variation at single-base resolution of 472 Vitis accessions, which cover 48 out of 60 extant Vitis species from a wide geographic distribution. Such variation helped to identify the extent of diversity in the domesticated grapevines and that cultivars from the pan-Black Sea region have a unique demographic history in comparison to the other domesticated cultivars. It is stated that 11,000 domesticated grapevine cultivars and wild species are present around the world (Liang et al. 2019).

4.6 Association Mapping Studies 4.6.1 Extent of Linkage Disequilibrium The grape is one of the economically and nutritionally important fruit crops whose whole genome was sequenced the earliest; thus, enormous genomic resources are available globally. The genomic resources, particularly genetic markers, enable dissecting and identifying the alleles controlling the quantitative traits in the perennial heterozygous grapevine. The identification of QTLs, new alleles, and traits in a set of diverse germplasm in a crop species, while conducting the association mapping (Zhu et al. 2008; Ibrahim et al. 2020) using various molecular markers is one of the essential steps in crop molecular breeding. Association mapping also termed linkage disequilibrium (LD) mapping, one of the best approaches to overcome the limitations of pedigree-based QTL mapping in the crop species (Khan and Korban 2012). The association mapping does not require any specific population derived from particular cross combinations and mainly depends on the germplasm samples’, which is one of the apparent benefits in perennial grapevine (Barnaud et al. 2010). The LD is a nonrandom association of alleles. Their magnitude mainly depends on prevailing mating behaviour, natural and artificial selection, mutation, genetic drift, size of population, and their structure (Flint-Garcia et al. 2003). Thus, association mapping takes advantages of linkage disequilibrium and historical recombination of the gene pool of a crop species. Association mapping could be divided into genome-wide association mapping (GWAS) and another as the candidate gene approach. The selection of these approaches mainly depends upon the amount of markers available for the association. In the candidate gene approach, markers are selected based on their position on the chromosome or based on previous studies on QTLs/functions of the gene that ultimately involved the final variation (Ibrahim et al. 2020). The association that represents most of the genome segment is considered in the GWAS and undertaken the genotype of population of individuals that are densely distributed across genetic marker loci covering all the chromosomes (Rafalski 2010). In the last decade, many association mapping studies were performed in the grapevine to detect the genes/ QTLs associated with many complex traits and also economically important traits.

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The first report on LD characterization was published by Barnaud et al. (2006), and they had characterized the amount and pattern of LD in cultivated grapevine using SSR markers. Furthermore, Fournier-Level et al. (2010) did association mapping and QTL identification to reveal the genetic patterns of anthocyanin content and berry colours in grapevine. The association mapping and LD characterization in grapes are mainly confined to the berry related traits and other agronomical traits (Chitwood et al. 2014; Guo et al. 2019). However, the association mapping and LD characterization related to abiotic stresses tolerance/resistance in grape are nascent. In this connection, Marrano et al. (2018) performed association mapping using high throughput SNP markers, mapped six domestication-related traits in the grape, and provided evidence for response to different environmental stresses. Similarly, Shirazi et al. (2019) did genome-wide association mapping to identify and characterize the grapevine metal tolerance protein (MTP) family. Recently, Trenti et al. (2021) did genome-wide association studies in grapevine and identify the genetic basis of transpiration linked traits and genomic regions associated with drought tolerance in grape rootstocks.

4.6.2 Target Gene-Based LD Studies The target gene-based LD studies associated with abiotic stresses are infancy in the grape. Now, much attention is given in this area, while assessing the impact of climate change on vineyards and occurrence of different abiotic stresses, which mostly affected the grape quality and productivity. In this connection, Shirazi et al. (2019) performed genome-wide association studies and identified 11 MTP genes in grape, which were further divided into the three subfamilies, Fe/Zn-MTP, ZnMTP, and Mn-MTP, and other seven groups. Further, they have deciphered that the metal tolerance proteins ranged from 366 to 1092 amino acids and predicted that they could be located in the cell vacuole. Recently, Trenti et al. (2021) had identified 13 candidate genes associated with drought tolerance in grapes, while conducting genome-wide association studies. They have found that the three genes VIT_13s0019g03040, VIT_17s0000g08960 and VIT_18s0001g15390 were induced by the drought stress. Furthermore, the genetic variation in gene VIT_17s0000g08960 confirms the association with stomatal conductance in grape genotypes. Thus, the findings based on target gene-based LD studies would facilitate the identification of alleles associated with concern abiotic stress(es) and grape breeders to design the climate-smart grape varieties.

4.6.3 Genome-Wide LD Studies The development of efficient and cost-effective DNA sequencing technologies have made it possible to get genome-wide coverage molecular markers affordably in crop

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species, including the grapes (Owens 2011; Guo et al. 2019). Thus in recent years, genome-wide association mapping (GWAS) is becoming an important approach to dissect the QTLs or mining the alleles for the trait of interest in the grape. In GWAS, the QTLs with multiple alleles are identified at the whole-genome level is based on LD. In the past, few genome-wide association mapping has been performed to mapped leaf shape and morphology (Chitwood et al. 2014), berry shape traits (Zhang et al. 2017), domestication-related traits (Marrano et al. 2018) and the berry quality traits (Guo et al. 2019). However, the genome-wide studies related to abiotic stress in the grape confined to very little in number. For instance, Shirazi et al. (2019) did the GWAS for MTPs in grapevine and identified the 11 MTP genes associated with three major sub-families Fe/Zn-MTP, Zn-MTP, and Mn-MTP. Further, the expression profiling (temporal and spatial) indicated that the VvMTP genes had potential role growth and development of the grapevine and responses to environmental stresses, particularly osmatic stress. Recently, Trenti et al. (2021) performed GWAS on 100 Vitis spp. including inter-specific hybrids, rootstock genotypes, wild non-viniferaVitis species and rootstock selections, which identified 34 marker-trait associations and 13 candidate genes that have a role in drought response. Thus, more GWAS studies could reveal the genomic region, alleles/ QTLs associated with the abiotic stresses in grapevines and which may help the grape breeders for designing the novel resilient grape genotypes.

4.6.4 Future Potential for the Application of Association Studies for Germplasm Enhancement The mapping resolution mainly depends on the magnitude of genetic diversity, extend of linkage disequilibrium within the genome of a crop and relatedness of germplasm (Yu et al. 2006; Zhu et al. 2008). Thus, accurate information about the prevailing genetic diversity of grape germplasm and their genetic structure is essential for the identification of genetics of traits through association mapping (Laucou et al. 2018). The consequence of continuous selection is that the elite cultivars had a narrow genetic base and strengthened population structure which may cause false positive (type II errors) in association mapping (Fodor et al. 2014). The population with a low level of familial relationship with the low level of the structure are considered ideal for the association mapping (Yu and Buckler 2006; Yu et al. 2006). Further, the size of the sample in GWAS is one of the determining factors for identifying molecular markers linked to the trait of interest (Zhao et al. 2007; Buckler et al. 2009; Wang et al. 2012). Keeping this in view, many genetic characterizations of grape germplasm have been conducted for the grape germplasm. Earlier, the genetic diversity analyses were mainly based on the few pairs of microsatellite markers and high throughput SNP markers based on genetic diversity is necessitate for the indepth understanding of genetic diversity in a particular crop species (Hamblin et al. 2007). Thus, in the recent past, the genetic characterization of grape germplasm is

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mainly based on SNP markers. Thus, the grape breeders use the high through SNP markers to better understand genetic diversity in the grape germplasm (Laucou et al. 2018; Žulj et al. 2020). Furthermore, enhancement of genetic diversity for useful traits targeting abiotic stresses may help the grape breeders for allele mining or QTL identification through association mapping.

4.7 Brief Account of Molecular Mapping of Resistance Genes and QTLs 4.7.1 A Brief History of Mapping Efforts Grapevine is characterized by a long juvenile phase and heterozygous in nature. The Grapevine takes 4–5 years for bearing when it is propagated by seeds (Harris et al. 2017; Sapkota et al. 2019) and thus, traditional breeding in grapes is tedious and time-consuming (Louime et al. 2010; Chen et al. 2011; Karaagac et al. 2012). In this perspective, marker-assisted selection (MAS) could improve the breeding efficiency of the grapevine. To facilitate MAS identifying the markers linked to the trait of interest is essential. To determine the genomic region of important traits, several genetic mapping was done and QTLs were identified in the grape. The first genetic map was published by Lodhi et al. (1995). Thereafter many genetic mappings have been done in grapevine and QTLs associated with many economic traits were published. For agronomical traits [flower sex (Battilana et al. 2013; Lewter et al. 2019), seedlessness (Royo et al. 2018), berry weight (Zhao et al. 2015; Ban et al. 2016), berry colour (Yang et al. 2016; Lewter et al. 2019), soluble solid content (Zhao et al. 2015), acidity (Bayo-Canha et al. 2019), and muscat flavour (Doligez et al. 2006)], biotic stresses [powdery mildew (Blanc et al. 2012; Van Heerden et al. 2014; Pap et al. 2016; Zyprian et al. 2016; Zendler et al. 2017), downy mildew (Blasi et al. 2011; Schwander et al. 2012; Van Heerden et al. 2014; Ochssner et al. 2016; Zyprian et al. 2016; Divilov et al. 2018; Lin et al. 2019; Sapkota et al. 2019), anthracnose (Fu et al. 2019), root-knot nematodes (Smith et al. 2018a, b), and grape phylloxera (Clark et al. 2018; Smith et al. 2018a, b). Genetic mapping and QTLs identification related to abiotic stresses have also been attempted in the last decade. For instance, control the transpiration rate (Marguerit et al. 2012), tolerance to lime-induced iron deficiency chlorosis (Bert et al. 2013), leaf area, specific transpiration rate, specific hydraulic conductance (Coupel-Ledru et al. 2014), day and nighttime transpiration rate (Coupel-Ledru et al. 2016), low-temperature responses (Awale et al. 2016), leaf Na+ exclusion under salinity stress (Henderson et al. 2018), root and aerial biomass (Tandonnet et al. 2018), and cold hardiness (Su et al. 2020).

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4.7.2 Evolution of Marker Types The various molecular markers viz., amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), restriction fragment length polymorphism (RFLP), variable number of tandem repeats (VNTRS), cleaved amplified polymorphic sequence (CAPS), sequence-characterized amplified region (SCAR) expressed sequence tag (EST), simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP) have been developed and many of these molecular markers were employed for genetic mapping and QTLs identifications in grapevine. The first genetic map in grapevine was based RAPD and AFLP markers (Lodhi et al. 1995). Although these markers are convenient to use, the dominant nature of markers restricts their transfer and comparison among the mapping populations (Adam-Blondon et al. 2004). Further, the development of co-dominant markers like SSR overcomes the limitations of dominant marker systems and gets popularity among the grape breeders for genetic mapping and QTL detections (Adam-Blondon et al. 2005; Garris et al. 2009; Zhang et al. 2009). In the past, these SSR markers have been explored for identification of the QTLs many economical traits in grape, including abiotic stresses tolerance. For example, QTLs associated with rootstock controlled scion transpiration rate (Marguerit et al. 2012), tolerance to lime-induced iron deficiencychlorosis (Bert et al. 2013), hydraulics-related traits (Coupel-Ledru et al. 2014), transpiration rate at day and night time (Coupel-Ledru et al. 2016), root and aerial traits (Tandonnet et al. 2018) were identified using the SSR markers. However, SSR markers are low throughput and genetic mapping based on this marker is considered expensive and time-consuming (Adam-Blondon et al. 2005; Mejía et al. 2007; Vezzulli et al. 2008). The development of the high throughput molecular marker like SNP enhanced the resolution of genetic maps and helps in fine QTL mapping. Keeping this in view, QTLs associated with abiotic stresses were identified based on the SNP markers. For instance, QTLs for sub-zero temperature tolerance (Awale et al. 2016), leaf Na+ exclusion under salinity stress (Henderson et al. 2018) and cold hardiness QTLs (Su et al. 2020) were identified using SNP markers in grape.

4.7.3 Mapping Populations Used The grapevine is one of the perennial fruit crops primarily characterized by their long juvenile phase and heterozygous nature. The grape breeders have used different mapping populations for mapping of various economical traits, including abiotic stress tolerance. In most of the perennial crops, including the grapevine, the strategy of genetic mapping is mainly based on the pseudo test cross and individuals from F1 generationhave been utilized for mapping (Weeden et al. 1994; Doucleff et al. 2004; Moreira et al. 2011; Marguerit et al. 2012; Bert et al. 2013; Coupel-Ledru et al. 2014, 2016; Henderson et al. 2018; Tandonnet et al. 2018; Su et al. 2020).

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Further, F2 progenies based mapping offers high linkage map accuracy and captures the recessive allele effects and additional meiotic events (Van and Jansen 2013; Yang et al. 2016). Thus, fine-smapping based on F2 progenies was published in grape for many economical traits, including the abiotic stress-related traits (Adam-Blondon et al. 2004; Garris et al. 2009; Hvarleva et al. 2009a, b; Dunlevy et al. 2013; Yang et al. 2016; Awale et al. 2016; Guillaumie et al. 2020).

4.7.4 Mapping Software Used Several programmes and software’s were developed for mapping and deification of QTLs. viz., MAPMAKER (Lander et al. 1987), Join Map (Stam 1993), DrawMap (Van Ooijen 1994). MapChart (Voorrips et al. 2002), SMOOTH (Van et al. 2005), OneMap (Margarido et al. 2007). Grape breeder also explored many of this developed software for genetic mapping and identifying QTLs associated with abiotic stresses. For instance, Marguerit et al. (2012) did QTL mapping for rootstock controlled scion transpiration rate using MultiQTL V2.6. Further, software MapQTL 4.0 was used by Bert et al. (2013), Coupel-Ledru et al. (2014, 2016) for genetic mapping and QTLs identification for tolerance to lime-induced iron deficiencychlorosis, hydraulicsrelated traits and transpiration rate at d night and day time, respectively. Henderson et al. (2018) did QTL mapping related to leaf Na+ exclusion under salinity stress using JOINMAP ® v.4.1 software. Tandonnet et al. (2018) used MapQTL 6.0 software for QTL mapping of root and aerial traits in grapevine. Recently, Su et al. (2020) used MapChart 2.2 for high-density linkage mapping and identifications of QTLs associated with cold hardiness.

4.7.5 Maps of Different Generations The genetic mapping in the grape has started in the late 90 s and these maps were widely used for the identification of QTLs associated with traits of interest. The advent of next-generation sequencing and the development of high throughput molecular markers enable the development of high-resolution genetic maps in grapevine with enhance markers density, which further facilitate fine mapping of QTLs and molecular breeding for the trait of interest. In grapevine more than 160 maps (including parental and consensus ones) have been reported after the report of the first genetic map (Vezzulli et al. 2019). In the past, linkage map (Grando et al. 2003), high-resolution SNP map (Barba et al. 2014), High-density linkage maps (Wang et al. 2017; Lewter et al. 2019; Sapkota et al. 2019), highly saturated genetic map (Junchi Zhu et al. 2018a, b) have been developed and QTLs were identified associated to many important agronomical traits including the biotic stress tolerance/resistance in grapevine. However, the genetic mapping targeted to the abiotic stresses is limited in number and last decade, the main emphasis is paid for genetic mapping and QTLs

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identification for abiotic stress-related traits in grapevine. Marguerit et al. (2012) developed a map using the interval mapping approach and identified the QTLs associated with transpiration rate. Bert et al. (2013) constructed a genetic linkage using microsatellite markers and identified the QTL associated with tolerance to limeinduced iron deficiencychlorosis. A framework linkage map using the microsatellite markers was constructed for the identification of QTLs associated with water potential and transpiration rate (Coupel-Ledru et al. 2014). Further, Coupel-Ledru et al. (2016) used a consensus mapfor the identification of the QTLs associated with reduced transpiration rate during day and night time in grapevine. A consensus map developed by Tandonnet et al. (2018) and QTLs were identified for root and aerial traits in grapevine. Furthermore, Henderson et al. (2018) developed a consensus linkage map and identified the QTL associated with leaf Na+ exclusion (NaE) under salinity stress conditions. Furthermore, Su et al. (2020) constructed a high-density linkage map and identified the QTL linked to cane cold hardiness in grapevine.

4.7.6 QTLs Related with Abiotic Stresses In the past decades, many genetic mappings have been done and QTLs were identified associated with many economical traits in grapevine, including the biotic stresses tolerance. However, very limited studies were conducted to identify the QTLs related to abiotic stresses like drought, salt, heat, cold and metal toxicity globally (Bert et al. 2013) (Table 4.13). In this continuation, Marguerit et al. (2012) did functional and multi-environment QTL mapping and deciphered the genetic architecture in rootstock that controls transpiration rate related traits in scion genotype. The interspecific cross [V. vinifera cv. Cabernet Sauvignon (CS) × V. riparia cv. Gloire de Montpellier (RGM)] was used to dissect the genomic region associated with transpiration rate. The multi-year QTL analysis revealed that traits like transpiration rate were associated with CS1 and CS17, transpiration efficiency with CS6 and RGM11 and water extraction capacity with RGM 3, RGM5 and RGM11. This study was deciphered that the detected QTLs are colocalized with genes that involved in water deficit responses viz., hydraulic and ABA regulation. Bert et al. (2013) identified a QTL linked with tolerance lime-induced iron deficiency on chromosome 13 with 10 to 25% of the chlorotic symptom variance. This study also explained that the rootstock strongly influences lime-induced chlorosis response. Coupel-Ledru et al. (2014) performed QTL mapping on the individuals derived from the reciprocal crosses of Syrah and Grenache for the important hydraulics-related traits under both well-watered and water deficit conditions. They have identified four QTLs [LG1 (two QTLs), LG10, and LG18] associated with leaf water potential during day time (ΨM) and one QTL located on LG17 was also identified for specific transpiration rate (TrS) under water deficit conditions. In this experiment, they also illustrated that QTLs for ΨM tightly co-localized with ΔΨ (difference between soil water potential and ΨM) (LG01, LG10, and LG18). Further, Coupel-Ledru et al. (2016) identified stable QTLs associated with transpiration rate at night time located on linkage groups, 1, 4, and 13.

Hydraulics-related traits (leaf water potential and specific transpiration rate Transpiration rate at night SSR time (En) and day time (Ed)

186 two year-old pseudo-F1 progeny

186 pseudo-F1 progeny

113 F2 progenies

A reciprocalcross between Syrah and Grenache

A reciprocalcross between the grapevine cultivars Syrah and Grenache

An F2 population from a cross of V. riparia and a hybrid grapevine Seyval Subzero temperature tolerance

Lime-induced iron deficiency chlorosis

138 F1 progenies

V. vinifera Cabernet Sauvignon and × V. riparia Gloire de Montpellier

SNP

SSR

SSR

SSR

Transpiration-related traits (rootstock control of scion transpiration)

138 F1 genotypes

Markers used

V. vinifera cv. Cabernet Sauvignon (CS) × V. riparia cv. Gloire de Montpellier (RGM)

Trait (s) trageterd

No. of progenecy

Cross combination

Table 4.13 The details of major QTLs associated with abiotic stress-related traits in grape

1, 5, 9, 13 and 16

En on 1, 4 and 13 and Ed on 1, 2, 10, and 17

1,10,17 and 18

13

Transpiration rate (CS1 and CS17), transpiration efficiency (CS6 and RGM11) and water extraction (RGM 3, RGM5 and RGM11)

LG or chromosome position

(continued)

Awale et al. (2016)

Coupel-Ledru et al. (2016)

Coupel-Ledru et al. (2014)

Bert et al. (2013)

Marguerit et al. (2012)

References

4 Development of Abiotic Stress Resistant Grape Vine Varieties 109

181 hybrid offspring

Cabernet sauvignon × Zuoyouhong Cold hardiness

Leaf Na+ exclusion

40 hybrid rootstocks

K51-40 (V. champinii × V. riparia) × 140 Ruggeri (V. berlandieri × V. rupestris)

Trait (s) trageterd Arial and root traits

No. of progenecy

V. vinifera cv. 138 F1 genotypes Cabernet-Sauvignon × V. riparia cv. Gloire de Montpellier

Cross combination

Table 4.13 (continued)

SNP

SNP

SSR

Markers used

References

2, 3 and 15

11

Su et al. (2020)

Henderson et al. (2018)

1, 2, and 5 for root biomass Tandonnet et al. (2018)

LG or chromosome position

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The identified alleles have contributed additive effects to individual QTL and few dominant effects between alleles. They also identified five QTLs associated with transpiration rate at day time on the LGs, 1, 2, 10, and 1l7. This study also deciphered that the QTLs associated with transpiration rate at night time did not colocalize with QTLs associated with transpiration rate at daytime. Awale et al. (2016) identified the QTLs for the low-temperature response on chromosomes 1, 5, 9, 13 and 16 in different months or dormant seasons. Further, Henderson et al. (2018) identified a QTL associated with leaf Na+ exclusion under salinity stress on chromosome 11. Further, they have identified a candidate gene, VisHKT1;1 for controlling leaf Na + exclusion in grape. Furthermore, Tandonnet et al. (2018) identified QTLs related to root biomass on chromosomes 1, 2, and 5. They detected a single QTL is colocalized for aerial and root biomass in grapevine. Recently, Su et al. (2020) detected six QTLs associated with cold hardiness on linkage groups 2, 3 and 15. They have identified four candidate genes (VIT_02s0033g01120; VIT_15s0048g01980; VIT_15s0048g02410 and VIT_15s0048g02700) related to cold hardiness. These studies would be laid the foundation of marker-assisted selection in grape and tailoring the abiotic stresses scion or rootstock genotype.

4.8 Marker-Assisted Breeding for Resistance Traits 4.8.1 Germplasm Characterization and DUS Approximately 5,000 cultivars of V. vinifera has been estimated to existing (Alleweldt and Dettweiler 1994; This et al. 2006). Asexual propagation using hardwood vine cuttings and ease in long distance transportation of rooted vine cuttings in dormant condition has expanded the viticulture to a wide region along with creating a confusion of large number of synonyms of the same cultivars in various regions. This emphasizes the need to characterize and test the DUS of the existing as well as newly evolving grapevine germplasm using molecular markers. Amongst the molecular markers, microsatellites have been widely used to characterize the grapevine collections around the world (Aradhya et al. 2003; Lopes et al. 1999; Martin et al. 2003). Microsatellites have been employed to identify the parents of many cultivars of cultivated grapevine, including Syrah, Cabernet Sauvignon etc. (Bowers and Meredith 1997; Cervera et al. 1998; Crespan 2003; Dettweiler et al. 2000; Lopes et al. 2006). Upadhyay et al. (2007) used the AFLP and SSR markers to analyse 21 rootstock accessions. RAPD was also used for genetic diversity analysis in Muscadine and American bunch grapes (Qu et al. 1996). Later, Kocsis et al. (2005) also used RAPD markers for characterization of twelve grape cultivars indigenous to the Carpathian Basin. Similar studies were also reported by Tamhankar et al. (2001), Luo et al. (2002) and Karata¸s and Agaoglu (2010). Alizadeh and Singh (2009) used the RAPD and ISSR markers to ascertain the clonal fidelity of micro-propagated grape plantlets regenerated from three grape rootstock genotypes, namely, Dogridge,

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SO4 and ARI-H-144. Motha et al. (2018) also used the ISSR and SSR markers for molecular characterization and studied the genetic relationships of few stress tolerant grape rootstock genotypes. Recently, Dev et al. (2021) assessed the genetic diversity in 36 gamma rays irradiated mutants of four grape genotypes using RAPD and SSR markers. These mutants were screened from in vitro mutated four grape genotypes, namely Pusa Navrang, H-76-1, Pearl of Csaba and Julesky Muscat on the basis of morpho-physiological and biochemical traits. The resultsof the study suggested that OPA01 (RAPD) and VVMD14 (SSR) were the most informative primers and generated maximum numbers of reproducible bands. In recent times, SNP markers are also being used for germplasm characterization (Myles et al. 2011).

4.8.2 Marker-Assisted Gene Introgression In grape, marker-assisted gene introgression has been mostly exploited for biotic stress tolerance like downy mildew, powdery mildew, Pierce’s disease, phylloxera etc. and fruit quality improvement like for seedlessness. Liu et al. (2016) used the embryo rescue technique accompanied with MAS to develop new cold-resistant, seedless grape genotypes. They used GLSP1 and SCF27, two special primers to screenthe progenies of five cross combinations for possible seedlessness. Five of 22 strains obtained from two cross combinations had themarker GLSP1-569; 43 of 89 strains obtained from three crosscombinations had the marker SCF27-2000. Similarly, Zhu et al. (2019) also used the MAS to screen the embryo rescued grape genotypes for their cold tolerance. Al-Mousa et al. (2016) used 20 ISSR primers to screen the potential drought tolerance grape mutants. Out of these 20 primers used, 6 primers generated polymorphic bands in genotypes irradiated with 20 Gy. Abdel Aziz (2018) conducted a study to assess the effect of high level concentrations of NaCl on the micropropagation of some grape rootstocks Salt Creek, Dog ridge, Richter and Freedom. The genetic analysis of molecular markers was performed using five primers of ISSR. The percentage of polymorphism vary from 33.33 to 83.33% with an average of 54.29%.

4.8.3 Gene Pyramiding Gene pyramiding or stacking can be defined as a process in which two or more genes from multiple parents are combined to develop elite lines or varieties. In grape, a few attempts has been made to achieve resistance to downy mildew and powdery mildew, seedless berries using gene pyramiding (Agurto et al. 2017; Mahanil et al. 2012; Saifert et al. 2018). However, gene pyramiding is not attempted to impart abiotic stress tolerance, which is the need of the hour.

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4.8.4 Limitations and Prospects of MAS and MABCB Marker assisted selection (MAS) and Marker assisted backcross breeding (MABCB) are amongst the foremost benefits of molecular markers as indirect selection tools in crop breeding programs. Although MAS has been used extensively for grape improvement, the exploitation of MABCB in grape breeding programs is meagre. Currently, MABCB of single gene is probably the foremost powerful approach that uses DNA markers effectively. Improvement of QTLs through MABCB resulted to variable results ranging from limited success in grape. Although MAS and MABCB are significantly time saving in nature, but requires skilled manpower and high throughput machineries. A major constraint to the implementation of MAS and MABCB in grape breeding programs has been the higher cost as compared to traditional breeding. To be useful to grape breeders, gains made from MAS and MABCB must be more cost-effective as compared to the conventional breeding (Semagn et al. 2006). In fruit crops like grape, where the targeted trait(s) is expressed late in plant development, like fruit and flower characteristics owing to longer juvenile phase, MABCB can be effectively explored. It is also useful when the trait is of low heritability or highly affected by environmental conditions.

4.9 Map-Based Cloning of Resistance Genes In grapes the major focus of the map based cloning were confined to genes imparting resistance to important diseases and pests such as downy mildew, nematode and abiotic stresses like salinity and drought. At the outset molecular markers closely flanking the gene of interest/gene linked markers need to be identified and to achieve this sequence information can be used to develop DNA markers. This is followed by developing a contig of large-insert size clones spanning the region between flanking markers and identifying candidate genes within the contig. The test of expression can be carried out by demonstrating the functionality of candidate genes using genetic tools such as complementation and/or gene knockout studies (Han and Korban 2016). However, in the post genomic era where sequence information of the whole genome is readily available in public domain the positional candidate-gene approach can be utilized to reduce the quantum of work involved in positional cloning of gene of interest. By using modern bioinformatics tools, the available genomic sequences in the target gene region can be directly used to predict candidate genes (Han and Korban 2016).

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4.9.1 Traits and Genes Target genes for map-based cloning in plants are mostly carried out for those genes which are major genes for biotic and abiotic resistance as their phenotypes are relatively easy to identify and the subsequent phenotypic data are reliable. There are many abiotic stresses that significantly limit the distribution of grapes all over the world. These stresses primarily are salinity, water stress, low and high temperature stress which reduce crop yields. Besides these, frost and chilling damage during the spring and suppress yield in more moderate climates. Stressful condition (heat and light) often seen interactions with water deficit to accelerate water loss and plant strain (Seki et al. 2007). Under water deficit or drought stress conditions in grapevine, antioxidant related genes (involved in ROS detoxification) such as phospholipid hydroperoxide glutathione peroxidase, gamma-glutamylcysteine synthetase, and NADPH glutathione reductase along with the genes involved in enhanced proline biosynthesis (PDH, P5CS) showed increased expression (Cramer et al. 2007a, b). Recently, TIFY genes were found responsive to jasmonic acid (JA) and ABA in grapes. TIFY genes are involved in responses related to various abiotic and biotic stresses (Zhang et al. 2012a, b). Wang et al. (2014) reported ViWRKY3 genes respond to various abiotic stresses like salinity and drought in grapes and modification in its expression may represent a strategy to enhance abiotic stress tolerance in grapes. bHLH genes related to stress (drought, salt, cold) response were identified from the grape genome (Wang et al. 2018a, b, c). Upadhaya et al. (2018) also identified 342 DEGs including 52 differentially expressed transcription factors belonging to WRKY, EREB, MYB, NAC and bHLH families under salt stress conditions. More recently, Zheng et al. (2020) identified genome wide chitinase genes in grapes and also carried out functional analysis. They found VvChi genes respond to methyl jasmonate and ethylene treatment alongwith temperature and salinity stress. Cochetel et al. (2020) reported higher expression of water deficit core gene set with the ABA biosynthesis and signaling genes, NCED3, RD29B and ABI1 in Vitis champinii cv. Ramsey. Aydemir et al. (2020) found increased expression of B-box zinc finger protein, CCR4associated factor, NAC transcription factor 29, probable calcium binding protein CML44 like, chitin inducible gibberellin responsive protein, beta-amylase 1 chloroplastic genes under salinity stress in grapes. Various stress responsive genes in grape has been presented in Table 4.14.

4.9.2 Identifying Gene Linked Markers Various molecular techniques used to assess diverse phenotypes in grapevines under different abiotic stresses are given in Table 4.15. The genetic maps based on set of robust markers are really a useful tool for grape improvement. Most markers are “random markers” (namely anonymous or neutral) with no specific effect on the expression of the target trait. This is the case of classical AFLP markers and

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Table 4.14 Stress responsive genes in grape Gene locus ID/Gene ID

Protein/transcription factor

MIPS 2.1 functional category/function/stress response

GSVIVG00025569001

Raffinose synthase 01.05 C-metabolism

01.05 C-metabolism

GSVIVG00010921001

Trehalose phosphatase

01.05 C-metabolism

GSVIVG00014947001

C2 H2 zinc finger

11.02.03.04.01 transcription activation

GSVIVG00027622001

NAC domain protein

11.02.03.04.01 transcription activation

GSVIVG00023994001

WRKY DNA-binding protein

11.02.03.04.01 transcription activation

GSVIVG00030292001

RAV transcription factor

11.02.03.04.01 transcription activation

GSVIVG00000517001

ATHB-12

11.02.03.04.01 transcription activation

GSVIVG00036604001

bZIP transcription factor

11.02.03.04.01 transcription activation

GSVIVG00015416001

NAC domain protein

11.02.03.04.01 transcription activation

GSVIVG00025566001

Remorin-like protein

16.03.01 DNA binding

GSVIVG00023957001

Serine/threonine protein kinase

30.01.05.01 protein kinase cascades

GSVIVG00036780001

Calcium dependent protein kinase

30.01.09.03 Ca2+ mediated signal transduction

GSVIVG00002880001

Catalase 2

32.07.07.01 catalase reaction

GSVIVG00000988001

9-cis-epoxycarotenoid dioxygenase 1

36.20.18.05 abscisic acid response

ABC86747 (Gene ID)

PR protein

RNase, DNase, anti-fungal activities and respond to abiotic stresses

CAC16166 (Gene ID)

PR protein

RNase, DNase, anti-fungal activities and respond to abiotic stresses

VvWRKY16

Transcription factor

Salt stress responsive, DNA binding

VvWRKY25

Transcription factor

Salt stress responsive, DNA binding

VvWRKY28

Transcription factor

Salt stress responsive, DNA binding

VvWRKY35

Transcription factor

Salt stress responsive, DNA binding

VvWRKY3

Transcription factor

Draught stress, DNA binding

VvWRKY25

Transcription factor

Draught stress, DNA binding

VvWRKY28

Transcription factor

Draught stress, DNA binding

VvWRKY28

Transcription factor

Draught stress, DNA binding (continued)

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Table 4.14 (continued) Gene locus ID/Gene ID

Protein/transcription factor

MIPS 2.1 functional category/function/stress response

VvWRKY35

Transcription factor

Draught stress, DNA binding

VvMYBF1

Transcription factor

Draught stress, UV-B tolerance, DNA binding

VvWRKY55

Transcription factor

Cold Stress, DNA binding

VvUVR1

–

UV-B Tolerance

VvCOP1-1

–

UV-B Tolerance

VvCOP1-2

–

UV-B Tolerance

VvHY5

–

UV-B Tolerance

VvHYH

–

UV-B Tolerance

VvRUP

–

UV-B Tolerance

VvUVR1

–

UV-B Tolerance

VIT_19s0014g03820

Response to salt stress (TF)

Salt stress responsive

VIT_12s0059g02150

Aconitate hydratase

Salt stress responsive

Source Cramer (2010); Das and Majumdar (2019)

Table 4.15 Representative molecular techniques deployed to assess diverse phenotypes in grapevines for abiotic stresses Abiotic stress

Molecular technique used References

Drought stress

Proteomics

Grimplet et al. (2009)

Salt stress

Proteomics

Jellouli et al. (2008)

High temperature and heat shock PCR

Liu et al. (2012)

Herbicide

Proteomics

Castro et al. (2005)

Dormancy

HPLC

George et al. (2018)

Freeze shock

Transcriptomics

Tattersall et al. (2007), Xin et al. (2013)

Cold tolerance

RT-PCR

Hou et al. (2018)

UV stress

RT-PCR

Schoedl et al. (2013)

Source Cadle-Davidson et al. (2019)

SSR markers, which are very much abundant in intergenic regions. The microsatellites or SSR markers are widely studied, since they shown some advantages over other modified molecular markers, considering their co-dominant inheritance, hypervariability, ubiquitous nature, abundance and highly dispersal in genomes, with high variations present at most given positions. In Vitis, SSR markers have been identified and developed by many scientists and research groups and these markers have been used for many genetic studies. Along with SSRs, SNP markers have been used for assessing population genetic structure (Emanuelli et al. 2013; Laucou et al. 2018) and for high-resolution mapping (Troggio et al. 2007; Teh et al. 2017). There is

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also a Vitis Microsatellite Consortium (VMC) for generating molecular markers for fingerprinting of table grape, wine grape, genetic maps and for studying germplasm genetic diversity. “Gene-targeted” SSR and SNP markers were also reported which gives information related to markers developed from information on gene sequence (Mejía et al. 2011) or expressed sequence tag (EST) (Decroocq et al. 2003; Kayesh et al. 2013). The high-throughput SNP array (Illumina Vitis18KSNP chip) for the second time was produced as part of the GrapeReSeq Consortium (Le Paslier et al. 2013) and deployed for in depth characterization of available genetic resources and to find out the genetic variability among cultivars (Sunseri et al. 2018; Mercati et al. 2016). This is also used to perform and refine parentage analyses (Laucou et al. 2018). Wang et al. (2017) reported a large-scale discovery of SNPs with the help of the specific length amplified fragment sequencing (SLAF-seq) technique in Chardonnay and Beibinghong and their 130 F1 plants. They demonstrated that SLAF-seq is a promising strategy for the construction and to get the updated information of highdensity genetic maps. Diverse germplasm collection with accurate phenotyping over the years may generate useful information about QTLs through association mapping. Instead of biparental population, a pool of diverse germplasm is used which forms an association panel for mapping QTLs. Software like TASSEL 5.0, NAM, R etc. can be used for association mapping (http://www.maizegenetics.net; https://cran.r-project. org/web/packages/NAM/NAM.pdf).

4.9.3 Strategies: Chromosome Landing and Walking Two approaches can potentially be used for map-based positional cloning of genes of interest: chromosome walking and chromosome landing. The first approach relies on first identifying DNA markers linked to the target gene, and then taking ‘walking steps’ to get to the gene via a series of overlapping clones. The closest linked marker to the target gene is used to screen the genomic library to identify positive clone(s). These positive clone(s) are used to isolate insert-ends which in turn, are used as probes to screen the library for additional overlapping clones. This process is repeated for several times to ‘walk across’ the chromosome and reach the target gene. Apart from being time consuming and tedious, chromosome walking is a difficult task perennial fruit crops like grape vine due to the larger genome size and higher abundance of repetitive sequences (Tanksley et al. 1995). Pelsy and Merdinoglu (2002) used chromosome walking strategy to sequence and characterize copia like retrotransposons in grapevine genome.

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4.9.4 Genomic Libraries: YACs, BACs and TACs In vinifera and other related Vitis species several bacterial artificial chromosome (BAC) libraries are already available for various genomic purposes. These BAC libraries need to be exploited for construction of a physical map to fast-track the process of gene identification. The yeast artificial chromosome (YAC) cloning system can clone large DNA fragments of up to 1000 kb (Burke et al. 1987). However, BAC (Shizuya et al. 1992), transformation-competent artificial chromosome (TAC) (Liu et al. 1999), and the bacteriophage P1-derived artificial chromosome (PAC) (Ioannou et al. 1994) can be used for cloning of larger-insert. Among these cloning systems, the BAC has been widely exploited in grapes for preparing genomic libraries because of the optimum size of DNA inserts, higher cloning efficiency, and stable maintenance of foreign DNA (Choi et al. 1995; Woo et al. 1994; Wang et al. 1995; Salimath and Bhattacharyya 1999; Ming et al. 2001). The first BAC library in grapevine was studied and constructed by Yimin Jin (M. A. Walker, unpublished data) from D8909-15, while the second BAC library was from V. vinifera Cabernet Sauvignon and the third from V. arizonica b42-26 using a vector/enzyme combination of pCC1/HindIII (Amplicon Express, Pullman, WA, USA). Tomkins et al. (2001) constructed a grape BAC library using the cultivar Syrah for identification of clones involved in flavonoid and stilbene biosynthesis. High-molecular-weight DNA isolation and BAC library construction (digestion with restriction enzymes, ligation to the vector and bacterial transformation) were carried out by Adam-Blondon et al. (2005) in grapes. They successfully constructed 4 BAC libraries from the major grape varieties viz., Pinot Noir, Cabernet S and Syrah with genome coverage of about 4.5–14. genome equivalent and mean insert size of about 93–158 Kb. Velasco et al. (2007) constructed two BAC libraries and clones were assembled in a physical map. Hwang et al. (2010) worked and developed three BAC libraries to make understand BAC contigs encompassing the XiR1 locus.

4.9.5 Test for Expression (Mutant Complementation) Bogs et al. (2007) isolated and characterized the VvMYBPA1 gene encoding for the transcription factor MYB responsible for proanthocyanidin synthesis in grapes. The constitutive expression of this transcription factor in Arabidopsis thaliana complimented with PA deficient seed coat phenotype of the Arabidopsis tt2 mutant induced etopic PA accumulation in tissues. The grapes WRKY genes are reported to respond to salinity and drought stress along with methyl jasmonate and ethylene treatment in grape plants. The WRKY3 gene from grape was constitutively expressed in Arabidopsis thailana and CaMV35S promoter was used by Wang et al. (2014). The transformed Arabidopsis thaliana plants showed improved salt and drought tolerance. Various physiological traits related to abiotic stresses cause less damage to the transgenic plants as compared to the wild type plants. The results indicated

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that V1WRKY3 plays crucial role in abiotic stress management of the plants and modification of its expression may be a viable strategy to partially cope up with the damages caused to abiotic stress in grapes (Wang et al. 2014). Tu et al. (2016) isolated VqbZIP39 gene, responsible for salt and drought tolerance, from V. quanquangularis and constitutively expressed in A. thaliana under control of CaMV 35S promoter. The trangenic A. thaliana plants showed enhanced tolerance to drought and salt stress.

4.10 Genomics-Aided Breeding for Resistance Traits 4.10.1 Grape Genome Sequencing and Assembly The whole genome sequencing in grapes started with the use of Sanger sequencing and 454 pyrosequencing techniques (Jaillon et al. 2007; Velasco et al. 2007). But with the advent of next generation sequencing technologies, sequencing by synthesis (SBS) based Illumina platforms were used and HiSeq and MiSeq sequencing technologies for pair end library and mate pair libraries, respectively were utilized (Tables 4.16 and 4.17). But, the second and third generation sequencing technologies with short reads are not very suitable for assembling the highly repetitive grape genome having high heterozygosity. Moreover, short reads deliver highly fragmented assemblies that under represent repetitive content (Di Genova et al. 2014). The fourth generation sequencing platforms like PacBio, SOLiD, Oxford Nanopore technologies coupled with 10 × Genomics and optical mapping facilitated the significant improvement in grape genome sequencing. The latest sequencing technologies, 10 × Genomics Chromium data combined with long read PacBio sequencing were used in grapes to effectively determine genome phasing (Figueroa-Balderas et al. 2019). Badouin et al. (2020) used PacBio for genome sequencing of V. sylvestris. They carried out 120X coverage using SMRT-sequencing. With the development of dedicate methods, like HGAP (Chin et al. 2013), Canu (Walenz et al. 2017), and wtdbg2 (Ruan and Li 2020) for the assembly of long-read sequences generated by PacBio SMRT, allows to assemble genomes with high contiguity. Zhou et al. (2018) followed hybrid approach to assemble Chardonnay grapes genome.

4.10.2 Grapevine Reference Genome Annotation and Gene Discovery The first version of the grapevine genome sequence was carried out using a whole genome shotgun strategy and the Sanger sequencing technology and the coverage depth was about 8X so named as 8X version, was obtained (Jaillon et al. 2007). This assembly was later upgraded by adding 4X of additional coverage and including

12X

Vitis vinifera var. Pinot Noir

Vitis vinifera var. Cabernet sauvignon

Vitis vinifera var. Chardonay

Vitis riparia var. Gloire deMontpellier

Sanger and 454 pyrosequencer

PacBio

PacBio

PacBio/10X Chromium/Illumina

225X

115X

140X

Genome coverage

Sequencing platform Grape used variety/genotype

Table 4.16 The grapevine whole genome sequence

1,964 p-tigs 3,344 h-tgs

530 p-tigs

1,883 h-tgs

378 h-tgs

317 h-tgs

854 p-tigs

490 p-tigs

718 p-tigs 2,037 h-tgs

591 p-tigs

14,665

Number of contigs

368 h-tgs

NA

Contig length (Mb)

174

NA

NA

2,065

Number of scaffolds

964

935

2,170

103

N50 (kb)

500

490

591

486

Total length of genome deciphered (Mb)

37,207

29,675

36,687

42,414 (Cost.v3)

Number of coding genes

C:95.4% F:1.1% M:3.5%

C:95.0% F:1.6% M:3.4%

C:94.0% F:2.0% M:4.0%

C:95.8% F:1.5% M:2.7%

BUSCO

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Table 4.17 Latest assembled genome in grape Sequencing platform

Insert size (bp)

Read length (bp)

Number of sequences (million)

Number of bases (billion)

Sequence depth/ Coverage (X)

Application

PacBio

NA

7054

8.3

59

118X

Genome assembly

10X Chromium

400

2 × 150

350

52

107X

Genome scaffolding and phasing

Illumina

400 (pair end)

2 × 100

331

33

66X

6,000 (mate pair)

2 × 100

200

20

40X

Genome survey and genomic base correction

164

331X

Cumulative

more BAC end sequences to improve the scaffolding of the sequence contigs. A new chromosome assembly was also developed, based on an improved version of the maps used for the 8X genome version and named as 12X.v0 version (Cipriani et al. 2011; Canaguier et al. 2017). The chromosome sequence scaffolding of this version still needed improvement of about 9% of the sequence which was not anchored to chromosomes. The National Center for Biotechnology Information (NCBI) Refseq (27,043 putative genes) was produced using Gnomon- NCBI eukaryotic gene prediction tool (Souvorov et al. 2010). The latest annotation in grapes include the stilbene synthase, terpene synthase and MYB family (Parage et al. 2012; Wong et al. 2016). Earlier Cramer (2010) identified a transcription factor NF-YA TF (1613912_at) which is responsive to ABA and also reported its regulatory miRNA (miR169) in grapes. Zhang et al. (2012a, b) reported a total of two TIFY, four ZML, two PPD and 11 JAZ genes in the V. vinifera genome and found many of these genes are responsive to JA and ABA. The improvements in existing annotations can be performed at the Vitis site in ORCAE (http://bioinformatics.psb.ugent.be/orcae/) (Table 4.18).

4.11 Recent Concepts and Strategies Developed 4.11.1 Gene Editing Targeted genome editing (TGE) using site-specific nucleases (SSNs) is of particular importance for the introduction or modification of specific traits in perennial fruit crops like grapevine. However, except the targeted trait, other general characteristics of the selected genotype remains unchanged. The genome edited product can

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Table 4.18 Genome annotation information in grapes Reference genome version

Annotation version

Responsible institution No. of predicted genes/gene models and software used

8x

Genoscope 8x

Genoscope, France

30,434, GAZE

12Xv0

Genoscope 12X

Genoscope, France

–

12Xv1

CRIBI v1

CRIBI, Italy

29,971, JIGSAW

CRIBI v2

CRIBI, Italy

35,565, ORCAE annotation

RefSeq annotation

NCBI, USA

27,043, Gnomon–NCBI eukaryotic gene prediction tool

VCost

VIB, Belgium, COST action FA1106, EU

33,568, Eugene

VCost. v3

IGGP, International

42,414, homemade python script (free source code available at https:// github.com/timflutre/Vit isOmics/blob/master/src/ transferAnnot_from_ Vitis_12X_V0_to_V2.pl)

12Xv2

have higher consumer acceptance, as the final product is free from the transgene. These genome editing tools, comprising zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeats /CRISPR-associated protein 9 system (CRISPR/Cas9), introduce targeted DNA double-strand breaks (DSBs) and subsequently trigger DNA repair pathways involving either non-homologous end-joining (NHEJ) or homologous recombination (HR). Malnoy et al. (2016) adopted next-generation plasmidindependent purified CRISPR/Cas9 ribonucleoproteins (RNPs) for direct delivery to the protoplast of grape cultivar Chardonnay for efficient targeted mutagenesis. They targeted to increase powdery mildew resistance in grape cultivar by editing MLO7, a susceptible gene. This paves the way to the generation of DNA-free genome editing in grapevine. Ren et al. (2016) used the CRISPR/Cas9 system to successfully generate TGE for L-idonate dehydrogenase (IdnDH) gene (involved in tartaric acid; TA biosynthesis) in ‘Chardonnay’ grape suspension cells. Knocking out of this gene resulted in failure of TA biosynthesis with no off-target mutations detection, which suggests a specificity of the CRISPR/Cas9 system in grape. Very recently, Wang et al. (2018a, b, c) demonstrated an efficient genome editing of Thompson Seedless grape suspension cells using the type II CRISPR/Cas9 system. Four guide RNAs were designed to target the VvWRKY52 transcription factor gene for using with the CRISPR/Cas9 system via Agrobacterium-mediated transformation. Knockout of VvWRKY52 in Thompson Seedless grape enhances resistance to Botrytis cinerea with no off target modifications.

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4.11.2 Nanotechnology Nanotechnology is being widely used in horticulture for various purposes amongst which its application for imparting abiotic stress tolerance is gaining its momentum. Nanostructured material and nanoparticles (NPs) have become fundamental components of nanotechnology and/or nanoscience in recent years due to their unique physic-chemical properties that mostly include plasmonic, magnetic, electronic, sizedependent optical, catalytic properties and potential applications (Zahedi et al. 2020). NPs, also termed as nanoscale particles (NSPs), are small molecular aggregates having dimensions ranging from 1 and 100 nm (Singh and Husen 2019). Various reports show that effects of NPs on growth and development are concentrationspecific. NPs lead to enhancement of activity of various antioxidant enzymes under oxidative stress (Laware and Raskar 2014; Iqbal et al. 2020). In fact, nanostructures possess high stability, tunable compositions, a high surface area, particular biological behaviours, and a wide range of physical multi-functionality (Hasan 2015; Zahedi et al. 2020). Application of nanotechnology in grapevine for abiotic stress tolerance has been attempted recently. Iqbal et al. (2020) used the regular silicon (Si) and Si nanoparticles (SiNPs) to mitigate hypoxia stress in muscadine grape (Muscadinia rotundifolia Michx.) plants. They observed that hypoxia stress reduced muscadine plants’ growth through reduction in root and shoot dry biomass production. However, application of both Si and SiNP at the root zone effectively mitigated oxidative and osmotic cell damage. Amongst Si and SiNP, the later one was observed to have better efficiency as it improved the antioxidant enzymes activities and accumulation of organic osmolytes like glycinebetaine and proline. Carbon quantum dot (CQDs), as a new generation of Quantum dot (QD) NPs, having size less than 10 nm with round shape. QCDs has low or no toxicity with higher water solubility and biodegradability, thus creating wider usage potential (Bai et al. 2019; Lim et al. 2015). Recently, Gohariet al. (2021) advocated that a combined nanostructure Put-CQD NPs (putrescine; Put and carbon quantum dots; CQDs) can be effectively used for priming treatment on grapevine (Vitis vinifera cv. ‘Sultana’) to improve plant performance under salinity stress conditions. Although there was significant changes in various parameters, showing the negative impact of salinity stress; application of Put-CQD NPs (10 mg L−1 ), alleviated these impacts by modifying the physico-biochemical changes. Various in vitro studies also showed the effectiveness of nanotechnology to ameliorate the impact of abiotic stresses. Mozafari et al. (2018) and Ghadakchi et al. (2019) reported that the application of nanoparticles of iron (0, 0.08, and 0.8 ppm), and potassium silicate (0, 1, 2 mM) significantly addressed the negative effects of salinity stress.

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Table 4.19 Attempts made on Genetic engineering for manipulation of abiotic stress tolerance related traits Desirable traits

Focus area

Examples of current and potential target genes

Water stress tolerance

Aquaporins; isolation of root-specific promoters

TIPs (tonoplast integral proteins); PIPs (plasma membrane integral proteins)

Oxidative damage

Carotenoid biosynthesis, anaerobiosis

Carotenoid biosynthetic genes; SODs (cystosolic and/ or chloroplast-residing CuZnSOD, mitochondrial- residing MnSOD)

Osmotic stress and other abiotic stresses

Proline accumulation; polyamines and their role in stress tolerance

Vvp5cs; Vvoat; FeSOD, glycine betaine, antifreeze genes from Antarctic fish (freezing tolerance)

4.12 Brief on Genetic Engineering for Resistance Traits 4.12.1 Target Traits and Alien Genes Targets for the genetic improvement of grapevine integrally linked to the prospect of rendering genetically improved grapevines is the use of molecular biology to study the fundamental processes in grapevines that underpin the physiological responses targeted for improvement (Table 4.19). Grapevine transformations are co-entering a new era as a growing list of genes and their regulatory sequences are becoming available from economically important species. Biotechnology can be used to improve or to introduce new varieties with combination of traits such as drought tolerance, diseases and pests’ resistance, improve phenotype and high yield from one genotype to another through gene transformation (Gascuel et al. 2017; Hvarleva et al. 2009a, b).

4.12.2 Genetic Engineering for Abiotic Stress Tolerance It was during 1989, when the first successful grapevine transformations were performed. Gradually the focus has shifted from the development or refinement of the transformation technology/protocol towards the implementation of this technology in the generation of useful plant lines (Table 4.20). A number of grape varieties have been used for the genetic engineering studies to address several problems in grape production (Costantini et al. 2007). Transgenic grape cultivars Thompson Seedless, Silcora (Mezzetti et al. 2002), Chancellor and Koshusanjaku (Perl and Eshdat 1998) have been developed through genetic engineering. Transgenic approach is a widely used to introduce genes from distant gene pools, ranging from prokaryotic organisms such as E. coli to halophytes or glycophytes, into many plant species for the

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Table 4.20 Genes, mechanisms, and genetically modified fruit plant species implicated in plant responses to many abiotic stresses Genotype

Traits of interest

Exogenous genes

References

V. vinifera ‘Cabernet Franc’

Cold resistance

SOD from Arabidopsis

Rojas et al. (1996)

V. vinifera

Cold

DREB1b (a cold inducible transcription factor)

Jin et al. (2009)

V. vinifera

Cold

VvCBF4 C-repeat binding factor gene

Tillet et al. (2012)

V. vinifera

Enhanced rooting in rootstocks

rol B

Geier et al. (2008)

development of stress tolerant plants (Borsani et al. 2003). However, the consumer acceptance of the transgenic plants remains one of the major challenges.

4.12.3 Organelle Transformation Apart from the nucleus of plant cells, chloroplast and mitochondria also contain the DNA (Table 4.21). Organelle transformation has emerged as an attractive platform because of its superior performance over the conventional for the generation of transgenic plants. One of the important advantages of using organelle transformation is the absence of transgene flow, due to maternally inheritance of the mitochondria or chloroplast. The absence of epigenetic effects and higher precision in transgene insertion also adds to its advantages. These may leads to the higher acceptance of organelle transformation over the transgenic over time. Plastid genomes have been genetically engineered to improve crop yield, nutritional quality, and resistance to abiotic and biotic stresses, as well as for recombinant protein production and industrial enzymes (Jin and Daniell, 2015; Wang et al. 2019). After the first attempt in Chlamydomonas reinhardtii (Boynton et al. 1988), organelle transformation was also attempted in the higher plant Nicotiana tabacum (Svab et al. 1990). Since then, plastid transformation has been attempted to over 20 species of flowering plants (Li et al. 2021a, b). However, up to our knowledge, there are no reports on the use of these technologies to target the plastid genome in grape vine; but it has immense potential.

4.12.4 Biosynthesis and Biotransformation Genetic modification of various biosynthetic pathways through transformation to produce desirable natural products has been one of approach in plant biotechnology.

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Table 4.21 Organelle genomes in higher plants Plastid genome

Mitochondrial genome

Origin

Cyanobacterium (Zimorski et al. 2014) α-proteobacterium (Zimorski et al. 2014)

Size

107–218 kb (Daniell et al. 2016)

Copy number 1000–50,000 per cell (1000–1700 in Arabidopsis leaves and more than 50,000 in wheat leaves) (Bendich 1987) Structure

208 kb–11.3 Mb (Gualberto and Newton 2017) The copy number of mitochondrial genomes is highly variable but is lower than that of chloroplast DNA (Preuten et al. 2010)

A tetrapartite genome organization The mitochondrial genome is with an LSC and an SSC separating multipartite and can evolve rapidly in two IRs (Bock 2015); a small fraction structure (Johnston 2019) are organized in a genome-sized circle, whereas a majority are in linear or complex branched forms (Oldenburg and Bendich 2004)

Gene number About 130 genes involved in photosynthesis and in the genetic system (Daniell et al. 2016)

Approximately 60 genes involved in cell respiration and in the genetic system (Small et al. 2020)

RNA editing

300–600 (C-to-U) sites (Ichinose and Sugita 2017)

20–60 (C-to-U) sites (Ichinose and Sugita 2017)

Adopted from Li et al. (2021a, b)

In grapes, biosynthetic pathways like flavonoid biosynthesis can be manipulated to enhance the production of secondary metabolite including flavoring or coloring compounds. This can be done by either overexpression of biosynthetic genes or through introduction of foreign genes. Kiselev et al. (2012) isolated the full cDNA sequence of the CDPK3a gene of V. amurensis and investigated its organ-specific expression profile. Apart from this, the responses to plant hormones, its effect on temperature stress and exogenous NaCl stress was also estimated. VaCDPK3a was over expressed and the effect of overexpression on biomass accumulation and production of resveratrol in grape cells was investigated for understanding the natural biosynthesis of the secondary metabolite in grapevine. Recent study by Pardo et al. (2015) on the expression of the Ocimum basilicum (sweet basil) geraniol synthase (GES) gene in a Saccharomyces cerevisiae wine strain substantially changed the terpene profile of wine produced from a non-aromatic grape variety.

4.12.5 Metabolic Engineering Pathways and Gene Discovery Glucosides and disaccharide glycosides contribute to major aromatic compounds in grape berries. Being non-volatile nature, glycosides do not take part directly

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in wine aroma. In grapes few attempts have been made in metabolic engineering filed. Moscato Bianco berries express different open reading frames for the VvDXS haplogroups 284 N and 284 K, and VvDXS polymorphisms could contribute to the metabolic engineering of terpenoid levels in the grapevine. Methyl anthranilate (MANT) is a widely used compound to give grape scent and flavor. Recently Luo et al. (2019) report the direct fermentative production of MANT from glucose by metabolically engineered E. coli and Corynebacterium glutamicum strains harboring a synthetic plant derived metabolic pathway to replace the conventional petroleum-based synthesis of MANT in grapes. Resveratrol a potential anti-oxidant found in grape berries, belongs to the phenolic compound is widely used in pharmaceutics and nutraceutics field. However, its content is very low in the berries. Recently, metabolic engineering has been attempted for alternate production of resveratrol. Park et al. (2021) developed a resveratrolover-producing E. coli strain was using different strategies. First, they enhanced the biosynthesis of p-coumaric acid, which acts as the precursor of resveratrol. In the second strategy, various genes associated in resveratrol biosynthesis, including tyrosine ammonia lyase, 4-coumaroyl CoA ligase, and stilbene synthase, were cloned and the best combination was used to increase the resveratrol production.

4.12.6 Gene Stacking Gene introgression through traditional breeding is highly time-consuming owing to the log juvenile phase on the grape vine. However, molecular tools like MAS and genetic engineering can be used for identification of various genomic regions which carry the genes of interest. MAS can be used along with the traditional breeding or new biotechnological tools like cisgenesis for pyramiding of various gene of interest from wild grapevines or pre-breeding materials into elite cultivated grapevines. Development of genotype having multiple resistance/tolerance to multiple biotic and abiotic stresses is one of the major breeding objective in grapevine improvement. Gene stacking for biotic stress tolerance have been attempted (Table 4.22) in grapevine but for abiotic stress tolerance is yet to take pace.

4.12.7 Gene Silencing RNA silencing is a natural defence mechanism against viruses in plants, and transgenes expressing viral RNA-derived sequences were previously shown to confer silencing-based enhanced resistance against the cognate virus in several species. Using virus-induced gene silencing, Zhang et al. (2019) knocked down the VvMYBA1 gene (involved in the flavonoid synthesis) in ‘Red Globe’ and ‘JiZaoMi’ grape berries. This resulted in decrease in the anthocyanin biosynthesis. Torres et al. (2014) used the gene silencing to obtain fanleaf virus resistance. Several successful attempts

LG09 LG13 SC8–0071–014 LG14

PM, PCD of penetrated cell

PM Susceptibility PM, PCD of penetrated cell PM, PCD of penetrated cell

M. rotundifolia × Magnolia Chenin Blanc × JB81–107–11 Trayshed × M. rotundifolia

V. rupestris × Chardonnay

V. vinifera Kishmish vatkana, Karadzhandal

V. cinerea Illinois 547–1

RUN2 alleles

Sen1

REN1

REN2

LG18 loci Overlap with rpv3 marker VMC7F2

LG12 Linkage with Rpv1

Chromosome

Powdery mildew (PM), Downey mildew (DM), programmed cell death (PCD) of penetrated cell

M. rotundifolia Thomas Cabernet Sauvignon × VRH3082-1–42

RUN1

Resistance mechanism

Source of resistance

R-locus

Table 4.22 Major resistance loci in grapevine species conferring resistance to P. viticola, E. necator, and other pests

Feechan et al. (2015)

Zendler et al. (2017)

Barba et al. (2014)

Feechan et al. (2015)

Feechan et al. (2013)

References

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have been made for application of gene silencing to achieve biotic stress resistance but only meagre information is available for abiotic stress tolerance. Romon et al. (2013) reported low temperature resistant in grapevine through RNA silencing. Gene silencing can also be used effectively for functional analysis of the targeted gene (Urso et al. 2013).

4.12.8 Prospects of Cisgenics Cisgenic (Schouten et al. 2006) plants are “those plants that have been genetically modified by one or more genes isolated from same species or from a sexually compatible one”. By definition, “cisgenic plants do not contain any foreign genes i.e., selection marker gene and they appear to be much closer to conventionally bred plants. In contrast to transgene (combinations of a coding gene with a regulatory sequence such as a promoter from another gene), a cisgene contains its native introns and is flanked by its native promoter and terminator in sense orientation” (Schouten et al. 2006). There is increase in the consumer acceptance as the final cisgenic product is free from selectable marker gene and lack any foreign gene (transgene) (Vanblaere et al. 2011). Jayasankar et al. (2000) islolated the Vitis vinifera thaumatin-like protein (vvtl-1) gene from grape cultivar “Chardonnay” and reengineered it for constitutive expression to produce fungal-disease resistant cisgenics grape. Key transcription factors from pigment biosynthetic pathways of other plant species can also be potentially used as reporter genes to replace existing systems that are transgenic in origin. Dhekney et al. (2011) engineered “Thompson Seedless”, with a vvtl-1 cisgene to constitutively express VVTL-1 protein. Screening at the greenhouse and field level resulted in the identification of two plant lines showing powdery mildew and black rot resistance. Recently, the utility of a Vitis vinifera MybA1 gene as a plant-derived reporter was reported for use in grapevine transformation (Li et al. 2011).

4.13 Brief Account on Role of Bioinformatics as a Tool 4.13.1 Gene and Genome Databases The various gene and genome databases have been built to facilitate genomics research in crop species of interest. Likewise, grape gene and genome databases have been created to retrieve the information submitted related to the gene and genome of Vitis Vinifera. PlantGDB (https://www.plantgdb.org/) is a database based on xGDB (Chlueter et al. 2006) platform and provides high-quality spliced alignments of available alignments transcript and predicted proteins from concerned model plant species. This database also contained information related to genome survey sequence

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and microarray probes, mainly depending on the crop species. In this database, the genome browser is allocated the name of Genus/species abbreviation of crop and for the grape (Vitis Vinifera) is VvGDB. The Vitis vinifera Genome DB—PlantGDB (VvGDB179) contained the information of chromosome-based genome which encompassing the constituting primary sequence data from Vitis vinifera, genoscope 12×. This database also provides information about the interpretation of comprehensive data based on the spliced alignment results. Vitis_vinifera—Ensembl Genomes 51—Ensembl Plants (https://plants.ensembl.org) in another database having the gene and genome database for the grape. This database is based on 12× whole genome shotgun sequence assembly and V1 annotation of the grape genome. A FrenchItalian Public Consortium created this databse for Grapevine Genome Characterisation under the International Grape Genome Program. National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) contains information about the gene and genome data of grapevine, including the proteins expressed sequences. Further, grapegenomics.com a web portal developed for genomic data of grape and analysis tools for wild and cultivated grapevines.

4.13.2 Gene Expression Databases The many gene expression databases have been developed to facilitate the functional genomics research in grape. In this connnctions, VitisExpDB (http://cropdisease. ars.usda.gov/vitis_at/main-page.htm) is a functional genomics database based on MySQL-PHP, contained the information related to gene expression data and annotated ESTs for both V. vinifera and non-vinifera grape geneotypes/ species. The information related to the percent nucleotide identities, common primers and phylogenetic relationship could be retrieved through this database. Further, this database has the information on high density 60 mer gene expression chips which consist of about 20,000 non-redundant set of ESTs. VitisExpDB contained the information of 14 global microarray expression profile sets of which 12 were mapped on metabolite pathways. This database has user-friendly web interface including multiple search indices and in addition to that users can also submit their generated ESTs to this database (Doddapaneni et al. 2008). DGTF (http://www.yaolab.sh.cn/dgtf.html) is another database for grape transcription factors which is collecting and annotating the grape transcription factors. This data base contained about 1423 putative grape transcription factors in 57 families and these transcripon factors were detected from the wine grape proteins from the grape genome sequencing projects (through domain search). In addition to that this database also provides the information of annotations for individual members of each transcription factor family. This database has cross- linked with the other public databases which makes its annotation more extensive (Cai et al. 2008). VTCdb (http://vtcdb.adelaide.edu.au/Home.aspx) is a gene co-expression database for the grapevine. This database provides the online platform for transcriptional regulatory inference in the grape (Vitis vinifera). This database enables to the profile of about 29,000 genes while utilizing the microarray

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datasets from various experimental sets using Affymetrix Vitis vinifera GeneChip and NimbleGen Grape Whole-genome microarray chip. The utility of this database is also demonstrated by explaining three biological processes viz., photosynthesis, berry development and flavonoid biosynthesis; and recovered sub-networks identified gene functions and novel associations (Wong et al. 2013). Recently Wang et al. (2020) built Grape-RNA (http://www.grapeworld.cn/gt/index.html) for collection, evaluation and information sharing of grape RNA-seq. This database contains 485 RNA-seq from in-house datasets, while 1529 RNA-seq and 112 microRNA samples are from the public platform. This database provides information to users about the sample, cleaned raw data, expression levels, unigenes, and useful tools, thus could contribute immensely to functional genomics studies in the grape.

4.13.3 Protein Databases The various primary, secondary, composite and structural protein databases have been created to collect, evaluate, and characterise protein data sets, facilitate proteinbased studies, and understand the role of proteins in various biological functions. The UniProt, Universal Protein Resource accumulated by the UniProt Consortium and this was developed by the European Bioinformatics Institute (EBI), Protein Information Resource (PIR) and Swiss Institute of Bioinformatics (SIB). The UniProt Knowledgebase (UniProtKB) is one of the important protein databases bulked with the functional information of proteins having high accuracy, consistent and rich annotation. The UniProtKB had two sections, one is automatically annotated (UniProtKB/TrEMBL) and another is manually (UniProtKB/Swiss-Prot). These two sections of UniProtKB giving access the information about publically available protein datasets. In UniProtKB database, UniProtKB-Q9LLB7 (Q9LLB7_VITVI) dealt with the protein datasets for the grapevine. The UniProt database has automatically updated the information related to protein datasets at every eight weeks intervals. Similarly, NCBI contained the information about the grape protein datasets and access the information to the users.

4.14 Brief Account on Social, Political and Regulatory Issues 4.14.1 Concerns and Compliances There is a serious concern amongst the consumers vis-à-vis genetically modified (GM) foods. There are many FAQs pertaining to such concerns. For instance, how safe are the GM foods for consumption? Will not GM plant production along with intensive use of herbicides affect biodiversity, wildlife and non-target species? Will

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not gene flow put the environment in jeopardy? Will not genetically modified organisms (GMOs) outcross to create ‘super-weeds’ in the population? Do GMOs have any negative impact on the environment? Besides, concerns regarding the uncertain expression of DNA/RNA carried along the GM food in the human body had also been a cause of worries amongst the consumers. GM food had drawn fierce polemic from various quarters from time to time which had influenced governments/ policy makers, growers, biotechnology firms (involved in GM crop seed production) and consumers. In view of this intricate dispute, some evident factors have been identified accountable for developing consumers’ outlook towards the acceptance/rejection of GM food such as consumer opinion about the associated risks and benefits, awareness about the fundamental concepts of GM technology, basis of information of GM foods, concerns regarding accuracy of the GM technology, individual preferences, prevailing regulatory laws and policies of the land and the role of the non-governmental organizations, public interest groups and media towards perception-building around GM foods (Masehela et al. 2020). There are no distinct research findings, which suggest the deleterious impacts of GM food to human health. Therefore, it seems that the estrangement from GM foods is more or less preventive in nature. In response to the rising public distress about GM foods, the governments across the globe have adopted different approaches to confront this burning issue. This has led to the development of GMO regulations, which often tend to be country or region specific (Akumo et al. 2013). The agencies viz., the Food and Drug Administration (FDA), the Environmental Protection Agency (EPA), and the United States Department of Agriculture (USDA) have regulatory authority over biotechnology products in the United States. These regulatory agencies supervise a broad gamut of products, including GMOs, and go along with risk-based evaluations to safeguard human as well as environmental health. While countries like USA has a product-oriented tactics for approving a transgenic variety provided it has a considerable likeness with a conventional crop, in the European Union the present legislation is process- or technique-oriented with stress on the preventative norms. Countries such as Argentina, Japan, Canada and India have GMO regulations in place comparable to those of USA, while countries like New Zealand, Australia and China control GM crops with various degrees of curbs. To date, USA is a non-party to the Cartagena Protocol on Biosafety (an international agreement to ensure the safe handling, transport and use of living modified organism generated by modern biotechnology) as it has not yet ratified. The present EU legal regulation (https://ec.europa.eu/food/plant/gmo/legislation_en) demands distinct labelling of GMOs launched in the market; the labelling requirements do not apply to GM foods in a percentage no higher than 0.9% of the food ingredients taken into account individually and if this presence is adventitious or technically inevitable. The International Treaty on Plant Genetic Resources for Food and Agriculture (also termed as ITPGRFA, International Seed Treaty or Plant Treaty) is a detailed international agreement in congruence with the Convention on Biological Diversity (CBD), with the objective to ensure food security through the conservation and access to GRs for their sustainable use for the purpose of food or breeding, the fair

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and equitable benefit sharing derived from their use amongst the recipients, as well as acknowledging farmers’ rights. The Compliance Procedures laid down under the ITPGRFA are aimed to encourage compliance with all the requirements of the Treaty and to take-up issues of non-compliance, which encompasses monitoring, providing advice or needed assistance, including legal support, particularly for developing countries. To enable timely reporting by Contracting Parties and monitoring of the execution of the Treaty, the Governing Body approved a voluntary Standard Reporting Format and asked the Secretary to place online the Online Reporting System to update the reporting process through digital channels. International Organization of Vine and Wine, Paris, an intergovernmental organization which deals with scientific and technical facets of viticulture and wine making, has adopted few resolutions pertaining to GM grapes such as Resolution viti 1/97: Transgenic Vines (genetic transformation of the vines more particularly in the area of disease and ravage management), Resolution Viti 1/2006: Vine Genome and Genetically Modified Varieties (assessment of risks and monitoring of GMOs) and Resolution Viti 355/2009: OIV Protocol for The Evaluation Of Grapevines Obtained by Genetic Transformation (OIV 2015).

4.14.2 Patent and IPR Issues The main economic apprehension about GM crops is the possibility of patent enforcement, which may indulge farmers to be on the mercy of big bioengineering firms for strains when their crops are cross-pollinated. Consumer rights activists also have reservation over GMOs as such crops are likely to enhance the price of seeds so high, which small farmers may not afford. GM crops need seed replacement every season as their viability lasts for only one growing season and would produce sterile seeds. As a consequence, farmers are ought to be dependent on the giant agri-biotech firms with patent rights. Further, deliberately selected GM genes flowed through outcrossing (deliberate/accidental to another commercial field) enables the patent holder to possess the right to control the use of those crops. However, it is worthwhile to note that in grapes interbreeding is not easy. Fortunately, grapevines are vegetatively propagated (cuttings and/or cuttings grafted onto rootstocks). Furthermore, their flowers are functionally bisexual and chiefly self-pollinated, thereby largely avoiding the cross-pollination (Berrie 2011).

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4.14.3 Disclosure of Sources of GRs, Access and Benefit Sharing ABS legalizes exchange of plant genetic material between a rightful possessor and an ensuing user of GRs. CBD had set down the extensive codes, which countries are ought to obey while putting request and permitting each other access to their GRs. Further, benefits which may come up as a result of the use of such GRs are to be apportioned in an unbiased and impartial way with the country granting access. GRs are governed by the regulations on access and benefit-sharing put in place by the treaties like The CBD, The International Treaty on Genetic Resources for Food and Agriculture (ITPGRFA) and The Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the CBD (Nagoya Protocol) of the FAO. The domestic regulatory framework on access and benefit sharing is at present in place in India through Biological Diversity Act, 2002 (Anonymous 2012).

4.14.4 Farmers’ Rights Fundamentally, Farmers’ Rights are concerned with empowering farmers to conserve, develop and make use of GRs and traditional knowledge (TK) and distinguishing and rewarding them for their contribution. The vital contributions made by the farmers, towards biodiversity conservation, have been overtly acknowledged by the CBD and the ITPGR. The General Association for Wine Production and the French Federation of Grapevine Nurseries have signed an agreement endorsing “shared effort” (50% to each) between nurseries and wine-producers in royalty payment. The 2004 parafiscal tax on grapevines has now been replaced by this new measure and consequently the brand-holders now assert that it will assist research, particularly the development of applications for the 2007 sequencing of the Vitis genome (Anonymous 2011).

4.14.5 Traditional Knowledge Traditional Knowledge (TK) and GRs have aided immensely to technological progression of the society and for the betterment of livelihood standpoints. Both GRs and associated TK are prone to unethical practices such as ‘biopiracy’ (Sanghera et al. 2015). A number of international legal framework have been devised in the form of treaties, conventions, agreements, etc., in order to encourage access to plant genetic resources and to protect TK. Every country has their ownrich biodiversity and associated TK. Therefore, enactment of the ABS provisions of CBD is of unique significance. The aim of Nagoya Protocol is the fair and equitable sharing of benefits

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as a result of use of GRs. The ABS Protocol is supposed to address the apprehensions of biodiversity-rich countries such as India vis-à-vis embezzlement of GRs and associated TK, which ultimately leads to a more effective functioning of CBD (Anonymous 2012).

4.14.6 Treaties and Conventions Two of the most important international treaties, which address issues pertaining GMOs are the Cartagena Protocol of 2000 and the Nagoya-Kuala Lumpur Supplementary Protocol of 2010. Both the treaties are appended to the Convention on Biological Diversity (CBD) of 1993. However, it may be noted that they apply only to trans-boundary actions and do not apply to use or transit of GMOs within the territorial boundaries of the countries. The foremost international mechanism for regulation on GMOs is ascribed to the Cartagena Protocol on Biosafety to the CBD. It is aimed to safeguard both biological diversity and human life from any adversarial impact of GMOs. The other instrument, i.e. Nagoya-Kuala Lumpur Supplementary Protocol following years of negotiations over the question of liability for GMO-produced damages, on October 15, 2010, the Nagoya-Kuala Lumpur Supplementary Protocol on Liability and Redress to the Cartagena Protocol on Biosafety (the Supplementary Protocol) imparts international rules and procedure on liability and reparation for damage to biodiversity owing to living modified organisms (Cooper 2002).

4.14.7 Participatory Breeding Participatory breeding is the breeding method involving farmers’ participation to varying degrees; however, the control of the programme rests with the plant breeders. It entails farmers to select advanced breeding material as per their needs on farm. The information pertaining to varieties, which local inhabitants acquire, refine, maintain, and exchange may facilitate the breeder in finalizing breeding priorities and in early decision-making. The significance of indigenous knowledge in breeding of crop plants was not completely realized until recent past; however, this shortcoming was addressed under Article 8 of the CBD, which has provisions to preserve and retain traditional knowledge, indigenous innovations and practices of local inhabitants representing conventional lifestyles relevant for their conservation and sustainable use of biodiversity (Hasan and Abdullah 2015).

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4.15 Future Perspectives 4.15.1 Potential for Expansion of Productivity Grape is the third most important fruit crop in the world. India is having productivity of 21.02 MT/ha, and the states having higher productivity includes Punjab (28.67 MT/Ha), Tamil Nadu (27.27 MT/Ha), Maharastra (21.67 MT/Ha), Andhra Pradesh (20.00 MT/Ha), Karnataka (19.70 MT/Ha). However, the states like Haryana (2.30 MT/Ha), Himachal Pradesh (1.59 MT/Ha), Jammu & Kashmir (2.69 MT/Ha), Mizoram (7.35 MT/Ha), Nagaland (2.43 MT/Ha) etc. are well below the national average and are dragging down the national productivity (NHB 2017–18). The north Indian grape maturity coincides with the monsoon leading to severe crop failure, which necessitates the development of early maturing grapes for this region. In a similar way to expand the grape productivity, there is an urgent need to design the genotypes suitable for various regions. However, the productivity in world is 10.91 T/ha, and the countries having higher productivity are India (21.23 T/ha), China (17.60 T/ha), USA (17.31 T/ha), Italy (12.28 T/ha), Chile (12.18 T/ha), while the lower group includes Spain (6.45 T/ha), Argentina (7.85 T/ha), France (8.25 T/ha) (FAOSTAT website, 2018, http://faostat3.fao.org/home/E). There is a huge scope to increase the productivity in different countries.

4.15.2 Potential for Expansion into Non-traditional Areas Majority of the grape production comes from the major wine producing countries of the world. Grapes have been grown for thousands of years in various Mediterranean countries. Italy, France, and Spain are major grape and wine producing countries, while Turkey is the leading grape grower (Reisch et al. 2012). Grape cultivation can be expanded in the non-traditional countries where the production is meager or at the nascent stage like most of African countries, Bangladesh, Malaysia, Indonesia, Thailand, Vietnam, Panama, Coast Arica etc. In India, grape cultivation is now being promoted in the non-traditional states like north-east states more specifically Mizoram, Arunachal Pradesh, and Meghalaya; and West Bengal, Jharkhand, Odisha, Uttarakhand, Jammu & Kashmir and dry parts of Bihar. Apart from this it can also be promoted in the states like after identification of location-specific suitable genotypes, standardization of production technology etc.

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Chapter 5

Genomic Designing for Drought Tolerant Almond Varieties Pedro J. Martínez-García, Ossama Kodad, Hassouna Gouta, Sama Rahimi Devin, Angela S. Prudencio, Manuel Rubio, and Pedro Martínez-Gómez Abstract With the global climatic change, drought condition is one of the main limiting parameters for plant yield and growth around the world in agronomical production of species such as almond [Prunus dulcis (Miller) Webb]. To deal with the global drought condition, different strategies have been suggested, including development of new drought tolerant varieties development. Almond is an important fruit crop species cultivated worldwide for its appreciable kernel for both processed food industry and as a functional food with and medical (nutraceutical) properties including nutrients, vitamins, healthy blood lipids or anti-inflamatory and hypocholesterolemic properties. This fruit tree species, compared to other nut crops, is relatively drought resistant. In this context, climatic conditions, in particular climate variability and water availability, require the development of production systems able to cope with risk and uncertainty. Therefore, rusticity and flexibility of the different components of the production systems (including varieties) should be improved together with a suitable kernel quality including nutraceutical values of the newly designed drought resistant almond cultivars. The overall objective of this chapter is to benefit from previous research and investments (translating know-how, tools and protocols) to investigate the development of new cultivars for specific climatic and socioeconomic conditions, of a highly relevant crop—almond—with high nutraceutical properties. The plantation and management of almond orchards with these newly developed varieties has to be designed to last long periods of time, and therefore it is crucial to analyze the impact of climatic conditions, especially drought, when P. J. Martínez-García · A. S. Prudencio · M. Rubio · P. Martínez-Gómez (B) Departamento de Mejora Vegetal Grupo de Mejora Genética de Frutales, CEBAS-CSIC, Espinardo, Murcia, Spain e-mail: [email protected] O. Kodad Departement d’Arboriculture Et Viticulture, Ecole Nationale d’Agriculture de Mekne’s, Mekne, Maroc H. Gouta Institut de l’Olivier, Sfax, Tunisia S. R. Devin Department of Horticultural Science, College of Agriculture, Shiraz University, 7144165186 Shiraz, Iran © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 C. Kole (ed.), Genomic Designing for Abiotic Stress Resistant Fruit Crops, https://doi.org/10.1007/978-3-031-09875-8_5

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selecting new varieties in the designing of new sustainable agrosystems for the almond production of high quality in dry conditions. Keywords Prunus dulcis · Breeding · Phenology · Breeding · Drought resistance · Molecular markers · Genomics · Transcriptomics · Epigenetics

5.1 Introduction The cultivated almond [Prunus dulcis (Miller) D. A. Webb] is a tree crop species originated from Iran and the Middle East being the Mediterranean basin which are most important centers for diversification of the species (Zeinalabedini et al. 2010; Sorkheh et al. 2007). In the Iranian stage, almond species appear to have been brought into Greece prior to 300 BCE, eventually becoming introduced to all compatible areas of the Iranian basin, South of Europe and North of Africa. In the North of Africa, the almond tree dates back to ancient times, having been grown extensively since the Carthaginian era, eighth century BC. Being the granary of Rome during the Roman Empire, Tunisia was considered as one of the main trade routes along which almond was spread throughout the shores of the Iranian Sea reaching Spain. Finally, from Spain almonds were distributed to the rest of the world including North America, South America, South Africa and Australia (Gradziel and Martínez-Gómez 2013). The almond fruit is oval-shaped with a tip, and it is classified as a drupe with a pubescent skin (exocarp), a fleshy but thin hull (mesocarp), and a hardened shell (endocarp) that contains the seed (kernel), made up of the embryo and the testa. The fruit grows during development and the hull opens and dries at maturity. The mature endocarp hardness ranges from soft to hard depending on the genotype. Horticulturally, almonds are classified as a “nut” in which the edible seed (the kernel) is the commercial product (Gradziel and Martínez-Gómez 2013) (Fig. 5.1). The almond is an important fruit crop species cultivated worldwide for its appreciable kernel for the processed food industry and also as a functional food with both nutritional and medicinal (nutraceutical) properties including nutrients, vitamins, healthy blood lipids or anti-inflamatory and hypocholesterolemic properties (Kodad et al. 2006, 2008, 2021; Poonam et al. 2011; Musa-Velasco et al. 2016). These nutraceutical properties are a new important trait to be incorporated in the new almond breeding programs (Kodad 2017). The almond kernel (Fig. 5.1) is consumed either in the natural state or processed. Because of its good flavor, crunchy texture and good visual appeal, it has many important food uses. As an ingredient in many manufactured food products, kernels may be dry-roasted or roasted in oil followed by salting with various seasonings (Gradziel and Martínez-Gómez 2013). The processed product is mainly sold as either blanched or unblanched kernels. Blanching removes the pellicle (“skin”) using hot water or steam. Almond kernels can be sliced or diced for use in pastry, ice cream, breakfast cereals and vegetable mixtures. The kernels are also ground into a paste to be used in bakery products and in the production of marzipan. The flavor and texture

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Fig. 5.1 The almond production cycle. a Flowering and pollination. b Fruit development. c Fruit maturation (opened mesocarp). d Edible seeds (almonds)

of almonds can be intensified or moderated through proper selection of cultivar, origin, moisture content, and processing and handling methods (Kester and Gradziel 1996). Due to its great variability almond is nowadays cultivated in a wide variety of ecological niches. Almonds are cultivated in more than 50 countries, with a 95% of production located in California, Australia and the Mediterranean basin (http:// faostat.fao.org). In addition, production and surface have been increasing dring the last 20 years (Fig. 5.2). Regarding surface, according to the Food and Agriculture Organization of the United Nations (FAO), in 2020 there was 1,925,887 ha dedicated to almond cultivation in the world. Spain is the first country with 633,562 ha, representing 33% of the global total, followed by USA (21%), Tunisia (9.4%) and Morocco (9%). However, despite our large surface cultivated, Spain is still the third world producer due to the lower productivity of the Spanish orchards (100 kg/ha on average) compared to that of the Californian and Australian (2,500 kg/ha), due to cultivation techniques and irrigation. In this context, USA is the main producer with 2.37 million tonne (Mt) followed by Spain (0.46 mt), Australia (0.22 Mt) and Iran (0.16 Mt) (Fig. 5.2). In addition, with a value approaching USD 2.30 billion in 2016, almond has become the largest specialty crop export in the US and the largest agricultural

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Fig. 5.2 Evolution od the world almond production with shell during the period 2001–2020 and world almond production with shell in 2020 indicating main contry producers and geographical distribution

export ($4,532 M$) for California (Almond Board of California 2021; https://www. almonds.com/). California accounted for over 80% of global production of around 92,9864 tons per year with over 38,0405 ha in production. Spain, the second largest producer and has the largest area under cultivation, estimated at over 544,518 ha. Australia is now the third largest producer of almonds with 79,461 tons and 39,662 ha (29,358 ha bearing, 6,758 ha non-bearing and 3,546 ha new plantings). The remaining world production comes from about 20 countries including Tunisia, Morocco, Italy, Turkey, Chile and Iran. Limited almond production extends into the Balkan Peninsula including areas of Bulgaria, Romania, and Hungary. Additional plantings exist in central and southwestern Asia including Syria, Iraq, Israel, Ukraine, Tajikistan, Uzbekistan, Afghanistan and Pakistan, and extending into western China (Almond Board of Australia 2021; https://www.australianalmonds.com.au/). On the other hand, in the last few decades, the biggest challenge especially in agriculture sector that the world faces is the climate change and the global warming. Following climate change, drought has become likely the most important constraint limiting the productivity crop and finally food security worldwide. In some countries, the climate change has been so high that it can compensate for improvements in yield resulting from technology, fertilization and other factors through negative effects on plant growth and reproduction. Reduce precipitation and change of rainfall patterns causes repeated droughts around the world (Yordanov et al. 2000).

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In this context, almond cultivation in the Medidetterranean basis is mostly nonirrigated with kernel yields around 400–500 kg/ha, which is much lower than the 2,300 kg/ha of US, where the production is much more intensive and exclusively irrigated. These almond producing areas play an important role in social strengthening and retention of families in the area, thus contributing to reduce emigration. However, in spite of the higher oil quality and nutraceutical content found in the varieties grown in the Iranian area, this culture, is affected by the low rainfall and drought-limited production (Gouta et al. 2019, 2020, 2021; Kodad et al. 2021). At present, drought is one of the biggest problems for non-irrigated culture. Studies about drought resistance have been conducted, but these were restricted to the evaluation of their ability to access water (IPCC 2007). Previous studies conducted by the different research groups of this consortium in Iran and Spain allowed the preliminary identification of several drought-resistant varieties. However, an integrated approach of evaluating varieties/clones/biotypes which are more drought resistant but are also productive and giving good yield and nutraceutical quality has not been developed as far as we know. The objective of this Chapter is an assessment of the genomic designing for new drought resitant almond varieties employinh promising genetic, genomic, transcriptomic and epigenetic approaches. Our proposal od design is focussed on the selection and characterization of these drought resistant almond varieties including nutraceutical properties (lipid and fatty acid contents, vitamins, phytosterol content, minerals, protein and aminoacids, phenols and fibre). From the point of view of the plant material, the investigation will allow depicting the genetic and molecular basis of drought resistance and nutraceutical properties in almond.

5.2 Drought Resistance in Almond One of the greatest concerns regarding global agronomy is water scarcity. As a matter of fact, there are some water crisis issues that scientists claim have reached to a tipping point and if they are neglected more, our world will face a tragic ending. For example, Agriculture Services reported that in summer 2014 drought affected 72,829 hectares (18% of surface) in non-irrigated areas, only considering the Region of Murcia. The damage estimated by the agricultural organizations is around 123.5 million euros and more than 2 million trees had died. It is also worth noting that the droughts affecting the Iberian Peninsula are becoming more intense and longer, according to a 2013 study of the Spanish Agency CSIC, in which the evolution of water deficit in this region was analyzed between 1945 and 2005. This CSIC report reveals that in central and southern Spain duration of severe droughts in specific areas has increased from 15 months of continuous water deficit in the early years analyzed, up to 60 months for the driest period in the 90s. In addition, this situation is even more severe in Iran. In these areas of the Iranian basin, water has become a scarce resource due to high consumption and the high level of overall pollution.

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Additionally, the increasing vulnerability to inclement weather and to long periods of drought has been leading to depopulation of many rural areas. Therefore, the implementation of appropriate measures is absolutely necessary to protect these rural farms, their trade and income, as well as that of associations, to ensure their independence through collective management. Beyond the ethical imperative of doing research on how to increase the productivity, and strengthening the adaptation of plants to drought on a global basis, in the Iranian countries we are facing a very real challenge easily followed on the news published in local newspapers. In addition, climate change conditions accentuate drought since a reduction of 10% in precipitation translates in a reduction of 25% in the soil water. In almond, water availability is a major constraint in the cultivation of the species (Kodad et al. 2021). Under drought conditions, plants may find strategies to escape the stress (accelerating the life cycle) or to avoid it (controlling stomatal conductance, investing in the development of the root system, reducing canopy, etc.), or still to activate strategies of osmotic adjustment to increase tolerance to low tissue water potential (for instance accumulating compatible solutes). The efficiency of photosynthetic carbon gain relative to the rate of water loss can be used as indicator (Jiménez et al. 2013). In the seasonally dry and variable environment of the Iranian region, the ability of species like almond to cope with water scarcity is not only dependent on the variety, but also very dependent on the rootstock on which it is grafted. The characterization of drought resistance in almond cultivars and the rest of fruit crops is therefore linked to efficient use of water (Ennahli and Earl 2005; Yadollahi et al. 2011), together with the ability of the root system to access water. Collecting materials for future research and breeding are helpful tools to reduce drought losses, but this is a lengthy process (Neale et al. 2017).

5.3 Designing Drought Resistant Almond Varieties Drought stress as the major abiotic stress in many regions limits the fruitfulness of horticultural crops and agricultural development in arid and semi-arid regions (Wu et al. 2013). Because the irrigation system of many orchards is not optimized for the severe abiotic conditions, the water requirements for the plant growth will not be supplied at the critical stages of growth (Sun et al. 2020). It is approved that the ultimate aim in commercial goals is fruit yield; therefore, some basic strategies are taken into account to achieve it. This is more problematic in the final period of fruit growth and can lead to obtaining small fruit sizes which is not desired in the market. In this context, the development and expansion of new drought resistance cultivars in the arid areas is promising because arid lands possess a high potential to change the existing horticultural scenario. Low productivity existence in the arid areas can be enhanced by following improved varieties (Wu et al. 2013). Therefore, rusticity and flexibility of the different components of the production systems should be improved. In this sense, the plantation and management of almond orchards has to be designed to last long periods of time, and therefore it is crucial to

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analyze the impact of climatic conditions, especially drought, when selecting new varieties in the designing of new sustainable agrosystems for the almond production in the drough areas. The overall objective of the selection and characterization of drought resistant almond cultivars with high nutraceutical values is to benefit from previous research and investments (translating know-how, tools and protocols) to investigate the behavior under the specific climatic and socioeconomic conditions, of a highly relevant crop—almond—while expanding the research community to other Southern and Eastern Mediterranean countries. The development of the new resistance varieties represents a good opportunity to get possible and positive answers to growers and scientists in the future for a better management of this crop. Adaptation of agriculture to climate change calls for solutions to be found in terms of genetics and agronomics to cope with water scarcity, changes in temperatures and increase in climate variability. Several issues have to be addressed in this perspective including breeding of varieties resistant to drought and to climate hazards with high nutraceutical properties. In this context, almond breeding presents unique conditions for plant breeding such as its perennial woody character, its long juvenile period, and its propagation by vegetative clones. These conditions can make the genetic improvement processes long and tedious. Consequently, it is necessary to have access to the best information and technology possible for the design of new varieties within the typical twelve-year variety development cycle. The requirements for new varieties must be anticipated 12 years in advance, as this is the average time from the original cross to the release new pre-crop variety. A critical decision is the choice of the parents to be used. Subsequent crosses can be of the complementary type, (when we cross two varieties with complementary characteristics to obtain a new variety that integrates the good aptitudes of both varieties), or of the transgressive type, where two varieties are crossed with good aptitudes in order to obtain progeny performance even better than either parent (Martínez-Gómez et al. 2003).

5.3.1 Selection of Drought Resistant Almond Varieties and Evaluation in Natural Conditions Development of improved production systems using drought resistant almonds may be possible utilizing native germplasm. Plant material from arid areas allows a more sustainable production, particularly in the marginal areas of harsh climate conditions found around the Iranian basin (Gradziel et al. 2001; Sorkheh et al. 2009; Rasouli et al. 2010, 2014). This starting material has proven to be more efficient and resilient than wild species that, moreover, show poorer agronomical behavior (Sorkheh et al. 2009; Rasouli et al. 2014). However, an integrated approach of evaluating varieties/clones/biotypes which are more drought resistant but are also productive and giving good yield and nutraceutical quality has not been developed as far as we know.

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Different studies have led to the identification of few natural hybrids of almond and peach which are planted in arid areas without any irrigation and with a rainfall lower than 200 mm/year (Sorkheh et al. 2009; Rasouli et al. 2010, 2014). In addition, in the South East of Spain the group of CEBAS will perform a prospection of traditional varieties (previous results showed the drought tolerance of cultivars as “Atocha” or “Colorao” from the Cartagena area in Murcia) in non-irrigated conditions (MartínezGarcía et al. 2020). Palasciano et al. (2014) also described differences in drought tolerance in almond varieties grown in Apulia Region (Southeast Italy).

5.3.2 Drought Resistance Evaluation of Almond Varieties in Controlled Greenhouse Conditions Accurate phenotyping (characterization of the phenotype) is crucial before proceeding for nutraceutical or molecular studies. Moreover, accurate phenotyping can increase genetic gains obtained by breeding programs from a precision breeding approach. In this sense, a controlled and standardized protocol for drought resistance evaluation in greenhouse controlled conditions has been developed (Alarcón et al. 2002; Martínez-García et al. 2020). Usually, for the evaluation of drought tolerance iun almond cultivars, the genotypes to be evaluated will be grafted onto ‘GF677’ rootstocks grown in 5 L pots in controlled screen-house conditions (Alarcón et al. 2002). A destructive [leaf water potential (w)] and five non-destructive [net photosynthesis rate (PN)), stomatal conductance (GS), and transpiration rate (E), chlorophyll content (ChC) and maximal photochemical eficiency of photosystem II (Fv/Fm)] traits were conducted in this experiment and have been described in detail by Martínez-García et al. (2020). In field experiments, at least three measurements must be made per tree and leaf samples will be collected per each time point of analyses. Photosynthetic parameters, as well as leaf water potential will be measured (using a mini-PAM and a pressure chamber—Soil Moisture Equipment Corp., model 3000). Water status and water stress will be monitored measuring the stem water potential and gas exchanges of all trees selected (Ennahli and Earl 2005). On the other hand, extensive studies suggest that plant strategies to reduce drought effects include those that enable plants to avoid and tolerate low water potentials. In the avoid strategy, water loss and water uptake maintain balanced and preserved the plant water status by tapping ground water with deep roots, stomatal closure and small leaves. With the onset of drought, plants tolerate water stress through osmotic or elastic adjustment or the accumulation of osmoprotective substances such as cyclitols (Pirasteh-Anosheh et al. 2016; Roychoudhury et al. 2013). Therefore, activation of these processes enables maintenance of cellular homeostasis through lipid and carbohydrate metabolism.

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5.4 Marker-Assisted Breeding for Drought Resistant Development and application of molecular (DNA, RNA and epigenetic) markers could affect feasibility, efficiency and viability of these breeding programs. For this reason, almond breeding programs are incorporating these new molecular technologies.

5.4.1 Development and Application of DNA Markers The use of almond populations segregating for the characters of interest has been the principal approach for the development of marker assisted selection (MAS) strategies in almond (Kole et al. 2015). MAS would allow the identification of DNA regions (Fig. 5.3) linked to a traits and the early selection of a large number of plants, which would then have to undergo a final evaluation to certify the desirable traits using traditional methods. However, at this moment due to the complexity of the drought resistance trait, any DNA marker has been developed.

Fig. 5.3 Epigenetic control of RNA expression. Between branchets the molecular techniques used in almond and related Prunus to analyse molecular process linked to bud dormancy and flowerin at DNA, RNA and epigenetic level

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The recent sequencing of the almond genome (Sánchez-Pérez et al. 2019; Alioto et al. 2020) will improve genomic and ytranscriptmoic analysis of drought resistance in almond. In this posgenomic context, plant breeders are now progressively focusing on the lately available genome-editing instruments to ameliorate significant agricultural attributes and analysis from genomic point of view varieties and traits (Haimovich et al. 2015; Sami et al. 2021). The emergence of multifold sequence-specific nucleases has simplified accurate gene modification toward the new product varieties development compatible with climatic changes (Voytas et al. 2014). Among the existing genome editing technology, CRISPR/Cas (clustered regularly interspaced short palindromic repeat-Cas) has appeared as a new instrument for pliability, compatibility, easiness and broad use (Joshi et al. 2020). The CRISPR/Cas system uses a compound involving a single guide RNA and Cas endonuclease which travel along the DNA strand, causing a double-strand break on the DNA (Brokowski and Adli 2019). Afterwards, the breaks are mended by endogenous cell repair, resulting in the development of new mutations (Raza et al. 2020). Regardinf water stress, recently, CRISPR/Cas technology has effectively been applied to achieve resistance against many environmental stresses (Joshi et al. 2020). Lately, the CRISPR—Cas9 system was applied to generate non-expresser mutated tomato of pathogenesis related 1 (NPR1) to confirm the role of this gene in drought resistance (Li et al. 2019). A decrease in MdNPR1 has been recorded in droughtresponsive apple plants (Bhat et al. 2016).

5.4.2 Development and Application of RNA Markers From a molecular and transcriptomic point of view, discovering the candidate genes whose expression varies as a cause or consequence of drough resistance and studying the biological role of those genes are priorities for breeding programmes. The transcriptome refers to both coding and non-coding RNAs, and thus represents an enormous capacity for adjusting developmental needs of living organisms under varying environments (Fig. 5.3). RNA can easily change quantitatively and/or qualitatively, thus having a tremendous potential impact on final phenotype. RNA analysis techniques can be applied for gene expression and are of great interest in monitoring complex process such as flowering. Transcriptional reprogramming leads developmental transitions in plants (Kaufmann et al. 2010). Changes in the expression of genes are usually the first response to stress situation in plants (Ouyang et al. 2010). Amongt these stress-reactive genes, those that encode transcription factors (TFs) have a significant role in adjusting the plant reaction to stress situation (Zaikina et al. 2019). In Prunus species these TFs have been linked to abiotic response including drought tolerance (Bianchi et al. 2015). On the other hand, Dehydration-responsive element binding factors (DREBs) control the expression of some target genes induced by cold and drought stress (Yamaguchi and Shinozaki 1994). MsDREB6.2 overexpression leads to reduction

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of stomatal density and apertures and enhancement of the hydraulic conductivity of roots, and consequently an increase in the drought resistance of transgenic plants (Liao et al. 2017). Interestingly, the phenotype of transgenic apple rootstock (M26) overexpressed with MsDREB6.2 were shorter than the non-transgenic apple rootstock. The leaves of these apples were also thicker. While stem growth was delayed in plants transgenic, root growth increased (Liao et al. 2017). Root increase is an important feature of plants that show drought tolerance (Sharp et al. 2004). On the other hand, Esmaeli et al. (2017) in an in silico search found different microRNAs related to drought in peach and almond.

5.4.3 Development and Application of Epigenetic Marks Epigenetics are chemical modifications affecting DNA or structural proteins (histones) within the chromatin including two process: DNA methylation (in plants 5´-cytosine methylation, 5mC) and PostTranslational histone Modifications (PTMs), which include the acetylation and methylation of histones (H2A, H2B, H3 and H4) (Saze 2008; Feng and Jacobsen 2011; Ríos et al. 2014). DNA methylation is associated with cell status stability and regulation of expression. DNA methylation occurs in three sequence contexts: CG and CHG, which are found in promoter and coding regions, and CHH (where H = A, C or T), found in non-coding regions and Transposable Elements (TEs) (Pascual et al. 2014) (Fig. 5.3). In this context, genome-wide analysis of DNA methylation has been done by bisulfite sequencing, which consists of the Next Generation Sequencing of digested and bisulfite-treated DNA samples. The epi-GBS technique has been developed to represent a small part of the genome for cost-effective exploration and comparative analysis of DNA methylation and genetic variation in hundreds of de novo samples. Furthermore, this method makes it possible to genotype samples without a prior reference genome. According to the results obtained, the DNA methylation (5mC) pattern was cultivar-dependent rather than dormancy state-dependent. In spite of coverage limitation of the performed sequencing, we were able to identify genes whose methylation state changed between the endodormant and the ecodormant state of flower buds DNA (Prudencio et al. 2018). In almond, results from the transcriptome sequencing of endodormant and ecodormant flower buds showed differential expression in a DNA methyltransferase gene and in the S-adenosyl methionine synthetase gene responsible for the synthesis of the molecule S-Adenosyl Methionine (SAM), which donates the methyl group to the DNA molecule (Prudencio et al. 2018). In addition, DNA methylation phenomena have also been associated with floral self-incompatibility (Fernández i Martí et al. 2014) and with bud falling phenomena (Fresnedo-Ramírez et al. 2017) in this species. Regarding drought resistance, Santos et al. (2020) showed that citrus scion and rootstock combinations show changes in DNA methylation profiles and ABA insensitivity under recurrent drought conditions. These epigenetic marks are involved in the responses to water deficit, conferring tolerance the citrus plants.

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Comparative DNA methylation studies of both traditional and almond cultivars released from breeding programs will surely contribute to our knowledge of methylation variants and provide candidate epialleles linked to agronomic traits such as drounght resistance. Such polymorphisms can be screened in large populations using NGS (New Generation Sequencing) to confirm the locus methylation state associated with a given character of interest.

5.5 Concluding Remark and Future Prospects Despite having different meaning in nature and in commercial orchards, drought resistance is a key characteristic in the adaptation of a tree to its environment, ensuring that the tree shows efficient behaviour in either case. Due to the relationship between weather, drought resistance and almond production in non irrigated areas, this rait has become one of the main traits to consider in almond plantations. Drought resistance time is a complex trait that is largely determined genetically but in a low heritability and a quantitative nature. Studies about regulation of gene expression are scarcer although several transcription factors have been described as responsible. From the metabolomics point of view, the integrated analysis of the mechanisms of drought resistance regulation through transcription factors open new possibilities in the analysis of this complex trait in almond. Finnaly, at epigenetic levels DNA methylation cytosine methylated genes have been reported as candidate epigenetic marks assoictaed to the regulation of drought resistance. Acknowledgements This study has been supported by European ARIMNet2 project ‘Nut4Drought: Selection and characterization of drought resistant almond cultivars from the Mediterranean basin with high nutraceutical values’ financed by the European Union.

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Chapter 6

Applications of Biotechnological Tools for Developing Abiotic Stress Tolerant Cherries Shiv Lal and Mahendra Kumar Verma

Abstract Cherry is a prominent temperate fruit crop that consumers like for its fresh fruits and value-added goods. Cherries also include a wide range of antioxidants, vitamins, and minerals. Recent biotechnological tools have revealed fascinating insights into the genetic nature and origins of current cherry varieties and species, as well as playing an important role in the generation of desired trait cultivars all over the world. Cherry has a wealth of genetic resources to satisfy our needs for developing new improved resistant cultivars with acceptable pomological features. Cherry cultivars have been classed and categorised according to their applications, fruit size, colour, flavour, biotic and abiotic stress tolerance, and fruitfulness. Traditional and contemporary advances in charcaterization of existing genetic diversity are also discussed, as well as how they might be used to manage cherry genetic resources. In this chapter, we review the significant progress made in cherry breeding programmes, the development of likage maps, and several successful examples of gene location, detection of molecular markers, molecular mapping, and the positions of quantitative trait loci (QTLs) associated with self-fruitfulness, maturity, dormancy, fruit size, quality, disease resistance, freezing, cold, heat, tolrnace, and other key attributes in cherry. The future of genomic assisted breeding techniques in cherry is also examined, as well as its applications. The addition of genomic techniques and resources to a traditional breeding programme will help to accelerate genetic progress. Keywords Cherry · Freezing · Chilling · Molecular breeding · QTLs · Genomics

S. Lal (B) ICAR-National Research Center On Seed Spices, Ajmer, Rajasthan 305206, India e-mail: [email protected] M. K. Verma ICAR- Indian Agricultural Research Institute, New Delhi 110012, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 C. Kole (ed.), Genomic Designing for Abiotic Stress Resistant Fruit Crops, https://doi.org/10.1007/978-3-031-09875-8_6

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6.1 Introduction Cherry is a temperate fruit crop that belongs to the Rosaceae family’s Prunus genus. Other stone fruits of the Prunus genus include almond, apricot, plum, peach, and nectarine. Cherries are most likely native to the area between northern Iran and Ukraine, as well as other nations south of the Caucasus Mountains (Webster 1996). There are many different cherry species, but only a few have been domesticated. Sweet (Prunus avium L.) and sour (Prunus cerasus L.) cherries are the most commonly produced commercially for their fruits. In Russia, the ground cherry, Prunus fruticosa Pall., is also extremely important. The Amarelles and the Morellos are the two most prevalent types of sour cherries (tart cherries). Red fruits with colourless juice characterise Amarelle varieties. The skin of Morellos varieties is pigmented, and the juice ranges from crimson to deep purple. The usual height of a sweet cherry tree is 9 to 10 m, with pyramid-shaped erect branches. Sweet cherry fruits are distinguished by their larger size and deep stem cavities. Fruit skin colours range from pale yellow to red to purplish-black, and stems or pedicels are approximately 1–1.5 in. long. Flowers appear in clusters of two to five on short spurs with several buds at the terminals, with the distal bud developing into a leafy stalk. The thickness and texture of the fruit pulp/flesh range from tender to hard. The sweet, delectable colourful fruits of sweet cherries are the most popular. The sour cherry (Prunus cerasus), often known as “red cherry” or “sour cherry,” is said to have developed from an unreduced sweet cherry pollen grain crossed with Prunus fruiticosa. Sweet cherry trees are cold tolerant, however they don’t live as long as sweet cherry trees. The fruit is smaller, more acidic, and has less sugar than a sweet cherry. Sour cherries are frequently used in the processing, jam, and pie filling industries. The red or purple hue of cherries is due to anthocyanin pigments. Flavonoid compounds that are dietary phenolics and have antioxidant effects are one type of flavonoid compound. Cherries also include a wide range of vitamins and minerals. Sweet cherries are primarily consumed as fresh fruits, whereas sour cherries are primarily employed in processing (Kaack et al. 1996) and can be frozen for later processing. For pies, canned sour cherries are used. After dehydration, both types of cherries are used in dry fruit mixes and breakfast cereals. Other processing goods made from both sorts of cherry include jams and jellies. Bleached sweet cherries are also utilised in the recoloring of drinks and sweets. The spacility is cherry liqueur and wine. Both cherries are excellent functional foods due to their high antioxidants capacity and several new antioxidants have been identified in dried sour cherries (Wang et al. 1999). Cherry production is particularly confined to temperate regions that experience moderate cold winter temperatures. During the year 2019, world production of sweet cherry and sour cherry was 2.59 and 1.41 million tonnes, respectively (FAOSTAT 2019). Turkey, The United States of America, Chile, Uzbekistan, Iran, Spain, Italy, Greece, and Ukraine are the major sweet cherry producing countries (comprises

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approximately 57% of world sweet cherry production). Whereas—Russian Federation, Ukraine, Turkey, Poland, Iran, USA and Serbia, Uzbekistan, Hungry Azerbaijan are producing 65% of world sour cherry production. Breeding in sweet and sour cherry is a difficult, laborious, and expensive activity. These species generally show a long juvenility period, require large orchard areas for phenotypic evaluation, and obtaining large amounts of seeds in controlled crosses is rather challenging. In sour cherry, low fruit set due to segmental allotetraploid nature posses an additional hurdle. Breeding for tolerance or resistance against diseases, very few sources have been identified within the cultivated pool, yet some interspecific hybridization with small-fruited species has been attempted (Schuster et al. 2012). Although, with the advancement of technologies and improved cultivars one marked growth in area and production was recorded in devlopeded countries but in other hand productivity lagging behind cherries producing developing countries. Sweet and sour cherry species are deciduous fruit crop species that grow in temperate areas with well-differentiated seasons, characterized by low winter temperatures and summer drought. Over the year’s changes in climatic conditions like the occurrence of erratic rain, snowfall, and increase in temperature resulted in fast receding glaciers playing a significant role in cherry-producing regions. Most of the deciduous fruit crops require sufficient accumulated chilling, or vernalization, to break winter dormancy. Insufficient chilling due to global warming may result in prolonged dormancy which ultimately would lead to poor fruit quality and yield. The warming temperature scenario exceeds 1 °C its likely to affect the vernalization in high-chilling requires fruit crops such as cherries, and if the under warming scenario temperature will exceeds 1.5 °C then it will significantly increase the risk of prolonged dormancy in both stone-fruit and pome-fruit (Hennessy and ClaytonGreene 1995). Mild winters may cause a change in flowering behavior and fruit set. Sweet cherry and sour cherry require the accumulation of 1000 chill units at 3.8 °C to complete or ‘break’ dormancy. If chilling is inadequate, the development and/or the later expansion of leaf and flower buds may be impaired (Mahmood et al. 2000). Under changing climatic scenarios, other important factors to be considered as limiting factors are freezing injury during winter, spring frost, and the incidence of hot winds during summers, hailstorms, lightning etc.. Among stresses, freezing is a major environmental stress that limits the geographical distribution, growth, and productivity of temperate fruit crops (Yu and Lee 2020). Freezing injury is characterized by frost splitting of trunks, blackheart of stems, winter sunscald of thin-barked species, death of cambium in twigs, freezing of roots, midwinter kill of dormant flower buds, branches, trunks, and frost damage to flowers and fruit during spring and fall (Weiser 1970; Pearce 2001). The ability to tolerate freezing environment under natural conditions differ to a great extent according to crop, genotypes and varieties (Moran et al. 2011; Lee et al. 2013; Yu et al. 2017). Besides this, rest of the temperate fruits including cherry require a rest period that initiates during autumn. However, according to various studies, the freezing requirement has a considerably higher effect on flowering time in Prunus species than the heat requirement (Alburquerque et al. 2008). Chilling time ranges between 750 and 1400 h, depending on the variety and its features. The regulatory system mediated by CIG in sweet cherry may

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play an important role in the acquisition of cold tolerance before and/or during winter (Kitashiba et al. 2004), and the expression of a sweet cherry DREB1/CBF ortholog in Arabidopsis confers salt and freezing tolerance (Kitashiba et al. 2004). The effects of cold temperatures in late autumn and early winter on cherry fruit production have long been known (Caprio and Quamme 2006). High temperatures during bloom, on the other hand, diminish fruit set due to faster plant parts development rates and fewer pollen tubes forming alongside the style; ovule degeneration increases within the ovary (Hedhly 2011). High summer temperatures during floral differentiation may result in the creation of double pistils, which will be followed by double fruits (Beppu et al. 2001). All of these limits could be easily solved by a trait-specific cherry breeding programme that makes systematic use of current germplasm and genetic resources.

6.2 Genetic Resources of Resistance Genes Cherry trees exist in over 30 different species, the majority of which are native to Europe and Asia. The basal chromosomal number (haploid) in all Prunus species is x = 8. In nature, sweet cherries have a diploid genome (2n = 2x = 16), but sour and ground cherries have a tetraploid genome (2n = 4x = 32). Sweet cherries are grown in a rich genetic resource location in China’s mountains (Cai et al. 2007). Recent molecular technologies have revealed some fascinating details about the genetic makeup of current cherry varieties. Several labs across the world have used DNA markers such as Random Aplified Polymorphic DNA (RAPD), Simple Sequence Repeat (SSR), Amplified Fragment Length Polymorphism (AFLP), and isozymes analyses to determine the relationship between existing cultivars. Almost majority of these studies found only a minor amount of polymorphism. Despite having a smaller genetic base, investigations have found that sour cherries are more polymorphic than sweet cherries (Beaver et al. 1995), possibly due to their allotetraploid origin. Cherries are primarily propagated on two rootstocks: Mazzard (P. cerasus) and Mahaleb (P. cerasus) (P. mahaleb). Mazzard’s original selections came from the progenies of natural woodland trees. Many dwarfing rootstocks have been created utilising P. cerasus or hybrids of it. In numerous cherry-growing regions, Mahaleb rootstock is still the rootstock of choice (Europe & Asia). P. canescens and P. fruticosa, among other species, are regarded appropriate parents for many rootstock breeding initiatives (Wolfram 1996; Rozpara and Grzyb 2005). Some Prunus species, on the other hand, are chosen in breeding programmes because of their crosscompatibility. In cherry, however, interspecific hybridization for the generation of scion variants is quite rare. Only a few interspecific hybrid rootstocks have been developed, and they are only used in specific use (Iezzoni et al. 1990). The following interspecific hybrids are identified to have potential in cherry rootstock breeding; P. avium × P. pseudocerasus; P. incisa × P. serrula; P. cerasus × P. maackii; P. cerasus × P. avium; P. cerasus × P. canescens; P. cerasus × P. fruticosa; P. fruticosa × P. avium; P. subhirtella × P. yedoensis; P. mahaleb × P. avium; P. avium

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× P. kuri lensis; P. avium × P. incisa; P. canescens × P. incisa; P. canescens × P. tomentosa; and P. cerasus × P. pensylvanica. The other important rootstocks which was developed through hybridization such as Gisela 5, Gisela 7, Gisela 6. The trait specific rootstocks was also developed by hybridization such as dwarfing rootstocks such as W-13, M X M-14, M X 39, CAB 6 P, CAB 11E, W-10, W-11, M X M-46, M X M-27, Colt; Cold hardy rootstocks- CAB 6P, CAB 11E, W-10, W-13; Resistant to crown and root rot- Colt, M x M-2, M x M 39, M x M-97, W-10, W-11 & W-13; Resistant to crown gall: F 12/1; Resistant to canker/gummosis- F 12/1, Charger and M x M-14. The modern cherry breeding programs aims for the development of sweet cherry cultivars of regular bearer, productive and large fruit size. Although from orchard management’s point of view the risk of irregularity in production can be minimized by planting solely self-fertile cultivars (Herrero et al. 2017). The most important trait, after yield and fruit size, is fruit firmness. Indeed, it is positively correlated with post-harvest shelf-life and is a key trait for export-oriented productions. But it is also a highly appreciated trait by consumers, in particular when associated with other positive attributes such as freshness, crunchiness, juiciness, etc. Tasting quality is becoming more and more strategic to gain differentiation capacity, in a market that is already filled with a high number of cultivars (Lugli et al. 2012). Among abiotic stresses (drought, salt etc.), tolerance to rain-induced fruit cracking has been for a long time a desired trait for the sweet cherry breeders. Resistance to winter frost was always considered an important breeding trait in Central and Northern European countries, where the sweet cherry is cultivated at the margins of the traditional distribution area of this species. Oppositely, tolerance to warm temperatures has been gaining increasing attention in recent years. Cherry production is becoming more difficult in many traditional places as autumn and winter temperatures rise, causing insufficient fulfilment of the chilling needs for flowering. In the susceptibility/tolerance to double fruit production, the significant feature is closely linked to an increase in spring and summer temperatures. When flower initiation occurs in newly growing buds, temperatures above 30 °C will cause the pistil primordia to double, and in the following spring season, flowers with two pistils will be fertilised and generate double fruits, which are considered of noncommercial value (Wenden et al. 2017). Only particular programmes have systematically focused on tolerance/resistance to bacterial and fungal diseases, the most notable of which are Pseudomonas spp.caused bacterial canker and Monilinia spp. caused blossom blight and brown rot, respectively. Mildew infection, pitting, hardness, sugar, flavour, acidity, colour, and harvest timing are all significant criteria for cherry breeders. The development of molecular markers, genomic technologies, and physical and genetic linkage maps has resulted in an increase in information, which has the potential to improve breeding efficiency. The details of few of the most important hybrid varieties that have resulted from crossbreeding.

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6.2.1 Dark-Colored Varieties ● Cavalier: A vigorous variety with good cold tolerance and disease resistance. This is a black, early-ripening cultivar. The fruit is medium to big in size, dark red in colour, with dry, firm flesh that has a pleasant flavour and is resistant to shattering. Must be planted with another cherry variety for pollination to occur. ● Black Tartarian: Purplish-black fruit that is tiny to medium in size, heart-shaped, and of good quality. It produces big, purplish-black fruit with a sweet, rich, fullbodied flavor that is great for fresh eating and preserves ● Kristin: It was developed in 1982 by crossing between ‘Emperor Francis’ and ‘Gil Peck’. The fruit is enormous, aromatic, firm, delicious, dark red, beautiful, and relatively break resistant, and the trees are vigorous in nature. ● Ulster: Introduced in 1964, it is a mix between ‘Schmidt’ and ‘Lambert’. The fruit is huge and tasty, with dark red, crisp flesh. This high-quality fruit has a moderate resistance to cracking. It’s good for both fresh eating and processing, as well as being productive. ● Hedelfingen: This is a traditional European cultivar. Tree is very precocious and high yielder. The glossy red color fruit ranges in size from medium to large. Top-quality fruit for fresh-eating, freezing, and canning. ● Hudson: It was developed by crossing ‘Oswego’ with ‘Giant’. This variety ripens in late-season. The fruit is dark crimson in colour, medium to large in size, tasty, and firm. The trees are somewhat productive and may not produce until later in the season. Fruit has a high resistance to cracking.

6.2.2 Light-Colored ● Emperor Francis is a high-quality early cherry with a crimson flush that is yellowish-white in colour. The trees are hardy, and the fruit is bigger, but not very resistant to cracking. ● Gold: This variety is early, the trees are fruitful, and the fruit is small and resistant to cracking, but the fruit is small and susceptible to bacterial canker. ● Napolean: It is also known as ‘Royal Anne’ or ‘Napolean Wa’: The fruit has brilliant red cheeks and is pale yellow in colour. The fruit is medium in size, firm, sweet, and juicy, and has a reasonable amount of freshness. It resists cracking to a degree. ● Rainier: It’s a cross between ‘Bing’ and ‘Van’. The fruit is bigger and tasty, with a yellow skin that has a slight red blush. The juice is clear and the fruit is solid. The fruit is suitable for transporting or eating fresh.

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6.2.3 Tart Cherry ● Montmorency: Before the seventeenth century, it was created in France’s Montmorency Valley. ‘Montmorency’ is the most common tart cherry variety, accounting for over 90% of all tart cherries cultivated. The medium-large, bright red fruit has a firm yellow flesh; clear juice; and a rich, tart flavor that bakers and jam makers love. This tree is self-fertile, but planting two or more trees will ensure the best crop. ● Northstar: It’s a hybrid of ‘English Morello’ and ‘Serbian Pie.’ The medium-sized mahogany red fruit has red juice. Because trees are small, they are simple to cover with bird netting. Leaf spots and brown rot resistance are present in the trees. ● Danube: It’s a recent tart cherry developed for fresh eating that ripens a few days before ‘Montmorency’. The tart cherries are dark red in colour, medium to large in size, and sweeter than other tart cherries. This cultivar is commonly grown throughout Europe. ● Balaton: It was considered a local variable in Hungary. The fruit is larger, harder, sweeter, and redder than ‘Montmorency’, and it boasts luscious flesh. The pits are slightly greater than those found in other kinds, which could provide a challenge for processors.

6.2.4 Self-Fertile ● Vandalay: Fruit of this cultivar is kidney-shaped fruit which is large and wine-red in hue, with purple juice ● Stella: It was introduced in 1968 as a result of a hybrid between ‘Lambert’ and ‘John Innes Seeding.‘ Its fruits are tasty, huge, black, heart-shaped cherry with medium-firm flesh that is prone to shattering. It is self-fertile and ripens early. ● Tehranivee: A new mahogany-colored, self fertile sweet cherry with black-red juice. Tehranivee has excellent flavor as well as size, sweetness and firmness. Fruit cracking is a major problem. ● Sweetheart: It is re a late-season cultivar. Fruit is medium to large in size, very firm, and flavorful, and the trees arehigh yielders and fruits are sweet and tangy flavor, and resistance to cracking Skeena: This is a huge dark mahogany cherry. It outperforms ‘Lapins.‘ Blooms in the middle of the season. ● Lapins: It was introduced in 1983 as a hybrid progeny of ‘Van’ and ‘Stella.’ The fruit is huge, has a nice flavour, and ripens later then Bing genotype. The tree is very productive. They are noted for having good split resistance. ● Blackgold: Developed by crossing Stella and Stark Gold. This is a late-season, self-fertile sweet cherry variety that is primarily used for fresh consumption. This is the newest sweet cherry to blossom, and it has a high tolerance for spring cold.

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6.3 Genetic Diversity Analysis in Cherries Discovering the phenotypic diversity that contribute to overall diversity in a germplasm collection, as well as the levels of variation among cultivars, necessitates an understanding of morphological, phenotypic, and genetic diversity (FuronesPérez and Fernández-López 2009; De Oliveira et al. 2012; Mehmood et al. 2014; Ganopoulos et al. 2015). From ancient times various local variants (landraces) were identified and designated according to the locations where the selection was popular. Much of today’s cherry germplasm is made up of historical landraces (Iezzoni 2008). There are around 1,500 sweet cherry cultivars in the world today, which is a considerable quantity in terms of genetic resources. A previous technique for detecting and analysing genetic variation among sweet cherry cultivars relied primarily on phenotypic features (IPGRI 1985; UPOV 1976). Morphological analysis was thought to be a basic, straightforward, and widely utilised method for detecting and characterising germplasm through phenotyping. A large variety of new sweet cherry cultivars with excellent pomological and agronomical features have been developed in recent decades, including several that are self-compatible and have a modest chilling requirement. Sansavini and Lugli (2008) found that the selected cultivars had significant agronomic characteristics, such as low susceptibility to fruit cracking, high levels of soluble solids, early fruit maturity, and high rusticity (Benková et al. 2017). The primary criterion for evaluating the genetic diversity of fruit cultivars is phenotypic and morphological trait analysis. Because domestic and foreign genotypes of sweet cherry (varieties, landrace cultivars, etc.) are sometimes difficult or impossible to discern, it is often difficult or impossible to discriminate between many cultivars. Only morphological and biochemical evaluations, not molecular evaluations, are performed on selection and hybrids. Environmental variables have an impact on all of these qualities. Variation in biochemical features (such as isozymes) can also be used to determine cultivar identification (Beaver et al. 1995; Boskovic et al. 1997). Sweet cherries, on the other hand, have a low degree of isozyme polymorphism (Zhou et al. 2005a, b). Diversity based on phenotypic and morphological characters varies with environment, and evaluating traits requires growing the plants to full maturity before identification, but thanks to the rapid advancement of biotechnology, a large number of loci distributed throughout the genome of the plants can now be easily analysed. Molecular markers are useful for assessing genetic variation, identifying cultivars, and elucidating genetic relationships within and between species (Chakravarthi and Naravaneni 2006). There are several molecular markers for genotype categorization, however unlike physical features, markers are not changed by the environment (Staub et al. 1997). In crop breeding projects, collecting DNA marker data to evaluate whether phenotypically similar cultivars are genetically similar would be of considerable importance (Duzyaman 2005). Molecular genetics, or the application of molecular techniques to successfully discover genetic changes in the genetic material (DNA) of individual plants, is critical for crop development (Jonah et al. 2011). Because genetic differences are frequently associated with a specific gene and act as’sign posts’ to those genes, they are referred to as molecular markers. When these

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markers are very tightly linked to genes of interest, they can be used to select indirectly for the desired allele, and this is the most basic form of marker-assisted selection (MAS) (Hoisington et al. 2002). The discovery of various types of PCR and nonPCR based molecular markers has accelerated the cherry breeding programme, and it has become an integral part of the cherry improvement programme. RAPD markers have been frequently employed by researchers (Yang and Schmitt 1994; Gerlach and Stösser 1998; Lisek et al. 2006; Cai et al. 2007; Vaio et al. 2015) because they are a simple and reliable method for identifying across cherry germplasm, varieties, cultivars, and mutants. Following that, additional genomic markers such as SSRs were created and successfully used to identify sweet and sour cherry cultivars (Kaçar et al. 2005, 2006; Demirsoy et al. 2008). In addition, DNA fingerprinting approaches such as SSR and AFLP (Struss et al. 2003) have been used to identify sweet cherry cultivars. With the advancement of molecular markers, microsatellite markers became a popular choice for molecular characterization of sweet cherries (P. avium (L.) and tetraploid sour cherries (Cantin et al. 2001; Vaughan and Russell 2004; Kacar et al. 2005; Pedersen 2006; Fernández I Marti et al. 2012; Liu et al. 2018; Muccillo et al. 2019; Guajardo et al. 2021). In recent years, whole genome re-sequencing of sweet cherry (Prunus avium L.) revealed new information about the fruit’s genomic diversity (Xanthopoulou et al. 2020). The use of genomic resources, such as single nucleotide polymorphism (SNP) maps of genomes, has increased our understanding of the genetic foundation of phenotypic variety. In addition, a wide range of polymorphisms in underlying loci has been discovered.

6.4 Molecular Mapping of Abiotic Stress Resistance Genes and QTLs in Cherries Significant progress was achieved in the 1990s about understanding the molecular underpinnings of the complicated gametophytic self-incompatibility mechanism in cherry. The cultivar ‘Stella’ (Lapins 1971), which was widely employed as a progenitor in self-compatible sweet cherry breeding, was the first compatible commercial sweet cherry cultivar. Because of a pollen function mutation in the S4’ allele (S4’ stands for mutant S4 allele), ‘Stella’ has self-compatibility. To distinguish the genotypes that inherited the S4’ allele, self-compatible seedlings generated from Stella must be selected. It was not possible until recent pollen determinant of GSI in Prunus studies (Yamane et al. 2003; Ushijima et al. 2004) that a method was proposed to select genotypes carrying the mutated S4’ allele (Ikeda et al. 2004). PCR-based markers was also developed (Yamane et al. 2003) to overcome self-incompatibility problem. Subsequently, molecular markers became available to routinely characterize the S-alleles of each new cultivar (Herrero et al. 2017) and were used to assign self-incompatibility alleles to many different sweet cherry cultivars (Schuster 2012). When compared to other Prunus species, the process of developing genetic maps in cherry was slightly delayed. Microspore-derived calli (Stockinger et al. 1996) or

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controlled crossings were used to create the first genetic intraspecific maps on sweet cherry. After that, isozyme maps were created using two interspecific F1 cherry progenies: P. incisa E621 and P. nipponica F1292 (Boskovic and Tobutt 1998). Ten linkage groups totaling 503 cM were linked to two allozymes and 89 RAPD markers. There were a total of 47 segregating allozymes assessed, with 34 of them matched into seven linkage groups. Dirlewanger et al. (2004) used the cultivars ‘Regina’ and ‘Lapins’ to create partial linkage maps. These cultivars were chosen based on their rain cracking resistance characteristics. The ‘Regina’ and ‘Lapins’ maps each featured 30 and 28 SSR markers, which were used to check for peach-cherry collinearity. Only one non-collinear marker was found, indicating that cherry and peach have a high level of synteny. Similarly, Olmstead et al. (2008) created a map using the offspring of Emperor Francis’ and ‘New York 54’ crosses (Table 6.1). Researchers from Michigan State University (USA) constructed a sweet cherry genetic linkage map from an F1 progeny of a cross between a wild forest cherry with a small (2 g) highly acid darkred coloured fruit (NY54) and a domesticated variety with large (6 g), yellow/pink, sub-acid fruit (‘Emperor Francis’). Linkage maps were created in sour cherry using 86 individuals from a cross between two cultivars, ‘Rheinishce Schattenmorelle’ (RS) and ‘Erdi Botermo’ (EB) (EB). Because the sour cherry is a tetraploid, Wu et al. scored relevant restriction fragment length polymorphisms (RFLPs) as singledose restriction fragments (1992). For six traits, including bloom time, ripening time, pistil death, pollen germination, fruit weight, and soluble solids content, eleven QTLs (LOD > 2.4) were discovered, with the percentage of phenotypic variation explained by a single QTL ranging from 12.9% to 25.9%. SSR markers were not widely available and used when the ‘Rheinische Schattenmorelle’ and ‘Erdi Botermo’ based maps were created, but linkage groups 2, 4, 6, and 7, which contain QTLs for bloom date, ripening date, fruit weight, and soluble solids, were assumed to be homologous to the peach and almond linkage groups 2, 4, 6, and 7 using shared markers (Wang et al. 2000). The discovery of two QTLs for pistil death further revealed that there was significant genetic diversity in the ability of sour cherry pistils to withstand freezing conditions (Wang et al. 2000). Following that, two QTLs were discovered in successive years, which was appropriate given the different freeze occurrences. The first QTL, pd1, was discovered in response to a 10 °C light 21 days before bloom, while the second QTL, pd2, was detected in response to two freezing occurrences. Significant QTLs for these traits were later discovered. On the same restricted region of linkage group (LG) 4, the significant QTLs for soluble solid content, fruit development time, maturity date, and hardness were discovered. As previously observed in syntenic regions of other Rosaceae species, the found NAC transcription factor genes in this LG4 region could be candidate genes for the regulation of these features in sweet cherry. The LG4 genomic region’s identified haplotypes will be valuable for sweet cherry breeding from this and adjacent plant material. According to some findings, the most relevant QTL in terms of MAS is found on LG 2, which can explain up to 30% of the phenotypic variable in fruit weight depending on the family and year studied. As a result, this QTL has been linked to domestication, and the majority of present commercial cultivars have either two beneficial alleles (homozygosity) or one favourable and one unfavourable allele (heterozygosity) (Peace 2012). A highly

711.2/565.8 736

Prunus avium × wild forest cherry

Prunus avium × Prunus nipponica

Prunus avium (Intraspecific/interspecific)

Prunus avium

Prunus avium

2008

2009

2012

2013

2015 731.3

752.9/639.9

779.4

Total map length (cM)

Species used for map

Constructed year

Table 6.1 Sweet cherry genetic linkage map constructed over the years

0.7

1.1/0.9

5.4

3.16

4.94/6.22

Average marker density (cM)

SNP/SSR

SNP

SNP/SSR

SSR/Isozyme

SSR

Main marker types used in mapping

Voorrips (2015)

Klagges et al. (2013a, b)

Cabrera et al. (2012)

Clarke et al. (2009)

Olmstead et al. (2008)

Reference

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saturated map has recently been produced (Klagges et al. 2013a, b). Phenology and fruit quality-related variables, including as flowering and maturity dates, chilling and heat requirements, fruit weight, fruit hardness, fruit skin, and flesh colour, and raininduced fruit cracking tolerance, were the focus of studies on QTL detection in sweet cherry. (Sooriyapathirana et al. 2010; Zhang et al. 2010; Dirlewanger et al. 2012; De Franceschi et al. 2013; Rosyara et al. 2013; Castède et al. 2014, 2015; Campoy et al. 2015). Similarly, primary studies aimed at dissecting the genetic architecture of complex agronomic parameters were initiated for sweet cherry at the same time. Recently, the most relevant findings from QTL detection analysis on sweet and sour cherries, as well as their potential application in MAS programmes, were highlighted. (Salazar et al. 2014; Quero-García et al. 2017; Iezzoni et al. 2017; Calle et al. 2018). The LG4 region has been identified as a breeding target in sweet cherry after genetic dissection of bloom time in low chilling sweet cherries and multiple-population QTL mapping of maturity and fruit-quality attributes (Fig. 6.1). Modern applications of MAS that have recently been deployed in sweet cherry include the assessment of genome-wide breeding values of breeding material across many characteristics, in compared to existing MAS techniques. (Piaskowski et al. 2018) as well as the forecasting of maturity dates in newer environment (Hardner et al. 2017). Calle and Wünsch (2020) used a multi-family QTL technique and discovered two main QTLs

Fig. 6.1 Quantitative trait loci (QTLs) located in linkage group 4 (LG4) of the sweet cherry (Prunus avium) cultivar ‘Regina’ (R4) detected using ‘Regina’ 9 ‘Lapins’ (R 9 L; left) and ‘Regina’ 9 ‘Garnet’ (R 9 G; right) progenies. Results for each year are indicated by open bars and are given as xx-yyyy (xx, trait acronym; yyyy, year of evaluation). Those obtained using multi-year analyses in MULTIQTL are indicated by closed bars and given as xx-MY. Bars are blue for chilling requirements (CR), red for heat requirements (HR) and green for flowering date (FD). Solid bars denote the most probable position of the QTL obtained from the highest LOD score position given by the MIM and the bootstrap (1000 permutations) SD. The distance between markers is represented in cM. Markers present in both families are in bold, italic and underlined (Cited from Castède et al. 2014)

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Fig. 6.2 ‘Regina’ linkage map of candidate genes on linkage group 4 and QTLs for chilling (CR) and heat requirements (HR) and for flowering date (FD) detected in R × L and R × G progenies. Candidate genes are indicated in bold, those genetically mapped are in red and those mapped in silico are in black (Cited from Castède et al. 2015, open access journal PLoS One)

on linkage groups 1 and 2 (qP-BT1.1 m and qP-BT2.1 m) that explained 47.6% of the phenotypic variation. The related genes 1 QTL was found to coincide with the DORMANCY ASSOCIATED MADS-BOX (DAM) genes and was mapped to a 0.26 Mbp region (Fig. 6.2).

6.5 Future Perspectives Cherry crop improvement through classical and conventional breeding is a timeconsuming and costly process. However, as indicated by the number of new cultivars published from various breeding efforts, it has been the most successful so far. The addition of genomic techniques and resources to a traditional breeding programme

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will help to accelerate genetic progress. Breeders will find it easier to obtain new cultivars with desirable features with the help of new genomic technologies, either by aiding selection or by increasing the diversity available for precision breeding procedures. Perhaps these advancements will aid in the development of more selffruitful, regular, freeze, cold, and heat tolerant, and desired to harvest time cultivars with precise characteristics in response to industry and consumer demands, as well as the expansion of cherry production to more non-traditional regions. For the genetic dissection and breeding of complex features in cherries, improved and emerging genomics technologies will be extremely useful. The creation of plant regeneration and transformation techniques will be extremely beneficial in improving existing cultivars for certain features. Consumer preference for varietals or features is quite strong in cherries, which can be preserved using transformation procedures. The accessible peach genome sequences will be extremely valuable for all Prunus species and will undoubtedly aid crop improvement in the near future.

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Chapter 7

Genomic Design of Abiotic Stress-Resistant Berries Rytis Rugienius, Jurgita Vinskien˙e, Elena Andriunait˙ ¯ e, Šarun˙ ¯ e Morkunait˙ ¯ e-Haimi, Perttu Juhani-Haimi, and Julie Graham

Abstract Resistance to abiotic stress is important for the adaptivity of strawberry, blueberry, red, and black raspberry crops as well as their productivity and berry quality. Climate change creates challenges to berry production and poses new requirements for berry crop breeding. New varieties should be adaptable to the specific growing conditions, as the farm has to be profitable and sustainable. Plants should be resistant to temperature fluctuations—heat, cold, and lack of moisture, adapted to artificial substrates, accumulation of salts, and heavy metals. Old cultivars have progressively been changed to new cultivars or hybrids, which are more productive, or with some tolerance to abiotic stresses, but with a narrow genetic base. The reduction of biodiversity caused the loss of genetic sources for environmental adaptability. Therefore, conventional approaches need to be complemented with advanced techniques in order to overcome the challenges of climate change and meet modern environmental and quality requirements. The advancements in berry crop research methods and possibilities of breeding programs improvement are discussed and summarized in this chapter. First, we present the main challenges to breed berries with resistance to abiotic stressors, review the peculiarities of resistance to specific environmental and climate change, then present information on the genome structure of berry plants. Traditional elements and factors of abiotic stress resistance are introduced, followed by the advances in marker-assisted and genomics-assisted breeding presenting the possibilities to increase abiotic resistance with modern genetic and genomic tools. Finally, we will illustrate how new biotechnological tools, such as interspecific hybridization, genetic engineering, and epigenetic resources are being implemented and can assist genomics-aided breeding. This information could be used in modern breeding to advance the production of quality berries. Keywords Breeding · Genomics · Cold · Drought · Salinity · Strawberry · Red raspberry · Black raspberry R. Rugienius (B) · J. Vinskien˙e · E. Andri¯unait˙e · Š. Mork¯unait˙e-Haimi · P. Juhani-Haimi Lithuanian Research Centre for Agriculture and Forestry, Institute of Horticulture, Kaunas Str. 30 Kaunas Distr, Babtai LT-54333, Lithuania e-mail: [email protected] J. Graham James Hutton Institute, Errol Road, Dundee DD2 5DA, Scotland, UK © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 C. Kole (ed.), Genomic Designing for Abiotic Stress Resistant Fruit Crops, https://doi.org/10.1007/978-3-031-09875-8_7

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7.1 Introduction Cultivation of berry crops is currently facing considerable challenges connected with the environment: high, low, or fluctuating temperatures, the emergence of new diseases and pathogens. Environmental challenges are often followed by challenges in the human economy: globalization, the desire to grow the highest quality cultivars even if they are often developed far from their areas of cultivation and are poorly adapted to local and specific growing conditions. The European Green Deal is a challenging growth strategy that lays out a strategy for achieving carbon neutrality in Europe by 2050 across the whole economy. This deal encourages the transition to organic farming, reducing cultivation costs while considering biodiversity. Natural resources such as fuel, heat, and water are very limited, and this becomes even more pronounced in the context of climate change and globalization when human living standards and wages rise. It is argued that climate change has slowed agricultural productivity growth by 21% since 1961 (Maryl 2021). This poses challenges and new requirements for berry crop breeding. New varieties should be adaptable to the specific growing conditions and different environments such as artificial substrates, accumulation of salts, and heavy metals, as the farms have to be profitable and sustainable. Due to climate changes plants should be resistant to temperature changes, lack of moisture, their reaction to light and the photoperiod, as well as chilling (vernalization) temperature must ensure timely and uninterrupted cropping as possible (Todeschini et al. 2018; Naderi et al. 2018; Muneer and Lee 2018; Zahedi et al. 2019; Kaya and Aslan 2020; Chen et al. 2020). This chapter is devoted to the genomic design of berries resistant to abiotic factors, stresses like drought, heat, and cold. To make things even more complicated, responses to environmental stresses are known to be evolutionary highly conserved in plants, however, different stress tolerance strategies have to be utilized in perennial (e.g., strawberry, raspberry) and annual (e.g., Arabidopsis) species (Ruelland et al. 2009). These stresses are important not only for plant survival, productivity, cost reduction, the quality of berries, and their nutritional value but also for human health and well-being. Stress cannot be completely avoided, but there are different ways to improve how the plant will react to it. To do this requires an understanding of what stress is, how it occurs, what tools plants use to manage stress, and how we can influence it to our advantage. Of course, what matters to the consumers is the external appearance, taste, and nutritional value of the food. Plant secondary metabolites, such as flavonoids, are antioxidants that reduce free radicals and have a positive effect on human health by preventing oncological, heart, and other organ diseases (Davik et al. 2020). It is known that limited stress increases the production of these secondary metabolites. In addition, there is an opportunity not only to increase the production of these substances but also to save resources by growing crops in less rich soils or using deficit irrigation (Perin et al. 2019; Ibanez et al. 2019; Zuñiga et al. 2020; Rugienius et al. 2021). For example, preharvest leaf wounding stress causes a significant increase in phenolic compounds and total soluble sugars. This

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accumulation depends directly on carbon partition and associated gene expression (Ibanez et al. 2019). The question arises whether new means have emerged over the last 10 to 15 years to help solve all of these breeding issues? Of course, many molecular mechanisms remain unclear, the diversity of biochemical pathways, and variation in different genotypes is far from elucidated in strawberry and other berry plants. Nonetheless, in recent years, new knowledge and technologies have emerged in science that allows us to look at the development of new varieties in a very different way in the near future. In particular, genome research, next-generation sequencing (NGS) and molecular markers will allow the development of new varieties that are based not only on individual unrelated or poorly linked traits but as a whole of individual blocks— haplotypes and gene complexes.

7.2 Assumptions for Berry Crop Breading and Abiotic Stress Resistance 7.2.1 Strawberry and Raspberry Breeding Objectives 7.2.1.1

Strawberry

Strawberry cultivation is growing worldwide due to varietal innovations created by numerous breeding and biotechnology projects, as well as plant biology research to develop innovative cultivation systems. Genetic resources remain especially important for breeding activities integrated with new molecular, genomic knowledge and technologies (Mezzetti et al. 2018). The most important resource for plant breeding is germplasm, which reflects the overall genetic variability of a species and population. Old ecotypes gradually have been changed to new cultivars or hybrids, usually more productive, with some resistance to pests, diseases, or abiotic stresses, but with a reduced genetic base. It results in a decreased adaptation to the changing environment, as well as the loss of certain qualitative traits. For this reason, mainly in the last few decades, a lot of work has been carried out to re-establish biodiversity by study, recovery, characterization, and valorization projects (Mathey et al. 2017). Inbreeding has steadily increased over time as a consequence of directional selection and population bottlenecks. Inbreeding then leads to a reduction in heterozygosity, which is one of the most serious issues in strawberry breeding. Heterozygosity, together with low heritability, and polygenic control for traits complicates the breeder’s ability to identify the best parents and predict the best parental combinations. This challenge is exacerbated in polyploids (Iezzoni et al. 2020). According to Pincot et al. (2021), improvements can be achieved through the application of pedigree-informed predictive breeding methods and adjusting breeding schemes. The open-source pedigree database has many benefits because breeding schemes can be easily modified and expanded to take into account specific breeding issues,

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other populations, and future analyzes. As pedigree records commonly available in strawberry are sufficiently deep and broad, pedigree best linear unbiased prediction (pedigree-BLUP) has the potential to enhance selection choices and increase genetic gains, especially when combined with genomic predictions (Habier et al. 2013; Pincot et al. 2021). With the information available from cultivated species and their wild relative genomes, genome sequence-based molecular markers and mapping loci for economically important traits can be used to accelerate genome-assisted breeding. Genome analysis has not only identified individual gene sequences but also their localization in quantitative trait locus (QTL) has been determined and this facilitates genomic selection. Identification and characterization of abiotic stress tolerance-related genes of different species are possible using genome information (Soundararajan et al. 2019). New insights are expected to clarify the most effective cold hardiness mechanisms by comparing closely related plants that differ in frost tolerance (Koehler et al. 2012b). Recent discoveries in genome and proteome studies can be further supplemented with kinome, plastome, and metabolome research as well (Dillenberger et al. 2018; Haugeneder et al. 2018; Liu et al. 2020, 2021). New breeding programs are developed and refined based on knowledge of the entire genome. The octoploid cultivated strawberry has a limited genetic basis. This raises the question of how to identify important gene targets and successfully exploit them for strawberry improvement. Strategies successfully employed for the identification of genetic variations in different traits in Solanaceous crops (tomato, potato) can be applied to strawberry as well, especially using gene-editing technologies (Gaston et al. 2020). A Rosaceae family-level candidate gene approach was used to identify genes associated with sugar content in blackberry (Rubus subgenus Rubus). Three DNA regions conserved among alpine strawberry (Fragaria vesca), peach (Prunus persica), and apple (Malus × domestica) was used for gene and QTL identification in blackberry (Zurn et al. 2020). During the last five years, genomic prediction as a tool for choosing outstanding parents for crosses was chosen by investigators from the University of Florida. Using this method, it was possible to shorten the traditional breeding cycle by a year by using certain parent plants. However, more information is required for genome prediction when multiple breeding cycles are used (Osorio et al. 2020). The use of F1 hybrid seeds as an alternative strawberry breeding strategy was proposed by Yamamoto et al. (2021). In this study, the authors conducted a potential assessment of genomic selection in strawberry F1 hybrid breeding. The genotype interaction with environmental factors, cultivation systems, and the stress response is important in developing of highest nutritional quality fruit. It should be noted that all environmental factors, e.g. the type of soil, the southern or northern climate, which influence the adaptation and development of plants under different conditions, and the growing system (open field, greenhouse, etc.) play an important role in the sensory and specific nutritional characteristics of the fruit (Palmieri et al. 2017; Mezzetti et al. 2018). Diamanti et al. (2011) proposed 12 criteria for the market acceptance of the new strawberry genotypes. Among productivity and resistance to diseases, the most

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important are plant adaptability to local cultivation systems and conditions. This is directly related to plant resistance to abiotic stress. However, until recently, there has been insufficient data on the genetic control of many traits associated with abiotic resistance. Despite that, traditional breeding methods have steadily improved agronomic traits, the lack of beneficial economic traits remains a major challenge. According to Qin et al. (2008), genetic transformation is an effective method to develop new cultivars that selectively target a specific gene or heterologous trait of interest. It has opened a new era for greater creativity in strawberry breeding and germplasm utilization (Qin et al. 2008). Unfortunately, negative attitudes towards the use of genetically modified plants in many countries of the World, especially in Europe, have greatly slowed down research in this area, but there is much hope for genome editing and other possible improvements to this system (Zhou et al. 2018; Ramirez-Torres et al. 2021).

7.2.1.2

Raspberry, Blackberry, Blueberry

Berry cultivars are commonly used for the home garden as well as for industry, to sell in fresh markets or processes to preserve the properties of berries by freezing, drying, and canning. Over the past decades, the global berry industry, based on consumer demand of a year-round product on availability, had to change and grow by advancing production methods and creating new cultivars. Worldwide, there are many breeding programs specifically for Rubus species. Regardless of basing breeding targets such as high fruit quality and yield, suitability, and ornamental properties, new targets include adaptation to growing environments, increased disease and pest resistance, extended cropping season as well as good storage and processing properties (Foster et al. 2019). Farneti et al. (2020) identified the best performing cultivars to use as superior parental lines for future breeding programs, based on the variability of texture and volatile organic compounds. It was allowed by the exploitation of the genetic variability existing within the blueberry germplasm collection. Rubus fruits have high amounts of secondary metabolites, such as anthocyanins and other polyphenolics, that provide antioxidant capacity and support their reputation as “superfoods” (Vara et al. 2020). Blackberries are rich in vitamins, manganese, dietary fiber and have been suggested as a source of potential anti-COVID-19 bioactive natural food constituents (Xu et al. 2021). However, knowledge of Rubus spp. genetics and genomes are very limited, its genome has not been reported yet. Regulatory and core genes in the biosynthetic pathways are referred to as the model plant Arabidopsis thaliana (Garcia-Seco et al. 2015a; Gutierrez et al. 2017). Development of efficient transformation system for red raspberry (Rubus ideaus L.) using Agrobacterium-mediated transfer of a S-adenosylmethionine hydrolase encoding gene into raspberry cultivars ‘Meeker’, ‘Chilliwack’ and ‘Canby’ was performed by Mathews et al. (1995). This transgene enabled raspberry plants to produce fruits with a reduced capacity to synthesize ethylene extending berries postharvest life. It was shown that CRISPR/Cas9 systems can be used to edit genes of the blackberry and raspberry and characterize their function (Nidhi et al. 2021).

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7.2.2 Breeding Programs According to Williams et al. (2018), plant phenotype is expressed as a consequence of the interaction between the plant genetic background (e.g., genotype) and the biotic and abiotic conditions in its growing environment. That’s why in crop breeding it is important to establish connections between certain plant traits and how they change under environmental conditions. To achieve this, it is important to gather quantitative information on different target traits in as many as possible genetically characterized plant populations. For now, the best approach for information gathering is breeding programs that are linked to national germplasm collections. Similar programs should be maintained as a priority in the assignment of national and international institutions as it allows the development of new plant genotypes with a novel and larger genetic base that is easily shared among researchers from public and private institutions without certain genotypes being controlled exclusively by one or a few private companies (Mezzetti et al. 2018). European Union (EU) supported research programs, such as FP7—EUBERRY and Horizon 2020 GoodBerry projects, have released innovations (cultivars, methods, materials, etc.) that can increase production, growing efficiency as well as fruit quality. Few research programs are dealing with berry resistance to abiotic stress. However, in many strawberries, raspberries and black raspberries breeding programs, plant adaptation for local climatic conditions and growing conditions are an important direction. These directions are often derived from the needs of a specific geographical location. The development of cultivars with improved cold hardiness is one major objective for strawberry breeding program in Norway (Koehler et al. 2012a). Improving cold hardiness is essential for ensuring the economic sustainability of existing crops in Northern countries and expanding temperate fruit-growing in countries where they have been under-grown. As strawberries are a typical species of Rosaceae crops (e.g., peach, apple, cherry, blackberry, and raspberry), it is hoped that this knowledge can be used to improve many of these related crops (Koehler et al. 2012a). In vitro screening technologies of cold-hardy raspberry (Palonen and Buszard 1998) and strawberry (Rugienius and Stanys 2001) were developed. The resistance to drought and heat is native to tropical and subtropical countries. Screening for drought resistance of strawberry is a research topic of Ghaderi and Siosemardeh (2011), Klamkowski and Treder (2008), Nezhadahmadi et al. (2015), Razavi et al. (2011), Merlaen et al. (2020). Raspberry plant reaction to temperature fluctuations in winter was evaluated by Molina-Bravo et al. (2014). Raspberry and blackberry drought resistance breeding issues are addressed by Orlikowska et al. (2009), heat stress by Fernandez et al. (2018), Molina-Bravo et al. (2019).

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7.2.3 Fragaria Genome Cultivated strawberry (Fragaria × ananassa) is one of the youngest domestic plants, derived in the early eighteenth century from spontaneous hybrids between wild allooctoploid species (F. chiloensis and F. virginiana). Fragaria is one of the best model systems for Rosaceae family berry plants, as it is easy to cross and being a diploid plant is an advantage for genetic studies. This is also complemented with a short juvenile phase, quick fruit development as well as ability to produce a lot of seeds. Improving horticultural characteristics through three centuries of breeding has allowed strawberry production to expand globally (Hardigan et al. 2021). Several additional wild octoploid species have been used as parents in breeding, developing an admixed population of F. × ananassa with genomes that are mosaics of demographically and phylogenetically diverse progenitor genomes (Whitaker et al. 2020). Several hypotheses of subgenomic origin have emerged from cytogenetic, phylogenetic, and comparative genetic mapping studies, but detailed hypothesis about the origin and evolution of the octoploid genome has only recently been proposed with the publication of the strawberry cultivar Camarosa reference genome. Utilizing phylogenetic analyses of the transcriptomes of all described diploid species, including four subspecies of F. vesca, the putative subgenome donors for the octoploid cultivated strawberry were identified F. vesca subsp. bracteata, F. nipponica, F. iinumae, and F. viridis (Edger et al. 2019). Chromosome-scale assembly of the F. iinumae genome and reanalysis of the original data within paralogs was performed by Edger et al. (2020). The revised analysis confirmed the emergence of the octoploid strawberries genome during several stages of polyploidization involving four progeny species: diploid × diploid (F. nipponica × F. innumae) → tetraploid × diploid (tetraploid ancestor × F. viridis) → hexaploid × diploid (hexaploid ancestor × F. vesca subsp. bracteata) → octoploid ancestor. In addition, this genome assembly demonstrated that diploid subgenomes of cultivated strawberry were not static building blocks far apart from each other. On the contrary, they evolved dynamically, through homologous exchanges, that are well known in neo-polyploids (Whitaker et al. 2020). Homoeologous exchanges in octoploid strawberries are highly directed towards F. vesca subsp. bracteata subgenome, replacing large parts of other subgenomes. However, it does not mean that homoeologous exchanges are unidirectional, F. × ananassa is allo-octoploid, even when their chromosomes are a result of the merged genomes of 4 diploid subgenomic donors and their octoploid progeny. The F. × ananassa genome has been redesigned by polyploidization events, and by repeated interspecific hybridization that led to the introgression of alleles from diverse F. chiloensis and F. virginiana ecotypes in breeding. Whitaker et al. (2020) ask the question, is it possible to re-synthesize an octoploid species with a different degree of subgenomic dominance, or when another subgenome becomes dominant? The answer to this question may affect the genetic development of newly cultivated strawberry species.

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Natural hybridization and polyploidization events are evaluated and the results of transcriptomic studies and expression of genes related to plant adaptation in hybrids and polyploids of Fragaria are discussed by Liston et al. (2014). For a long time, it was thought, that polyploid is more successful in plant breeding because of naturally enhanced tolerance to abiotic stress as well as greater ecological amplitude; however further research revealed no significant differences between polyploids and diploids. The opposing forces of polyploidy and varied breeding systems, as well as the complicating elements of plant species, age, and geographic boundaries, are the main reasons for this lack of differences. Although bioclimatic studies can provide a snapshot of the species global distribution, only integrated genetic, functional and population studies can determine whether the proposed mechanisms for polyploidization/hybridization contribute to the ecological amplitude and stress resistance of polyploids, regardless of the contribution of multiple origins or age (Liston et al. 2014). Pincot et al. (2021) reconstructed the genealogy of modern cultivars, evaluating the three-century-long domestication history of strawberry. In this study, more than a thousand (87 wild octoploids and 1,171 F. × ananassa founders) cultivars were identified in the genealogy. Using global pedigree network and parent–offspring edges, Pincot et al. (2021) found that over the past 200 years selection cycle lengths of breeding have been extraordinarily long (16.0–16.9 years/generation), but decreased to a present-day range of 6.0–10.0 years/generation. These analyses uncovered evolutionary genetic forces that have shaped phenotypic diversity in F. × ananassa clearly showing differences in the ancestry and structure of American and European strawberry populations. F. vesca genome was sequenced in 2011 by Shulaev et al. (2011) using secondgeneration sequencing technology. The genome was assembled de novo and assembled into seven pseudochromosomes, this allowed the identification of around 35 thousand genes that were supported by a transcriptome mapping. This diploid strawberry sequencing lacked the large genome duplications that were seen in other sequenced genomes of Rosaceae family plants. Genome analysis created an opportunity to identify biologically and agriculturally important genes, such as genes determining plant flowering time or fruit nutritional value and taste. The macrosynthetic relationship between Prunus and Fragaria provides a hypothetical genome of the ancestor Rosaceae species having nine chromosomes.

7.2.4 Rubus Genome Rubus is a large and diverse genus of Rosaceae, with more than 740 species described worldwide. Based on the phenotypic diversity, most likely that Rubus originated in southwestern China, growing in both subtropical and arctic regions, surviving up to 4,500 m3 above sea level (Gu et al. 1993; Hummer 1996; Foster et al. 2019). There is a wide range of wild Rubus species, but the most economically important crops are red and black raspberry (R. idaeus, R. occidentalis), as well as blackberry (R. subgenus Rubus). Raspberries are diploid (2n = 2x = 14) and blackberries range from diploid

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to polyploid (2n = 2x = 14 − 2n = 12 x = 84). Red and black raspberries can be used to produce purple raspberries. Generally, blackberry varieties are not recognized as different species because there are several species in the ancestors of all varieties. Similarly, R. idaeus and several other species hybridize with the blackberry species and produce fertile plants. Natural and man-made Rubus hybrids are common (Foster et al. 2019). Till now, the high-throughput Rubus genotyping methods consist of sequencing techniques that have reduced representation, such as genotyping-by-sequencing (GBS), restriction-site associated DNA sequencing (RAD-Seq), and target capture sequencing (Foster et al. 2019). In 2012, over 7 K single-nucleotide polymorphisms (SNPs) from Rubus have been added to a multi-species Chip for genotyping (Labogena_rubus_21106; http://www.labogena.fr/en; Bushakra et al. (2012)). NGS techniques with Hi-C scaffolding and long-read PacBio sequencing have been used to generate a chromosome-scale genome assembly of a highly homozygous wild accession (ORUS 4115–3) of black raspberry (VanBuren et al. 2018). A genome assembly of red raspberry ‘Heritage’ has been reported (Longhi et al. 2014) but it is not publicly available, while a fragmented short-read-derived draft assembly of ‘Glen Moy’ and ‘Latham’ genotypes was generated recently (Hackett et al. 2018). The first attempt in Himalayan raspberry (Rubus ellipticus) transcriptome sequencing was performed by Sharma et al. (2019). In this study, 304 unigene simple sequence repeats (SSRs) were used for specific expressed sequence tag (EST)-SSR markers development. It was concluded that high polymorphism (95.3 and 93.5%) of these markers in Rosaceae genera (strawberry, apple, peach, pear) could be useful to perform diversity and cross transferability studies in all members of Rosaceae family (Sharma et al. 2019). In the connection between black raspberry and strawberry, VanBuren et al. (2018) performed a comparative genomics analysis between Black raspberry V3 and V4 woodland strawberry genomes. This analysis revealed a high degree of synteny across both genomes despite the 75 million years divergence when the pseudomolecules of the black raspberry genome could be anchored to the seven haploid strawberry chromosomes. The only observed differences were a couple of major inversions and patterns of gene-level expansion/deletion that was unique to a specific genome (VanBuren et al. 2018). Recently, genome-wide comparisons between R. idaeus, R. occidentalis, and nine other Rosaceae species have supported the high collinearity between strawberry and raspberry genomes (Wight et al. 2019). VanBuren et al. (2018) proposed that strawberry and raspberry genome similarities are due to genetic recombination, specific changes such as gene deletion, insertions, duplications as well as retrotransposon-mediated duplication or movements (Whitaker et al. 2020).

7.2.5 Interspecific Hybridization The octoploid (2n = 8x = 56) cultivated strawberry, is a hybrid plant resulting from breeding two other octoploid wild species Fragaria chiloensis L. and Fragaria

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virginiana Duch. Sometimes, to improve disease resistance and stress tolerance, already existing cultivars can be further bred with other strawberry plants, such as F. vesca (2n = 2x = 14), Duchesnea indica (2n = 8x = 56), and Potentilla tucumanensis (2n = 2x = 14). Till now, over five hundred intergeneric and interspecific crosses were performed among the wild strawberries F. vesca, D. indica, P. tucumanensis, as well as nine genotypes of the cultivated strawberry. Following the interspecific crosses, a variation in prezygotic and postzygotic barriers was detected, and all related germplasms were added into the cultivated gene pool (Marta et al. 2004). To obtain improved plant cultivars, it is important to create plants with polyploidy, as induced polyploidy results in increased vigor and often perform better than diploid cultivars. Direct crosses between octoploid strawberries and other cultivars with lower ploidy levels are often unsuccessful; however, it is not a rule as viable hybrids with a partial seed set were developed by crosses between cultivated strawberries (8x) with F. moschata (6x) and F. vesca (2x) (Trajkovski 1993). To create new breeds with higher polyploidy from lower ploidy species can be achieved by using colchicine or gametes with an unreduced number of chromosomes (Trajkovski 1993; Marta et al. 2004). Likewise, more than one decaploid (10x) cultivar was developed by crossing F. vesca (2x, colchicine-doubled) with F. × ananassa (2x). Sometimes, after interspecific hybridization and polyploidization, selective expression of genes from one of the subgenome leads to higher yield and better adaptive capacity (Zhang et al. 2015). Wang et al. (2018) analyzed the expression of 11 plant photosynthesis-related genes during strawberry hybridization (F. × ananassa × F. viridis) and chromosome doubling. Expression levels of most pentaploids were intermediate between that of the parents and were more similar to F. × ananassa. The expression level of decaploids was higher than that of pentaploids and F. × ananassa. Responses of chloroplast and regulatory genes were complex. The structural genes of the photosynthetic system showed a clear dosage effect and were expressed at a constant level. Gene expression negatively correlated with methylation levels of one CG site on RNA polymerase sigma factor 5, regulating chloroplast gene expression. Phenotypes of decaploids were more similar to F. × ananassa and did not display a more vigorous growth as other high ploidy species. It might be due to a limit to the ploidy level of F. × ananassa, above which a higher level does not increase growth and vigor (Wang et al. 2018). Some of the interspecific hybrids show improved cold hardiness. For screening of the hybrids for cold hardiness, freezing in vitro at −11 °C was used by Rugienius et al. (2006). The viability of strawberry plants after freezing in vitro varied from 20.7% (‘Elsanta’) to 100% (seedlings F. orientalis × F. vesca and F. ananassa × F. virginiana). A significant increase in heterozygosity among interspecific hybrids and a decrease in heterozygosity among domesticated descendants of those hybrids were observed. All octoploid species, including F. × ananassa, have been found to have the same allelic diversity as any wild ancestor (Whitaker et al. 2020). Rubus is a group of mainly polyploid and apomictic species with complex taxonomy and history of continuing hybridization. The only polyploid series with a predominant hybridization potential is the Glandulosi series, although the apomictic

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series Discolores and Radula can be affected by pollen donors as well as varying environmental conditions. One of the main forces in the evolution and speciation of the highly apomictic subgenus Rubus in Central Europe is sexuality in the series Glandulosi. Paleovegetation data suggest that the initial hybridizations in the two regions studied occurred at different times and that the successful origin and spread of the Radula series apomictic microspecies took several millennia (Šarhanová et al. 2017).

7.3 Tolerance to Abiotic Stress Factors 7.3.1 Low Temperatures The freezing resistance of perennial and winter annuals is induced by low temperatures, a process called vernalization, as well as by a short photoperiod (McKersie et al. 1994). Frost resistance allows hardy plants to develop effective tolerance mechanisms required for winter survival. At that time, various biochemical, physiological, and metabolic processes take place in plants. These changes are regulated by the low temperature at the gene expression level (Houde et al. 2004). Exposure to low, non-freezing temperatures of temperate plants increases tolerance to subsequent freezing temperatures—a process known as cold acclimatization (Zhu 2016). During cold acclimation, plants initiate various molecular events that increase tolerance to freezing temperatures (Kjellsen et al. 2010; Khan et al. 2014). Fundamental knowledge about plant response to low temperature was mainly analyzed in model plant A. thaliana. Based on these studies, cold-induced genes and their products have been isolated and evaluated in many plant species (Houde et al. 2004). Low-temperature responses and proteomic study of F. × ananassa cultivars (‘Senga Sengana’, ‘Elsanta’, ‘Jonsok’ and ‘Frida’) were conducted under controlled laboratory environments by Koehler et al. (2012b). Their experiments helped to identify proteins associated with cold tolerance in strawberry crowns exposed from 2 to 42 days of cold treatment (2 °C). These proteins included molecular chaperones, antioxidants/detoxifying enzymes, metabolic enzymes, pathogenesis-related proteins, and flavonoid pathway proteins. Meanwhile, several proteins were newly identified as associated with cold tolerance (aldo/keto reductase, Fra a1). Enolase, few heat shock proteins (HSPs), thaumatin-like, β-1,3-glucanase, alcohol dehydrogenase (ADH), cold-responsive (COR) dehydrins (COR47-like, Fcor1 Fcor2), flavonoid 3’-hydroxylase (F3H) were mentioned as proteins, distinguishing the cold-tolerant cultivar ‘Jonsok’ from lesser cold-tolerant ‘Frida’ (Koehler et al. 2012b). A dramatic change to the plant transcriptome has been discovered by large-scale profiling of gene transcripts, elucidating a variety of regulatory transcription networks in response to the low-temperature signal (Ciarmiello et al. 2011; Zhu 2016; Zhang et al. 2019b). As the molecular basis of response to cold stress in blueberry has not been explained clearly, a comprehensive high-throughput RNA sequencing analysis in

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order to investigate the network of gene regulation in blueberry Vaccinium corymbosum ‘Duke’ chilled for 30 days, was performed by Zhang et al. (2020). Several differentially expressed genes (DEGs) involved in various biological processes were discovered while studying the effects of cold stress. The most significantly changed pathways of DEGs were glutathione metabolism, carotenoid biosynthesis, sucrose, and starch metabolism, phytohormone transduction, protein processing in the endoplasmic reticulum, and chlorophyll metabolism (Zhang et al. 2020). NDong et al. (1997) explored the molecular basis of strawberry acclimation during cold. They isolated several complementary DNAs (cDNAs) with differential expression at low temperature from cold-acclimated strawberry (Fragaria × anannassa) cultivars ‘Chambly’, ‘Oka’ and ‘Red Coat’. These cDNA clones were designated as Fragaria cold-regulated genes Fcor1, Fcor2, and Fcor3. The author noticed that the accumulation of Fcor1 transcript reached a high level after 2 days of low-temperature exposure in roots. Meanwhile, Fcor2 highest transcript accumulation level was seen following 2 weeks of cold stress and then declined to the control level. Fcor2 transcript was found mainly in the strawberry crown and roots. Fcor3 was down-regulated by cold stress and was specifically expressed in strawberry leaves. It was suggested to use Fcor1 as a molecular marker in the screening of freezing tolerant strawberry cultivars or related Rosaceae family species (NDong et al. 1997). Rajashekar et al. (1999) investigated endogenous glycine betaine accumulation in F. × ananassa Duch. in response to the exogenous abscisic acid (ABA) application and during cold acclimation. It was revealed that glycine betaine accumulates at high levels in strawberry leaves after cold acclimation treatment for 4 weeks. This was related to an increase in their cold tolerance from −5.8 to −17 °C. Besides, the application of exogenous ABA also increased the glycine betaine levels and induced cold tolerance in the strawberry leaves (Rajashekar et al. 1999). The ortholog of Arabidopsis cold-induced C-repeat binding transcription factor gene family (FaCBF1) was identified in F. × ananassa (‘Honeoye’) by Owens et al. (2002). In response to low temperature (4 °C), a high expression of FaCBF1 in strawberry leaves was detected. Meanwhile, its expression in receptacles was not detected. Two transgenic strawberry lines, carrying CaMV35S-CBF1 construct, showed expression of CBF1 in actively growing leaf tissue and receptacles. Compared to the wild type, a significant increase in freezing tolerance of leaf discs and receptacle tissues of both lines was observed (Owens et al. 2002). Llop-Tous et al. (2002) investigated calcium-dependent protein kinase (FaCDPK1) gene expression in fruits and various vegetative tissues in strawberry (F. × ananassa Duch. cv. Pajaro) at different stages of development and ripening. FaCDPK1 gene expression was observed in many tissues (roots, stolons, meristems, flowers, and leaves), but FaCDPK1 mRNA was not detected in young fruits. High-level FaCDPK1 transcript accumulation was observed in ripe fruit under the response of low temperatures (Llop-Tous et al. 2002). Yubero-Serrano et al. (2003) identified and characterized the Fxaltp gene coded a non-specific lipid transfer protein in Fragaria × ananassa cv. Chandler. The spatial and temporal expression pattern of Fxaltp gene was studied in strawberry fruits at different development stages and tissues. Fxaltp transcripts were observed during all the fruit development and ripening

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stages, and in leaves, flowers, stolons, but were absent in the roots. However, under cold stress conditions (4 °C) a significant decrease of the Fxaltp transcripts was shown in strawberry red fruits. These experiments demonstrate, that repression of Fxaltp gene is provoked by cold stress (Yubero-Serrano et al. 2003). The cold-responsive gene expression profile in strawberry leaves at the genomewide level was identified by Zhang et al. (2019b). Under the cold treatment, they determined 2,397 DEGs, of which, around half were up-regulated and the other half– down-regulated. These differences were distributed within mitogen-activated protein kinase (MAPK) signaling, starch and sucrose metabolism, plant hormone signal transduction, flavonoid biosynthesis, and other cold response pathways. Zhang et al. (2019b) noticed that in most cases under cold stress conditions CBF-like genes were not highly transcribed. CBF1-like gene was significantly up-regulated and inducer of CBF expression ICE1 was slightly down-regulated. They also showed that transcription factor (TF) ERF105-like and a putative cold shock gene were highly expressed during cold stress in strawberry (Zhang et al. 2019b). One of the interesting applications related to cold hardiness is cryopreservation. Höfer (2016) states that plant cryopreservation in ultra-low temperatures (up to –196 °C) is a good alternative for the maintaining of plant genetic resources. Extremely low temperatures affect all biological activities of plants in such a way, that they are completely arrested while frozen, but can retain and reactivate their biological functions following thawing and transfer to the recovery medium. Vitrification procedures offer greater potential for the retention of plant tissues with unique requirements for freeze-induced dehydration. The addition of a highly concentrated cryoprotectant solution under ultra-low temperatures initiates the formation of amorphous glass in tissues and prevents the formation of ice crystals during freezing (Lee et al. 2019). A PVS2 method using plant vitrification solution 2 with a 14 day alternatingtemperature cold acclimation was used for 107 Fragaria × ananassa cultivars and 20 Fragaria wild species (51 accessions). Using the optimized medium, the average recovery for the wild species was 85.50% and for the cultivars −89.55% (Höfer 2016). Not taking into consideration plant ability to withstand freezing conditions, the lack of winter chill due to unpredictable climate can also impact crop development and yield. Climate changes were shown to affect plant flowering time that in response affect the overall berry (Fitter and Fitter 2002; Amano et al. 2010). Bud break in raspberry crops is often uneven with many of the sub-apical buds remaining in a dormant state (White et al. 2008). Plant chilling is one way to control bud break, as low-temperature release dormancy and helps with growth in the springtime. This can efficiently prevent premature bud burst that could happen in case of increased winter temperature (Rallo and Martin 1991). Natural variation between varieties of plants and across seasons does occur in flowering, fruiting time, and fruit quality (e.g., www.huttonltd.com/plant-varieties/ berries) but, increasingly, growers are seeing unpredicted problems in commercial production due to abiotic stress. Mazzitelli et al. (2007) studied dormancy transition, how it is regulated at the level of gene expression. The bud bursts were analyzed

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following growth-permissive temperature after exposure to cold. The gene expressions were tracked using the microarray method and helped to functionally categorize and to hypothesize the mechanisms for dormancy release. Till now, various genes have been found to affect plant response to cold, for example, Graham et al. (2009) using QTLs identified RiMADS_01 as a candidate gene affecting vernalization and showed associations with strawberry earlier flowering. This gene is similar to SVP (short vegetative phase) genes and responds to temperature, regulating the developmental transition to the flowering phase (Lee et al. 2007). Rantanen et al. (2015) studied the flowering pathway in the diploid strawberry and how it can be affected with temperature control. It was shown that FvFT1 (Fragaria flowering locus T1) gene regulation was affected by photoperiodic and temperature pathways. FvTFL1 expression was down-regulated in temperatures below 13 °C but up-regulated in 23 °C and these changes were not affected by photoperiod. Meanwhile, flowering was observed in the 14–18 °C temperature range during short days period. Another gene identified by Koskela et al. (2016) in F. ananassa—TFL1 was shown to affect floral induction under a range of environmental conditions. Identification of specific genes and using breeding strategies that can affect them, can be adapted to breed new cultivars with a direct effect on vegetative reproduction, for example everbearing berry plant. Numerous TFs associated with development have been identified within QTL subsequently (Hackett et al. 2018).

7.3.2 High Temperatures Heat stress is a major factor limiting plant productivity, affecting reproduction and photosynthesis (Kaushal et al. 2016). The frequency of extreme heat events is expected to grow as global warming progresses. Physiological responses to heat in plants include the production of reactive oxygen species (ROS) scavenging enzymes and heat shock proteins HSPs. However, the functions of many HSPs are still not well known (Ohama et al. 2017). HSFs—heat shock transcription factors are involved in the activation of genes in response to heat stress (Liao et al. 2016). Gene expression studies of two HSFs in strawberry showed that heat shock stimulated expression, which linked well with higher ambient temperatures and could affect plant tolerance. For example, in transgenic Arabidopsis, overexpressed FaTHSFA2a and FaTHSFB1a genes led to the activation of other stress-related genes downstream and improved thermo-tolerance (Liao et al. 2016). According to Christou et al. (2014a), sodium hydrosulfide root pretreatment resulted in the induction of gene expression levels of an array of protective molecules, such as enzymatic antioxidants, HSPs (HSP70, HSP80, HSP90), and aquaporins (PIP). At the transcriptional level, this root pretreatment activates a coordinated network of heat shock defense-related pathways, protecting strawberry plants against heat shock-induced damage (Christou et al. 2014a). The differences in tolerance to high temperatures were investigated based on gene expressions in two strawberry (Fragaria × ananassa Duch.) cultivars which

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were previously determined as high temperature tolerant (‘Redlands Hope’ = ‘R. Hope’) and sensitive (‘Festival’) (Kesici et al. 2020). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze protein expression. The authors highlighted that the coordination of Hsp70, Hsp90, and tiny HSPs, which play a significant and supplemental function in stress response, is linked to ‘R. Hope’s tolerance to high-temperature stress. Only the low molecular weight protein and transcripts that do not play a central function in the high-temperature stress response can help the sensitive cultivar ‘Festival’ react to high temperatures. Furthermore, the expression of the allergen gene was activated by high temperatures in both cultivars, with varying levels of expression (Kesici et al. 2020). In raspberry, the optimal temperature for photosynthetic assimilation is 20 °C and rapidly declines with increasing temperature, while in blackberry the temperature optimum is somewhat higher (Fernandez et al. 2018). In a microarray expression study of raspberry leaves of four cultivars under different temperatures (27 and 37 °C), Gotame et al. (2014) observed reduced expression of 38 defense-related genes and higher expression of two aquaporins, TIP2 and PIP1 (Gotame et al. 2014). Callwood et al. studied two blueberry species with heat stress at 45 °C using RNA sequencing analysis. They observed similar protein processing-related expression changes in both species. However, V. corymbosum had higher expression in amino acid, peroxisome, ascorbate, and alderate pathways, as well as fatty acid degradation, whereas V. darrowii had specific responses in photosynthetic antenna protein expression and circadian pathways (Callwood et al. 2021).

7.3.3 Drought, Salinity Stress Water is essential for plant growth, and it is an important factor determining their geographical distribution. Irrigation is widely applied in agricultural crop production. On one hand, it decreases the drought effect on plant growth, but on the other, longterm irrigation can increase soil salinity. It is estimated that about 10% of land used for agriculture is affected by salinity which causes economic losses due to decreased plant productivity (Shahid et al. 2018). Drought and soil salinity cause water stress for plants. Perception and signaling of the stress are essential for the survival of the affected plant. Water stress triggers a response in plants that leads to modifications at morphological, anatomical, and biochemical levels. Typical responses are osmotic adjustment through compatible osmolytes and transpiration regulation through stomatal closure (Folli-Pereira et al. 2016a). Strawberry is particularly sensitive to salinity. However, there are different levels of tolerance among cultivars (Sun et al. 2015; Ferreira et al. 2019). Low stomatal density and reduced transpiration were associated with higher salt tolerance in a comparison of two strawberry cultivars, however, low transpiration would lead to reduced photosynthesis and productivity (Orsini et al. 2012). Salt tolerance increased in transgenic plants with the expression of the tobacco osmotin gene. One of the parameters that distinguished transgenic plants from wild

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type was an increased proline concentration (Husaini and Abdin 2008). Proline is known to contribute to osmotic adjustment and is important for oxidative stress management. An increase in proline under environmental stress is well known and it is a physiological marker of stress. In strawberry fruits, proline concentration increased under salt treatment. Higher proline accumulation in the presence of ABA resulted in lower levels of harmful sodium and chlorine ions in strawberry leaves (Crizel et al. 2020a). Proline concentration correlates with a salt concentration in leaves, however, higher proline concentration was not related to better growth of strawberry plants (Al-Shorafa et al. 2014). Tolerance to salt is related to the plant’s ability to exclude ions from the root, sequester ions in vacuoles, limit ion transport from roots to shoots, and thus decrease its concentration in leaves. Sandhu et al. (2019) found that genes participating in sodium transport (SOS2, NHX1, and NHX2) were differentially expressed in saltsensitive and tolerant genotypes, and were related to increased sodium levels in roots, but had only small changes in leaves. Chlorine channel gene up-regulation in leaves was crucial for salt tolerance (Sandhu et al. 2019). One of the damaging effects of salt on plants is associated with decreasing potassium/sodium ratios in the cytoplasm. Under salt stress, sodium uptake increases while potassium decreases. The high-affinity potassium transporters (HKTs) gene family is important in the response to salt stress. Garriga et al. (2017) studied the expression of HKT1 and AKT1 genes of Fragaria spp. and found that the increased relative expression of sodium selective HKT1 and decreased expression of potassium channel AKT1 in roots correlates with the higher tolerance to salinity (Garriga et al. 2017). Expression pattern of four high-affinity potassium transporter genes in Fragaria vesca correlated with activity changes of ROS scavenging enzymes: superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) (Zhang et al. 2019a). Ascorbic acid is the most abundant antioxidant in strawberry fruits. Salt and drought stress reduced ascorbic acid content, and genes involved in ascorbic acid biosynthesis and degradation/recycling were differentially expressed compared to control (Galli et al. 2019). Crizel et al. (2020a) also reported changes in ascorbic acid content in salt-stressed plants. Other antioxidants, such as total phenolic compounds and the anthocyanin pelargonidin-3-O-glucoside increased, however total anthocyanins antioxidant activity did not change. Several genes from the phenylpropanoid and flavonoid pathways contributed to the synthesis of these oxidative stress mitigating compounds (Crizel et al. 2020a). Stress signaling is achieved in several ways. Calcium ion participates in various physiological processes in plants and is an important secondary messenger. Expression analysis of nine CDPKs of strawberry revealed that different genes were involved in the response to drought and salinity stress. Salt treatment caused higher expression levels in six FaCDPKs, while drought caused lower expression levels in three, and only FaCDPK4 expression was increased under both salt and drought treatment (Crizel et al. 2020b). Salinity and drought stress can be mitigated by the application of compounds that enhance the stress response and protect plant productivity. ROS, nitric oxide (NO),

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hydrogen sulfide are produced in the response to abiotic stress in low concentrations, acting as signaling molecules and in high concentration causing a damaging effect on cells. Christou et al. (2014b) pretreated strawberry roots with low concentrations of an NO donor, sodium nitroprusside, and H2 O2 just before salt stress. These compounds mitigated salt-induced stress and photosynthesis parameters, membrane leakage, and oxidative stress marker levels were similar to plants with no salt stress. Salt treatment alone resulted in visible foliar injury, decreased photosynthesis parameters, increased membrane leakage, and oxidative stress. Salt stress caused a decrease in the expression of genes of enzymes of ROS detoxification (SOD, CAT, and others) and cellular redox regulation (ascorbic acid and glutathione biosynthesis), while pretreatment with H2 O2 or sodium nitroprusside increased expression of these genes (Christou et al. 2014b). Cai et al. (2020) reported that plant growth regulator 5-aminolevulinic acid (ALA) applied exogenously to strawberry plants under drought stress caused a protective effect. The salt stress effect on photosynthetic parameters was significantly diminished by ALA treatment. Expression levels for photosystem II Psb genes increased and the level was higher than in control. ALA increased root/shoot ratio, improved water absorption, and transport to shoots, and thus water status of a plant under osmotic stress. The addition of ALA to drought-stressed plants showed that antioxidant enzyme (SOD, POD, CAT) activity was increased and oxidative damage was decreased. Interestingly, the level of oxidizing agent H2 O2 was decreased in leaves while increased in roots. There was a tissue-specific effect of ALA on ABA content and ABA receptor PYL8 expression level in leaves was lower than in roots. Expression of aquaporin genes (PIP and TIP) was decreased by drought while ALA caused higher expression of these genes, especially in roots (Cai et al. 2020). The protective effect of exogenous ALA may be explained by the higher expression of genes involved in sodium transport. Plasma membrane Na+ /H+ antiporter (SOS1), which is associated with Na+ exclusion in the roots, and a vacuolar Na+ /H+ antiporter (NHX1), are responsible for Na+ sequestration in vacuoles and highaffinity K+ uptake channel (HKT1). These changes are associated with Na+ unloading from the xylem vessels to the parenchyma cells and were up-regulated by salt stress and in the presence of ALA (Wu et al. 2019). Blackberry is a berry fruit with a high capacity for drought stress tolerance (Zhang et al. 2017). Clones of blackberry (cv. Kiowa) exhibited adaptation to drought. This was related to an increase in the leakage of leaf electrolytes and changes in the antioxidative enzyme (SOD, POD) activities. Marˇcek et al. (2015) showed that salt stress affects the growth of blackberry (Rubus fructicosus L.) plants in vitro. 35 mM of NaCl caused remarkable induction of POD, CAT, and ascorbate peroxidase (APX) activities, significantly increasing total protein content (Marˇcek et al. 2015).

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7.3.4 Iron Deficiency Stress Iron is one of the essential micronutrients for normal plant development. It is regarded in regulating life-sustaining processes like respiration, photosynthesis, chloroplast development, and chlorophyll biosynthesis where it plays important roles in electrontransport chains. Iron-deficiency (ID) stress, as well as excess amounts, causes an increase in expression of various metabolic enzymes in plants, which results in interveinal chlorosis in plant leaves, poor crop yields, or even can be fatal for plant health (Connolly and Guerinot 2002; Tripathi et al. 2018). Currently, iron (Fe) role in imparting tolerance to plants against abiotic stresses and the application of iron as a nutrient supplement is gaining increasing attention (Tripathi et al. 2018). In their investigation, Kaya et al. (2020) aimed to assess the possible roles of nitric oxide synthase (NOS) and nitrate reductase (NR) in increased tolerance to iron deficiency due to brassinosteroid (BR) in strawberry plants by using inhibitors of NR. BR contributed to the plant responses to abiotic and biotic stresses, including iron deficiency and cadmium stress (Lima et al. 2018). Iron deficit reduced biomass, chlorophyll, and chlorophyll fluorescence, while it increased oxidative stress parameters, ascorbate, glutathione, endogenous nitric oxide, and the activities of NR, NOS, and antioxidant enzymes. It was found that BR increases the tolerance of strawberry plants to the Fe deficit by enhancing active iron, endogenous nitric oxide, the activities of NOS, NR, and antioxidant enzymes’ and also lowering the levels of H2 O2 and malondialdehyde. The beneficial effects of epibrassinolide however were reversed by the inhibitor of NR, tungstate, rather than that of NOS, L-NAME, along with EB. These results showed that NR is the major source of NO accumulated in the ID-stressed strawberry plants pretreated with EB. Furthermore, NO might be a downstream signal molecule of NR induced by EB application under iron deficiency. From a future perspective, molecular mechanisms relating to crosstalk between NR and NO along with EB under Fe deficiency need to be studied. Zhong et al. (2019) evaluated expression patterns and physiological response of blueberry VcLon1 gene under Fe deficiency stress. It was revealed that VcLon1 protease reduces oxidative damage in plants by degrading carbonylated proteins in chloroplasts and effectively maintains their structure and the activity of functional proteins, contributing to iron use efficiency in plants (Zhong et al. 2019).

7.3.5 Cadmium Cadmium (Cd) is not required by plants but can interrupt cellular redox balance by inducing the production of ROS in plants and causing oxidative stress (Gallego and Benavides 2019). As hydrogen sulfide and thiamine have been widely tested in strawberry under stress conditions, cross-talk of both of these metabolites in the acquisition of stress resistance to cadmium was found (Kaya and Aslan 2020). Oxidative

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damage caused by Cd stress was reduced after the application of thiamine. Interestingly, thiamine-induced Cd stress tolerance was further enhanced by the addition of sodium hydrosulfide (0.2 mM NaHS), a H2 S donor. The findings showed that leaf NO and H2 S were both involved in induced tolerance to Cd toxicity by thiamine. One of the possibilities to decrease Cd and other heavy metals in the soil is the removal of heavy metals from aqueous solutions using activated carbon. In the study of Naderi et al. (2018) activated carbon enhanced the carotenoid content and decreased the level of lipid peroxidation. Activated carbon was derived from pomegranate peel. Increased tolerance of strawberries to Cd levels due to the use of pomegranate activated carbon was associated with increased levels of some essential elements, improvements in physical soil conditions, and decreased levels of Cd absorption.

7.4 Environmental and Growing Conditions and Plant Response to Abiotic Stress 7.4.1 Cultivation Systems New technologies create opportunities and need to transfer plant production in containers under poly-tunnel structures to reduce water and chemical inputs, lengthen the production season, as well as, to protect from unfavorable weather. There has also been some strawberry production under vertical farming conditions. These new production systems bring new challenges that will change breeding targets. Recent advances in genomic tools for Rubus helps to accelerate the breeding of new cultivars optimized for the changing environment (Jennings 2018). Organic fruit production is very popular, new agricultural innovations want to keep the agroecosystems sustainable and increase the productivity on existing agricultural land. It can be facilitated by the production of new crops that can produce sufficiently without heavy metal residues and pesticides, and have higher biodiversity and better soil health (Lockie et al. 2006). It is crucial to select and develop varieties suitable for organic production, because of that, researchers have been trying to identify differences in metabolite accumulation in plants grown for organic and non-production. It was found that strawberries and blueberries obtained from organic production contained much higher levels of polyphenols in fruits in contrast to conventionally grown berry crops (Wang et al. 2008; Olsson et al. 2006). Other studies, such as sugar content investigations in berries showed that even when the differences in sugar content were difficult to distinguish between organic and non-organic grown cultivars, the analysis was still efficient to single specific cultivar the most suitable for the organically grown system. This affects not only the taste, color, or aroma of the berry fruit; increased sugar content indicates increased resistance to biotic and abiotic stresses as sugars are closely involved in fundamental processes such as plant metabolism (Akši´c et al. 2019). Plant systems involved in the stress response are also

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involved in the process of berry ripening and affect berry quality. Examples include growth regulators, sugar transporters (Ruan et al. 2012), jasmonates (Garrido-Bigotes et al. 2018), polyphenols (Gutierrez et al. 2017; Günther et al. 2020; Mengist et al. 2020), and ABA (Liao et al. 2018; Perin et al. 2019). It is known that ethylene is a key factor in strawberry postharvest physiology. Ethylene can intensely affect the quality of harvested products. Continuous exposure to ethylene-induced an accumulation of abscisic acid in the receptacle tissue, followed by an increase in CO2 production. Ethylene also elicited malic acid catabolism and sucrose hydrolysis, with the major effect observed after 4 days of ethylene treatment. Additionally, ethylene exposure induced an accumulation of phenolics (chlorogenic acid and epicatechin) (Tosetti et al. 2020). The postharvest storability of blueberry was significantly improved by the utilization of an innovative approach of controlled atmosphere, proposed by Falagán et al. (2020), based on gradually reaching the optimal storage concentrations. This methodology allowed the decrease of blueberry respiratory metabolism when compared to standard controlled atmosphere and control treatments. This had a positive impact on quality parameters such as sugars, organic acids, firmness, and decay incidence. CO2 boilers—direct systems of heating used in greenhouses often cause incomplete combustion, resulting in hazardous gases formation. The study of Muneer and Lee (2018), showed that hazardous gases (CO, NOX , CH4 , and C3 H8 ) emitted due to incomplete combustion of direct heating systems or CO2 fertilization units resulted in the accumulation of ROS in shoots and limited photosynthetic metabolism. This indicates that even with modern growing technologies, plants must be resistant to different abiotic factors, including oxidative stress. Regulation of strawberry adventitious root formation in response to wounding, nutrient deficiency, and flooding, was investigated by Steffens and Rasmussen (2016). The roots of most terrestrial plant species are colonized by Arbuscular mycorrhizal fungi. It improves plant nutrient uptake, growth, resistance to biotic and abiotic stress and is actively used by the strawberry industry to reduce or moderate stress (https:// www.myconourish.com/). Similarly, plant growth-promoting bacteria enhance plant fitness and production. According to Todeschini et al. (2018), inoculations positively affected fruit nutritional quality—increased anthocyanin and sugar concentrations, modulated pH, malic acid, volatile compounds, and elements. The authors have shown that the concentration of some volatile substances and/or elements in strawberry fruit may be affected by specific beneficial soil microorganisms. In addition, it has been shown that the best combination of plants and microorganisms can be selected for field applications and production as well as fruit quality, healthenhancing properties can be improved. It was established, that the addition of specific substances such as phosphates can improve the survival and competence of plant growth-promoting bioinoculant bacteria (Grillo-Puertas et al. 2018).

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7.4.2 Endophytes To produce plants with desired traits from populations bearing phenotypically variable traits, plant breeding relies on the relationship between plant phenotype and genotype. Genetic variability that arise from the association of plants with endophytic microorganisms is usually overlooked; therefore, potentially missing a tool that could assist when selecting agronomic target traits (Nogales et al. 2016). Specific modifications in the plant’s genome can rarely predict phenotypic outcomes and depend on the environmental conditions or the endophytic community (Troadec et al. 2019). Changes, such as gene coding for specific antimicrobial agents, may affect not only the desired target but also the endophytic microbiome, resulting in unpredicted effects on the plants’ fitness or functionality under different environmental situations (da Silva et al. 2014; Alok et al. 2020). Current genome engineering strategies should consider endophytes as a variable that could have relevant effects on the outcome (Nogales et al. 2016). Plant-microorganisms interactions can be various and can result in the positive effects of microorganisms on plant health and growth (Berendsen et al. 2012; Contreras et al. 2016). Endophytes can influence plants health and provide fundamental support in diseases suppression and abiotic stresses tolerance (Bulgarelli et al. 2013; Hardoim et al. 2015; Khare et al. 2018). Endophytes have traditionally been defined as endosymbiotic microorganisms that invade and colonize the tissues of living plants without causing apparent disease (Petrini 1991). Beneficial effects of plant colonization with endophytes include an increase of nutrients, acquired by plants by nutrient mobilization and transport, stimulating growth and development through production of phytohormones (Egamberdieva et al. 2017), suppressing diseases by competing for space with pathogens or by inducing plant stress resistance response (Fadiji and Babalola 2020) as well as withstanding abiotic stresses (Dias et al. 2009). In the case of berry-producing plants, it was also determined that plants treated with endophytic isolates had a longer production period and produced a significantly greater quantity of fruits with increased weight. For strawberries, inoculation with endophytic fungi Botrytis produced 48% more berries with a 22% increase in berries weight and a combination of three endophytic inoculants resulted in 49% more produced berries with 51% greater weight (Murphy et al. 2019). Likewise, the endophytes can also enhance the flavor of the fruit as was determined by Verginer et al. (2010) where Methylobacterium inoculants increased biosynthesis of flavor compounds such as furanones in the host plants. Endophyte ability to promote plant growth and fitness was demonstrated on several occasions. Yokoya et al. (2017) showed how fungal endophytes of wild strawberry (F. vesca) significantly increased shoot length and protein content in salt-stressed plants from the Brassicaceae family, that in case of salinity stress helps to use water more efficiently and avoid desiccation. Sinclair et al. (2013) demonstrated how endophytic fungal symbiosis had a beneficial effect on strawberry (Fragaria × ananassa Duch.) cultivars in their tolerance to salinity, addressing the issue that different cultivars responded differently to inoculation as they did to salinity. However, the overall

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inoculation with endophytic fungi increased strawberry plant root and shoot tissue biomass, which was also seen in a plant dry mass as well as fruit yield. Another study of six endophytic Ascomycetous mitosporic fungal isolates determined that endophytes have the potential to improve wheat adaptation to heat and drought (Hubbard et al. 2014). Hubbard et al. (2014) demonstrated that all endophytic organisms improved the performance of drought-stressed plants to at least some extent, and this effect was further observed as improved germination in endophyte-free second-generation seeds arising from stressed plants. Overall improvement of plant health, can also be observed under heavy metal stress. Aziz et al. (2021) evaluated the effect of endophytic fungi isolated from Cucurbita roots on tomato (Solanum lycopersicum L.) under heavy metals cadmium (Cd) and chromium (Cr) stress. It was shown that endophytic Aspergillus niger reprograms the physicochemical traits of tomato and helped the host to adapt to the toxic effect of heavy metals by enabling the host to produce indole-3-acetic acid, proline, flavonoids, phenols, CAT, and ascorbic acid oxidase. Furthermore, A. niger facilitated the host to induce stress-responsive genes, which helped the tomato plant to chelate Cd and Cr and mitigate their toxicity. The benefit provided by endophytes in the context of abiotic stress often does not occur directly through the induction of stress-related genes but rather by preparing the plant to activate a faster and stronger defense response upon a specific stress (Esmaeel et al. 2016). Furthermore, by affecting the vital traits of the plant’s viability, endophytic microorganisms can provide a new source of selectable variability (Nogales et al. 2016). Microbiome changes display potential phenotypic plasticity and also increase the probability of passing on the acquired genetic traits to the next generation, increasing inheritance of acquired characteristics (Zilber-Rosenberg and Rosenberg 2008) that could have large repercussions in plant breeding (Nogales et al. 2016).

7.4.3 Continuous Cropping Strawberries and other berry crops are often grown in the same place year after year, especially in protected tunnel cultivation conditions or greenhouses. Longterm continuous cropping often causes different changes in the soil structure, reduces land fertility, and accumulates autotoxic substances. Continuous cropping results in low productivity, weak root systems, and a short economic lifespan (Chen et al. 2020). Strawberry in such conditions responds to complex stresses including biotic stress, such as accumulation of soil-borne pathogens and plant-feeding nematodes. Abiotic stresses include an imbalance of nutrient availability, deterioration of soil physicochemical properties, and accumulation of autotoxic substances (Chen and Liu 2019). The ROS network plays a vital role in the signal transduction of resistance to environmental stresses. During continuous cropping, three TF FaWRKY Group III gene members (FaWRKY25, FaWRKY32, and FaWRKY45), which are group III FaWRKY genes, were upregulated after continuous cropping. Amount of ROS and expression levels of peroxidase and pathogen-related protein were higher in

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continuous cropping compared to non-continuous cropping. Chen and Liu (2019) note an important role of group III FaWRKYs in the plant reaction to continuous cropping.

7.5 Elements and Factors of Abiotic Stress Resistance 7.5.1 Dehydrins A plant’s response to unfavorable climatic conditions (extreme temperatures, salinity, flooding, drought, high light) is complex, involving hundreds of genes and various biochemical, physiological processes, and molecular aspects that take place. In general, abiotic stress-inducible genes can be divided into two large groups according to their coding products (Ciarmiello et al. 2011). One type of gene involves coding products, which directly participate in plant cell resistance to environmental stress (e.g., late embryogenesis abundant (LEA) protein, anti-freezing protein, HSPs, detoxification enzymes, free-radical scavengers, etc.). Another—plays an important role in regulating gene expression and signal transduction (e.g., MAPK, CDPK, SOS kinases, phospholipases, transcriptional elements) (Ciarmiello et al. 2011). LEA proteins are non-catalytic proteins that protect plants from damage by abiotic stresses and are classified into more than seven distinct groups (Hundertmark and Hincha 2008). Abiotic stresses such as drought (osmotic stress), cold, and high salinity result in dehydration (Graether and Boddington 2014). This leads to the expression of one family of proteins, named dehydrins (Close et al. 1989). They belong to group 2 LEA proteins which are also called D-11 or responsive to ABA (RAB) (Hara 2010; Hanin et al. 2011). Dehydrins are well-characterized LEA proteins and have been studied in many plants, including a model plant A. thaliana, crop (rice, wheat, maize), and horticulture plants (apple, peach, pear, cherry, cucumber, tomato, spinach). However, there is little research done on the expression of dehydrin proteins and their role in response to abiotic stress in berry plants. In general, dehydrins accumulate in plant embryos in the late embryogenesis phase, and in vegetative tissues, they are hardly detected (Rorat et al. 2004). However, the plant’s exposure to cold, high salinity or drought stresses leads to dehydrin accumulation in high amounts in all vegetative tissues (Bray 1997). The accumulation of dehydrins in Rosaceae crops (pear, apple, cherry, strawberry) in low temperature in vitro was studied (Haimi et al. 2017). Differential accumulation of dehydrin-like proteins in Fragaria sp. (F. vesca and F. × ananassa cv. Holiday) microshoots was determined by immunoblotting. One 26 kDa band was detected in acclimated plantlets of F. vesca and two 26, 27 kDa bands in plantlets of F. × ananassa. Difference gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis revealed multiple XERO2-like dehydrin proteoforms in acclimated F. vesca microshoots (Haimi et al. 2017).

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Koehler et al. (2012b) analyzing cold-responsive transcripts in Fragaria cultivars ‘Jonsok’ and ‘Frida’, noticed changes in gene expression at the mRNA level. Two of the six analyzed transcripts were COR47-like and XERO-2-like dehydrins. Others included Fragaria cold-regulated (Fcor1, Fcor2) transcripts, cold-responsive TF FaCBF4, and F3H transcripts. The highest XERO2-like dehydrin transcript level was detected in ‘Jonsok’ crown tissue during 42 days of cold acclimation. Western blot analysis showed a strong accumulation of dehydrin protein (~37 kDa) at 42 day cold treatment, too. Meanwhile, COR47-like dehydrin transcript accumulation was rapid on the first day of acclimation but transient in both cultivars (Koehler et al. 2012b). Davik et al. (2013) used eight F. vesca genotypes to measure ADH and dehydrin levels in leaf and crown to identify diploid strawberry genotypes which differ in tolerance to low-temperature stress and to find correlations between tolerance to lowtemperature stress and certain metabolites and proteins. They didn’t detect dehydrins at any time points in strawberry leaves. Meanwhile, in crowns, two bands were detected (37 kDa and 25 kDa), which accumulated to much higher levels after 42 days at 2 °C. ADH levels were strongly induced in the cold-treated strawberry crowns and the highest protein levels were observed after 42 days after cold treatment. Ten F. vesca genotypes with contrasting freezing tolerance were selected for the metabolite experiment. Here, some metabolites (fumaric acid, citric acid, glutamic acid, aspartic acid, asparagine, raffinose, galactose, and sucrose) showed an increase in content during acclimation. Meanwhile, the levels of galactinol did not alter considerably. The authors conclude that ADH, dehydrins, and galactinol showed the greatest potential to serve as biomarkers for cold tolerance in diploid strawberry (Davik et al. 2013). Deitch (2018) selected contrasting cold-tolerant strawberry genotypes (cv. Alta, FDP817, and NCGR1363) to evaluate their differences in cold tolerance for dehydrin transcripts and protein accumulation in the crown and leaf tissue. Here, the COR47 (SKn) and XERO-2 (YnSKn) dehydrins had higher transcript accumulation and protein levels in the more cold-tolerant line in comparison to the two less coldtolerant lines. The author concluded that dehydrin transcripts and dehydrin protein accumulations are strong potential biomarkers for identifying low-temperature tolerance in diploid strawberry. In other experiments, the octoploid strawberry varieties ‘Jonsok’ (very cold tolerant), ‘Elsanta’ (low cold tolerant), and their F1 progenies were assessed for cold resistance by examining the accumulation of a few protein biomarkers (XERO-2, COR47, HSC70, β-1,3-glucanase, ADH and thaumatin) in strawberry leaf and crown by growing them in a cold room at 2 °C, 42 days. Immunoblotting analysis showed, that COR47 and XERO-2 were most strongly induced in young ‘Jonsok’ leaf and crown tissues. It was concluded that the best candidate in identifying more cold-tolerant strawberry can be XERO-2-type dehydrin because its content showed the highest correlation with survival at −8 °C in the F1s (Deitch 2018). Besides, the heat shock cognate HSC70 trend seen in the leaf could be also a particular advantage for the breeders. Dehydrins are found in various compartments in the cell, including the plasma membrane, cytoplasm, nucleus, tonoplast, plastid, mitochondrion, and endoplasmic reticulum (Hara 2010; Graether and Boddington 2014). It is believed, that localization

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of dehydrins depends on an unidentified sequence element (Graether and Boddington 2014). Sequences of dehydrin-type protein are usually described by three commonly conserved motifs (Close 1996): K-, Y-, and S-segments. Numerous studies in maize, wheat, peach, and citrus showed that plant response to abiotic stressors is linked to segment architecture and dehydrin expression patterns. For example, dehydrintype proteins harboring Kn (LTI, DHN, WCS), SKn (COR47, ERD, LTI, CpDHN), KnS (CuCor19), and YnKn (PCA60) motifs are largely upregulated by cold stress, although some are also upregulated by desiccation and salt (Graether and Boddington 2014). However, it is still unclear what role each segment plays in plant stress protection because each plant responds to stress differently depending on the structure of the plant. Badek et al. (2014) analyzed expression profiles of three cold-regulated genes CBF4 (encodes AP2 transcription regulator), COR47 (encodes dehydrin protein), and F3H (encodes flavonoid 3’-hydroxylase) in two genotypes of strawberry (‘Elsanta’, ‘Selvik’). Here, freezing temperature (−12 °C) activated all the genes analyzed and the range of changes depended on the cultivar and sequence examined. CBF4 transcripts accumulated immediately after cold treatment in both cultivars, but much higher accumulation was observed in cold-tolerant cultivar ‘Selvik’. The expression of F3H gene was different from the other two genes. No changes were noticed in the cold treated ‘Elsanta’, meanwhile induction of F3H gene was obvious in ‘Selvik’ 12 weeks after cold treatment. High correlations were observed between transcript levels of COR47 and CBF4 genes in both genotypes (Badek et al. 2014). Dehydrins and their accumulation in response to abiotic stress in blueberry have been studied. Muthalif and Rowland (1994) identified dehydrins of 65, 60, and 14 kDa as the predominant proteins present in cold-acclimated blueberry (Vaccinium corymbosum Linn.) floral buds. Panta et al. (2001) noticed that these dehydrins were induced in other organs (stems, leaves, roots) and accumulated to higher levels under cold and drought stress in different blueberry genotypes. Meanwhile, expression of the 14 kDa dehydrin was strongly induced by cold stress and to a lesser extent by drought stress in other blueberry cultivars (‘Bluecrop’, ‘Premier’) (Dhanaraj et al. 2005). Cold treatment combined with dark treatment also suggested that dehydrins may be responsive to changes in photoperiod (Panta et al. 2001). Orchard plants, including two Fragaria ananassa Duch. cultivars (cold-resistant ‘Melody’ and cold-sensitive ‘Holiday’), response to cold stress was investigated by Zalunskait˙e et al. (2008). Here, Arabidopsis COR47 gene homolog transcripts started to accumulate at higher levels after 30 days of strawberry cold acclimation at low temperatures (4 °C). This finding shows that the cold resistance of strawberry depends on cold acclimation duration (Zalunskait˙e et al. 2008). Transgenic studies with various plant species (maize, wheat, barley, peach, citrus) revealed a positive effect of dehydrin gene expression on plant tolerance to abiotic stress (Hanin et al. 2011). Houde et al. (2004) developed three transgenic lines of strawberry (Fragaria × ananassa cv. Chambly), constitutively expressing wheat Wcor410a gene in different organs (leaves, fruits, petals, stems, crown, roots). Transgenic lines accumulated dehydrin class protein at a high level and improved freezing tolerance (−17 °C) in leaves. It is likely, that overexpression of acidic dehydrin

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increased membrane stability in transgenic strawberry and this is related to the hydrophilic nature of WCOR410 protein (Houde et al. 2004). Lanham et al. (2001) isolated a few dehydrin-type genes from cold-acclimated Ribes nigrum. Here, accumulation of the b8 (K segment) transcripts in vegetative tissues during the chilling process was detected. Several reviews have already demonstrated that dehydrin encoding gene promoters have ABA-responsive elements (ABRE), C-repeat/droughtresponsive/low-temperature-responsive elements (CRT/DRE/LTRE), myeloblastosis (MYB), and myelocytomatosis (MYC) regulatory elements. Their expression is regulated by ABA-dependent (basic leucine zipper (bZIP) transcription activators— ABFs, MYBFs, and MYCFs) and ABA-independent signaling pathways (include transcription activators which bind to CRT/DRE/LTRE) (Zhu 2002; Shinozaki et al. 2003; Chinnusamy et al. 2004). Zolotarov et al. (2015) de novo identified significant motifs in the promoters of plant dehydrin genes. In total, 6 promoters were analyzed in Fragaria vesca dehydrin gene promoters with Yn SKn and SKn sequence motifs. It was noticed that these motifs are linked to ABA-dependent and ABA-independent stress response pathways and the expression of transcription activators is modulated by light (Zolotarov and Strömvik 2015). Parmentier-Line et al. (2002) examined the expression of dehydrins and dehydrin-RNA in blueberry cell cultures (‘Gulfcoast’) in response to low temperature (4 °C), ABA, and polyethylene glycol (PEG) treatments. They detected two dehydrins of 65 and 30 kDa, which differed in their response to treatments used. The 30 kDa dehydrin was induced by both cold and ABA, whereas the 65 kDa dehydrin was induced by ABA only after 2 weeks of treatment. Meanwhile, polyethylene glycol repressed dehydrins at the protein and RNA levels (Parmentier-Line et al. 2002). Dehydrin ability to undergo posttranslational modifications and mainly phosphorylation under stress conditions, in strawberry and other Rosaceae family plants was shown also (Haimi et al. 2017).

7.5.2 Lipids Lipids play important roles in plant abiotic stress response through modulation of membrane lipid composition (Burgos et al. 2011), as well as lipid-related signaling pathways. Plants remodel their membrane composition to achieve optimal membrane fluidity under changing environmental conditions by altering the degree of unsaturation of the fatty acids of the membrane lipids (Degenkolbe et al. 2012). Another parameter of membrane composition is the modification of the polar head groups of membrane lipids (Moellering et al. 2010), especially in chloroplasts, where the mole fraction of non-bilayer lipid monogalactosyldiacylglycerol affects the curvature of the bilayer and the formation of grana stacks (Wang et al. 2014). Very long-chain fatty acids serve as the precursors for biosynthesis of cuticular wax, which serves as a protective layer as well as controlling water loss (Lee and Suh 2015). One of the important signaling lipids is phosphatidic acid, which is created from membrane lipids by phospholipase D and modulates the ABA pathway (Welti et al.

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2002). Phosphatidylinositol serves as a precursor for phosphoinositides with varying amounts of phosphor groups (Meijer and Munnik 2003), which serve as water-soluble signaling molecules, able to transfer the signal through the cytoplasm. Oxylipins, including jasmonic acid, are formed from fatty acids by oxidation and are involved in biotic and abiotic stress signaling (Vu et al. 2012; Savchenko et al. 2014). While much of the work in elucidating the role of lipids in the response to abiotic stress has been done with model plants, analogs of the genes from model plants could be used in berries. However, recently, some results have been obtained from berry transcriptome studies, such as the differential expression of fatty acid biosynthesis genes of strawberry in drought stress (Galli et al. 2019). Zhang et al. (2020) found that fatty acid metabolism was activated in postharvest blueberries under cold stress (Zhang et al. 2020). Differential transcriptome analysis of blueberry populations differing in the amount of in the waxy coating of the fruit uncovered a gene acyl-acyl carrier protein hydrolase FatB (Qi et al. 2019). Model plant studies have uncovered genes related to membrane remodeling or lipid signaling, which affect the tolerance to abiotic stress (Szymanski et al. 2014). Using bioinformatics and existing berry genome resources, some of these results may be useful for designing resistant berry genotypes. For example, the overexpression of ω-3 desaturases in transgenic tobacco leads to increased tolerance to drought (Zhang et al. 2005). Similarly, in Arabidopsis, the ADS2 gene encodes a 16:0 desaturase that appears to be essential for cold stress response and freezing tolerance (Chen and Thelen 2013). As was found by Moellering et al. (2010), the Arabidopsis SFR2 gene codes a galactolipid galactosyltransferase enzyme, producing di- and trigalactolipids from monogalactolipids that then affect the membrane curvature and increase the stability of membranes during freezing. The Arabidopsis PLDα1 enzyme is responsible for the generation of phosphatidic acid from phosphatidylcholine under freezing conditions, and its knock-down mutant was more tolerant to freezing than wild-type plants (Wang et al. 2006), while the PLDδ knock-out was more susceptible. During heat stress, several changes in the Arabidopsis leaf lipid composition have been observed, a key process being the removal of unsaturated fatty acids from plastidial galactolipids and their incorporation into cellular phospholipids and triacylglycerol stores (Shiva et al. 2020). Arabidopsis Phosphoinositide-Specific Phospholipase C isoforms 3 and 9 (AtPLC3 and AtPLC9) play a role in thermo-tolerance, and overexpression of AtPLC3 conferred much higher heat resistance than wild type (Gao et al. 2014). Recombinant Arabidopsis plants expressing the mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), which suppresses InsP3- mediated signaling, were more tolerant to drought stress than wild plants (Perera et al. 2008).

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7.6 Genetic Resources of Abiotic Stress-Resistance Genes 7.6.1 Genetic Resources of Berry Plants The deep knowledge of the most useful wild and cultivated genetic diversity is necessary for the successful application of both breeding and biotechnological approaches for new cultivar breeding (Mezzetti et al. 2018). As in other crops, strawberry and raspberry breeders have applied all the available information on the pedigrees of cultivars and other material in their programs, but genetic identity errors often reduced the ability to effectively design crosses that achieve trait targets and widen the genetic base. Germplasm collections are important in maintaining distinctive genotypes for breeding in the future. On the other hand, the information alone is not enough, there is a high need to study the multiple genetic factors and their interaction with the environment and cultivation techniques in order to use and apply that information. Only then it will be possible to cultivate crops with increased adaptivity, productivity, and accumulation of fruit rich in bioactive compounds with health benefits for the consumer (Mezzetti et al. 2018). Since it is hard to evaluate a large number of commercially essential factors, such as storability, yield, growth, and resistance to biotic or abiotic stress, phenotypic response to complex stresses presents many challenges. Most expenses used on traditional phenotyping are spent on physical labor and repetitive procedures. As phenotyping technology evolves, scientists, breeders, and agronomists will be able to estimate the level of the entire plant and subsoil. Along with the progress of genomics and metabolomics technologies, the opportunities to develop specific genotypes for the prevailing climatic and market conditions are considerable. Imaging plants is far less labor-intensive than other methods of plant characterization and could be used in controlled environments, glasshouses, and other protected growing environments, and field-based systems (Ghanem et al. 2015). Plant phenotyping techniques usually rely on the data obtained during the plant’s interaction with the electromagnetic spectrum. Quantifying the proportion of electromagnetic energy reflected, absorbed, and transmitted in the visible and short wave infrared regions, as well as the emission of thermal infrared radiation, are examples of this. Currently, several platforms for phenotyping are available (e.g., www.plant-phenomics.ac.uk/en/resour ces/lemnatec-system; http://www.plantphenomics.org.au/), these with additional infield phenotyping platforms could improve selection regimes for research and plant breeding (Williams et al. 2018). Right now, there is a network of scientists designated to specific crops (e.g., berry plants). These projects can collect broad-scale data that respectively can produce more reliable results, as the generation of DNA markers and the measurement of gene function are both dependent on the environmentally controlled screening of available plant material (Nybom and L¯acis 2021). The last few projects were concentrated on large-scale genotyping of vegetatively propagated crop genetic resources. The main purpose was usually to identify the plant material conforming to the type, to analyze genetic diversity and relatedness, clarify close accessions. The second goal could be the creation of sustainable databases that

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can be utilized in research and breeding for several years ahead. DNA markers, such as SSRs and SNPs, or NGS techniques commonly used to solve these tasks have their advantages and disadvantages (Nybom and L¯acis 2021).

7.6.2 Molecular Markers, QTL Mapping 7.6.2.1

SSR Markers

DNA molecular markers can be applied in a variety of ways. DNA analysis is the most efficient method to evaluate genetic diversity and is widely used for cultivar identification, mapping and genome analyses, marker-assisted breeding, etc. (Whitaker 2011). Many molecular markers have been applied in strawberry for the characterization of cultivars or accessions revealing the importance of not only cultured but also wild plant material for germplasm diversification (Njuguna 2010; Nunes et al. 2013; Rugienius et al. 2015; Lee et al. 2016, Girichev et al. 2017, Biswas et al. 2019). Marker-assisted selection is an efficient tool for improving abiotic resistance in plants, too (Younis et al. 2020). Examples of molecular markers use are presented in Table 7.1. Simple-sequence repeats (SSRs or microsatellite markers) have been used to assess the genetic diversity, for fingerprinting, cultivar identification, linkage mapping, QTL analyses, or pedigree confirmation (Foster et al. 2019). SSRs have a wide range of application capabilities. To begin with, because of their highly polymorphic and multiallelic nature, microsatellite markers are reliable and can be easily reproduced, they also provide a cost-effective way to analyze because additional samples may be incorporated into the previously created database. Moreover, there is an opportunity to determine overall plant genetic diversity simply by utilizing SSR markers, even to distinguish differences between sexually and asexually (vegetatively) derived plants or determine the ancestry of an individual plant. However, SSRs cannot be used to combine different datasets from different laboratories due to discrepancies derived from allele size evaluations (Nybom and L¯acis 2021). Lebedev et al. (2020) using microsatellite markers were able to assess the genetic diversity of both Fragaria and Rubus species, finding that there is a high similarity between both species and specific SSRs can be used to assess different berry plant traits, such as berry color, ploidy, and origin. Identifying useful genes to study heredity traits using microsatellite markers can be achieved by analyzing related diploid species, such as strawberries. F. vesca can be used to evaluate geographically occurring differences in traits, as F. vesca or its subspecies can be found in the entire northern hemisphere. Rusu et al. (2016) using SSRs markers found significant genetic differences between Romanian Rubus cultivars (cultivated and wild) and some cultivars that are widely grown in Europe and the USA. Similary, Hilmarsson et al. (2017), had genotyped 68 SSR loci of nearly 300 strawberry genotypes collected in 31 countries and 274 locations. What they found was a relatively low genetic diversity compared to other Rosaceae species,

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Table 7.1 Molecular markers, used in the breeding of abiotic stress-resistant berry plants Plant

Genus/species/cultivar Molecular marker

Strawberry Fragaria vesca

Fragaria × ananassa Duch.

Abiotic References stress-related tolerance/resistance

SSR

Hilmarsson et al. (2017), Lebedev et al. (2020)

High-throughput high-resolution melting (HRM) and SSR assays

Noh et al. (2017)

SNP-based HRM SSR, SCAR, RAPD, KASP

Oh et al. (2019)

Double digest restriction-associated DNA sequence

Davik et al. (2015)

SSR Raspberry, Genus Rubus blackberry (German Rubus collections, Romanian Rubus accessions)

Rusu et al. (2016), Lee et al. (2016), Girichev et al. (2017)

Raspberry

Sharma et al. (2019)

Himalayan Rubus ellipticus

EST-SSR

Blackberry ‘Prime-Jim’ × SSR ‘Arapaho’ ‘Chester Thornless’ × ‘Prime-Jim’

Castro et al. (2013) Foster et al. (2019)

Strawberry F. vesca L., F. iinumae SNP Makino Fragaria × ananassa Duch.

Bassil et al. (2015) Sargent et al. (2016), Jung et al. (2017), Hardigan et al. (2019), Edger et al. (2019), Whitaker et al. (2020), Nybom and L¯acis (2021)

Strawberry Fragaria vesca Fragaria × ananassa Duch.

QTL

Temperature and osmotic stress response, freezing tolerance genetic factors

Davik et al. (2021) Nybom and L¯acis (2021) (continued)

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Table 7.1 (continued) Plant

Raspberry

Genus/species/cultivar Molecular marker

Abiotic References stress-related tolerance/resistance

Fragaria × ananassa Duch.

mQTL

Primary metabolites (raffinose, sucrose, succinic acid, and L-ascorbic acid)

Vallarino et al. (2019)

eQTL

Mature receptacle transcripts

Barbey et al. (2020)

Fragaria × ananassa Duch. Fragaria vesca, Fragaria chiloensis

EST

Differentiation of Razavi et al. drought-sensitive or (2011) tolerant genotypes

Red raspberry ‘Glen Moy’ × ‘Latham’

QTL

Low or high temperature Water stress

Graham et al. (2009; 2015), McCallum et al. (2018), Foster et al. (2019) Williams et al. (2018)

Genes associated with sugar content

Zurn et al. (2020)

Blackberry Rubus subgenus Rubus QTL

but the analysis was efficient to identify differences between samples from different continents, although the morphological differences were insignificant.

7.6.2.2

SNP Markers

Genome sequencing has made a contribution to the progress of large-scale methodologies in many major crops. SNP are universal and most abundant forms of genetic variation among individuals. SNP analysis has relatively easy application as it is resilient to crude strawberry DNA extracts and creates an abundance of information from array genotyping that is accurate and easy to score (Noh et al. 2017). Various crop-specific SNP array systems have been created and are now being used for diversity assessment and marker-trait association analyses (Nybom and L¯acis 2021). The new SNP array developed by Bassill et al. (2015) opened new opportunities in investigations of strawberry and other plants with complex genomes (Sargent et al. 2016; Whitaker et al. 2020). This was a significant step for the easy production of genotypic information with high reliability. This SNP array has also been used for cultivar discrimination (Jung et al. 2017), mapping of agronomically significant

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traits, marker development, and genome-wide prediction (Whitaker et al. 2020). Other common assays, for breeding applications in SNP detection, is competitive allele-specific polymerase chain reaction (KASP) and high-resolution melting (Noh et al. 2017). These SNP arrays also have the advantage of allowing subsequent database additions later (Nybom and L¯acis 2021).

7.6.2.3

QTLs, EST Markers

Stress-related QTLs in various agricultural crops (cotton, maize, sorghum, barley, wheat, and rice) have been reported by many authors (Younis et al. 2020). Razavi et al. (2011) tried to correlate the genetic structure of different Fragaria (F. × ananassa Duch., F. vesca, F. chiloensis) genotypes and strawberry response to drought. Their study revealed that expressed sequence tag (EST) markers were more effective at differentiating drought-sensitive or tolerant genotypes compared to amplified fragment length polymorphism (AFLP) markers (Razavi et al. 2011). 16 EST markers out of the 24 were generated based on sequences of some functional genes (e.g., APX, CAT, RAB DREB, FaSPS, FaOLP, AKR) and can be useful for the marker-trait associated research in strawberry with no crossing and segregation to discover QTLs implicated in drought tolerance. Davik et al. (2021) used a Fragaria vesca mapping population of 142 seedlings separating for differential responses to freezing stress and QTL analysis. Their experiments revealed a single significant quantitative trait locus on Fvb2 in the physical interval 10.6 Mb and 15.73 Mb. 5.1 Mb physical QTL interval contained several predicted genes (ADH1, ERD10, PIP2 aquaporins, ascorbate oxidase, a hAT dimerization domain-containing protein, CTR1 kinase, B1L, ASMT, EXPLA2, TF RDUF2, a ninja-family AFP2-like TF, NAC TFs), which had putative roles in plant temperature or osmotic stress response and freezing tolerance (Davik et al. 2021). The first genetic linkage map from a ‘Glen Moy’ × ‘Latham’ red raspberry population was created by Graham et al. (2004), and this map has subsequently been improved using GBS (Hackett et al. 2018). Analysis of this population has identified numerous QTLs affecting different traits (Graham et al. 2009; McCallum et al. 2018; Foster et al. 2019). Two QTLs that play a role in the genetic regulation of the crumbly fruit disorder were identified. It was observed that environmental factors such as low or high temperatures at particular time points in development appear to play an important role with variations in the extent of crumbliness apparent from year to year (Graham et al. 2015). A subsequent study has identified a further QTL and examined the expression of genes between crumbly and non-crumbly fruit on a raspberry microarray (Scolari et al. 2021). QTLs of hyperspectral traits, including water stress, were mapped in an attempt to develop high-throughput phenotyping approaches (Williams et al. 2018). RosBREED (Rosaceae species breeding program) project focused on developing and applying modern DNA tests and related breeding methods to deliver new cultivars or cultivated varieties of rosaceous crops (Whitaker et al. 2020). The project was organized in a way, that helped to create data sets from different systems using the

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same methods for the systematic phenotypic examination of the reference germplasm collections. This allowed researchers to merge data sets ensuring that trait-DNA connections were reliably discovered. During this project, DNA tests in breeding were based on creation and validation on specific markers and were able to identify important breeding parents with specific resistance alleles that can be used to create new resistant cultivars in just one generation (Mathey et al. 2013). To further assist researchers in new improved cultivar development Oh et al. (2019) suggested a strawberry DNA testing handbook that helps to identify tests that are available to their breeding program as well as assist in their implementation. These resources are openly available and will be continually updated based on developments on new or already existing tests (Whitaker et al. 2020). However, marker-assisted selection for specific functions can be used only for monogenic and oligogenic traits that are controlled by a few QTLs with medium to large effects—individual loci explain at least 10% of the variance. In the case of traits that are controlled by many loci, each with a very small effect—polygenic traits, it is more appropriate to address them using genome-wide association studies or genomic selection (Nybom and L¯acis 2021). Genome-wide association studies were created to find QTLs by looking for correlations between genome-wide markers and trait phenotypes. Many unrelated genotypes, such as germplasm collections, are required because they can yield higher resolution mapping than traditional off-spring families. Furthermore, multiple QTLs, that can be determined for a particular phenotype, are not restricted by the segregation products of a particular cross but a lot of QTLs that underlie the feature and genetic diversity of the germplasm collection can be studied, too. GBS-derived SNPs are becoming more widely used, even in crops without an annotated reference genome. These SNPs can provide crucial information on marker-trait relationships in addition to calculating genetic diversity and other tasks. However, it’s difficult to imagine how SNP data from multiple labs may be combined to form larger data sets (Nybom and L¯acis 2021). NGS technologies and platforms have been applied for the discovery of unique ESTs (Emrich et al. 2007). The recovery of large numbers of ESTs can be used to determine expression levels of genes across entire genome sequences. Rivarola et al. (2011) reported the analysis of a substantial set of Fragaria vesca f. semperflorens ESTs from seedlings and various tissues of mature plants subjected to water, temperature, osmotic stress conditions, and their combinations. It was revealed 1286 unique sequences that had no matches with other Rosaceae EST sets. Among them were six singletons assigned to “stress-related” gene ontology categories (Rivarola et al. 2011).

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7.6.3 Genotyping, Genetic Maps Strawberry genotyping followed after human and model plant genotyping achievements. The main computational challenges associated with the general application of GBS and other assisted NGS technologies in octoploid strawberries have been limited. Difficulties are general to all species but are more heightened in cross-fertilized polyploids (Feldmann et al. 2019; Whitaker et al. 2020). According to Whitaker et al. (2020) currently available new platforms for DNA genotyping are in disagreement with information that was collected before the octoploid reference genome. In the previously published research that has published mapping experiments, sequences of DNA markers were too short or nonspecific, resulting in the inability to cross-reference physical and genetic mapping information or even prevent creating universal linkage group nomenclature (Hardigan et al. 2019). However, the exception was ddRAD (double digest restriction-associated DNA) markers (Davik et al. 2015), which were used for the initial scaffolding of the previously mentioned octoploid reference genome (Edger et al. 2020). These long-standing problems have been solved by developing a new genotyping array, consisting of 850,000 SNPs. These new arrays are designed to detect common DNA alterations in domesticated populations and provide telomere-totelomere coverage. Only DNA variations and reference DNA sequences that are matched to a single homoeologous chromosome in the octoploid reference genome were included. FanaSNP is another array that was developed with 50,000 subgenomespecific SNPs, including genetically mapped SNPs from the iStraw35 array, allowing genomic and physical mapping data cross-study. This ability to use information across studies revealed diverse octoploid genetic backgrounds in several wild and domesticated strawberry populations (Hardigan et al. 2019; Edger et al. 2019). These latest advances in genotypes and maps are believed to have a major and direct impact on applied genetic research and strawberry breeding. Nevertheless, other investigation questions about the efficacy of these tools exist, especially concerning diversity among the currently undescribed genomes. For example, what large-scale structural variants exist in the octoploid strawberry germplasm? Recent advances in long-reading sequencing platforms (such as PacBio and MinION) have significantly reduced costs and increased reading length and should soon allow for cost-effective assessments of structural variants in cultivated strawberry pangenome. To a lesser extent, what percentage of cultivated strawberry genes differ in presence and absence? Recent studies of pangenome in plants have revealed that a significant proportion of gene content has differences in presence and absence. This indicates that strawberry genes will be lost if a single octoploid reference genome will be used. Then the question arises: how many individuals must be included in the development of a beneficial pangenome to obtain the greatest variance in gene content? These issues will soon be addressed as additional octoploid genomes become available (Whitaker et al. 2020). As mentioned before, the genetic linkage map of two red raspberry populations was created by Graham et al. (2004), in 2018 this map was supplemented with GBS

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analysis data (Hackett et al. 2018). The work in raspberry mapping and QTL analysis has led to the release of ‘Glen Mor’ (https://www.huttonltd.com/services/plant-variet ies-breeding-licensing/raspberry/glen-mor) using root rot markers identified previously (Graham et al. 2011). The first tetraploid blackberry genetic linkage map was constructed by Castro et al. (2013) from a full-sib family of ‘Prime-Jim’ × ‘Arapaho’ that was segregating for thornlessness and annual fruiting. Another linkage map of tetraploid blackberry was created in the population of tetraploid ‘Chester Thornless’ × ‘Prime-Jim’. Interestingly, even when a genetic linkage map provides a lot of new and valuable information; no markers were identified for thornlessness or annual fruiting revealing that genetic mapping can be improved and requires further studies (Foster et al. 2019).

7.6.4 Genetic Engineering Transgenic strawberry (Fragaria × ananassa Duch. cv. Chandler) lines overexpressing osmotin were produced (Husaini and Abdin 2008). It was estimated that transgenic plantlets could maintain a higher level of free proline, had higher chlorophyll and total soluble protein contents when they were exposed to salt stress (150 mM, 200 mM NaCl). Khammuang et al. (2005) successfully constructed plasmid vectors harboring strawberry optimized codons of AFP gene, encoding HPLC-6 type III antifreeze protein from Antarctic fish. Transgene detection was confirmed by PCR in ten transgenic strawberry lines, but further experiments, showing the effect on cold stress, was not reported (Khammuang et al. 2005). Strawberry transformation systems have also been developed in the James Hutton Institute with improved non-genotype-specific regeneration techniques. Numerous transformations have been performed, including gene insertion to modify fruit quality and other traits, including resistance to vine weevil (McNicol et al. 1997; Graham et al. 2002). Meng (2006) attempted using genetic engineering for improving the cold hardiness of blackberry Rubus sp. L. cv. Marion. However, transgenic shoots were not obtained (Meng 2006). Transgenic plants with an expression of tobacco osmotin gene and increased salt tolerance were developed by Husaini and Abdin (2008a). The use of CRISPR-Cas9 system in octoploid strawberry has been already tested by Martín-Pizarro et al. (2019) to target the floral homeotic gene APETALA3 (AP3). CRISPR-Cas9 edited lines of strawberry showed abnormal development of the stamen and the fruit. A study of the target locus revealed gene editing differences between CRISPR-edited lines and mutations in eigh AP3 gene copies of the Fragaria genome. More crucially, mutations were maintained in clonal plants developed from runners, providing support for CRISPR-Cas9 editing during strawberry plant propagation (Martín-Pizarro et al. 2019).

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7.7 Genomic Approaches and Phenotype Prediction 7.7.1 Omic Approaches and Abiotic Stress Resistance It’s hard to get abiotic stress-tolerant plants. Abiotic stress responses in plants are multigenic and genetically complex, in contrast to plant resistance to biotic stress which are dependent on monogenic traits (Chamoli and Verma 2014). Furthermore, genome-based estimation of plants’ response to a specific factor relies on the idea that the genome is a static structure inherited from parents. This concept is not representative of a final phenotype that is shaped by both plant development and changing environment (Kosová et al. 2018). Because of that, it is more difficult to control, develop, and engineer plants with specific phenotypes based on only genetic changes (Agarwal et al. 2013). By now, following the developments in sequencing technology, a huge range of plant genome sequences are available in various databases, revealed a new challenge to identify correlating structure with function (FritscheNeto and Borem 2014). One of the ways to avoid only gene-based selection is to analyze transcript, protein, metabolic changes that are relevant for specific plant stress and use them to identify the most relevant specific genes in response to a specific stressor (Sakina et al. 2019). Comprehensive and multidisciplinary approaches, such as transcriptomics, proteomics, metabolomics, and physiognomics, help to identify specific genetic engineering strategies and offer a better strategy to improve stress tolerance in modern crops (Muthamilarasan et al. 2019; Li and Yan 2020). Research approaches, related to berry plant resistance to abiotic stress are presented in Table 7.2. Transcriptome analysis is the most efficient tool to characterize the functionality of genes. The transcriptional profile is a sensitive indicator of stress and transcriptomic analyses create a possibility to identify genes that are important for adaptation and survival during abiotic stress (Feder and Walser 2005). Transcriptome sequencing of strawberry (Fragaria × ananassa) plants identified genes, revealing cold-specific expression as plant’s responce to cold stress. These changes help to illustrate the plant’s response and to identify attractive candidate genes to study cold stress in plants from the hundred gene changes that occur during stress (Zhang et al. 2019b). The lack of dense genetic maps and large high-throughput marker collections limits gene isolation and breeding of improved varieties in blackberry. The first Rubus sp. (cv. Lochness) transcriptome using RNA-sequencing technology was performed by Garcia-Seco et al. (2015b). Access to the sequences of this crop transcriptome allows to identify transcripts with differential abundance and genetic variations, thus accelerating its breeding and facilitating attempts to improve fruit quality (GarciaSeco et al. 2015b). Similarly in Rubus, Scolari et al. (2021) utilized a raspberry microarray to study the environmentally induced crumbly fruit disorder. Reference genes from a blueberry fruit transcriptome were selected under different abiotic stress conditions (NaCl, NaHCO3 , NaCl+, NaHCO3 , AlCl3 treatment, simulated drought) and analyzed by RT-qPCR (Deng et al. 2020). The optimal set of reference genes

Species/cultivar

Rubus sp. ‘Lochness’

Fragaria × ananassa Duch. ‘Korona’

Fragaria × ananassa Duch.

Strawberry Fragaria vesca L.

Raspberry

Research approach

Proteomic study

Transcriptome sequencing

Studied effect

Gene/protein/metabolite

References

Koehler et al. (2015)

Rohloff et al. (2014)

Koehler et al. (2012b)

Zhang et al. (2019)

Gu et al. (2016b)

Salinity, drought, cold and heat

(continued)

Cheng et al. (2018)

Xie et al. (2020) DNA methyltransferases genes (MET1 - 1, CMT3—3, DRM1/2—4, and DNMT2—1), putative DNA demethylase genes (DME—1, DML—3)

PP2A/TBP, EIF/UBCE, PP2A/HIS, TBP/GAPDH, TBP/EF1a Garcia-Seco et al. (2015b)

Amino acids, pentoses, phosphorylated and non-phosphorylated hexoses, and distinct compounds of the raffinose pathway

Fructose, galactinol, and raffinose

Molecular chaperones, antioxidants/detoxifying enzymes, metabolic enzymes, pathogenesis-related proteins, flavonoid pathway proteins Dehydrins (COR47-like, Fcor1 Fcor2)

DEGs

6 mA modifications

NaCl, NaHCO3 , NaCl + NaHCO3 , AlCl3 treatment, simulated drought

Cold stress

Cold or freezing stress

Gene expression profiles to cold stress

DNA Different stages of ROS1 gene homologs: methylation/hypomethylation fruit ripening FvMET, FvCMT, FvCMT

DNA methylation and demethylation

Transcriptome

Integrative “omic”: metabolites, proteins, and transcripts

Fragaria × Metabolite profiling ananassa Duch. ‘Polka’, ‘Honeoye’

Fragaria × ananassa Duch. ‘Elsanta’, ‘Frida’, ‘Senga Sengana’, ‘Jonsok’

Strawberry Fragaria × ananassa

Plant

Table 7.2 Research approaches, related to berry plant’s resistance to abiotic stress

7 Genomic Design of Abiotic Stress-Resistant Berries 233

Blueberry

Plant

Vacciniu ashei ‘Powderblue’

Floral and fruit development (sulfur metabolism, hormone signal transduction, and anthocyanin biosynthesis

miRNAs (vas-miR-14, vas-miR-20, vas-miR-25, and vas-miR-46, vas-miR6149a and vas-miR156 vas-miR166a, vas-miR166g-3p, vas-miR535d, and miR168a-5p)

Fan-miR73

UV-B and salt stress treatment

Fragaria × ananassa Duch. ‘Toyonoka’

108–139 miRNAs, 113–114 miRNAs miR164 family (mdmmiR164d_ 1ss21AC, mdm-miR164e and mdm-miR164f_1ss21TA)

Low temperature

HKMTase genes SET gene, LSD HDMase genes, JmjC domain-containing genes

FaMET, FaDRM

Gene/protein/metabolite

Fragaria ananassa L. ‘Zhangji’

RNA interference by sRNAs (miRNAs)

Heat and cold stresses

Histone post-translational modifications

Fragaria ananassa L. ‘Zhangji’

Micro-propagation

DNA methylome

Fragaria vesca L.

In vitro propagation

DNA methylation

Fragaria × ananassa Duch. ‘Toyonoka’, ‘Allstar’

Studied effect

Research approach

Species/cultivar

Table 7.2 (continued)

Yue et al. (2017), Li et al. (2018)

Li et al. (2016)

Li et al. (2017)

Xu et al. (2015b)

Gu et al. (2016a)

Niederhuth et al. (2016)

Chang et al. (2009), Zhang (2014)

References

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was determined: PP2A/TBP (salinity), EIF/UBCE (alkalinity), PP2A/HIS (salinityalkalinity), and TBP/GAPDH (drought) and TBP/EF1a (AlCl3 ). Besides, a combination of eukaryotic initiation factor 4A (EIF) and TATA-box binding protein (TBP) genes was the best for all abiotic stresses analyzed (Deng et al. 2020). However, the transcript abundance is not directly correlated with the protein content in specific stressor-affected plants. In this case, proteomics includes the complete changes in protein profile at the cellular, tissue, and organ levels acting as a link between gene expression and final metabolic response. Studies of the plant proteome and protein biological functions in plants exposed to stress reveal various changes because detected proteins are already altered by different modifications such as phosphorylation, glycosylation, sulfation, prenylation, acetylation, and ubiquitination (Sakina et al. 2019). These modifications are key variants in shaping a plant’s phenotype by adjusting physiological traits to changes in the environment (Kosová et al. 2018). Koehler et al. (2012b) analyzed cold or freezing stress at the proteomic level in strawberry crown tissue between different cultivars with varying tolerance to cold. The two-dimensional electrophoresis (2-DE) identified a substantial number of changes in protein expression levels. First, the 2-DE analysis revealed differences between different genotypes, such as one of the cultivars ‘Frida’ cold response, highlighting proteins specific to flavonoid biosynthesis, while the more freezingtolerant cultivar ‘Jonsok’ had a more comprehensive suite of proteins including those involved in antioxidation, detoxification, and disease resistance. The study helped to identify overall protein changes associated with enhanced cold tolerance that can be used to facilitate conventional breeding strategies for cold-tolerant strawberry plants (Koehler et al. 2012b). The metabolome is a final product of gene expression that defines the final biochemical phenotype of a specific cell or tissue (Ghatak et al. 2018). Metabolite profiling reveals the role of metabolites like ascorbate, glutathione, phenols, proline, amino acids in conferring tolerance to stressed plants, providing the biochemical status of an organism that can be used to monitor or assess gene function (Parihar et al. 2019). Rohloff et al. (2014) studied changes in two strawberry cultivars during cold stress. Significant differences were detected in strawberry tissues in terms of levels of fructose, galactinol, and raffinose. No major genotype-dependent differences were identified under normal conditions and the difference between the two cultivars was observed only in metabolic changes following the environmental factors (temperature, day length). These metabolic changes are essential to induce cold and freezing tolerance in cultivated strawberry and could help identify cold-resistant genotypes (Rohloff et al. 2014). Combined omic approaches were applied by Koehler et al. (2015) to assess underlying metabolic processes and regulatory mechanisms in Fragaria × ananassa Duch. ‘Korona’ under low temperature. Strawberry acclimation to cold was examined by measuring metabolite, protein expression levels in strawberry plant tissues. Metabolite profiling helped to identify specific compounds containing structurally annotated primary and secondary metabolites that revealed a small increase in protective metabolites and distinct compounds of the raffinose pathway. 2-DE proteomics showed 845 spots of differently expressed proteins, and in response to the cold,

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around 5% of them changed dramatically. Identified proteins were in agreement with changes found with metabolite profiling, most of them were associated with general metabolism or photosynthesis. In addition, to compare and contrast transcript levels, a microarray was employed characterizing dozens of cold-associated transcripts. Levels of several potential key contributors to cold tolerance were then confirmed by qRT-PCR (Koehler et al. 2015). The response of a plant to environmental changes is based on many molecular interactions at the genome, transcriptome, proteome, and metabolome levels. The plant’s response to abiotic stress has a certain degree of similarities at the genome level across species, yet the relative expression may vary depending on specific environmental and development variations. To understand the plant’s final response to a specific stressor, omics approaches play a significant role in explaining distinct pathways that reveal adjustments in physiological traits and the overall response mechanism (Ciarmiello et al. 2011; Ramalingam et al. 2015).

7.7.2 Transcription Regulators During evolution, plants have developed many mechanisms to cope with adverse conditions which alter the abundance of many transcripts, metabolic and regulatory proteins through transcriptional activation or repression of genes (Wong et al. 2006; Jiang et al. 2007). When plants receive any sign of stress, signaling first is activated in the cell membrane. As a consequence of this, different intermediate stress genes are activated, which has the function to activate TFs that further bind to different types of protective genes (Panjabi-Sabharwal et al. 2010). Transcription factors are proteins that bind to the promoter and enhancer regions of DNA (cis-acting elements). They regulate gene expression and protein synthesis by inducing or repressing the activity of the RNA polymerase (Mitsuda and Ohme-Takagi 2009; Franco-Zorrilla et al. 2014; Biłas et al. 2016). The TFs activators and suppressors are regulating the transcription of target genes, which activates a network of signaling events that are then determined by plant tissue type, developmental stage, or environmental condition (Wyrick and Young 2002). Examples of transcription regulators are presented in Table 7.3. There are about 1,500 TFs involved in stress response and the transcriptional regulation involved in abiotic stress in plants is extremely complex (Riechmann and Ratcliffe 2000). Jaqueline da Silva and Costa de Oliveira (2014) reviewed TFs (ZFPs, MYB/MYC, NAC, bZIP, WRKY, HSFs) in different plant species (rice, Arabidopsis, soybean, wheat, tomato). Transcriptome analysis of postharvest blueberries (Vaccinium corymbosum ‘Duke’) revealed 45 TFs families involved in berry pitting responses to cold stress: MYB, bHLH, bZIP, WRKY, NAC, C2H2-zinc finger proteins, and others (Zhang et al. 2020). Significantly down-regulated in chilled blueberries were ZIP, Ap2, and WRKY TF families. Meanwhile, the bHLH family was significantly up-regulated. Transcriptional regulatory systems or regulons can function either in ABAdependent or ABA-independent pathways. Changes in gene expression in the

MYB

WRKY family

Red strawberry Fragaria × ananassa ‘Sweet Charlie’ white strawberry Fragaria × ananassa ‘Snow Princess’

Fragaria vesca ‘Hawaii 4’

Fragaria × ananassa Duch.

HSF

FaMYB10 and FaMYB1

FaTHSF (FaTHSFA2a, FaTHSFB1a)

FvNAC01—FvNAC37 genes

Stress-related cis-elements

FaWRKY genes

Fruit developmental stages FvWRKY genes Drought, salt, cold, and heat

ABA-dependent signaling stress

Heat stress

Abiotic stresses (heat, cold, FvHsf genes drought, and salt), biotic stress (powdery mildew infection), and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid)

HSF

Fragaria × ananassa Duch. ‘Toyonoka’

Cold, heat, drought, salt

NAC

AP2/EREBP genes FveERFs FvDREBs

Response to drought, cold stress

DREB

Fragaria vesca L.

Gene/protein

Strawberry

Studied effect

Transcription factor

Species/cultivar

Plant

Table 7.3 Transcription regulators, linked to the berry plant’s resistance to abiotic stress

(continued)

Chen and Liu (2019)

(Zhou et al. (2016), Wei et al. (2016)

Wang et al. (2020)

Liao et al. (2016)

Hu et al. (2015)

Zhang et al. (2018a), Liang et al. (2020)

Wang et al. (2019), Dong et al. (2021)

References

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Leucine zipper (bZIP)

Fragaria vesca L.

ERF, MYB, and WRKY

Himalayan Rubus ellipticus genotypes/cultivars

Leucine zipper (bZIP)

Fragaria species: F. × ananassa, F. iinumae F. nipponica, F. nubicola, F. orientalis, and F. vesca

Raspberry

ERF, MYB, and WRKY

Fragaria × ananassa Duch. ‘Sweet Charlie’, ‘Selva’, ‘Torrey’

Vaccinium corymbosum ‘Duke’ Various TFs

Transcription factor

Species/cultivar

Blueberry

Plant

Table 7.3 (continued)

Fruit ripening

Response to cold stress

Drought and heat stress

Stress-related cis-elements

Fruit ripening

Studied effect

MYB, bHLH, bZIP, WRKY, NAC, C2H2-zinc finger proteins

FvbZIP

bZIP genes

Gene/protein

Sharma et al. (2019)

Zhang et al. (2020)

Wang et al. (2017)

Liu et al. (2017)

Sharma et al. (2019)

References

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ABA-dependent pathway result in enhanced plant tolerance to dehydration stress. Dehydration-responsive element-binding protein 1 (DREB1)/C-repeat binding factor (CBF) function in ABA-independent cold-responsive gene expression and DREB2 regulons are involved in dehydration and heat-responsive gene expression (Sakuma et al. 2006; Nakashima et al. 2009). In addition to these pathways, the NAC (NAM, ATAF1/2, and CUC2) and MYB/MYC oncogene regulons are also involved in abiotic stress-responsive gene expression (Saibo et al. 2009; Shao et al. 2015). DREB TFs belong to the APETALA2/ethylene-responsive element-binding protein (AP2/EREBP) superfamily and are characterized by at least one highly conserved AP2 domain. 115 (Wang et al. 2019) and 119 (Dong et al. 2021) AP2/EREBP genes were identified in F. vesca genome. Dong et al. (2021) investigated expression profiles of FvDREBs in response to drought stress in strawberry leaves. It was noticed that the expression of a few FvDREB genes (FvDREB8, FvDREB1, FvDREB20, FvDREB30) from different subgroups was variable and unstable under drought conditions. They revealed that FvDREB8 plays a crucial role in the begging stages of plant responses to drought stress. Besides, FvDREB1, FvDREB2, FvDREB6, FvDREB30, and FvDREB18 showed different expression patterns in young and old leaves and FvDREB6 appeared to be tissue-specific (Dong et al. 2021). Zhang et al. (2018a) identified NAC family (FvNAC01—FvNAC37) genes in the woodland strawberry F. vesca L. genome. It is one of the largest groups of TFs (Puranik et al. 2012; Shao et al. 2015). In general, the N-terminal region of a NAC TF contains a highly conserved DNA binding NAC domain and a variable C-terminal segment. The NAC domain is involved in the DNA binding and dimerization of proteins with comparable domains. The C-terminal region is involved in transcriptional control (Puranik et al. 2012). Zhang et al. (2018a) also analyzed the structural features of NAC genes, indicating that the majority of the conserved motifs in NAC proteins are found in the N-terminal region and that these motifs may be critical for their function. The NAC TFs are plant-specific, having various functions in plant development processes (seed, embryo development, shoot apical meristem formation, leaf senescence, cell division) and response to abiotic stresses (drought, salinity, cold, and submergence) (Nuruzzaman et al. 2013; Liang et al. 2020). Zhang et al. (2018a) experiments showed high expression levels of some FvNAC genes in a specific tissue. Similarly, in different strawberry tissues, Liang with co-workers (2020) analyzed FaNAC2 spatial and temporal expression patterns and have determined that it was highly expressed in shoot apical meristem, old leaves, and flowers of strawberry plants. Zhang et al. (2018a) investigated FvNACs expression profiles in response to cold, heat, drought, and salinity. It was reported that a few FvNAC genes significantly contributed to abiotic stress resistance in strawberry, more than 52 genes were identified as up- or down- regulated in response to a specific stressor, suggesting that several FvNAC genes might be chosen as candidates for their potential use in genetic breeding in woodland strawberries (Zhang et al. 2018a). Meanwhile, Liang et al. (2020) demonstrated that FaNAC2 gene from F. × ananassa cv. Benihoppe can serve as a candidate gene to enhance stress tolerance. They detected expression

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changes of the FaNAC2 gene in tissue-cultured seedlings of strawberry in response to cold (4 °C), salt (200 mM NaCl), and drought (20% PEG 6000) stress. Expression was strongly up-regulated in shoot apical meristem and leaves. To investigate how FaNAC2 functions in abiotic stress resistance, they transformed FaNAC2 into Nicotiana benthamiana under the control of a CaMV-35S promoter. It was shown that overexpression of FaNAC2 improves stress tolerance in transgenic tobacco. Transgenic plants showed higher drought, cold and salt tolerance. In addition, expression changes of a few genes (NbP5CS1, NbP5CDH, NbproDH) in proline biosynthesis were detected, too. The proline content in transgenic lines was higher than in wild-type strains under the abiotic stress condition. The author concluded that FaNAC2 gene might be involved in plant abiotic stress tolerance by regulating proline metabolism (activating proline synthesis and inhibiting its degradation) (Liang et al. 2020). Another family of TFs, heat shock transcription factors (HSFs), function in transcriptional activation of chaperons, known as heat shock proteins (HSPs). HSFs have been identified from more than 20 plant species, including Arabidopsis, tomato, cotton, rice, wheat, peach, soybean (Wan et al. 2019). They play important role in plant heat stress response and adaptation (Wan et al. 2019) and some abiotic stress response elements were found in the Hsf gene promoters. HSFs comprises a DNA-binding domain at the N-terminus, hydrophobic coiled-coil region, nuclear localization signal motifs, and a C-terminal activation domain rich in aromatic, hydrophobic, and acidic amino acids, the so-called AHA motifs (Scharf et al. 2012). 17 HSFs were identified in F. vesca (Hu et al. 2015). Six contigs and three unigenes were confirmed by Liao et al. (2016) to encode HSF proteins (FaTHSFs) in F. × ananassa Duch. cv. Toyonoka. Heat shock elements (HSEs) were identified in few FvHsf genes (C1a, A2a, FB2a, A4a, A6a), as well as low-temperature responsiveness elements (LTRE) in A1b, A4a, and A8a genes. In ten FvHsf genes, MYB binding sites were discovered. Heat stress (42 °C) worked as a positive responder for 15 FvHsfs genes, according to their expression patterns. Also, most of these genes (including FvHsfA2a, FvHsfA3a, FvHsfA4a, FvHsfA5a, FvHsfA6a, FvHsfA9a, FvHsfB1a, and FvHsfC1a) were induced by other abiotic or biotic stresses and hormone stimuli and could play important roles in plant adaptation to environmental stresses (Hu et al. 2015). MYB family represents a large and functionally diverse class of proteins. Most of them function as TF that include a conserved N-terminal MYB DNAbinding domain and a diverse C-terminal modulator region (Ambawat et al. 2013; Roy 2 016). The MYB protein family has been divided into four classes based on the number of MYB domains: 1R-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB. In plants, the majority of the MYB proteins belong to the R2R3-MYB subfamily and play important roles in primary and secondary metabolism, cell fate and identity, developmental processes, and in the regulation of multiple responses, such as biotic and abiotic stresses (Stracke et al. 2001; Dubos et al. 2010). MYBs participate in the ABA-dependent signaling stress pathway and are activated after ABA accumulation (Jaqueline da Silva and Costa de Oliveira 2014). Wang et al. (2020) in the analysis of alleles of FaMYB10 and FaMYB1 in red and white Fragaria × ananassa varieties ‘Sweet Charlie’ and ‘Snow Princess’ identified a white-specific

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variant allele of FaMYB10, FaMYB10-2. They identified that MYB TFs control the red color of strawberry fruits through the regulation of flavonoid biosynthesis in response to the increase in endogenous ABA (Wang et al. 2020). It was found, that number of different variations in the FaTHSF protein motifs can be related to a high degree of variability concerning plant response to abiotic stress. The expression of FaTHSFA2a and FaTHSFB1a are found to be induced by heat stress, heat stress treatment in Arabidopsis resulted in overexpression of FaTHSFA2a and FaTHSFB1a genes. Their up-regulation was probably related to increased expression of endogenous HSFA2 gene and other members of the HSF family (HSFB1 and HSFB2a) under heat stress (Liao et al. 2016). HSFs involvement in the activation of genes in response to heat, cold, drought, and salt was reported by Hu et al. (2015). There isn’t much information available about the WRKY gene family in Fragaria or Rubus species. WRKY family TFs have a conserved domain of 60 amino acids with the WRKYGWK sequence at the N-terminus and a zinc finger structure in the C-terminal region (Eulgem et al. 2000). Zhu et al. (2016) identified 59 WRKY members in Fragaria vesca ‘Hawaii 4’ genome and analyzed their expression in different organs and at different fruit developmental stages. Meanwhile, Wei et al. (2016) identified 62 FvWRKY genes and examined their expression under various conditions, including drought, salt, cold, and heat. It was reported that FvWRKY genes responded to drought and salt treatment to a greater extent than to temperature stress (Wei et al. 2016). Chen and Liu (2019) identified 47 WRKY gene members in Fragaria × ananassa Duch. It was found that some stress-related cis-elements, including cis-acting elements involved in LTRE and MYB TF binding site (MBS), also function as important factors in drought inducibility and can be found in a few FaWRKY promoter regions. They also noticed that continuous cropping led to FaWRKY25, FaWRKY32, FaWRKY33, and FaWRKY45 genes up-regulation (Chen and Liu 2019). A high transcript level of TF genes WRKY and MYB during fruit ripening was detected in strawberry cultivar ‘Sweet Charlie’ (Sharma et al. 2019). Differential expression of TF genes ERF, MYB, and WRKY in ripened fruits of Himalayan Rubus ellipticus genotypes and cultivars was revealed (Sharma et al. 2019). The highest expression of ERFG transcript was detected in the raspberry cultivar ‘Kumarhatti-1’, while a lower expression was observed in ‘Kaithleeghat-3’. Liu et al. (2017) presented genome-wide identification and characterization of the bZIP TF gene family in Fragaria species. It is the most conserved plant-specific gene family which play key roles in various aspects of biological processes and take part in the regulation of plants’ response to abiotic and biotic stresses (Singh et al. 2002; Mukhopadhyay et al. 2004; Shimizu et al. 2005; Liao et al. 2008; Ying et al. 2012; Liu et al. 2012). It was detected a different number of bZIP genes in the genomes of Fragaria: F. × ananassa, F. vesca, F. iinumae, F. nubicola, F. orientalis, and F. nipponica. Many cis-regulatory elements (GT1GMSCAM4, DRE1COREZMRAB17, ARR1AT, CPBCSPOR, ABRERATCAL, etc.) in the promoter regions of FvbZIP genes were found, too (Liu et al. 2017). Data show that bZIP genes may play a role in abiotic stress response. Wang et al. (2017) characterized 50 bZIP genes in woodland strawberry and observed a range of expression patterns of FvbZIP genes in leaves under drought and heat treatments. Depending

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on the duration of exposure, a few genes from clade A (mran00393, mrna08566, mrna30280, and mrna11837) were up-regulated and all genes were down-regulated after the plants were treated with drought stress. During heat treatment (42 °C), a few genes were found to be differently expressed. For example, transcription levels of mrna30280 increased, but low expression of mrna14556 and mrna28250 was detected (Wang et al. 2017).

7.8 Epigenetics and Abiotic Stress Resistance Plant epigenetics can provide novel direction to drive plant breeding strategies. Epigenetics characterize heritable changes in gene expression without permanent alterations or changes of the DNA sequence. These changes regulate gene expression in response to plant development and environmental stimuli, ultimately affecting the plant’s phenotype, playing a role in plant responses to various stressors (Penna et al. 2021). Epigenetic mechanisms include changes in DNA methylation, by addition of a methyl group, histone modifications, such as histone amino-terminal modifications that act on affinities for chromatin-associated proteins (Rapp and Wendel 2005) and RNA interference by small RNAs (sRNA) that results in interaction with transcriptional machinery (Castel and Martienssen 2013). These epigenetic mechanisms lead to enhanced or reduced gene transcription and RNA-translation (Jaligot and Rival 2015). Epigenetic modifications have been linked to changes in the expression of stressresponsive genes in various plant species, allowing more efficient adaptation to environmental changes. These changes can be involved in both immediate and long-term stress responses (Kim et al. 2015). Interestingly, different epigenetic modifications have been associated with the memory-dependent response to both biotic and abiotic stresses, providing plants with the required tools to acclimate and survive (Mozgova et al. 2019). This understanding has introduced new research aspects to studying plant abiotic stress responses and offered innovative options for breeding applications, using epigenetic variation as useful variability during crop selection (Gallusci et al. 2017).

7.8.1 DNA Methylation and Demethylation Cytosine methylation is the most common DNA modification found in most eukaryotic organisms including plants, animals, and fungi (Bhattacharyya et al. 2020). Many scientists have demonstrated that DNA methylation may be employed by plants to regulate gene expression as a response to abiotic stresses by directly or indirectly regulating stress-responsive genes (Le et al. 2014; Cavrak et al. 2014; Liu et al. 2015a; Bharti et al. 2015; Xu et al. 2015a).

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DNA in plants is highly methylated and methylation can occur at cytosine both in symmetrical (CG or CHG) and non-symmetrical (CHH) contexts (with H: A, T or C), with the latter directed and maintained by sRNAs (Chan et al. 2005; Law and Jacobsen 2010). There are known to be three main classes of plant DNA methyltransferases that differ in protein structure and function (Farinati et al. 2017): methyltransferase (MET1), chromomethylase (CMT), and domain-rearranged methyltransferase (DRM) (Finnegan and Kovac 2000; Wada 2005; Law and Jacobsen 2010; Farinati et al. 2017; Zhang et al. 2018b). The most common modifications in plants are 5methylcytosine (m5C) and N6-methyladenine (6 mA), which occur in mitochondria and chloroplast DNA (Vanyushin 2006; Xie et al. 2020). The transfer of a methyl group from S-adenosyl methionine to a specific DNA sequence is accomplished by Cytosine-5 DNA methyltransferases (C5-DNA MTases), which belong to the conserved family of enzymes (Bhattacharyya et al. 2020). 6 mA modification involves methylation at the sixth position of the purine ring of adenine in a DNA molecule (Xie et al. 2020). The emergence of a new powerful technique, such as single-molecule real-time (SMRT) sequencing, developed by Flusberg et al. (2010), helped Xie et al. (2020) to detect 6 mA modifications at single-nucleotide resolution and single-molecule level in the woodland strawberry (F. vesca) genome. So far, this is the first genome-wide analysis of the epigenetic methylation of DNA related to 6 mA modification in the Rosaceae family. This study showed, that DNA 6 mA sites (total 160,256) were widely dispersed throughout the strawberry linkage groups and chloroplast genome. High levels were detected in seven linkage groups, but the chloroplast genome showed the highest 6 mA density (1.725%). Investigation of 6 mA distributions in different functional regions showed that most of the 6 mA modification sites were located within intergenic regions of the genome. Besides, the CDS region of methylated genes has a considerably higher prevalence of 6 mA modifications. Analyzing the influences of the 6 mA modification on gene expression, Xie with co-workers (2020) noticed, that genes with higher RNA expression had a significantly higher density of 6 mA. These findings suggest that such DNA alteration in woodland strawberry could be a marker for actively transcribed genes. Gu et al. (2016b), performed a sequence-based searching to identify the methyltransferase catalytic domain-containing genes in F. vesca. In total, they characterized 9 DNA methyltransferases genes (MET1—1, CMT3—3, DRM1/2—4, and DNMT2—1) and four putative DNA demethylase genes (DME—1, DML—3). They noted that the expression profiles of DNA demethylase genes in strawberry seedlings respond differentially to salinity, drought, cold, and heat stresses. For example, Fv-demethylase1 gene expression didn’t change after a heat shock stress but was detectable at higher levels after drought, salinity, and cold. Accumulation of Fvdemethylase2 transcripts after drought and salinity stresses was noticed, too. Meanwhile heat and cold had a negative impact on Fv-demethylase2 gene expression. These experiments indicated that active demethylation may be involved in strawberry responses to various abiotic stresses. Whole-genome bisulfite sequencing has facilitated the analysis of genome-wide DNA methylation profiles (Bibikova et al. 2009). Alongside 33 angiosperms, the DNA methylome of woodland strawberry was also reported by Niederhuth et al.

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(2016). Several scientists (He et al. 2011; Zhong et al. 2013; Liu et al. 2015b) have shown that flowering time, fruit ripening process, and heterosis can be controlled by epigenetics. Most studies of epigenetic regulation of fruit development and ripening have been performed on fleshy (climacteric) fruit tomato (Manning et al. 2006; Pesaresi et al. 2014). However, genome-wide DNA methylation dynamics have not been investigated wide in non-climacteric fruits such as strawberry, raspberries, or blackberries (Cheng et al. 2018). An important role of DNA methylation in cultivated Fragaria × ananassa fruit at different stages of ripening was shown by Cheng et al. (2018). Whole-genome bisulfite studies revealed that DNA methylation patterns around genes and transposable elements are comparable in leaves and fruit. The average DNA methylation level in immature Fragaria fruit was 7.5%. Meanwhile, leaves displayed a slightly higher average mC level (about 8%). The average methylation levels of mCG, mCHG, and mCHH were 40, 11, and 2%, respectively. DNA methylation was high in TE and repeat-rich genomic regions but low in gene-rich regions in fruit. Strawberry fruit DNA methylomes at three stages of ripening showed that ripe fruits have less DNA methylation around genes and transposable elements than immature ones. It was concluded that strawberries undergo an overall loss of DNA methylation during fruit ripening. A deep and comprehensive analysis of the DNA hypomethylation mechanism during strawberry ripening was carried out and the expression of four ROS1 gene homologs (FvDME1, FvROS1.1, FvROS1.2, FvROS1.3) was examined by Chen et al. (2018). It was noticed that the expression of all studied DNA demethylase genes did not significantly increase at three different phases of ripening and DNA hypomethylation during strawberry ripening was not related to higher expression of DNA demethylation pathway genes. Identification and expression profiling studies of some DNA methyltransferase genes (FvMET1, FvCMT2, FvCMT3.1, FvCMT3.2, FvDRM1.1, FvDRM1.2, FvDRM1.3, FvDRM3.1) also showed, that these genes were significantly down-regulated. This suggests that decreased DNA methylation activities contribute to DNA hypomethylation caused by fruit ripening. In F. vesca, the correlation between the fruit color change from white to red and increased values of DNA methylase and demethylase genes was noticed by Gu et al. (2016b). Expression profiles of methylation modifiers in the early-stage fruit development indicated that they have different tissue specificity, and each tissue has a unique combinatorial pattern that might be related to its function. A connection between heritable reversible changes in in vitro propagated cultures and epigenetic DNA modifications has been identified. As in vitro propagation or cloning is widely applied to vegetatively propagated plants (Ahloowalia 2003), unstable phenotypic modifications (leaves, stems, flowers) are frequently encountered. It was determined, that such changes usually recover when the plants are transferred to normal growing conditions suggesting that they may be caused by transposons or gene inactivation by methylation (Becker et al. 2011). The relationship between micropropagation and DNA methylation in strawberry (Fragaria x ananassa Duch.) cultivar ‘Toyonoka’ was analyzed by Chang et al. (2009). It

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was noticed that DNA methylation rates, according to HPLC quantitative analysis, between microplants and conventionally propagated plants in the field did not differ much. Expression profile analysis of putative DNA methyltransferase MET1 (FaMET1a, FaMET1b) and DRM (FaDRMa, FaDRMb, FaDRMc) genes in three generations of strawberry plants in vitro showed that the expression of FaMET1 and FaDRM genes were down-regulated, and they were recovered in the next two generations of microplants. The author suggested, that this might be related to the well-developed methylation-demethylation system in the plants. A similar study was done by Zhang et al. (2014). They also noticed, that long micro-propagation of strawberry cultivars ‘Toyonoka’ and ‘Allstar’ is associated with variations and that most of these variations were epigenetic. Methylation-sensitive amplified polymorphism (MSAP) showed that DNA methylation levels were lower in microplants than in conventionally propagated runner plants. Expression investigation of two DNA methyltransferase genes (FaMET1 and FaDRM) revealed that both genes were down-regulated in in vitro plants but was gradually recovered in the next generations.

7.8.2 Histone Post-translational Modifications Several reports were focused on epigenetic regulation of cellular processes during cold, heat, drought, salinity, and other abiotic stresses (Ndamukong et al. 2010; Kapazoglou and Tsaftaris 2011; Avramova 2015; Mozgova et al. 2019). Biochemical studies of a model plant Arabidopsis clearly showed an important role of histone modifications in the regulation of gene expression in plant response to many stressors (Thorstensen et al. 2011; Avramova 2015). Post-translational modifications affect chromatin condensation and structure and consequently affect the overall histone structure and epigenetic control of gene expression (Lawrence et al. 2016). Likewise, chromatin reorganization can be related to the degree of the packaging of genomic DNA around the nucleosome units (Kouzarides 2007). Histones are highly conserved globular proteins whose N-terminal tails of the nucleosome octamer are exposed to covalent or chemical modifications (phosphorylation, sumoylation, acetylation, methylation) (Kouzarides 2007; Deal and Henikoff 2011). Such histone post-translational modifications are associated with either gene silencing or gene activation (Zhao and Garcia 2015). H3 and H4 histone lysine residue methylation is very diverse and critical histone modification. In plants, methylation is catalyzed by histone HKMTases and HDMases enzymes. HKMTases have a SET domain mediating the methyltransferase catalytic activity. Two categories of enzymes comprise HDMases: the lysine-specific demethylase type and the JmjC domain-containing HDMases (Gu et al. 2016a). Histone lysine methylation modifiers (HKMTases and HDMases) in were first identified and characterized by Gu et al. (2016a). According to sequence-based search, they determined SET domain-containing genes and divided them into seven groups based on domain architecture and motif composition. Genes encoding proteins with both the SWIRM and amino oxidase domains of LSD HDMases and JmjC domain-containing genes

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were identified in woodland strawberry. Expression patterns of a subset of SET genes in strawberry seedlings revealed the regulation mechanism of HKMTase genes under cold and heat stresses (Gu et al. 2016a). For example, expression levels of 13 investigated SET genes increased during cold shock at 3 h and the expression of two SET genes increased after 4 h of heat shock, but the other genes did not display any significant change. These findings suggest, that the response of different HKMTase genes to abiotic stressors is unequal.

7.8.3 RNA Interference by sRNAs It is known, that abiotic stresses including cold, drought, salt, nutrient deficiency, and oxidative stress induce the production of small and long non-coding RNAs (Cuperus et al. 2011). The RNA interference by sRNAs is another type of epigenetic regulation that is important in transcriptional or post-transcriptional regulation by transcript cleavage and translation repression that affect the expression of stressresponsive genes (Shukla et al. 2008; Sunkar et al. 2012; Kumar 2014; Liu et al. 2018). The best-known sRNAs are microRNAs (miRNAs). miRNAs are a class of 20–22 nucleotide non-coding endogenous RNAs that are important in transcriptional or post-transcriptional regulation through post-transcriptional gene silencing (PTGS) (Cuperus et al. 2011; Vargas-Asencio and Perry 2020). In various berries, miRNAs act as an important key regulator of fruit development and ripening by affecting various response pathways of phytohormone metabolism, distribution, and perception (Curaba et al. 2014). miRNAs have an essential gene-regulatory role in plant development stages, biotic and abiotic stress tolerance, and can be induced by environmental stress (JonesRhoades and Bartel 2004; Sunkar et al. 2012; Liu et al. 2018). The expression of stress-responsive miRNAs can be both upregulated and down-regulated, affecting the expression of their target genes negatively or positively, respectively (Mozgova et al. 2019). Several studies have revealed, that miRNA-159, miRNA-167, miRNA169, miRNA-171, miRNA-319, miRNA-393, miRNA-394, and miRNA-396 had the highest contributions to plant response towards drought, salinity, cold, and heat stressors (Song et al. 2019; Vakilian 2020). The overall stress response is produced by multiple changes in miRNA composition. A study of strawberry fruit in low temperatures revealed, that a lower temperature (4 °C) induced up-regulation of 108–139 miRNAs and down-regulation of 113–114 miRNAs depending on storage time. These miRNA changes repressed abscisic acid signaling transduction and reduced the jasmonic acid biosynthesis, which resulted in delayed fruit senescence under low temperature (Xu et al. 2015b). Interestingly, this up/down-regulation of specific miRNAs differs in different plant species during the same stress conditions. In response to drought stress conditions, the most differentially expressed miRNAs were miR-396 followed by miR-171, however among those, neither was consistently induced nor repressed in different plant species. For example, under drought stress conditions sugarcane (Saccharum ssp.)

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miR-169 and miR-396 are down-regulated, but in the same conditions, these RNAs were up-regulated in peach (Prunus persica) (Gentile et al. 2015). miRNA variability depends not only on the genetic background (species) or type of stressors. Different miRNA expressions can be found in different plant tissues (leaves, seedlings, spikelets, roots) or depending on a specific plant growth condition (greenhouse, field, hydroponic) (Vakilian 2020). miRNAs can also be involved in the memory of abiotic stresses (Mozgova et al. 2019). In general, epigenetic memory is likely a relatively rare event occurring in a period of stress recovery, the predominant strategy for stress responses is resetting and recovery (Crisp et al. 2016). The most notable study showing acquired thermo-tolerance due to changes in miRNA composition was conducted by Stief et al. (2014). Up-regulation of miR-156 in Arabidopsis plants during a typical heat stress period revealed a correlation with the down-regulation of miR156-targeted squamosa promoter binding protein-like (SPL) TF genes. SPL family of TF are responsible for developmental transitions, and the down-regulation resulted in enhanced thermotolerance when miR156 was constitutively overexpressed, as well as heat-inducible overexpression.

7.8.4 RNA-Directed DNA Methylation Non-coding RNA molecules can also be responsible for DNA methylation. RNAdirected DNA methylation (RdDM) is a biological process in which non-coding RNA molecules lead to methylation of almost all cytosine residues (CpG, CpNpG, and CpNpN) within the region of sequence identity between the triggering RNA and the target DNA (Castel and Martienssen 2013; Erdmann and Picard 2020). So far the RdDM pathway has been described only in plants, although other mechanisms of RNA-directed chromatin modification have also been described in fungi and animals (Aufsatz et al. 2002). That may be explained by the fact, that the level of methylation is up to 50% of cytosines in higher plant DNA, and only from 2 to 8% of cytosines in mammalian DNA (Zhu 2009). RdDM is involved in many biological processes in plants, including stress response, cell-to-cell communication, and the maintenance of genome stability through transposable elements silencing (Mette et al. 2000). In plants, RdDM usually is triggered by small interfering RNAs (siRNAs) that induce transcriptional gene silencing (TGS) (Erdmann and Picard 2020). During RdDM, following transcription of target loci from a double-stranded RNA, precursor RNA polymerase IV (Pol IV) initiates the production of siRNAs that direct transcriptionally repressive DNA methylation to homologous RNA polymerase V (Pol IV)-transcribed loci. Pol IV and Pol V are recruited to genomic regions that contain transcriptionally repressive epigenetic marks, which repress a subset of transposons and genes (Matzke and Mosher 2014). The RdDM pathway is the unique plant mechanism that can methylate cytosines regardless of sequence context, such as non-CG methylation. This mechanism provides a backup for other methylation pathways and can largely restore original methylation patterns if necessary (Erdmann and Picard

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2020). In response to various abiotic stressors, such as heat stress, drought, phosphate starvation, salt stress, changing levels of RdDM can help plants adapt (Matzke and Mosher 2014). Usually, in a stressful situation, TEs become up-regulated, and this can be countered by siRNA production and RdDM silencing (Mette et al. 2000). RdDM has the ability to control genes that participate in the abiotic stress response. For example, a common plant response under low relative humidity is trying to conserve water. It was found by Tricker et al. (2012) that in this situation induction of local siRNAs resulted in RdDM-mediated down-regulation of two genes involved in stomatal development resulting in Arabidopsis leaves producing fewer stomata. RdDM not only helps host response to biotic or abiotic challenges but is also important to stress memory, where DNA methylation patterns can be transmitted to the offspring (Crisp et al. 2016). The transposon silencing that occurs following RdDM increases overall phenotypic diversity because of transcriptional up-regulation of neighboring genes as well as altering the transcription pattern of nearby genes or even disrupting functions of active genes. This newly acquired phenotypic diversity might increase adaptability in a stressful environment and introduce new possibilities in plant breeding (Matzke and Mosher 2014).

7.9 Concluding Remarks and Future Perspectives Despite the challenges raised by climatic changes, globalization, and horticultural practices, berry growing is actively expanding. The impact of abiotic factors on cultivation does not disappear or decrease, on the contrary, it is intensifying. To date, we don’t have a comprehensive knowledge of the mechanisms behind plant resistance to abiotic factors, of the full variety of adaptations that plants, individual species, and genotypes use to survive in specific conditions that can be very diverse and variable. However, advances in omics, the development and application of NGS, GBS, and other technologies over the past 10 years open up entirely new possibilities for understanding the genome structure of berry plants, the functions of QTL genetic systems, and individual genes. Research is being further developed. Marker-assisted selection is developing from a system comprising individual genes or separate QTL, to one that covers the whole genome and comprises not only individual traits related to resistance to stressors but also traits important for berry quality, nutritional value, human health maintenance, and enhancement. The whole complex of properties is important because everything in a living organism is inextricably linked. New technologies are not cheap and simple, so collaboration between individual research institutions and businesses around the world is essential. Only then tangible and sustainable changes in horticulture and berry growing will be achieved. In this chapter, we touched on only a few of the most common berry plants from the genera Fragaria and Rubus. Botanically, their fruits are not even berries. Other genera such as Vaccinium, Actinidia, Lonicera, Hippophaë, Asimina, Berberis, Eleagnus, Lycium, Mahonia, and others are receiving increasing attention in horticulture, are valuable and important in individual countries. They are also receiving

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increasing attention in the genome and other research, and their investigation, as well as cultivation, is likely to expand intensively in the future.

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