Plant Male Sterility Systems for Accelerating Crop Improvement 9811938075, 9789811938078

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Abhishek Bohra · Ashok Kumar Parihar  Satheesh Naik SJ · Anup Chandra Editors

Plant Male Sterility Systems for Accelerating Crop Improvement

Plant Male Sterility Systems for Accelerating Crop Improvement

Abhishek Bohra • Ashok Kumar Parihar • Satheesh Naik SJ • Anup Chandra Editors

Plant Male Sterility Systems for Accelerating Crop Improvement

Editors Abhishek Bohra Crop Improvement Division, Block B Indian Institute of Pulses Research Kalyanpur, Kanpur, Uttar Pradesh, India

Ashok Kumar Parihar ICAR-Indian Institute of Pulses Research Kanpur, India

Satheesh Naik SJ ICAR-Indian Institute of Pulses Research Kanpur, India

Anup Chandra ICAR-Indian Institute of Pulses Research Kanpur, India

ISBN 978-981-19-3807-8 ISBN 978-981-19-3808-5 https://doi.org/10.1007/978-981-19-3808-5

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 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 Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

In the eighteenth century, the pioneering work of Joseph Gottlieb Kölreuter at the University of Karlsruhe, Germany, laid the foundation of concepts of pollination and sterility in plants. In subsequent years, the phenomenon of heterosis or hybrid vigour was reported by Dr. Beal and Dr. G. H. Shull as the superiority of offspring (F1) over parents. Male sterility is the formation of non-functional pollen grains. Various types of male sterility systems exist in plant species under the influence of nuclear, cytoplasmic and environmental factors described as genic, cytoplasmic and environment-sensitive male sterility, respectively. Feeding 34% more people projected to be added over next 30 years worldwide demands the current crop production rate to increase by nearly 37% in hotter agricultural settings. The challenge is further exacerbated by a growing competition for water, energy and land. Accelerating development of hybrids with high yield and resilience traits is one of potential breeding approaches to boost global productivity rates of food crops. In this context, the utility of the male sterility as a system to circumvent the need for cumbersome emasculation procedures has bolstered the hybrid seed production in different crops. Occurrence of male sterility has been observed in different plant species. For instance, cytoplasmic male sterility (CMS) is reported from over 150 plant species. Given the significance of the topic, the book entitled, Plant male sterility systems for accelerating crop improvement is convenient and user-oriented and could create a room for exchange of ideas and communication among the researchers involved in development and improvement of food crops, ultimately resulting in higher crop productivity. We, editors, express deep gratitude to our organization, the Indian Council of Agricultural Research (ICAR) which has fostered all the way and made us capable of taking up this prestigious book project. We are very much thankful to Dr. Trilochan Mohapatra, Director General, ICAR and Secretary (DARE), Ministry of Agriculture and Farmers’ Welfare, Government of India, and Dr. TR Sharma, Deputy Director General (Crop Science) who at the helm are continuous in paving the way for the development of agriculture. We are highly indebted to Dr. Shiv Sewak, Director, and Dr. NP Singh, Former Director, ICAR-Indian Institute of Pulses Research (IIPR), Kanpur, for rendering institutional support and cultivating scientific insights into us. We would like to thank Dr. Farindra Singh, Head, Division of Crop Improvement

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and Dr. Bansa Singh, Head, Division of Crop Protection, ICAR-IIPR, Kanpur, for their constant support and guidance during the entire journey of the work. Largely, the present accomplishment would not have been realized without the painful dedication and tireless efforts from the contributing authors. We feel highly obliged to all authors who have made tremendous efforts in the synthesis of their chapters. Ten chapters focusing on male sterility techniques, heterosis and hybrid development in globally important crops: rice, maize, pearl millet, sorghum, sunflower, pigeonpea, soybean, brassica, safflower and vegetables are written by experienced researchers in this field. Significance of insect pollinators in efficient hybrid seed production is also highlighted. We show our sincere thanks to the team springer, especially Shruthi Radhakrishnan, Rhea Dadra and Ashok Kumar, for their endurance and for what they delivered us the fullness of time for editing and finalization of this book project. Editors convey their extreme sense of thanks to their respective family members for moral boost ups, assistance and cooperation received during the entire work occupancies. In this regard, AB is thankful to his wife Renu Upreti and son Ayushman Bohra, who allowed their time to be taken away to fulfill editorial responsibilities. AP is grateful to his wife Santosh Parihar and children (Abhimanyu Parihar and Aaradhya Parihar) for their help and moral support in performing editorial responsibilities. AC is thankful to his wife Asha and children (Somil and Siya) for allowing their family time invested in this book project. SNSJ is thankful to his wife Kumada and children (Vanshika and Vashisht) for their continuous support and encouragement throughout the completion of this book Finally, we strongly believe that the book will be read and cited extensively. The chapters certainly provide new insights to its readers and would be of great resource to the scientific community including scientists, university faculties and students working in this field. Kanpur, Uttar Pradesh, India

Abhishek Bohra Ashok Kumar Parihar Satheesh Naik SJ Anup Chandra

Contents

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Male Sterility and Hybrid Technology for Sustainable Production: Status and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . S. J. Satheesh Naik, Abhishek Bohra, Ashok Kumar Parihar, and Anup Chandra Advances in Male Sterility Systems and Hybrid Breeding in Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ashutosh Kushwah, Sheetal Raj Sharma, K. B. Choudhary, and Suruchi Vij

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Male Sterility in Maize: Retrospect, Status and Challenges . . . . . . . Subhash Chander, Bhupender Kumar, Krishan Kumar, Sonu Kumar, Chayanika Lahkar, Brijesh Kumar Singh, Shankar Lal Jat, Chittar Mal Parihar, and Ashok Kumar Parihar

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Male Sterility Technologies to Boost Heterosis Breeding in Pearl Millet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. B. Choudhary, H. R. Mahala, and Vikas Khandelwal

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Sorghum Improvement: Male Sterility and Hybrid Breeding Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. B. Choudhary, Vikas Khandelwal, and Sheetal Raj Sharma

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Advances in Male Sterility Systems and Hybrid Breeding in Sunflower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. P. Meena, M. Sujatha, and A. Vishnuvardhan Reddy

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Discovery and Application of Male Sterility Systems in Pigeonpea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Abhishek Bohra, S. J. Satheesh Naik, Abha Tiwari, Alok Kumar Maurya, Shefali Tyagi, and Vivekanand Yadav

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Achievements, Challenges and Prospects of Hybrid Soybean . . . . . 167 Subhash Chandra, Shivakumar Maranna, Manisha Saini, G. Kumawat, V. Nataraj, G. K. Satpute, V. Rajesh, R. K. Verma, M. B. Ratnaparkhe, Sanjay Gupta, and Akshay Talukdar vii

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Contents

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Recent Progress in Brassica Hybrid Breeding . . . . . . . . . . . . . . . . . 195 Javed Akhatar, Hitesh Kumar, and Harjeevan Kaur

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Cytoplasmic Male Sterility: A Robust and Well-Proven Arsenal for Hybrid Breeding in Vegetable Crops . . . . . . . . . . . . . . 221 Pradip Karmakar, B. K. Singh, Vidya Sagar, P. M. Singh, Jagdish Singh, and T. K. Behera

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Male Sterility and Hybrid Breeding Strategies in Safflower . . . . . . 251 Vrijendra Singh, Nandini Nimbkar, and C. V. Sameer Kumar

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Insect Pollinators and Hybrid Seed Production: Relevance to Climate Change and Sustainability . . . . . . . . . . . . . . . . . . . . . . . 265 Anup Chandra, Gopalakrishnan Kesharivarmen Sujayanand, Revanasidda, Sanjay M. Bandi, Thejangulie Angami, and Manish Kanwat

Contributors

Javed Akhatar Punjab Agricultural University, Ludhiana, Punjab, India Thejangulie Angami ICAR Research Complex for NEH Region, AP Centre, Basar, Arunachal Pradesh, India Sanjay Maruti Bandi ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India T. K. Behera ICAR—Indian Institute of Vegetable Research (IIVR), Varanasi, Uttar Pradesh, India Abhishek Bohra ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India Subhash Chander ICAR—National Bureau of Plant Genetic Resources, New Delhi, India Anup Chandra ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India Subhash Chandra ICAR—Indian Institute of Soybean Research, Indore, India K. B. Choudhary ICAR—Central Arid Zone Research Institute (CAZRI), Jodhpur, Rajasthan, India Sanjay Gupta ICAR—Indian Institute of Soybean Research, Indore, India Shankar Lal Jat ICAR—Indian Institute of Maize Research, New Delhi, India Manish Kanwat Krishi Vigyan Kendra (ICAR-CAZRI), Bhuj, Gujarat, India Pradip Karmakar ICAR—Indian Institute of Vegetable Research (IIVR), Varanasi, Uttar Pradesh, India Harjeevan Kaur Punjab Agricultural University, Ludhiana, Punjab, India Vikas Khandelwal ICAR—All India Coordinated Research Project on Pearl Millet, Jodhpur, Rajasthan, India Bhupender Kumar ICAR—Indian Institute of Maize Research, New Delhi, India ix

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Contributors

Hitesh Kumar Banda University of Agriculture and Technology, Banda, Uttar Pradesh, India Krishan Kumar ICAR—Indian Institute of Maize Research, New Delhi, India Sonu Kumar ICAR—Indian Institute of Maize Research, New Delhi, India G. Kumawat ICAR—Indian Institute of Soybean Research, Indore, India Ashutosh Kushwah Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, Punjab, India Chayanika Lahkar ICAR—Indian Institute of Maize Research, New Delhi, India H. R. Mahala ICAR—Central Arid Zone Research Institute (CAZRI), Jodhpur, Rajasthan, India Alok Kumar Maurya ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India H. P. Meena ICAR—Indian Institute of Oilseeds Research, Hyderabad, India V. Nataraj ICAR—Indian Institute of Soybean Research, Indore, India Nandini Nimbkar Nimbkar Agricultural Research Institute, Phaltan, Maharashtra, India Ashok Kumar Parihar ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India Chittar Mal Parihar ICAR—Indian Agricultural Research Institute, New Delhi, India V. Rajesh ICAR—Indian Institute of Soybean Research, Indore, India M. B. Ratnaparkhe ICAR—Indian Institute of Soybean Research, Indore, India A. Vishnuvardhan Reddy ICAR—Indian Institute of Oilseeds Research, Hyderabad, India Revanasidda ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India Vidya Sagar ICAR—Indian Institute of Vegetable Research (IIVR), Varanasi, Uttar Pradesh, India Manisha Saini Division of Genetics, ICAR—Indian Agricultural Research Institute, New Delhi, India C. V. Sameer Kumar Department of Genetics and Plant Breeding, College of Agriculture, PJTSAU, Hyderabad, India Satheesh Naik SJ ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India

Contributors

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G. K. Satpute ICAR—Indian Institute of Soybean Research, Indore, India Sheetal Raj Sharma Division of Plant Breeding and Genetics, RARI, Jaipur, Rajasthan, India M. Shivakumar ICAR—Indian Institute of Soybean Research, Indore, India B. K. Singh ICAR—Indian Institute of Vegetable Research (IIVR), Varanasi, Uttar Pradesh, India Brijesh Kumar Singh ICAR—Indian Institute of Maize Research, New Delhi, India Jagdish Singh ICAR—Indian Institute of Vegetable Research (IIVR), Varanasi, Uttar Pradesh, India P. M. Singh ICAR—Indian Institute of Vegetable Research (IIVR), Varanasi, Uttar Pradesh, India Vrijendra Singh Nimbkar Agricultural Research Institute, Phaltan, Maharashtra, India M. Sujatha ICAR—Indian Institute of Oilseeds Research, Hyderabad, India G. K. Sujayanand ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India Akshay Talukdar Division of Genetics, ICAR—Indian Agricultural Research Institute, New Delhi, India Abha Tiwari ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India Shefali Tyagi ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India R. K. Verma ICAR—Indian Institute of Soybean Research, Indore, India Suruchi Vij Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, Punjab, India Vivekanand Yadav ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India

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Male Sterility and Hybrid Technology for Sustainable Production: Status and Prospects S. J. Satheesh Naik, Abhishek Bohra, Ashok Kumar Parihar, and Anup Chandra

Abstract

Male sterility in plant is a reproductive physio-morphological inability to articulate incompetent or inoperative male reproductive parts in flowers deterring sexual reproduction. The phenomenon of male sterility in plants is governed by both genetic and/or environmental conditions, which results in plant reproductive biology abnormalities ranging from hampered microsporogenesis, non-functional stamens to production/release of viable pollens. Existence of male sterility has been reported in many cereals, pulses, oilseeds and horticultural crops. Nevertheless, male sterility is a constraint for natural gene flow in plants. But the phenomenon of natural male reproductive inability of the plants is a gift to the breeders to harness the heterosis through hybrid technology. Heterosis is the manifestation of higher vigour in the offspring pertinent to the yield and its attributing traits, resistance to biotic and abiotic stresses and enhanced nutritional quality traits. Several reports are available in cereals, pulses, oilseeds and in horticultural crops, stating the yield plateau due to repeated cultivation of inbreed varieties in spite of being cultivated using the best management practices. In this regard, hybrid technology is the way ahead to break the yield plateau and achieve sustainable productivity increase and resilience in different agricultural crops. Keywords

Breeding · Fertility restoration · Heterosis · Hybrid · Male sterility

S. J. Satheesh Naik (*) · A. Bohra · A. K. Parihar · A. Chandra ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_1

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1.1

S. J. Satheesh Naik et al.

Introduction

Male sterility in plants is a floral physio-morphological and reproductive inability to show normal micro-sporogenesis, stamens development and/or to release functional pollen grains (Bohra et al. 2017). Evolutionarily, male sterility in plants is considered as a constraint for natural gene flow. However, the invasion of systemic plant breeding and the understanding of heterosis phenomenon made it as a gift to enhance the hybrid vigour in cultivated crops. Several reports stating the existence of male sterility in different crops, namely, maize, wheat, sorghum, rye, barley, rice, pearl millet, pigeon pea, mustard, sunflower, flax, sugar beet, cotton, onion, spinach, carrot, asparagus, celery, cucurbits, tomato, pepper, eggplant, leek, fennel, radish, cabbage, cauliflower, broccoli, turnip, chicory, etc. Duvick (1966) stated that, nuclear male sterility can probably be found in all diploid species. However, the cytoplasmic male sterility has been reported in more than 140 plant species (Laser and Lersten 1972). Apart from the spontaneous nature of its occurrence, male sterility can be created through experimental means like induced mutations, wide/ inter-specific hybridization, protoplasmic fusion and genetic engineering. Male sterility in plants is manifested by means of producing malformed male reproductive organs, complete failure to produce micro-sporogenous tissues in anthers, abnormal micro-sporogenesis leading deformed pollens, unmatured pollens, non-dehiscent anthers and unreachable pollen tube to ovules. Besides being a breeding tool for population improvement, male sterility traits facilitate inter-specific hybridization to perform genetic inheritance studies, but the most important and the commercially exploited is efficient hybrid seed production. After successful demonstration of heterosis in hybrid maize (Shull, 1914), hybrid breeding methods are the now new normal breeding tool for improving vigour in various field crops, vegetable and ornamental crops. As research towards male sterility-oriented hybrid breeding progressed in the last 108 years, the possible hurdles for scaling up of hybrid technologies were eliminated. Plant breeders have introduced different kinds of male sterility into plant populations and used them to circumvent the restrictions to large scale controlled hybridization imposed by flower morphology and breeding systems. Presently hybrid breeding technology is a US $billion business worldwide.

1.2

An Account of Male Sterility in Crop Plants

The chronology of male sterility invention and its application begins after the report of anther abortion in intra- and inter-species hybrids of plants by Joseph Gottlieb Koelreuter in 1763. Naturally male sterility is more prevalent than female sterility, may be due to male sporophyte and gametophyte being less protected from influencing environmental factors than the ovule and embryo sac. The ease of male sterility detection, due to abundant pollens available for experimentation and wellestablished swift staining techniques (caramine, lactophenol or iodine) in

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Male Sterility and Hybrid Technology for Sustainable Production:. . .

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comparison to female sterility, where it requires crossing. Male sterility has propagation potential, therefore can still set seed in nature. Male sterility can be broadly divided into two groups, viz., genetic where the sterility is governed by genes present in nucleus and mitochondria, and non-genetic, which is momentarily induced through stresses (Kaul 1988). Genetic male sterility may be either spontaneously isolated from nature or artificially induced using mutagenesis or incorporated through protoplast fusion or genetically engineered in the targeted genome. Mode of origin or inheritance of genetic male sterility system led to divide it broadly into, nuclear male sterility (Genic, Genetic, Mendelian) and cytoplasmic male sterility (Chondrial). Nuclear male sterility governed by the genes present in the nucleus, which is monogenic or polygenically controlled. While CMS is a result of altered cytoplasmic organellar genomes, mostly mitochondria, non-genic male sterility (environmentally conditioned, chemically induced) is temporal and spatial in nature induced by the researcher using chemical hybridizing agents or gametocides.

1.2.1

Genic or Genetic Male Sterility (GMS)

Pollen sterility in GMS is governed by nuclear gene(s) and it is reported to have occurred in all diploid species, including cereals, pulses, oilseeds and vegetables (Kaul 1988; Duvick 1966). In most of the plant species, GMS is generally inherited through recessive nuclear gene(s), for example, broad bean (Vicia faba [L.]), grass pea (Lathyrus sativus [L.]), groundnut (Arachis hypogea [L.]), sunhemp (Crotalaria juncea [L.]), soybean (Glycine max [L.] Merr.), pea (Pisum sativum [L.]), common bean (Phaseolus vulgaris [L.]0] and alfalfa (Medicago sativa [L.] spp. sativa) etc., nevertheless, there are some exceptional cases like in white clover (Trifolium repens [L.]), cabbage, broccoli and genetically engineered male sterility where dominant genes are involved in male sterility (Kaul 1988; Williams et al. 1997). The occurrence of GMS credited to a spontaneous mutation in any of the gene(s) that either involves in stamen development or pollen development (microporogenesis) or micro-gametogenesis. There are certain mutants reported, which even though produce viable pollen, are unable to self-fertilize either due to lack of pollen dehiscence or special flower morphology. These types of mutants are frequently termed as functional male sterile, for example, genotypes with exerted stigma or positional sterility in tomato (Georgiev 1991; Atanassova 1999), non-dehiscence of pollen grain in brinjal (Kaul 1988). Spontaneous mutations may alter male fertility controlling dominant (Fr) nuclear gene, which makes it to recessive form under the influence of some natural forces. Subsequently, natural selfing of heterozygotes (Frfr) leads to the appearance of the male-sterile genotypes (frfr) within the population. These male sterile genotypes, if not cross-pollinated by fertile pollen, then are gradually eliminated from their parental population. Therefore, nature does not permit the creation of a population where all plants are male sterile. As a result, the maximum male sterility that can be realized in a population is 50% only, which is obtained in the backcross frfr  Frfr. Further, the male sterile genotype elimination

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process depends on the rate of natural out-crossing in a given population (Dundas 1990; Lasa and Bosmark 1993). Though, in majority of plant species, male sterility is governed by the single recessive gene. However, less frequently, more than one recessive gene (Driscoll 1986), polygenes (Athwal et al. 1967) or by dominant genes (Mathias 1985) operate male sterility in plants.

1.2.2

Cytoplasmic Male Sterility (CMS)

Male sterility in CMS is due to complex interplay between the specific genetic elements residing in nucleus and cytoplasmic organelle (mitochondria) (Bohra et al. 2016). Cytoplasmic male sterility (CMS) is a maternally inherited trait as the mitochondrial genome is accountable for the expression of male sterility and the mitochondria are generally barred from the pollen grain at the time of fertilization. Incompatibility between recessive nuclear gene and specific male sterile cytoplasmic genome results in male sterility in CMS plants. It is reported to occur in several crop plants and is frequently linked with chimeric mitochondrial open reading frames. CMS has been observed in over 140 plant species (Laser and Lersten 1972). Availability of CMS along with sources in a few important crops is listed in Table 1.1. CMS originated due to spontaneous cytoplasmic or nuclear mutation are referred as autoplasmic CMS (Prakash et al. 2015). Interspecific hybridization involving wild relatives have led to occurrence of male sterility in different crops (Bohra et al. 2022). Autoplasmic CMS have been reported in several field crops: T-cytoplasm of maize, pol cytoplasm of B. napus cultivar Polima and the S-cytoplasm of onion, etc. CMS generated through inter-specific or inter-generic crosses, bringing into existence various nuclear–mitochondrial combinations, are called alloplasmic CMS. Thus alloplasmic CMS is an outcome of impaired nucleo-cytoplasmic compatibility between endogenous nucleus and alien cytoplasm. CMS system is an outstanding model to study the relation between nuclear and cytoplasmic genes, because fertility restoration depends on nuclear genes that suppress cytoplasmic abnormalities (Schnable and Wise 1998). When the dominant gene for fertility restoration (Rf) present in the nuclear genome and responsible for pollen fertility in a cytoplasmic male sterile line is recognized, it is called as cytoplasmic genetic male sterility or three line breeding system (A-sterile line, B-maintainer line and R-restorer line). Thus pollen sterility in cytoplasmic genetic male sterility is expressed when sterile mt-genome is located in cytoplasm and recessive allele for fertility restoration (rf) is located in the nuclear genome is present together, but none of them singly can govern sterility. Cytoplasmic male sterility in crop plants may develop in nature through distance hybridization (inter-specific/inter-generic), but it can be also induced artificially using mutagenesis or antibiotic effect (Kaul 1988). Because of their value in hybrid seed production, cytoplasmic male sterile systems have been reported and characterized in many crop species. Pollen sterility is induced by applying gametocides or by growing under conditioned environment. Non-genic male sterility gives higher degrees of freedom

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Table 1.1 List of sterile cytoplasm available in different crops Crop species Rice

Wheat

Maize

Sorghum

Pearl millet

Sterile cytoplasm CW-CMS

Source genotype Oryza rufipogon

BT-CMS LD-CMS CMS-WA CMS-DA IR66707A CMS-D (Dissi) CMS-TN RT102A RT98 A FA-CMS CMS-HL (Honglian) CMS-GA T-cytoplasm YA-type K-type V-type D2 type

Gambiaca Triticum timopheevi CA8057 Ae. kotschyii Aegilops ventricosa Aegilops crassa

CMS-T (Texas)

Golden June (Mexican OPV)

CMS-C (Charrua) CMS-S (USDA) A1 A2 A3

Charrua (Brazilian maize)

A4 A5 A6 9E A1 A2 A3 A4 CMS

IS 7920C IS 7506C IS 1056C IS 112603C Tift 23A IP 189 Amber grain stock Pennisetum glaucum subsp. monodii CMS plant of Large Seeded Gene Pool (LSGP-66) PT 819

A5 CMS Bellary cytoplasm

Chinsurah Boro II Lead Rice O. sativa sp. spontaneae O. sativa sp. spontaneae O. perennis Dianyu 1 A O. sativa O. rufipogon O. rufipogon O. rufipogon O. rufipogon

Teopod maize Milo IS 12662C IS 1112C

References Katsuo and Mizushima (1958) Shinjyo (1969) Watanabe et al. (1968) Lin and Yuan (1980) – Dalmacio et al. (1995) Tao et al. (2004) – Okazaki et al. (2013) Igarashi et al. (2013) Xian-hua et al. (2013) Rao (1988) Lin and Yuan (1980) Wilson and Ross (1962) Liu et al. (2006) Liu et al. (2011) Murai (2002), Chen (2003) Sasakuma and Ohtsuka (1979) Rogers and Edwardson (1952) Beckett (1971) Jenkins (1978) Stephens and Holland (1954) Schertz (1994) Schertz (1994), Tang et al. (2007) Schertz (1994) – – – Burton (1958) Athwal (1965) Athwal (1965) Hanna (1989) Rai (1995) Vetriventhan and Nirmala (2010) (continued)

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Table 1.1 (continued) Crop species Pigeon pea

Brassica

Sterile cytoplasm A1 A2 A3 A4 A5 A6 A7 A8 Ogura CMS Polima CMS

CMS-PET1 CMS GIG 1 CMS GIG 2 CMS 514A CMS PEF1 CMS RES1 CMS ARG 1 CMS-D2 CMS-D8 JA-CMS Peterson CMS CMS CMS

C. lineatus C. platycarpus C. reticulatus Japanese radish Polima spring variety of B. napus Sinapis arvensis Spontaneous CMS variants in Xiangyu (B. napus) Bronowski or Hokuriku 23 (B. napus) Helianthus petiolaris H. giganteus H. giganteus H. tuberosus H. petiolaris sp. fallax H. resinosus Helianthus argophyllus Gossypium harknesii G. trilobum G. hirsutum USDA P.I. 164835 1005 and 1006 C. frutescens

CMS CMS

Seungchon and Suwon CMS-pennellii

CMS CMS CMS CMS CMS

S. gilo S. kurzii S. violaceum S. virginianum S. aethiopicum Aculeatum Group S. anguivi S. grandifolium CV. Morton

Nsa CMS 681A Nap CMS Sunflower

Cotton

Chilli

Tomato Brinjal

Source genotype Cajanus sericeus C. scarabaeoides C. volubilis C. cajanifolius C. cajan

CMS CMS

References Ariyanayagam et al. (1993) Ariyanayagam et al. (1993) Wanjari et al. (1999) Rathnaswamy et al. (1999) Mallikarjuna and Saxena (2005) Saxena et al. (2010) Mallikarjuna et al. (2006) Saxena et al. (2013) Ogura (1968) Li et al. (2011) Yan et al. (2013) Liu et al. (2005) Budar et al. (2006) Leclercq (1968) Whelan (1981) Feng and Jan (2008) Liu et al. (2013) Serieys and Vincourt (1987) Ardila et al. (2010) Christov (1992) Meyer (1975) Stewart (1992) Yang et al. (2014) Peterson (1958) Shifriss and Frankel (1971) Csillery (1983), Woong Yu (1990) Anon (2006) Petrova et al. (1999), Radkova (2002) Fang et al. (1985) Khan and Isshiki (2009) Isshiki and Kawajiri (2002) Khan and Isshiki (2008) Khan and Isshiki (2010) Khan and Isshiki (2011) Hasnunnahar et al. (2012), Saito et al. (2009) (continued)

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Table 1.1 (continued) Crop species French Bean

Sterile cytoplasm CMS CMS CMS

Cole crop

CMS CMS CMS CMS

Radish

Carrot

Beet root

Onion

Ogura cytoplasm NWB cytoplasm CMS

Ogura cytoplasm Wisconsin| or Cornell cytoplasm Petaloid cytoplasm CMS

Owen cytoplasm CMS I-12CMS BMC-CMS S cytoplasm T cytoplasm CMS

Source genotype Accession line G08063 P. coceineus Cultivar “Kurodane Kinugasa” Japanese radish (Raphanus sativus) B. nigra

References Singh et al. (1980) Bannerot and Charbonnier (1987)

McCollum (1981), Dickson (1975), Hoser-Krauze (1987) Pearson (1972), Kaminski and Dyki (2007) Pradhan et al. (1991) Shinada et al. (2006) Chamola et al. (2013)

B. tournefortii D. muralis Erucastrum canariense and Moricandia arvensis Japanese radish

Ogura (1968)

Korean radish

Nahm et al. (2005)

Radish from Uzbekistan

Kim et al. (2007), Lee et al. (2008, 2009) Bang et al. (2011) Ikegaya (1986)

B. maurorum Japanese radish cultivar Kosena Wild carrots “Daucus carota carota”

Thompson (1961) McCollum (1966)

“Guelph” from wild carrot population. Daucus carota gummifer, D. carota maritimus and D. carota gadecaei Cultivar, ‘US1’

Wolyn and Chahal (1998)

A second source from wild beets collected from Pakistan Wild beet Beta maritime Cultivar Italian Red Cultivar Jaunepaille des Vertus Nasik White Globe

Mikami et al. (1985)

Nothnagel et al. (2000)

Owen (1945)

Mann et al. (1989) Jones and Clarke (1943) Berninger (1965) Pathak (1997)

to convert any agronomically superior line(s) as sterile line. Therefore, induction of male sterility by applying male gametocides holds immense potential in utilization of heterosis in crop breeding aside from nuclear encoded genetic male sterility and cytoplasmic male sterility. Wei et al. (2012) recorded SQ-1 (pyridazinone derivative)

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for male sterility induction in maize. The gametocide (SQ-1) treated plants were having viable female organs with more than 90% of male sterility. Therefore, as an alternative of GMS and CMS, the gametocide SQ-1 could be a substitution approach in maize breeding program considering its rapidity and flexibility. Similarly, environmental conditioned male sterility is modulated by temperature (TGMS) and photoperiod (PGMS). Chen and Liu (2014) have described the high temperature and long photoperiod as “Restrictive conditions (RC)” that create pollen sterility. The same line, however, behaves as male fertile under “Permissive conditions (PC),” represented by low temperature and short photoperiod in case of TGMS and PGMS, respectively. This is also known as two-line system where the environment conditioned line is propagated under PC and hybrid seed is produced under RC. Environment-conditioned male sterility has been reported in different crops. For instance, thermo sensitive genic male sterility (TGMS) was reported in Cabbage (Rundfeldt 1961), Brussels sprout (Nieuwhof 1968), broccoli (Dickson 1970), pepper (Daskalov 1972), tomato (Sawhney 1983), carrot (Kaul 1988) and pigeon pea (Saxena 2014). PGMS or photoperiod sensitive genic male sterility was reported in cabbage by Rundfeldt 1961.

1.3

Molecular Basis for Male Sterility and Fertility Restoration

At molecular level, the dysfunction is attributed to compromised crosstalk between cytoplasm and nucleus and is reported to cause CMS, and several mitochondrial CMS-associated genes (MCAGs) have been found in plants. The extensive rearrangement events in plant mitochondrial genomes results in production of new open reading frames (ORFs), chimeric proteins, extensive recombination and deletions in mtDNA; decrease or lack of RNA editing process induce sterility in plants (Bohra et al. 2016). The high-throughput sequencing methods have facilitated generation of whole genome sequences in different flowering plants, especially those with CMS cytoplasm and their corresponding restorer. A comparison of these mitochondrial sequences between CMS line and cognate maintainer line establishes the role of mitochondria-associated genes in CMS induction. Research on various CMS systems has shown that the CMS phenotypes are rescued by the action of nuclear genes called restoration-of-fertility (Rf) genes (Bohra et al. 2012). Pollen fertility is restored through reconciliation of the “lost” harmony between the two genomes with different inheritance. Researchers suggest a metaphorical “molecular arms race” between the newly evolving mitochondrial orfs and the corresponding Rf genes. Genomic locations of Rf genes responsible for CMS restoration have been determined in a wide range of plant species. Fertility restoration (Rf) genes have enormous importance from an economic point of view for efficient seed production of F1 hybrids and academic points of view for their scientific significance as a model system to explore nuclear control on mitochondrial gene expression and also for simultaneous evolution of the nuclear and mitochondrial genome (Chen and Liu 2014; Hanson and Bentolila 2004).

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1.4

9

Hybrid Technology for Economic Yields

The problem of yield stagnation in most of the cultivated crops in spite of the best management practices puts pressure on breeders to break the sealing of yield plateau using hybrid breeding technology. After the successful demonstration of the revolutionary work of G.H. Shull (1914) on the heterosis or hybrid vigour in maize, the popularization of hybrid breeding penetrated swiftly in all the crops. Heterosis is a well-proven genetic tool that has contributed significantly to enhance productivity in crops, and cytoplasmic male sterility is one of the most important mechanisms deployed for commercial hybrid development in cereals, pulses, oilseed and vegetable crops. At present, the most commercial varieties of field crops and vegetables are F1 hybrids that perform stably over a wide range of environments and offers a range of 15–100% of higher vigour in comparison to the popular inbreed line. Exploitation of male sterility system in crops significantly reduces the labour required for hybrid seed production and ensures high varietal purity (Chen and Liu 2014). The tendency for using F1 hybrid seed in crops is going up worldwide day by day not only in term of species/cultivars but the volume of seed required. In developed nations, hybrid varieties were developed and generally hybrid seeds were sown in maize, cotton, bajra, tomato, sweet pepper, eggplant, cucumber, squash, pumpkin, melon, watermelon, cabbage, cauliflower, broccoli, Chinese cabbage, radish and onion. The F1 hybrid varieties are more popular due to their vigour, uniformity, resistance to diseases, tolerance to environmental stress and expression of superior agro-morphological characters like earliness, long shelf-life and consistent high productivity. Development of F1 hybrid is a quick and handy means to combine desirable traits in a large number of crops.

1.5

Conclusion and Prospects

Enormous advances in recent years have expanded our understanding of the CMS and Rf system in several food crops (Bohra et al. 2016, 2021a, b, c; Mishra and Bohra 2018). CMS systems in staple crops need to be diversified and applied in agriculture to avoid genetic vulnerability in hybrid crop production that relies on a few CMS cytoplasm, as evident from T-CMS in maize. Innovative research tools and technologies may greatly support the basic research and applied breeding (Fig. 1.1). Scientists and breeders need to pay more attention to research on other systems, including EGMS, because of its great potential and advantages in hybrid seed production to meet the increasing demand for food. Hybrid technology offers tremendous potential for the much needed second green revolution; as area expansion is not possible, thus the need is to increase production/unit areas. Production of hybrid varieties is a major goal to improve crop plants for exploitation of heterosis. Cost of hybrid seeds is one of the major constraints in achieving more rapid adoption of hybrid technology (Saxena et al. 2015, 2021). Thus use of various genetic emasculation which are cost effective mechanisms is the need of the hour. Significantly higher performance of hybrids over the popular inbreed cultivars has become

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Fig. 1.1 A holistic approach to use male sterility for genetic research and breeding

evident from multi-location evaluation of different crop hybrids. However, growing demand for nutritious food will require more productive crop hybrids in combination with cost-efficient large-scale production of hybrid seeds.

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Advances in Male Sterility Systems and Hybrid Breeding in Rice Ashutosh Kushwah, Sheetal Raj Sharma, K. B. Choudhary, and Suruchi Vij

Abstract

Rice (Oryza sativa L.) is an important staple crop for a large portion of the world’s population. Male sterility trait leads to formation of non-functional pollen. Discovery of these male sterility systems facilitate the exploitation of heterosis in rice which leads to additional enhancement of yield. Over the years, research has uncovered the complexity of various male sterility systems in rice which helps to understand the molecular basis of pollen abortion and fertility rescue. In this article, we briefly describe various types of male sterility systems. We then highlight identification of cytoplasmic male sterility (CMS)-determining orfs and genes/QTL for fertility restoration (Rf) in rice. We discuss the growing role of innovative tools and technologies in discovering CMS and Rf genes for further exploration and exploitation of male sterility systems to improve hybrid breeding. Finally, we present the future perspectives for incorporation of omicslevel data to elucidate mitochondrial-nuclear crosstalk and implementation of genome-based predictions for improving the hybrid performance. Keywords

Fertility restoration · Hybrid · Male sterility · Mitochondria · Molecular mapping · Wild species

A. Kushwah (*) · S. Vij Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, Punjab, India S. R. Sharma Division of Plant Breeding and Genetics, RARI, Jaipur, Rajasthan, India K. B. Choudhary ICAR—Central Arid Zone Research Institute (CAZRI), Jodhpur, Rajasthan, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_2

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2.1

A. Kushwah et al.

Introduction

By 2050, the world population would exceed 10 billion, imposing a challenge on plant breeders for enhanced yield gains to meet the food, fodder, and fuel requirements from the same amount of arable land. Rice (Oryza sativa L.) being an important staple crop globally contributes towards 21% of the calorie supply to world’s population (Hu et al. 2016a, b). Thus global food security mainly depends on sustainable increase of rice yields. Hybrids, which harness the phenomenon of heterosis, have immense potential to help breeders tackle the challenge of achieving sustainable food production. Hybrids do exhibit superior yield and growth characteristics over its parental lines or best commercial check cultivar. Hybrid seed production can rely upon mechanical emasculation, chemical hybridizing agent, and male sterility systems (Zhou et al. 2005). Male sterility circumvents hardship of mechanical emasculation and difficulty associated with time and stage-specificity of CHA. Thus inherent male sterility is preferred by breeders, over manual emasculation and CHA, owing to costeffective, viable, and efficient hybrid seed production technology. Male sterility is a physiological inability characterized by incompetent or inoperative male reproductive parts of the plants, deterring sexual reproduction. Male sterility is an outcome of biological abnormalities ranging from hampered microsporogenesis to production/release of viable pollen. Male sterility is of the following types: Genic male sterility (GMS), cytoplasmic male sterility (CMS), environment sensitive male sterility, viz., photoperiod-sensitive male sterility and thermosensitive male sterility, genetically engineered male sterility, and chemically induced male sterility. In rice, all the male sterility systems have been exploited for hybrid development. Among these, cytoplasmic male sterility has contributed enormously to achieve remarkable productivity. CMS is a non-Mendelian maternally inherited genomic disagreement between mitochondrial and nuclear genomes where mitochondrial genome mediates and nuclear genome opposes male sterility. As male sterility is counteracted by nuclear gene(s), therefore such genes are termed restorer of fertility (Rf). Nuclear restoration is crucial for commercial exploitation of CMS-based hybrid seed development as restoration ensures male-fertile F1 progeny. More than 150 plant species reported CMS ready to get utilization (Laser and Lersten 1972). CMS can arise spontaneously as a result of mutation/mutation followed by selfing or can be created by means of wide hybridization (usually inter-specific), protoplasmic fusion, and genetic engineering (Bohra et al. 2016, 2017). CMS originated due to spontaneous cytoplasmic or nuclear mutation referred to as autoplasmic CMS (Prakash et al. 2009). Autoplasmic CMS has been reported in several field crops: T-cytoplasm of maize, pol cytoplasm of B. napus cultivar Polima, and the S-cytoplasm of onion. CMS generated through interspecific or intergeneric crosses, bringing into existence various nuclear–mitochondrial combinations (Leino et al. 2005), are called Alloplasmic CMS. Thus alloplasmic CMS is an outcome of impaired nucleocytoplasmic compatibility between endogenous nucleus and alien cytoplasm. The well-known CMS-WA (wild abortive) of rice was developed by wild rice (Oryza

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rufipogon) cytoplasm combined with Eejiunan 1 (indica) nucleus. BT-CMS of rice resulted from crossing of indica rice Chinsurah Boro II (cytoplasm) and japonica rice Taichung 65 (nucleus). In 1945, cytoplasmic sterility was discovered in rice for the first time (Sampath and Mohanty 1954). Sasahara and Katsuo developed first rice cultivar, Fujisaka 5, by using transfer the male nuclear cytoplasmic sterile rice genotype. But it did not become popular due to lack of stability and poor performance over environments. However, CMS became the most popular method to develop hybrid rice after successful utilization of heterotic cross into hybrid rice and started the research in China by Yuan Long Ping in 1964. A CMS, pollen-abortive wild rice cytoplasm plant (called wild abortive, i.e. WA) was developed in common wild rice (Oryza rufipogon Griff. L.) in China. As soon as WA male sterile cytoplasm was developed, it was successfully utilized to develop rice hybrid such as Erjiunan 1A, Zhenshan 97A, and V20A in China. Thus China has become most popular and the first country to bring about hybrid rice revolution. Hybrid rice has performed better than conventional varieties by over 20% in yield (Cheng et al. 2007). CMS-based hybrid development consists of three line system: the CMS line (A line), an iso-nuclear maintainer line with normal fertile cytoplasm (B line), and a restorer line (R line) having superior agronomic performance with one or two dominant nuclear Rf genes to yield male fertile F1-hybrids (Bohra et al. 2017). These restorer genes of nuclear origin counter the male sterility favouring mitochondrial transcripts thereby allowing the production of male-fertile F1-hybrids. Sterile cytoplasm of A line can be transferred to given genetic background following recurrent backcrossing of male sterile line with candidate background (nucleus donor) as the pollen parent (Atri et al. 2016). Discovery of potential fertility restorer lines demands extensive field testing of large-scale A
R progenies (Bohra et al. 2012) or R
R or transfer fertility-causing elements into wide genetic-diverse backgrounds. However, a restorer is recovered from inter-specific population and can be obtained from wild relatives (Feng and Jan 2008). Furthermore, the potential exploitation of R line could be slowed down due to undesirable linkage drag mending which involves considerable effort and time.

2.2

Hybrid Development and Heterosis

First attempt to utilize CMS for hybrid seed production was made by D.F. Richey and H.A. Wallace in maize soon after Rhoades described pollen sterility of maize tassel in 1931 (Duvick 1959). In India, the first CMS-based hybrid was developed in sorghum (CSH 1) in 1964, followed by pearl millet (HB-1). Performance of these hybrids prompted plant breeders to utilize CMS for hybrid production in many field and horticultural crops. A brief account on time line of hybrids developed in India is presented in Table 2.1 along with estimated heterosis. In India, first CMS-based rice hybrid APHR-1 was released by ANGRAU, Hyderabad in 1994 recommended for Andhra Pradesh region. APHR-1 out-yielded check variety Chaitanya and scored about 35% higher grain yield. CMS being

1994

2001 2004

2010

PKV Hy3

PGSH-51

PRH 10 GTH-1

ICPH 2671 (first commercial pigeon pea hybrid) MRSA-521

Cotton

Brassica (B. napus) Rice Pigeon pea

Safflower

1980

BSH-1

2006

1993

Year of release 1964 1965

CMS-based hybrid CSH 1 HB-1

Crop Sorghum Pearl millet Sunflower

46% yield advantage, 38% yield superiority over popular variety Maruti

40% higher yield 57% more yield over GMS hybrid AKPH 4101

Pusa 6A
PRR 78 GT 288 A (Cajanus scarabaeoides)
GTR11 ICPA 2043
ICPR 2671

–

10–15% lower than the conventional hybrid developed using the same parents 18% yield advantage over check

100% over open pollinated check variety and 88% yield increment over best local Indian cultivars 25–30% increase in seed yield over Armaviriski3497

Estimated heterosis (%)

CAK 32A
D -286-1R

CMS-234A
RHA-274

Pedigree of hybrid CK60A
IS 84 Tift 23A1
Bil 3B

Table 2.1 A brief timeline of CMS hybrid developed in India along with available estimates on heterosis

Saxena et al. (2013)

Saxena et al. (2010)

Downey and Chopra (1996)

Giriraj (1991), Meena et al. (2013) www.sac.org

References Athwal (1965) Serba et al. (2017)

20 A. Kushwah et al.

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Table 2.2 A brief timeline of CMS-based rice hybrids developed in India along with available estimates on heterosis Hybrid APHR-1 APHR-2 KRH 1 DRRH 1

Year of release 1994 1994 1994 1996

Yield (t/ha) 7.14 7.52 6.02 7.30

PHB-71 Sahyadri CORH-2 PA 6444 Pusa RH 10 Pant Sankar Dhan 3

1997 1998 1999 2001 2001 2004

7.86 6.64 6.25 6.11 4.35 6.12

DRRH 2 Ajay

2005 2005

5.35 6.07

JKRH 401 Indira Sona Sahyadri - 4 DRH - 775 DRRH- 3 CO (R) H-4 Hybrid CO 4 Arize Tej (HRI 169) (IET 21411) Arize Dhani Ankur 7434 Arize 6444 Gold Arize Tej NK 16520 JKRH 3333

2006 2007 2008 2009 2010 2011 2012 2012

6.22 7.0 6.80 7.70 6.07 7.34 7.34 7. 0

Check yield (t/ha) 5.27 (Chaitanya) 5.21 (Chaitanya) 4.58 (Mangala) 5.50 (Tellahamsa) 6.14 (PR-106) 4.89 (Jaya) 5.20 (ADT-39) 4.91(Jaya) 3.11 (PB-1) 4.99 (Pant Dhan12) 4.28 (PHD-1) 4.47 (Tapaswini) – – – – – – – –

2013 2014 2015 2015 2016 2017

– – – 6.5–7.0 6.1 –

– – – – – –

Estimated heterosis 35% 44% 31% 32% 28% 36% 20% 24% 40% 22% 25% 35% – – – – – – – – – – – – – –

Source-Directorate of rice development, Patna: Hybrid Rice Released/Notified in India, 2017 Singh Sanjeev (2013) Hybrid rice development: Two line and three line system. Biologix II(I), pp 178–195

economic over GMS was soon adapted by many plant breeders and, since then, a large number of hybrids have been released by various public and private sectors one after another suitable to specific locations and conditions. These hybrids exhibited remarkable yield gain over existing cultivars in addition to better abiotic and biotic stress resistance. Pusa RH 10, released in 2001, was the first superfine basmati quality rice having yield potential of 4.35 t/ha over check PB-1 (3.11 t/ha). CMS-based rice hybrids released in India has been listed in Table 2.2 indicating their estimated heterosis.

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2.3

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Diversity of Male Sterility-Inducing Cytoplasm

Limited CMS resources and lower variation of CMS lines lead to genetic vulnerability and impose threat to diseases and pests. Diversifying CMS sources hence becomes imperative and indispensable for a sustainable hybrid seed production system. More than 60 types of CMS related with various cytoplasmic resources have been documented in rice (Li and Yuan 2000). Of which, only 13 types of CMS were commercially utilized for hybrid production in rice which include Dwarf wild abortive, Boro II (BT), Dissi, Gambiaka (G), Dian1, Honglian, K52 (K), Indonesia Paddy, Luihui, NX, Maxie, Yegong (Y), and wild abortive (Huang et al. 2014). Similarly, in sunflower, more than 72 CMS sources have been reported (Serieys 2005), but not all have been commercially exploited. Commercial exploitation of CMS source in any breeding program depends on a number of factors like stability for male sterility, effect of CMS on agronomic traits, availability and frequency of restorer gene germplasm, stable and effective restoration, and commercially viable heterosis. These attributes of a CMS source to qualify for utilization in hybrid development program can be achieved through diversifying the existing CMS source in the genetic background of locally adapted genotypes. Till date, various cytoplasm imparting male sterility have been reported in several field crops (Table 2.3).

2.4

Mapping of Factors Related to Fertility Restoration

Cytoplasmic male sterility (CMS), widely known in higher plants, is a maternally inherited trait which causes inability to produce fertile pollen. Various types of CMS are mainly due to expression of aberrant chimeric genes located on mitochondrial genomes. In few CMS lines, a nuclear-encoded gene, which is also known as fertility restorer gene (Rf), is responsible for restoration of pollen fertility. These Rf genes are well known to regulate ectopic mRNAs (Hanson and Bentolila 2004), which is derived from a chimeric gene. Therefore, CMS is supposed to be the occurrence of incompatibility between nuclear and mitochondrial genomes. A lot of CMS-determining candidate genes have been already identified, for example, Pcf in Petunia, T-urf13 in maize, orf224 and orf138 in Brassica, and orfH79 and orf79 in rice. CMS fertility restorer genes have been cloned in petunia, radish, and rice. The gene symbol for representative fertility restorer genes (Rf) and CMS in some main crop species is shown in Table 2.4. A fertility restorer gene (Rf1) and CMS/Rf system or ms-bo-type CMS is utmost considered (Shinjyo 1969). The Southern blot analysis recommends that it contain two different circular molecules in BT type CMS mitochondrial genome. Although it is possible to have a similar structure (Kazama and Toriyama 2016). There are two categories for types of fertility restoration molecules: gametophytic type and sporophytic type. In a sporophytic manner, single dominant Rf gene is responsible for restoration of fertility, while in gametophytic type, pollen fertility of F1 plant produced due to crossing between a restorer line with a CMS line is considered possible by the genotypes of that sporophyte. It suggests that pollen is fertile

Maize

Wheat

O. sativa sp. spontaneae O. sativa sp. spontaneae O. perennis Dianyu 1 A O. sativa O. rufipogon

O. rufipogon

O. rufipogon

O. rufipogon Gambiaca K52 Triticum timopheevi CA8057 Ae. kotschyii Aegilops ventricosa Aegilops crassa Golden June (Mexican OPV) Charrua (Brazilian maize) Teopod maize

CMS-WA CMS-DA IR66707A CMS-D (Dissi) CMS-TN RT102A

RT98 A

FA-CMS

CMS-HL (Honglian) CMS-GA K-type T-cytoplasm YA-type K-type V-type D2 type CMS-T (Texas) CMS-C (Charrua) CMS-S (USDA)

(continued)

Rao (1988) Lin and Yuan (1980) Huang et al. (2014) Wilson and Ross (1962) Liu et al. (2006) Liu et al. (2011) Murai (2002), Chen (2003) Sasakuma and Ohtsuka (1979) Rogers and Edwardson (1952) Beckett (1971) Jenkins (1978)

Xian-hua et al. (2013)

Igarashi et al. (2013)

Lin and Yuan (1980) – Dalmacio et al. (1995) Tao et al. (2004) Athwal and Virmani (1972) Okazaki et al. (2013)

References Katsuo and Mizushima (1958) Shinjyo (1969) Watanabe et al. (1968)

Crop Rice

Cross Chinese wild rice
Fujisaka 5 Chinsurah Boro II
Taichung 65 Lead rice (Burmese indica variety)
Fujisaka 5 O. sativa sp. spontaneae
Hsien (indica) Dwarf aborted
Xue Qin Zhao O. perennis
IR64 Dissi
Zhenshan 97 Taichung Native 1
Pankhari 203 W1125 (O. Rufipogon)
Taichung 65 (O. sativa) W1109 (O. rufipogon)
Taichung 65 (O. sativa) Dongxiang wild rice
Zhongzao 35 (indica) O. rufipogon
Lian Tang Zao Gambiaca
Chao yang 1 K52 (japonica)
Fenglongzao (indica) T. timopheevi
T. aestivum – – – – – – –

Table 2.3 List of cytoplasm discovered to date in various field crops

Source Oryza rufipogon Chinsurah Boro II Lead Rice

Advances in Male Sterility Systems and Hybrid Breeding in Rice

Cytoplasm CW-CMS BT-CMS LD-CMS

2 23

Brassica

Pigeon pea

Pearl millet

Crop Sorghum

A1 A2 A3 A4 A5 A6 A7 A8 Mori-cytoplasm Ogura CMS Polima CMS

A5 CMS Bellary cytoplasm

Cytoplasm A1 A2 A3 A4 A5 A6 9E A1 A2 A3 A4 CMS

Table 2.3 (continued)

Cajanus sericeus C. scarabaeoides C. volubilis C. cajanifolius C. cajan C. lineatus C. platycarpus C. reticulatus Moricandia arvensis Japanese radish B.napus

Source Milo IS 12662C IS 1112C IS 7920C IS 7506C IS 1056C IS 112603C Tift 23A IP 189 Amber grain stock Pennisetum glaucum subsp. monodii CMS of Gene Pool (LSGP-66) PT 819 Burton (1958) Athwal (1965) Athwal (1965) Hanna (1989)

Rai (1995) Vetriventhan and Nirmalakumari (2010) Ariyanayagam et al. (1993) Ariyanayagam et al. (1993) Wanjari et al. (1999) Rathnaswamy et al. (1999) Mallikarjuna and Saxena (2005) Saxena et al. (2010) Mallikarjuna et al. (2006) Saxena et al. (2013) Chandrasekhar et al. (2013) Ogura (1968) Li et al. (2011)

– – – – – – C. sericeus
advanced breeding line C. scarabaeoides
ICPL 85030 C. volubilis
cultivated type ICPW 29
ICPL 28 C. cajan
C. acutifolius C. lineatus
ICPL 99044 C. platycarpus
ICPL 85010 C. reticulatus
C. cajan Mori-cytoplasm into Brassica juncea – –

References Stephens and Holland (1954) Schertz (1994) Schertz (1994), Tang et al. (2007) Schertz (1994)

Cross Milo
Kafir – – –

24 A. Kushwah et al.

Cotton

Sunflower

Nsa CMS Hau CMS Line (00-6102A) 681A Nap CMS CMS-PET1 CMS-PET2 CMS I CMS GIG 1 CMS GIG 2 CMS 514A CMS PEF1 CMS RES1 CMS ARG 1 CMS-D2 CMS-D8 JA-CMS Liu et al. (2005) Budar et al. (2006) Leclercq (1969)

– – Helianthus petiolaris
H. annuus Helianthus petiolaris
H. annuus H. lenticularis
H. annuus H. giganteus
H. annuus H. giganteus
H. annuus H. tuberosus
Inbred line 7718B H. petiolaris sp. fallax
H. annuus H. resinosus
H. annuus Helianthus argophyllus
H. annuus G. harknessii
G. hirsutum G. trilobum
Gossypium hirsutum –

B. napus B. napus Helianthus petiolaris H. petiolaris H. lenticularis H. giganteus H. giganteus H. tuberosus H. petiolaris sp. fallax H. resinosus Helianthus argophyllus Gossypium harknesii G. trilobum G. hirsutum

Whelan (1981) Feng and Jan (2008) Liu et al. (2013) Serieys and Vincourt (1987) Ardila et al. (2010) Christov (1992) Meyer (1975) Stewart (1992) Yang et al. (2014)

Yan et al. (2013) Heng et al. (2014)

S. arvensis
B. napus –

Sinapis arvensis B. juncea

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Table 2.4 Examples of fertility restorer (Rf) genes and CMS in some important crop species Crop Rice

Maize

Sunflower B. napus

B. juncea

B. tournefortii Sorghum

Wheat

CMS type BTCMS LDCMS HLCMS CWCMS WACMS RT120CMS RT98CMS T-CMS

Associated ORF B-atp6-orf79

Protein property PPR protein

Rf Rf1

Mode of restoration Gametophytic

L-atp6-orf79

Glycin-rich protein

Rf2

Gametophytic

atp6-orfH79

PPR protein

Rf5,Rf6

Gametophytic

Orf307

Rf17

Gametophytic

rpl5-WA352

Acyl-carrier protein synthase Unknown

Rf3, Rf4

Sporophytic

rpl5-orf352

Unknown

Rf102

–

orf113-atp4cox3 urf13-atp4

Unknown

Unknown

–

Rf1, Rf2

Sporophytic

S-CMS C-CMS PET1CMS OguCMS PolCMS NapCMS CMSHau CMSorf220 TourCMS A3CMS A1CMS APCMS

orf355-orf77 atp6-C atp1-orf522

Aldehyde dehydrogenase Unknown Unknown Unknown

Rf3 Rf4 Rf1

Gametophytic Sporophytic Gametophytic

orf138-atp8

PPR protein

Rfo

Sporophytic

orf224-atp6

Unknown

Rfp

Sporophytic

orf222nad5corf139 atp6-orf288

Unknown

Rfn

Sporophytic

Unknown

Unknown

Sporophytic

orf220

Unknown

Unknown

–

atp6-orf263

Unknown

Unknown

Sporophytic

orf107

Unknown

Rf3

Gametophytic

Unknown

PPR protein

Rf1, Rf2

Gametophytic

orf256

Unknown

Unknown

–

irrespective of absence or presence of restorer gene participates in the fertilization of F1 plant. In F2 generation, a quarter of total plants is to be sterile. If restoration of fertility performs in gametophytic manner, genotype of pollen grain governs its own fertility. Consequently, pollen having no restorer gene in F1 plant is incapable of germinating on to the stigma, and F2 plants are anticipated to be fertile. Several

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fertility restorer genes function gametophytically on fertility restoration, except those for WA-CMS.

2.4.1

Molecular Mapping of Rf Genes

Molecular mapping of the fertility restorer genes of rice has been done by a few research groups. Fertility restoration is conferred by one of two loci in WA-CMS: Rf4 on chromosome 10 and Rf 3 on chromosome 1 (Zhang et al. 1997). The Rf4 is revealed to be firmly linked to DNA marker adjacent to Rf1 locus. In a research, two HL-CMS fertility restorers, i.e. Rf5 and Rf6(t), were mapped (Liu et al. 2004), both on chromosome 10. The Rf5 was recognized in region of 10 cM from Rf1, while Rf6 (t) was tightly linked to Rf1. In another study on Dian-type 1 CMS, fertility restorer gene Rf-D1(t) is situated in a region analogous to Rf1, Rf4, Rf5, Rf6(t) (Tan et al. 2004). It cannot be considered as casual coincidence, but probably derives from co-evolution of fertility restorer locus and CMS cytoplasm. The occurrence of the PPR gene cluster located in Rf1 region stipulates a hint of frequent duplication and recombination inside this region. Besides from fertility restorer genes located on chromosome 10, Rf17 for CW-CMS on chromosome 4 and Rf2 for LD-CMS on chromosome 2 were mapped (Fujii and Toriyama 2005). By knowing the role of fertility restorer proteins, we can understand the mechanisms of CMS restoration from Rf1-mediated case.

2.4.2

Molecular Cloning of Rf Elements

High density molecular mapping and cloning of Rf1 was completed by numerous research workers (Akagi et al. 2004). On the basis of sequence of Rf1, the protein of Rf1 was revealed to be a penta-tricopeptide repeat protein (PPR). PPR proteins are known as a penta-tricopeptide repeat as they are distinguished by occurrence of tandem arrays of degenerated 35 amino acids (Small and Peeters 2000). Rf1 protein contains18 repeats of PPR motif and 26 amino acids of N terminal sequence of mitochondrial targeting sequence, but does not include other identified motif. In Arabidopsis, organelle gene expression is regulated by several genes encoding PPR proteins. For example, processing of plastid psbB-psbT-psbH-petB-petD operon required PPR protein HCF152 in Arabidopsis thaliana (Meierhoff et al. 2003). In Arabidopsis thaliana plastid ndhD gene CRR4 is involved in the editing of a specific base (Kotera et al. 2005). PPR genes involve a large family, having about 480 members in rice and 450 members in Arabidopsis thaliana and involved for alteration of organelle mRNAs (Lurin et al. 2004). ORFs containing a mitochondrial targeting signal and PPR motif have been searched after mapping of Rf1, as restorer genes have been earlier cloned in petunia and in Kosena radish and shown to encode PPR proteins ((Koizuka et al. 2003). Identification of Rf and PPR containing genes in petunia and radish, respectively, proposed that mapping of PPR motif genes located close to identified restorer loci can be a useful tactic for searching of Rf genes in other

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species, counting rice. Three PPR genes (PPR8-1, PPR8-2, and PPR8-3) with mitochondrial targeting sequence were identified by Kazama and Toriyama (2003) in a fertility restorer line (Milyang 23). They were pretty analogous to each other and situated in a tandem array. Nucleotide sequence of PPR8-1 confirms 93.2% and 93.7% identical to PPR8-2 and PPR8-3, respectively. Four PPR genes (PPR683, PPR762, PPR791, and PPR794,) identified by Komori et al. (2004) are present in Rf1 region, out of which, PPR791 was known to be Rf1 in another fertility restorer line, IR24. PPR762 sequence was not present in other restorer lines like MTC-10R and Milyang 23 and Rf1 is identical with PPR8-1, Rf1-A, and PPR791 (Kazama and Toriyama 2003). Using fine-resolution mapping of Rf1, Wang et al. (2006) cloned two different fertility restorer genes, i.e. Rf1a and Rf1b from 105 kb region of classical Rf1 locus on an elite line of fertility restorers, Minghui 63. The Rf1a gene contains 18 PPR repeats, whereas Rf1b encodes 11 PPR repeats and restores fertility to BT-CMS. It is evident from Kazama and Toriyama (2003) that Rf1a is capable of processing dicistronic B-atp6–Orf 79 mRNAs. A transgenic BT-CMS expressing Rf1b does not have B-atp6 and orf 79 mRNAs, suggesting that Rf1B promotes degradation of the entire B-atp6-orf 79 mRNA instead of just processing intergenic sequences. Based on the finding that a line having both Rf1a and Rf1b has a similar pattern of B-atp6 transcription factor as like in the line carrying Rf1a alone. Wang et al. (2006) anticipated that Rf1a locus is epistatic to Rf1b locus. The researchers concluded that the processed mRNA cannot be degraded by RF1B, if B-atp6–orf 79 mRNA is processed by RF1A.

2.5

Progress Towards Deciphering Male Sterility Determinants

The mitochondrial genome of Nipponbare is reported to contain 35 genes for proteins, 2 pseudo-ribosomal protein genes, 3 ribosomal RNAs, 5 pseudo-tRNAs and 17 tRNAs (Notsu et al. 2002). Many genes contain multiple exons and are distributed all over the mitochondrial genome which trans-spliced to form functional mRNAs. All genes exist in rice mitochondrial genome sequences, except the predicted ORFs (Asaf et al. 2016). Through illegitimate homologous recombination, sequence complexity of mitochondrial genomes in plants sometimes creates new sequences and ORFs that lead to CMS. Researchers have mapped genes that cause CMS (Igarashi et al. 2013). Comparing the gene structures and expression profiling of CMS and normal mitochondria led to the identification of CMS-associated genes. During rice breeding, CMS lines are derived through cytoplasmic substitution through backcrossing in which cytoplasmic donor carries Rf genes in its nuclear genome. Cytoplasmic donor has CMS associated gene and Rf gene, thus Rf gene suppresses CMS-associated gene, hence no male sterility. Northern blots technique is most common for screening CMS lines and fertility restorer lines for identifying CMS-associated genes (Hanson and Bentolila 2004). Mitochondrial chimeric genes, first discovered in BT-CMS, were possibly involved CMS in rice. Pollen abortion starts after pollen mitosis in the BT-CMS

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line. Nevertheless, restoration of Pollen fertility can be achieved by gene product of Rf1, single dominant gene. Mitochondrial sequence of BT cytoplasm includes two copies of atp6 gene, which encodes subunit 6 of ATPase complex. First copy is similar to Nipponbare atp6 (N-atp6) and the other copy has an altered 30 UTR sequence (B-atp6). In the B-atp6 genes, a unique sequence (orf79) encodes 79 amino acid ORFs and a putative transmembrane protein. The findings of B-atp6 and orf79 were crucial for understanding the complexity of CMS in rice. Researchers found that an HL-CMS line has a gene structure analogous to B-atp6-orf79, wherein orfH79 is present adjacent to atp6 (Yi et al. 2002). Conversely, association of orfH79 with restorer genes (Rf5, Rf6) for HL type CMS still remains unclear. Additionally, LD-CMS line contains the homologous B-atp6–orf79 gene L-atp6–orf79, but does not carry N-atp6. In B-atp6–orf79 gene, Rf1 cleaves L-atp6–orf79 mRNA into 0.45 and 1.5 kb fragments, and restores the fertility of LD-CMS lines. Although, fertility restorer gene of LD-CMS line, Rf2, is not involved in the processing of L-atp6–orf79 mRNA. Rf2 also plays a role in partial fertility restoration for BT-CMS; however Rf2 inhibits the processing of B-atp6–orf79 mRNA. These studies depicted that Rf1 and Rf2 play distinct roles in fertility restoration. In addition, an orf similar to orf79, i.e. orf107 was detected in A3-type CMS line IS1112C in sorghum (Tang et al. 1996). In BT-CMS line of rice, C-terminal sequences are akin to C-terminus of ORF79 (Tang et al. 1999). Although progress has been made to understand the nature of BT-CMS rice, the factors associated with other rice CMS still remain unknown.

2.6

Approaches Towards Understanding of Molecular Mechanism for CMS Induction

There have been numerous reports of BT-CMS/Rf1 mechanism, but “How does B-atp6-orf 79 act in CMS?” has been questioned for more than a decade, while some of them were answered by Wang et al. (2006). In transgenic rice, orf79 expression causes gametophytic male sterility with normal cytoplasm. Using IPTG-induced promoter, orf79 causes lethal effects in E. coli bacteria and C terminus was crucial for this toxicity. In BT-CMS line, antibody rose against recombinant ORF79 showed accumulation of ORF79 protein exclusively in microspores. The production was concealed by RF1A and/or RF1B gene with unique mechanisms, i.e. endonucleolytic cleavage and dicistronic B-atp6–orf79 mRNA degradation, respectively. In the absence of an enzyme motif in either RF1A or RF1B, they are believed to identify and bind B-atp6-orf 79 mRNA-specific sequence to cooperate with important proteins required for RNA cleavage and degradation. In addition, they showed editing of atp6 mRNA with RF1A, although RF1B does not. Conclusively, Wang et al. (2006) stated that cytotoxic peptide was coded by orf 79 which is responsible for BT-CMS. Unexpectedly, the abnormal mitochondrial ORF, orf79 is cotrabcribed with unedited atp6 gene and causes toxicity in the development of microspores. Although, the mechanism of cytotoxicity caused by orf79 was not revealed till now. The transcript

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profile of normal and CW-CMS cytoplasm was analysed from mature anther using oligoarrays to find the nuclear genes responsible for pollen abortion (Fujii et al. 2007). In CW-CMS line, 58 genes were detected that was upregulated by more than three-fold, while downregulated genes were 82. Further, 20 genes from a different organ were studied, and five genes that also include alternative oxidase genes, were expressed whilst no expression was noticed in a fertility-restored CW-CMS line. Expression of CW-CMS-specific genes was traced specifically in mature anthers and not in other plant part, signifying nuclear gene expression with mitochondrial retrograde regulation. Several nuclear genes that are regulated by mitochondrial genome require specific nuclear-mitochondrial interactions for their expression. Thus, mitochondrial gene has a vital role in development of normal pollen. Recently, next-generation sequencing is being widely used to recognize CMS-associated genes. New ORFs are being identified by comparing CMS rice mitochondrial genome which are absent in reference genome sequence, viz., Nipponbare (Kazama and Toriyama 2016). Later, CMS-linked genes were chosen that are chimeric to mitochondrial genes or peptides containing transmembrane domains, since the documented CMS-linked genes have those characteristics. Further comparison of candidate gene expression was conducted in presence and absence of Rf gene(s) to determine the pattern. However, presently it’s not possible to alter mitochondrial genome by knock-in and out of DNA fragment and specific ORFs, respectively. Thus, CMS-causative gene has no absolute evidence with CMS-inducing gene.

2.7

Marker-Assisted Selection and Hybrid Breeding

Conventional hybrid breeding suffers from longer generation time, selection of parents having narrow genetic diversity, resulting in poor heterosis on-farm seed production to assess genetic purity (grow out test), which slows down breeding efficiency and selection speed as these activities have to be performed through visual selection and analysing data based on morphological characteristics. MAS is being increasingly employed in hybrid breeding for several purposes like characterization of genetic resources, assessment of collection redundancies and gaps, simultaneous selection for multiple traits governed by multiple genes, selection without intensive laboratory work or laborious fieldwork, selection for environment sensitive traits (such as temperature/photoperiod sensitivity, seed traits, quality traits and abiotic and biotic stresses) at an earlier breeding stage, construction of core collection, precise gene introgression, assessment of hybrid performance and scrutinizing of seed quality in seed production. Applications of molecular marker-assisted selection in rice hybrid breeding have been presented in Table 2.5. MAS has been successfully utilized for genetic purity assessment in rice using STS and SSR markers by Yashitola et al. 2002. CAPS marker has been used to differentiate CMS line from maintainer line in rice with ease and certainty (Ngangkham et al. 2010). MAS has successfully improved the existing parental lines in terms of biotic or abiotic stress tolerance in rice. Restorer line RPHR 1005 of

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Table 2.5 Applications of molecular marker-assisted selection in rice hybrid breeding MAS applications Gene tagging

Marker SSR

Accelerated conversion of B line into A line

SSR and ISSR

Improvement of the existing restorer line by gene stacking Gene pyramiding

SSR

Simultaneous multi-trait improvement

Assessment of genetic purity (parents/hybrids)/ unambiguous identification of hybrids (alternative to tedious grow out test

Fast-track recovery of potential R lines

Early/rapid discrimination among parental lines Discriminating A and B lines

Construction of high heterotic groups

Traits Four Rf genes (Rf 3, Rf 4, Rf 6, Rf 7) were tagged on different rice chromosomes Transfer of CMS into maintainer line by markerassisted backcrossing (MABC) method RPHR-1005 improved for bacterial blight and blast Xa21 and Xa33 incorporated in DRR17B to confer bacterial blight resistance through MABB

References Bazrkar et al. (2008)

Rf34 and Rf44 (restorer genes), gs3, gw8, Wxg1 and Alk (grain quality genes), qBLAST11 (blast resistance gene) pyramided Bigger panicle, brown plant hopper resistance and fertility restoration

Dai et al. (2016)

Yashitola et al. (2002)

SSRs

Detection of off-types, extent of heterozygosity within parental lines of rice hybrids Differentiate parental lines of the hybrids, resolving the problems associated with seed certification program HL-CMS

CAPS

CW-CMS

CAPS AFLP

Differentiation of CMS line from its maintainer Differentiated A and B lines

SCAR

Differentiated A and B lines

SSR

Identifying foundational hybrid parents in germplasm pools to provide a reference for hybrid rice breeding Validation of heterotic patterns

Genespecific PCR markers and SSRs SSR

Genespecific PCR markers and SSRs SSR and STS STMS

SNP

Ahmadikhah et al. (2015)

Abhilash et al. (2016) Balachiranjeevi et al. (2018)

Fan et al. (2017)

Bora et al. (2016)

Huang et al. (2003) Fujii and Toriyama (2005) Ngangkham et al. (2010) Rajendran et al. (2007) Chao et al. (2013) Xie et al. (2014)

Wang et al. (2015)

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rice hybrid, DRRH-3 has been improved for blast and bacterial blight resistance through marker-assisted backcross breeding (MABB). Xa21 and Xa33 genes for blight and Pi2 gene for blast resistance were introgressed to RPHR 1005 along with simultaneous selection for fertility restorer genes (Rf3 and Rf4) using gene-specific functional markers and SSR markers (Abhilash et al. 2016). Xa21 and Xa33 have been incorporated in DRR17B, an elite maintainer line of rice hybrid development program through marker-assisted backcross breeding (MABB) to confer bacterial blight resistance using gene-specific PCR-based markers for foreground selection and SSR markers for background selection (Balachiranjeevi et al. 2018). Similarly xa5, xa13, and Xa21 genes imparting bacterial blight resistance have been incorporated in rice maintainer lines (Ramalingam et al. 2017). Gn8.1 (big-panicle gene), Bph6, and Bph9 (brown plant hopper-resistant genes) and Rf3, Rf4, Rf5, and Rf6 (fertility restorer genes) have been pyramided to develop wide spectrum restorer lines for BPH resistance through MAS. Restorer lines thus developed have broad spectrum recovery potential towards Wild abortive CMS, two-line GMS sterile lines and Honglian CMS along with high seed yield and disease resistance (Fan et al. 2017). Gene tagging is one of the significant contributions in MAS which can be efficiently utilized for pyramiding of numerous Rf genes mapped till date from several male sterility systems in the genetic background of restorer line to develop agronomically superior restorer line in rice (Bazrkar et al. 2008). In rice, four Rf genes (Rf 3, Rf 4, Rf 6, Rf 7) has been tagged on chromosomes 1, 7, 10, 12, respectively, employing simple sequence repeat markers (SSR) (Bazrkar et al. 2008), which would further facilitate identification of potential restorers in hybrid rice breeding program with lesser tedious workload. After the remarkable advancement in molecular markers, next generation markers like DNA methylation-specific epigenetic markers were developed (Bird 2007) which are able to mine variations beyond the nucleotide sequence level. Methylation sensitive amplification polymorphism technology (MSAP) has been recently introduced in wheat to analyse male sterile and maintainer lines associated to diverse cytoplasm such as S, K, and T (Ba et al. 2015). Similar technology can be extended to crops like rice. Marked variation comprising hypermethylation/demethylation can bring epigenetics into picture to resolve CMS complexity and to assist hybrid breeding in various crops.

2.8

Scope for Adoption of Modern Technologies

Modern technologies can provide comprehensive insight on molecular mechanism and help understand the regulation of male sterility and delineate the pathways involved in pollen abortion in rice. Advance omics technology can lead to identification of key genes involved and decipher regulatory networks affecting pollen fertility.

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Transcriptomic Profiling of Male Sterile Lines

The photoperiod sensitivity of pollen development in rice was expressed by the transcriptomic study that marked the upregulation of 11,726 differentially expressed genes (DEGs). Of the total, 177 were differentially expressed at six time points over a 24-h period. Genes were enriched in lipid and carbohydrate metabolism, transport, and phytohormone signalling. Four hundred and ninety-six hub genes were identified by co-expression network analysis, exhibiting high relativity to photoperiod-sensitive DEGs. These hub genes included Carbon starved anther and UDP-glucose pyrophosphorylase, which have been earlier reported as photoperiod- and temperature-sensitive genes, respectively, affecting male fertility (Sun et al. 2021a, b). Rice alloplasmic sporophytic CMS lines analysed for microarray based transcriptome profiling expressed 622 DEGs in every CMS line. The set of upregulated DEGs was playing a pivotal role in lipid metabolism and cell wall structure and organization. Weighted gene co-expression network analysis differentially expressed a major quantum of hub genes in the CMS lines (Hu et al. 2016a, b). In a study, microarray based transcriptome profiling of TGMS-Co27 and wild type Heijang was carried out at high and low temperatures which revealed that 15462 probes representing 8303 genes have been differentially expressed. Series-cluster analysis of DEGs exhibited that low temperature induced the expression of a gene cluster which included many meiosis stage-related genes (Pan et al. 2014). RNA-Seq performed in rice TGMS line revealed 1070 DEGs at the microspore mother cell and meiosis stages. These DEGs were enriched in protein folding and binding, transcriptional regulation, TF activity, and metabolic processes. The study suggested hub genes UbL40s, DNA-directed RNA polymerase subunit, HSPs and kinases regulating fertility alteration in rice TGMS lines (Li et al. 2020). Transcriptional profiling of TGMS sterile rice panicles at dyad stage depicted an upregulation of 232 DEGs. Further qRT-PCR study of 20 selected DEGs (comparison was drawn between panicles of wild type rice and TGMS exposed to high and low temperatures), expressed six DEGs may be unique to TGMS, while the rest out of 20 DEGs had an overlapping response in both TGMS and wild-type type at dual temperature conditions (Khlaimongkhon et al. 2021). Whole transcriptome profiling of immature florets of IR58025A (WA-CMS line) and IR58025B (isonuclear maintainer line) revealed 774 differentially expressed transcripts (DETs), of which 496 were downand 278 were upregulated in CMS line compared to maintainer line. The expression of genes related to antioxidative defence was upregulated, whereas those associated with pectinesterase activity, cell wall modifications, and respiration were downregulated (Pranathi et al. 2019). These studies would be useful for gaining insight into molecular mechanisms controlling male sterility in rice in response to environmental alterations.

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Whole Genome Sequencing of Mitochondria

Tetep-CMS lines that were developed by successive backcrossing between rice subspecies japonica and indica were subjected to whole genome sequencing and de novo assembly of mitochondrial genome. Chimeric gene orf312 possessing transmembrane domain and overlapping with two mitotype-specific sequences (MSSs) were identified. Encoded peptide of orf312 contains COX11-interaction domains, considered functional major domain for WA352c in WA-CMS line. A QTL for Rf-Tetep (fertility restoration) has been identified on chromosome 10. Interactions of orf312 and Rf-Tetep confer male sterility and fertility restoration, respectively (Jin et al. 2021). CMS line RT102A and fertility restorer line RT102C, obtained by successive backcrosses between O. sativa Taichung 65 and O. rufipogon W1125, have been subjected to whole genome sequencing of mitochondria in RT102-CMS using next-generation pyrosequencing. A candidate ribosomal protein gene rpl5 was co-transcribed with CMS-associated chimeric gene orf352. The rpl5orf352 transcripts sized 2.8 kbs were processed into 2.6 kb transcripts in the presence of Rf gene (Okazaki et al. 2013). Similarly Tadukan-type CMS line (TAA) and its restorer line (TAR) were developed by successive backcrossing between O. sativa cultivars Taichung 65 and Tadukan. Whole-genome sequencing of mitochondria of TAA employing Illumina HiSeq, identified orf312, being differentially expressed in CMS line and restorer line (Takatsuka et al. 2021). Expression profiling of Boro-type mitochondrial genome using pyrosequencing indicated orf79 as unique CMS-associated gene in Boro-type mitochondria compared to standard japonica cultivar Nipponbare (Kazama and Toriyama 2016).

2.8.3

Micro RNA-Regulated Male Sterility

Micro-RNA (miRNA) are endogenously expressed small RNAs having 21–24 nucleotides. MiRNAs have been demonstrated in regulation of male sterility along with other developmental crop stages (Mishra and Bohra 2018). The miRNAs of photo-thermo-sensitive genic male sterile rice line Peiai64S anthers were sequenced through high-throughput sequencing at alternating high (PA64S-H) and low temperature (PA64S-L) conditions. A differential expression of total of 133 miRNAs was reported between PA64S-H and PA64S-L. Target genes were shown encoding MYB and TCP transcription factors and bHLH proteins related to pollen development and male sterility. KEGG pathway annotation influenced metabolic pathways of sphingolipid metabolism, starch and sucrose metabolism, hormonal signalling pathways, and arginine and proline metabolism (Wu et al. 2019). In expression profiling of miRNAs in PTGMS rice line, Wuxiang S (WXS) identified 497 known miRNAs and 273 novel miRNAs through RNA-seq. Twenty-six miRNAs exhibited significant differential expression between sterile and fertile lines. qRT-PCR validation depicted downregulation of 11 and upregulation of 15 miRNAs in sterile line compared to fertile line. miRNAs (osa-miR156a-j, osa-miR164d, and osa-miR528) were found to be negatively correlated with targets being related to pollen

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development and male sterility (Zhang et al. 2016). Small RNA library sequencing of anthers from PTGMS rice line Peiai64S and PA64S(F) deciphered 196 miRNAs, of which 166 were known and 30 were novel miRNAs. Thirteen pairs of miRNA/ target genes have been identified regulating male fertility of PA64S by affecting SPLs, lignin synthesis of anther walls, and flavonoid metabolism pathway (Sun et al. 2021a, b).

2.8.4

Proteomic Investigation of Male Sterile Lines

Proteomic analysis of anthers in rice AnnongS-1 (TGMS line) using tandem mass tag (TMT) subjected to high and low temperatures led to the differential accumulation of 89 proteins. Out of 89 proteins, 46 were upregulated and 43 were downregulated in abundance. Many of the differentially abundant proteins (DAPs) were enzymatic in nature, having their roles in photosynthesis, metabolic regulation, protein and carbohydrate metabolism, and antioxidative defence strategies (Wang et al. 2017). Translating ribosome affinity purification (TRAP)-based translatome analysis coupled with RNA sequencing of TGMS line in rice expressing FLAGtagged ribosomal protein L18 from germline-specific promoter MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1) was investigated. DEGs at the transcriptional and translational levels were highly correlated to pollen and anther development and involved in tapetum programmed cell death and lipid metabolism during pollen development and anther dehiscence (Liu et al. 2022).

2.9

Major Challenges and Potential Opportunities

Remarkable research milestones have been attained in the past decades, elucidating the complex mechanisms controlling male sterility in crop species arising as a result of interaction between nuclear and cytoplasmic genomes. Several ORFs associated with CMS and related Rf genes have been characterized. Findings from previous studies depict set of genes at transcriptional, post transcriptional, and translational levels which were involved in imparting male sterility in rice. However, mitochondrial signalling pathways conferring sterility and metabolome profiling of male sterility lines in rice needs greater attention in days to come for better comprehension. Additionally, mechanisms governing the induction and regulation of male sterility under different environmental conditions require further clarity. Recent advancements of new technologies can supplement and assist in resolving existing challenges. High throughput sequencing platforms enabling whole genome sequencing, whole transcriptome, proteome, and metabolome characterizations will facilitate the mining of Rf genes in CMS systems of various crops. Understanding the complexity of mitochondrial genome sequences of CMS lines may be a search tool uncovering novel CMS-associated ORFs. Exploitation of genome-wide association studies along with high throughput mapping of candidate genes will enable dissecting the genetic architecture of Rf genes with better resolution. Translatome

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characterization of male sterile lines in rice may further assist in enhancing our knowledge of genetic basis of sterility and help in genetic modification in rice hybrid breeding. Molecular cloning (map-based cloning, T-DNA or transposon tagging, or reverse genetic approaches) would add valuable information to our understanding on male sterility systems. Targeted mutagenesis employing RNA interference silencing will be an effective technique in functional characterization and validation of sterility determinants. Implementation of genome editing techniques like zinc finger nuclease, transcription activator-like effector nuclease, and CRISPR-Cas9 may provide functional confirmation of male sterility genes.

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Lin SC, Yuan LP (1980) Hybrid rice breeding in China. In: Innovative approaches to rice breeding. Selected papers from the 1979 international rice research conference. The International Rice Research Institute, Philippines, pp 35–52 Liu XQ, Xu X, Tan YP et al (2004) Inheritance and molecular mapping of two fertility restoring loci for Honglian gametophytic cytoplasmic male sterility in rice (Oryza sativa L.). Mol Gen Genomics 271:586–594 Liu Y, Dong ZS, Zhang GS et al (2005) Cytological study on growth of anther of CMS 212A in Brassica napus L. Acta Agric Boreali Occident Vin 14:33–37 Liu CG, Hou N, Liu LK, Liu JC, Kang XS, Zhang AM (2006) A YAtype cytoplasmic male sterile source in common wheat. Plant Breed 125:437–440 Liu H, Cui P, Zhan K et al (2011) Comparative analysis of mitochondrial genomes between a wheat K-type cytoplasmic male sterility line and its maintainer line. BMC Genomics 12:163 Liu Z, Wang D, Feng J, Seiler GJ, Cai X, Jan CC (2013) Diversifying sunflower germplasm by integration and mapping of a novel male fertility restoration gene. Genetics 193:727–737 Liu W, Sun J, Li J, Liu C, Si F, Yan B, Wang Z, Song X, Yang Y, Zhu Y, Cao X (2022) Reproductive tissue-specific translatome of a rice thermo-sensitive genic male sterile line. J Genet Genomics. https://doi.org/10.1016/j.jgg.2022.01.002 Lurin C, Andres C, Aubourg S et al (2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16:2089–2103 Mallikarjuna N, Saxena KB (2005) A new cytoplasmic nuclear male sterility system derived from cultivated pigeonpea cytoplasm. Euphytica 142:143–114 Mallikarjuna N, Jadhav D, Reddy P (2006) Introgression of Cajanus platycarpus genome into cultivated pigeonpea genome. Euphytica 19:161–116 Meena HP, Sujatha M, Varaprasad KS (2013) Achievements and bottlenecks of heterosis breeding of sunflower (Helianthus annuus L.) in India. Indian J Genet 73(2):123–130 Meierhoff K, Felder S, Nakamura T, Bechtold N, Schuster G (2003) HCF152, an Arabidopsis RNA binding pentatricopeptide repeat protein involved in the processing of chloroplast psbBpsbTpsbH-petB-petD RNAs. Plant Cell 15:1480–1495 Meyer VG (1975) Male sterility from Gossypium harknessii. J Hered 66:23–27 Mishra A, Bohra A (2018) Non-coding RNAs and plant male sterility: current knowledge and future prospects. Plant Cell Rep 37:177–191 Murai K (2002) Comparison of two fertility restoration systems against photoperiod-sensitive cytoplasmic male sterility in wheat. Plant Breed 121(4):363–365 Ngangkham U, Parida SK, De S, Kumar KAR, Singh AK, Singh NK, Mohapatra T (2010) Genic markers for wild abortive (WA) cytoplasm based male sterility and its fertility restoration in rice. Mol Breed 26:275–292 Notsu Y, Masood S, Nishikawa T, Kubo N, Akiduki G et al (2002) The complete sequence of the rice (Oryza sativa L.) mitochondrial genome: frequent DNA sequence acquisition and loss during the evolution of flowering plants. Mol Gen Genomics 268:434–445 Ogura H (1968) Studies on the new male-sterility in Japanese radish, with special reference to the utilization of this sterility towards practical raising of hybrid seed. Mem Fac Agric Kagoshima Univ 6:39–78 Okazaki M, Kazama T, Murata H, Motomura K, Toriyama K (2013) Whole mitochondrial genome sequencing and transcriptional analysis to uncover an RT102-type cytoplasmic male sterility associated candidate gene derived from Oryza rufipogon. Plant Cell Physiol 54:1560–1568 Pan Y, Li Q, Wang Z et al (2014) Genes associated with thermosensitive genic male sterility in rice identified by comparative expression profiling. BMC Genomics 15:1114. https://doi.org/10. 1186/1471-2164-15-1114 Prakash S, Bhat SR, Quiros CF, Kirti PB, Chopra VL (2009) Brassica and its close allies: cytogenetics and evolution. Plant Breed Rev 31:21–187 Pranathi K, Kalyani MB, Viraktamath BC, Balachandran SM, Hajira SK, Koteshwar Rao P, Kulakarni SR, Rekha G, Anila M, Koushik MBVN, Senguttuvel P, Hariprasad AS, Mangrautia SK, Madhav MS, Sundaram RM (2019) Expression profiling of immature florets of IR58025A,

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a wild-abortive cytoplasmic male sterile line of rice and its cognate, isonuclear maintainer line, IR58025B. 3 Biotech 9(7):278 Rai KN (1995) A new cytoplasmic-nuclear male sterility system in pearl millet. Plant Breed 114: 445–447 Rajendran N, Gandhimani R, Singh S, Palchamy K (2007) Development of a DNA marker for distinguishing CMS lines from fertile lines in rice (Oryza sativa L.). Euphytica 156(1):129–139 Rao YS (1988) Cytohistology of cytoplasmic male sterile lines in hybrid rice. In: Smith WH, Bostian LR, Cervantes EP (eds) Hybrid rice. Manila, International Rice Research Institute, pp 115–128 Rathnaswamy R, Yolanda JL, Kalaimagal T, Suryakumar M, Sassikumar D (1999) Cytoplasmicgenic male-sterility in pigeonpea (Cajanus cajan). Indian J Agric Sci 69:159–160 Rogers J, Edwardson J (1952) The utilization of cytoplasmic male inbred lines in production of corn hybrids. Agron J 44:8–13 Sampath S, Mohanty HK (1954) Cytology of semi-sterile rice hybrids. Curr Sci 23:182–183 Sasakuma T, Ohtsuka I (1979) Cytoplasmic effects of Aegilops species having D genome in wheat. I. Cytoplasmic differentiation among five species regarding pistilody induction. Seiken Ziho 27:59–65 Saxena KB, Sultana R, Mallikarjuna N, Saxena RK, Kumar RV, Sawargaonkar KL (2010) Malesterility systems in pigeonpea and their role in enhancing yield. Plant Breed 129:125–134 Saxena KB, Ravikoti VK, Tikle AN, Saxena MK, Gautam VS, Rao SJ et al (2013) ICPH2671—the world’s first commercial food legume hybrid. Plant Breed 132:479–485 Schertz KF (1994) Male-sterility in sorghum: Its characteristics and importance. In: Witcombe JR, Duncan RR (eds) Use of molecular markers in sorghum and pearl millet breeding for developing countries. Proc. Intl. Conf. Genet. Improvement. Overseas Development Administration (ODA) plant sciences research conference, 29 March–1 April 1993, Norwich, pp 35–37 Serba DD, Perumal R, Tesso T, Min D (2017) Status of global pearl millet breeding programs and the way forward. Crop Sci 57:2891–2905 Serieys H (2005) Identification, study and utilisation in breeding programs of new CMS sources in the FAO subnetwork. In: Proceedings of the 2005 sunflower subnetwork Progress report, Novi Sad, Serbia and Montenegro, 17–20 July 2005; FAO, Rome, Italy, pp 47–53 Serieys H, Vincourt P (1987) Characterization of new cytoplasmic male sterility sources from Helianthus genus. Helia 10:9–13 Shinjyo C (1969) Cytoplasmic-genetic male sterility in cultivated rice, Oryza sativa L. Jpn J Genet 44:149–156 Small ID, Peeters N (2000) The PPR motif—a TPR-related motif prevalent in plant organellar proteins. Trends Biochem Sci 25:46–47 Stephens JC, Holland RF (1954) Cytoplasmic male sterility for hybrid sorghum seed production. Agron J 46:20–23 Stewart JM (1992) A new cytoplasmic male sterile and restorer for cotton. Proc Beltwide Cotton Prod Res Conf, p 610 Sun SY, Wang DX, Li JB, Lei YQ, Li G, Cai WG, Zhao X, Liang WQ, Zhang DB (2021a) Transcriptome analysis reveals photoperiod-associated genes expressed in Rice anthers. Front Plant Sci 12:621561. https://doi.org/10.3389/fpls.2021.621561 Sun Y, Xiong X, Wang Q, Zhu L, Wang L, He Y, Zeng H (2021b) Integrated analysis of small RNA, transcriptome, and degradome sequencing reveals the MiR156, MiR5488 and MiR399 are involved in the regulation of male sterility in PTGMS rice. Int J Mol Sci 22:2260. https://doi. org/10.3390/ijms22052260 Takatsuka A, Kazama T, Toriyama K (2021) Cytoplasmic male sterility-associated mitochondrial gene orf312 Derived from Rice (Oryza sativa L.) cultivar Tadukan. Rice 14:46. https://doi.org/ 10.1186/s12284-021-00488-7 Tan XL, Tan YL, Zhao YH et al (2004) Identification of the Rf gene conferring fertility restoration of the CMS Dian-type 1 in rice by using simple sequence repeat markers and advanced inbred lines of restorer and maintainer. Plant Breed 123:338–341

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Tang HV, Pring DR, Shaw LC et al (1996) Transcript processing internal to a mitochondrial open reading frame is correlated with fertility restoration in male-sterile sorghum. Plant J 10:123–133 Tang HV, Pedersen JF, Chase CD, Pring DR (2007) Fertility restoration of the sorghum A3 malesterile cytoplasm through a sporophytic mechanism derived from Sudan grass. Crop Sci 47: 943–950 Tao D, Xu P, Li J, Hu F, Yang Y, Zhou J, Tan XL, Jones MP (2004) Inheritance and mapping of male sterility restoration gene in upland japonica restorer lines. Euphytica 38:247–254 Vetriventhan M, Nirmalakumari A (2010) Identification and screening of restorers and maintainers for different CMS lines of pearl millet (Pennisetum glaucum (L.) R.Br.). Electron J Plant Breed 1(4):813–818 Wang Z, Zou Y, Li X et al (2006) Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing. Plant Cell 18:676–687 Wang K, Qiu F, Larazo W, Paz MAD, Xie F (2015) Heterotic groups of tropical indica rice germplasm. Theor Appl Genet 128:421–430 Wang X, Li L, Yang Z et al (2017) Predicting rice hybrid performance using univariate and multivariate GBLUP models based on North Carolina mating design II. Heredity 118:302–310 Wanjari KB, Patil AN, Manapure P, Manjaya JG, Manish P (1999) Cytoplasmic male-sterility in pigeonpea with cytoplasm from Cajanus volubilis. Ann Plant Physiol 13:170–174 Watanabe Y, Sakaguchi S, Kudo M (1968) On the male sterile rice plant possessing the cytoplasm of Burmese variety ‘Lead Rice’. Jpn J Breed 18:77–78 Whelan EDP (1981) Cytoplasmic male sterility in Helianthus giganteus L. x H. annuus L. Interspecific hybrids. Crop Sci 21:855–858 Wilson JA, Ross WM (1962) Male sterility interaction of the Triticum aestivum nucleus and Triticum timopheevii cytoplasm. Wheat Inf Serv 14:29–30 Wu S, Tan H, Hao X, Xie Z, Wang X, Li D, Tian L (2019) Profiling miRNA expression in photothermo-sensitive male genic sterility line (PTGMS) PA64S under high and low temperature. Plant Signal Behav 14:12 Xian-Hua S, Song Y, Ren-liang H, Shan Z, Hong-lian X, Lin-jun S (2013) Development of novel cytoplasmic male sterile source from dongxiang wild rice (Oryza rufipogon). Rice 20:379–382 Xie F, He Z, Esguerra MQ, Qiu F, Ramanathan V (2014) Determination of heterotic groups for tropical Indica hybrid rice germplasm. Theor Appl Genet 127(2):407–417 Yan X, Dong C, Yu J et al (2013) Transcriptome profile analysis of young floral buds of fertile and sterile plants from the selfpollinated offspring of the hybrid between novel restorer line NR1 and Nsa CMS line in Brassica napus. BMC Genomics 14:26 Yang P, Han J, Huang J (2014) Transcriptome sequencing and de novo analysis of cytoplasmic male sterility and maintenance in JA-CMS cotton. PLoS One 9:e112320 Yashitola J, Thirumurugan T, Sundaram RM, Naseerullah MK, Ramesha MS, Sarma NP, Sonti RV (2002) Assessment of purity of rice hybrids using microsatellite and STS markers. Crop Sci 42: 1369–1373 Yi P, Wang L, Sun Q, Zhu Y (2002) Discovery of mitochondrial chimeric-gene associated with cytoplasmic male sterility of HL-rice. Chin Sci Bull 47:744–747 Zhang G, Bharaji T, Lu Y, Virmani S, Huang N (1997) Mapping of the Rf-3 nuclear fertility restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers. Theor Appl Genet 94:27–33 Zhang H, Hu J, Qian Q, Chen H, Jin J, Ding Y (2016) Small RNA profiles of the Rice PTGMS line Wuxiang S reveal miRNAs involved in fertility transition. Front Plant Sci 7:514. https://doi.org/ 10.3389/fpls.2016.00514 Zhou WC, Kolb FL, Domier LL, Wang SW (2005) SSR markers associated with fertility restoration genes against Triticum timopheevii cytoplasm in Triticum aestivum. Euphytica 141(1–2):33–40

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Male Sterility in Maize: Retrospect, Status and Challenges Subhash Chander, Bhupender Kumar, Krishan Kumar, Sonu Kumar, Chayanika Lahkar, Brijesh Kumar Singh, Shankar Lal Jat, Chittar Mal Parihar, and Ashok Kumar Parihar

Abstract

Male sterility in crops can be defined as failure in the production of functional pollens resulting from impairments in microsporogenesis process. This is the noteworthy crossbreeding tool in flowering plants. In the twentieth century, the cytoplasmic male sterility (CMS) system (T-CMS) was utilized for the first time in hybrid maize, which tremendously increased the efficacy of hybrid seed production. Hence, CMS became very much popular among breeders and seed companies due to its reliability and stability for seed production. However, during 1969–1970, Southern corn-leaf-blight disease critically affected corn possessing cms-T gene. Hence, the identification of diverse CMS systems in a wide array of staple crops is required to minimize the vulnerability of hybrids based on a single CMS source. Efforts are also needed for use of stable environmental-sensitive genic male sterility (EGMS) as it circumvents the need for a separate restoration source in producing hybrids. Concerning molecular mechanisms of the CMS-Rf system, numerous CMS-linked ORFs and respective Rf genes have been identified and described in maize. Recent research has offered new insights into cytoplasmic–nuclear communication. The next-generation sequencing technologies also offer an effective approach for identifying the unique ORF candidates for CMS by mitochondrial genome sequencing. S. Chander (*) ICAR—National Bureau of Plant Genetic Resources, New Delhi, India B. Kumar · K. Kumar · S. Kumar · C. Lahkar · B. K. Singh · S. L. Jat ICAR—Indian Institute of Maize Research, New Delhi, India C. M. Parihar ICAR—Indian Agricultural Research Institute, New Delhi, India A. K. Parihar ICAR—Indian Institute of Pulses Research, Kanpur, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_3

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Keywords

Cytoplasmic · Fertility restoration · Male sterility · Maize · Hybrid breeding · Seed production

3.1

Introduction

Male sterility in crops can be defined as failure in the production of functional pollens, which is the result of the failure in the completion of microsporogenesis process (Lasa and Bosemark 1993). It is noteworthy crossbreeding tools which prevent self-pollination and permit outcrossing in flowering plants. Various types of male sterility were revealed in >150 crops, viz., rice, wheat, barley, maize, sorghum, mustard, sugar beet, sunflower, rye, cotton, flax, onion, spinach, carrot, asparagus, celery, cucurbits, tomato, fennel, leek, radish, cauliflower, cabbage, broccoli, eggplant, chicory, turnip, pepper, etc. Male sterility can be induced by various means such as mutation, protoplasmic fusion, inter-specific or wide hybridization, and genetic engineering (Wang et al. 2013; Yamagishi and Bhat 2014; Singh et al. 2015). Cytoplasmic male sterility (CMS) was reported first time by Rhoades (1933) in corn, and it was categorized as cms-C (C ¼ Charrua), cms-S (S ¼ USDA), and cms-T (T ¼ Texas). Being one of the most important food crops of the world and considering its economic importance, the use of the CMS in maize is one of the discernible choices for the development of hybrid varieties. Also, it is widely used for genetic studies to determine the types of sterility. During the 1950s, maize hybrid was developed using cms-T system of male sterility for the first time and described as failure of anther protrusion, resulting in pollen-abortion, which greatly increased the efficacy of hybrid seed production and corn yield. This tool (cms-T) of male sterility has been comprehensively studied and extensively utilized for the production of hybrid seeds (during the 1950s and 1960s) to avert hand/ mechanical emasculation. It was very popular among breeders and seed companies due to its reliability and stability for seed production. During 1969–1970, Southern corn-leaf-blight (fungal disease), caused by Cochliobolus heterotrichus race T (synonym Helminthosporium maydis race T) had become an epidemic in the Southern region of the United States (Pring and Lonsdale 1989). This disease critically affected corn possessing cms-T gene, which occupied nearly 85% of the U.S. corn acreage. It was realized that cms-T is prone to Southern corn-leaf-blight, hence, massive utilization of this has been discontinued for the production of hybrid seeds. Another fungal pathogen (Phyllosticta maydis) was also found fatal to corn possessing male-sterile cytoplasm (cms-T). However, the disease epidemic triggered by Phyllosticta maydis was not as severe as that caused by C. heterotrichus as it was confined to the colder regions of the United States. Male sterile plants play a vital role in harnessing heterosis or hybrid vigour. Besides, male-sterile plants serve as study material for cytoplasmic and nuclear gene interactions as well as pollen development. Hence, researchers have been fascinated

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by the investigation at molecular and genetic levels of fertility restoration and malesterility in crop plants (Horn et al. 2014).

3.2

Hybrid Development and Heterosis

Heterosis or hybrid vigour can be defined as a phenomenon where the offspring obtained from the crosses of two inbred parents perform better than the parental inbreds. Hybrid crops can harvest 15–50% greater yields than their parental inbreds. Heterosis term was first used by G.H. Shull in 1914 in Maize. Originally, hybrid maize seeds were developed by manual/hand-operated detasseling of the maternal parents to avert self-pollination. Earlier than the mid-twentieth century, manual labour, chemicals remedies, or machines were involved in hybrid seed production, hence it was costly and even adversely affected the climate. In mid-twentieth century, the cms-T system of male sterility was, for the first time, utilized in hybrid maize, which tremendously increased the efficacy of hybrid seed production and improved the corn yield. Hence, Texas cytoplasm was in high demand—as it carries the trait for male sterility, which was inherited cytoplasmically—by the maize breeders to use the same in corn hybrid production for employing hybrid vigour.

3.3 1. 2. 3. 4.

Different Ways of Inducing Male Sterility

Cytoplasmic Genetic Male Sterility (CGMS) Genetic Male Sterility (GMS) Gametocides Cytoplasmic Male Sterility

3.3.1

Cytoplasmic Genetic Male Sterility System (CGMS) or 3-Line Hybrid Seed Production

This system was discovered by Jones and Davis in 1944 in onion. It is the outcome of the interaction among nuclear genes and specific sterility-inducing cytoplasm. A nuclear gene, known as the restorer gene, restores the fertility in the male sterile line. This system needed 3.0 lines, viz., CMS line (‘A’ line), maintainer line (‘B’ line), and restorer line (‘R’ line) (Fig. 3.1), hence, it is designated as the 3-line system of hybrid seed production. The sterile cytoplasm and recessive (rf) nuclear genes are necessary for getting male sterility expression. It is the only system being commercially exploited in India. CMS line (‘A’ line): this line is used as a female parent having male-sterile gene (S) in its cytoplasm, which is used for hybrid seed production. Hence, it is inevitable to devise a female parent as cytoplasmic male sterile (CMS) line. In this CMS system, the inheritance of male sterility is of maternal type.

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rr

RR

rr

S

F

F

Male sterile or ‘A’ line

Restorer or ‘R’ line

Maintainer or ‘B’ line

Fig. 3.1 Three lines of cytoplasmic genetic male sterility (CGMS) system

Maintainer line (‘B’ line): It is similar to the ‘A’line and maintained continuously for maintaining the ‘A’line. Since ‘A’ line is male sterile henceforth, we would not get seed in ‘A’ line without pollinating it with maintainer or ‘B’ line. Restorer line (‘R’ line): This line comprises the male parent (with restorer gene or R gene in its nucleus) for the hybrid seed production. The hybridization of this line with ‘A’ line results into hybrid seed having the fertility restorer gene in heterozygous condition (Rr) in the nucleus. Thus, produced hybrid seed is fertile and grown by the farmers for rr commercial cultivation as a hybrid crop. Maintenance of Parental Lines: As ‘R’and ‘B’lines are typically inbred lines, thus maintenance of these lines does not require any special techniques. Therefore, these lines are maintained in isolation as other inbred lines are maintained; however, distinctive techniques are required for the maintenance of ‘A’lines. Maintenance of ‘A’ Line: For the maintenance of ‘A’ line, both ‘A’ and ‘B’ lines should be planted in such a manner that more and more pollen is dispersed from ‘B’ to ‘A’ line resulting into higher seed setting in ‘A’ line. Thus, produced seeds are used in the hybrid seed production with the restorer line in the next year/season. Generally, row ratio of ‘B’ line to ‘A’ line is kept at 2:6 or 1:2 or 1:3 or 2:4, depending upon the pollen availability in male parents (Fig. 3.2). During maintenance of ‘A’ as well as ‘B’ lines, off-type plants should be discarded by regular rouging under special supervision. Further, special care should be taken for rouging of pollen shedder plants noticed in ‘A’ lines. During maturity, ‘B’ line has to be harvested first and ought to no longer be used for seed production. After that ‘A’ line should be harvested, threshed, dried, and stored appropriately to use it in the subsequent year for hybrid seed production (Fig. 3.3). During the production of hybrid seeds, ‘R’ line and ‘A’ line should be used and harvested seeds from ‘A’ line would be F1 hybrid. The ratio of male into a female in hybrid seed production of maize depends upon the pollen shedding ability of the male parents. Generally, 1 (male):2 (female); or 1 (male):3 (female) row ratios are used for the commercial production of hybrid seeds in maize (Fig. 3.3). Majorly, three CMS systems, viz., cms-C (Charrua), cms-S (USDA), and cms-T (Texas) have been described in maize (Pring and Levings III 1978), and through DNA restriction digestion profile, these can be extricated from one another (Borck and Walbot 1982). The Rf1 and Rf2 nuclear restorer gene responsible for the

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Fig. 3.2 Maintenance of ‘A’ line

Fig. 3.3 Hybrid seed production by utilizing A and R lines

restoration of cms-T were present on chromosome 3 and 9 (Rogers and Edwardson 1952), whereas Rf3 for cms-S and Rf4 for cms-C were present on chromosome 2 and 8, respectively, of maize (Jenkins 1978; Beckett 1971). The donors of cms-T, cms-S and cms-C were Golden June, Teopod maize, and Charrua, respectively. After the finding of CMS-T in Golden June, an open-pollinated variety (Rogers and

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Edwardson 1952), CMS began as an alternate and sustainable option to manual detasseling. However, due to the widespread susceptibility of the CMS-T system to Southern corn leaf blight, it was entirely discontinued from corn hybrid breeding programmes, which became noticeable during 1970.

Molecular Basis of CMS in Maize At the molecular level, this dysfunction is attributed to the production of chimeric proteins, extensive recombination and deletions in mitochondrial DNA (mtDNA), and decrease or lack of RNA editing process. Different type of new ORFs has been identified due to mt genome rearrangement resulting into chimeric proteins production. For instance, a unique mtORF, T-urf13 is concomitant with male sterility in T-cytoplasm. The considerable correlative proof advocates T-urf13 is responsible for CMS in cms-T corn. The gene is being expressed in cms-T mitochondria and respective transcripts are found in roots, tassels, leaves, ear shoot, and coleoptiles. The pollen abortion in maize (cms-T) is actually suppressed by the joint action of the dominant nuclear alleles of the Rf1 and Rf2 loci. Dewey et al. (1987) reported that Rf1 alters the transcriptional profile of T-urf13 and decreases the abundance of URF 13. In contrast, homozygous recessive genotypes (rf1rf1), could not modify the transcriptional profile of T-urf13, which creates sterility .

3.3.2

Genetic Male Sterility (GMS)

This male sterility arises due to the failure in the development of the pollen grains and it may be the result of the lesions produced in nuclear-encoded genes. This kind of male sterility has been identified in several crop species, such as rice, maize, soybean, tomato, pea, etc. The GMS mutants can be either dominant or recessive and typically follows Mendelian inheritance.

Molecular Basis of GMS Male sterile genes are generally recessive (ms); however, dominant genes (MS) are identified in safflower. These genes are rather variable as there are 70 different recessive genes in maize, 64 in tomato, 54 in pea, and 25 in rice. The recessive genes induce male sterility by way of diverse expressions. Some are male sex suppressors: ‘ms’ genes act during anther development. These genes resulted either into no anther formation or development of defective microsporangium, i.e. abnormal microsporogenesis and hence abnormal anther formation. All these changes have been differentiated into pre-meiosis, meiosis, and post-meiosis stages of anther development. The ms genes interrupt during these stages, due to which abnormal or abortive pollens are produced. After the first elucidation of genic male sterility by Eyster (1921) in corn, more than 40 loci influencing male fertility have been documented (Skibbe and Schnable 2005). And cytological investigation indicate that lesions in nuclear genes/genome influence the developmental stages of anthers from pre-meosis to fully developed pollen grains (Albertsen 1997; Chaubal et al. 2003).

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Hybrid seed production utilizing Genic male sterility requires less area and labour as the breeder has to maintain only ‘A’ and ‘B’ lines. However, this system is less stable, which produces 50% of fertile plants.

3.3.3

Use of Male Gametocides

Besides genic and cytoplasmic male sterility, the sterility induced by male gametocides has enormous potential to utilize hybrid vigour in crop breeding programmes. In maize, SQ-1 gametocide (pyridazinone derivative) was found successful for induction of male sterility (Wei et al. 2012). SQ-1 gametocide was used at 5.0 kg/ha in five corn varieties at the seedling stage of the plant (8–10 leaves), and it was found that female organs of SQ-1-treated plants remained intact and responsive to alien pollination (>90% of male sterility). The male sterility induced by SQ-1 was not scrupulously affected by diverse corn genotypes. Hence, this could be the alternative approach to GMS and CMS in maize breeding programme due to its rapidity and flexibility. At present, detasseling by hand is a predominantly used method for seed production through hybrids in maize, where the tassels of female lines are removed before anthesis.

3.4

Mapping Fertility Restoration Genes and Genomic Regions

Fertility restoration is governed by nucleus-encoded factors and genes, for the same have been mapped and subsequently cloned across different crop species (Bohra et al. 2016). The restorer gene in maize does not produce any heritable change in the cytoplasm, hence considered as suppressor of the CMS phenotype. As discussed earlier, three kinds of CMS have been originally identified in maize based on varying restoration responses in field experiments. In maize, Beckett (1971) reported that nuclear genes (Rf) are responsible for resotoration and discrimination of S, T, and C types of male sterility (Table 3.1). cms-T: In this T cytoplasm, male sterility is restored by the dominant alleles of the two loci rf1 and rf2 (Laughnan and Gabay 1983). This system of male sterility has sporophytic mode of restoration. Wise and Schnable (1994) mapped rf1 and rf2 with closely linked restriction fragment length polymorphisms (RFLP) markers in five different populations. The resultant consensus maps have rf1 between umc 97 and umc 92 on chromosome 3 and rf2 between umc 153 and sus1 on chromosome 9. The joint action of Rf2a and Rf8 or Rf* (nuclear gene) is responsible to restore partial fertility. The Rf8 is rare in maize germplasm and it has been reported that this gene is environmentally sensitive and incompletely penetrant (Dill et al. 1997). Pei (2000) has mapped Rf* and Rf8 close to the white-pollen (whp1) gene at chromosome-2L. A transposon-tagging strategy was used to clone the nuclear fertility-restorer gene and hence rf2 mutant (rf2-m) alleles has been isolated (Cui et al. 1996). In total 178,300 plants were screened and seven rf2-m mutant alleles have been isolated.

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Table 3.1 Type of male sterility, responsible nuclear restorer genes, mode of restoration, and their chromosomal locations Type of CMS cms-C cms-C cms-S

Nuclear gene for restoration Rf4 Rf5, Rf6 Rf3

Restoration mode Sporophytic Sporophytic Gametophytic

Chromosomal location 8 – 2L

cms-S cms-T

rfl1 (RfIII) Rf1

Gametophytic Sporophytic

– 3S

cms-T

Rf2 (Rf2a)

Sporophytic

9L

cms-T

Rf8, Rf*

Sporophytic

2L

References Gracen et al. (1979) Josephson et al. (1978) Laughnan and Gabay (1975, 1978) Laughnan and Gabay (1975) Duvick et al. (1961), Wise and Schnable (1994) Snyder and Duvick (1969), Cui et al. (1996) Dill et al. (1997)

cms-S: A single restorer gene, designated Rf3, located in chromosome 2 is essential for fertility restoration in cms-S (Beckett 1971; Laughnan and Gabay 1973). Liu 1991 reported that this system of male sterility possesses dominant gene and gametophytic mode of restoration. Further, evidence suggests that the transposable element may be associated with cms-S restoration (Laughnan and Gabay 1978). The rf3 gene has been found to be located on the long arm of Chromosome-2 with the help of RFLP markers (Kamps and Chase 1997). Thus, the mapping investigation has exhibited that rf3 locus found to be positioned approximately on 4.3 cM proximal to whp locus and 6.4 cM distal to the bnl17.14 locus. Zhang et al. (2006) have mapped Rf3 locus on the distal region of the chromosome-2 long arm by using amplified fragment length polymorphism (AFLP) technique. cms-C: Based on the proportions of male-fertile and male-sterile plants of a backcross and their F2 offspring, Kheyr-Pour et al. (1981) reported that Rf4 is responsible for restoring the cms-C. This Rf4 locus has been found on chromosome 8 (Table 3.1) with an approximate genetic distance of 2.0 cM from npi114A (Sisco 1991).

3.5

Marker-Assisted Selection (MAS) and Hybrid Breeding

There is enormous importance of MAS for introgression/transfer of desired genomic segment in an agronomical superior genetic background. It has been extensively utilized for the development of restorer lines in other crops (Bohra et al. 2016); however, this is not much exploited in maize specifically for the transfer of fertility restorer genes. Numerous DNA markers concomitant with Rf genes were used in the MAS system to assist accelerated retrieval of the R lines (possesses pertinent Rf gene) for varied sterile cytoplasm such as YAC-based probes in CMS-WA, SSRs in HL-CMS, CW-CMS, and WA-CMS; and cleaved amplified polymorphic sequence (CAPS) markers in CMS-CW (Zhang et al. 2002; Huang et al. 2003; Fujii and

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Toriyama 2005; Suresh et al. 2012). Various DNA marker techniques such as CAPS (Ngangkham et al. 2010), SSR (Sheeba et al. 2009) and RAPD (Sane et al. 1997; Ichii et al. 2003) assisted in distinguishing the parental line at early stage apart from exploring the R- lines. Wise and Schnable (1994) reported that RFLPs assisted in the selection of rf1 and rf2 genes for CMS-T in maize; however, the association of CAPS-E3P1 and SCAR-E12M7 was linked with Rf3 gene for CMS-S (Zhang et al. 2006). After the identification of the Rf3 and Rf4 genes using DNA-based markers, this technique became a powerful tool in hybrid rice breeding for discovering the pertinent Rf fragments as well as pyramiding/transferring Rf genes into promising agronomic background (Sattari et al. 2007). Bazrkar et al. (2008) pyramided the multiple Rf genes using SSR markers to attain a desirable combination of Rf genes as a unique R line.

3.6

Scope for the Adoption of Modern Technologies (Male-Sterile Systems) and Their Use in Agricultural Sector

The hybrid vigour was found to be the important phenomena which was used to enhance the grain yield of maize since the 1930s. The development of pollen in maize (female parent) is controlled by genetic, hand, or mechanical emasculation. Though mechanical emasculation is very effective; however, it enhances the cost of seed production process. Hence, this alternate approach (i.e. CMS) of regulating the pollen production in female parents laid the foundation for hybrid seed production. During the 1960s, T-cytoplasm (CMS system) became successful for the production of hybrid seeds in maize. Due to the severe epidemic of Southern leaf blight in maize, this T-cytoplasm has been discarded from maize breeding programme during 1969–1970 (Wise et al. 1999). The major problem associated with the use of genetic male sterility (sterile mutants) for hybrid seed production was costly, difficulty in the production of the sterile plant as well as discarding of the pollen-producing plants from female rows. In spite of the unwanted traits, the GMS still remained promising for hybrid production. Numerous stocks/lines, viz., internal deficiency and tertiary trisocmic have been utilized to produce complete male-sterile progeny (Burnham 1975); however, none has been found relevant for large-scale production of hybrid seeds. The advancement in molecular tools and techniques could significantly and efficiently contribute to the development of new restorer lines in various crops. Marker assisted backcrossing coupled with SSR and ISSR found to be useful for the accelerated conversion CMS line in rice (Ahmadikhah et al. 2015). Moreover, DNA-based applications were used for cytoplasmic male sterility-based hybrid breeding programme for production of superior hybrids in rice (Xie et al. 2013). In addition, with the understanding of the principles of CMS and GMS, these become leaders among the competent approaches of hybrid productions, and therefore had noteworthy impact in agricultural sector.

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Major Challenges and Potential Opportunities

Numerous CMS-linked ORFs and few of their respective Rf genes were recognized and described. This kind of research has offered some molecular understandings into plant mitochondrial signalling pathways. Conversely, the interfaces of different proteins of CMS and RFs, and retrograde signals of mitochondria has not been recognized for most of the CMS systems. Furthermore, the mechanisms of regulating the accumulation of male-specific CMS proteins and induction of male sterility continued to be unclear. Thus, the continuous progress in the latest technologies may facilitate in resolving the present challenges of studying the Rf/ CMS system of male sterility. The whole-genome sequencing (massive and complex genomes) in many crops such as wheat, sorghum, soybean, and maize will further fast track the identification of Rf genes in CMS systems. This next-generation sequencing facility also offers an effective approach for identifying the unique ORF candidate for CMS and determine the whole genome sequence (mitochondrial) of the CMS lines. With the help of said technique, many genomes will be sequenced, the blind facts may be uncovered and thus information about the evolution of the CMS/Rf system will be readily available. With the availability of the TALEN (transcription activator-like effector nuclease) technique, the targeted mitochondrial ORFs can be knocked out to corroborate the action of CMS-linked ORFs and revive the male sterility at the genetic level. Further, the recognition of the targeted genes of the non-coding RNA regulator (Mishra and Bohra 2018), and exploring their interactions with climatic factors and biological behaviour will reveal the molecular mechanisms intrinsic to the regulation of environmentally sensitive genic male sterility (EGMS).

3.8

Conclusion

Hybrid technology provides a remarkable potential for the much-needed second green revolution; as the expansion of area is not possible, hence we need to enhance production/unit areas. Production of hybrid varieties is a major goal to improve crop plants for exploitation of heterosis. The cost of hybrid seeds is one of the foremost constraints in attaining prompt adoption of hybrid varieties. Thus, the use of various genetic emasculation, which is cost-effective and an efficient mechanism, is the need of the hour. The research based on CMS/Rf has become static in the past few years as applied in some major cereal crops, such as corn, sorghum, wheat, etc. The identification of novel CMS system in a wide array of staple crops is urgently needed to minimize the vulnerability of hybrids as their genetic background depends on limited CMS sources. There is urgent need to draw more attention of the breeders towards EGMS as it has enormous potential and benefits in producing hybrids to deal with the growing need of foods.

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References Ahmadikhah A, Mirarb M, Pahlevani MH, Nayyeripasand L (2015) Marker-assisted backcrossing (MABC) using CMS-specific, SSR and ISSR markers to develop an elite CMS line in rice. Plant Genome 8:2 Albertsen MC (1997) Male sterile mutants. p 313 Bazrkar L, Ali AJ, Babaein NA, Ebadi AA, Allahgholipour M, Kazemitabar K, Nematzadeh G (2008) Tagging of four fertility restorer loci for wild abortive—cytoplasmic male sterility system in rice (Oryza sativa L.) using microsatellite markers. Euphytica 164:669–677 Beckett JB (1971) Classification of male sterile cytoplasms in maize (Zea mays L.). Crop Sci 11: 724–726 Bohra A, Jha UC, Adhimoolam P, Bisht D, Singh NP (2016) Cytoplasmic male sterility (CMS) in hybrid breeding in field crops. Plant Cell Rep 35(5):967–993 Borck KS, Walbot V (1982) Comparison of the restriction endonuclease digestion patterns of mitochondrial DNA from normal and male sterile cytoplasms of Zea mays L. Genetics 102:109– 128 Burnham CR (1975) Progress report on three possible methods producing an all male-sterile progeny. Maize Genet Coop Newsletter 49:119–121 Chaubal R, Anderson JR, Trimnell MR, Fox TW, Albertsen MC, Bedinger P (2003) The transformation of anthers in the msca1 mutant of maize. Planta 216:778–788 Cui X, Wise RP, Schnable PS (1996) The rf2 nuclear restorer gene of male-SterileT-cytoplasm maize. Science 272:1334–1336 Dewey RE, Timothy DH, Levings CS (1987) A mitochondrial protein associated with cytoplasmic male sterility in the T cytoplasm of maize. Proc Natl Acad Sci U S A 84:5374–5378 Dill CL, Wise RP, Schnable PS (1997) Rf8 and Rf* mediate unique T-urf13-transcript accumulation, revealing a conserved motif associated with RNA processing and restoration of pollen fertility in T-cytoplasm maize. Genetics 147:1367–1379 Duvick DN, Snyder RJ, Anderson EG (1961) The chromosomal location of Rf1, a restorer gene for cytoplasmic pollen sterile maize. Genetics 46:1245–1252 Eyster WH (1921) Heritable characters of maize. VII. male sterile. J Hered 12:138–141 Fujii S, Toriyama K (2005) Molecular mapping of the fertility restorer gene for rice ms-CW-type cytoplasmic male sterility of rice. Theor Appl Genet 111:696–701 Gracen VE, Kheyr-Pour A, Earle ED, Gregory P (1979) Cytoplasmic inheritance of male sterility and pest resistance. Proc 34th Ann Corn Sorghum Res Conf 34:76–91 Horn R, Gupta KJ, Colombo N (2014) Mitochondrion role in molecular basis of cytoplasmic male sterility. Mitochondrion 19:198–205 Huang J, Hu J, Xu X, Li S, Yi P, Yang D (2003) Fine mapping of the nuclear fertility restorer gene for HL cytoplasmic male sterility in rice. Bot Bull Acad Sin 44:285–289 Ichii M, Hong DL, Ohara Y, Zhao CM, Taketa S (2003) Characterization of CMS and maintainer lines in indica rice (Oryza sativa L.) based on RAPD marker analysis. Euphytica 129:249–252 Jenkins MT (1978) Maize breeding during the development and early years of hybrid maize. In: Walden B (ed) Maize breeding and genetics. Wiley, New York, pp 13–28 Josephson LM, Morgan TE, Arnold JM (1978) Genetics and inheritance of fertility restoration of malesterile cytoplasms in corn. In: Proc 33rd Ann Corn Sorghum Res Conf 33, pp 48–59 Kamps TL, Chase CD (1997) RFLP mapping of the maize gametophytic restorer-of-fertility locus (rf3)and aberrant pollen transmission of the nonrestoring rf3 allele. Theor Appl Genet 95:525– 531 Kheyr-Pour A, Gracen VE, Everett HL (1981) Genetics of fertility restoration in the C-group of cytoplasmic male sterility in maize. Genetics 98:379–388 Lasa JM, Bosemark NO (1993) Male sterility. In: Hayward MD, Bosemark NO, Romagosa I, Cerezo M (eds) Plant breeding. Plant breeding series. Springer, Dordrecht. https://doi.org/10. 1007/978-94-011-1524-7_15

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Laughnan JR, Gabay SJ (1973) Mutations leading to nuclear restoration of fertility in S male-sterile cytoplasm in maize. Theor Appl Genet 43:109–116 Laughnan JR, Gabay SJ (1975) An episomal basis for instability of S male sterility in maize and some implications for plant breeding, pp 330–349 Laughnan JR, Gabay SJ (1978) Nuclear and cytoplasmic mutations to fertility in S male-sterile maize. In: Walden DB (ed) Maize breeding and genetics. Wiley, New York, pp 427–47. 794 pp Laughnan JR, Gabay SJ (1983) Cytoplasmic male sterility in maize. Annu Rev Genet 17:27–48 Liu JL (1991) Maize breeding (in Chinese). Beijing Agricultural Press, Beijing Mishra A, Bohra A (2018) Non-coding RNAs and plant male sterility: current knowledge and future prospects. Plant Cell Rep 37:177–191 Ngangkham U, Parida SK, De S, Kumar KAR, Singh AK, Singh NK, Mohapatra T (2010) Genic markers for wild abortive (WA) cytoplasm based male sterility and its fertility restoration in rice. Mol Breed 26:275–292 Pei D (2000) Positioning the Rf8 and Rf* restorers of fertility loci in the maize genome. MS thesis, Iowa State University, Ames Pring DR, Levings CS III (1978) Heterogeneity of maize cytoplasmic genomes among male sterility cytoplasms. Genetics 89:121–136 Pring DR, Lonsdale DM (1989) Cytoplasmic male sterility and maternal inheritance of disease susceptibility in maize. Annu Rev Phytopathol 27:483–502 Rhoades MM (1933) The cytoplasmic inheritance of male sterility in Zea mays. J Genet 27(7):1–93 Rogers J, Edwardson J (1952) The utilization of cytoplasmic male inbred lines in production of corn hybrids. Agron J 44:8–13 Sane AP, Seth P, Ranade SA, Nath P, Sane PV (1997) RAPD analysis of isolated mitochondrial DNA reveals heterogeneity in elite wild abortive (WA) cytoplasm in rice. Theor Appl Genet 95: 1098–1103 Sattari M, Kathiresan A, Gregorio GB, Hernandez JE, Nas TM, Virman SS (2007) Development and use of a two-gene markeraided selection system for fertility restorer genes in rice. Euphytica 153:35–42 Sheeba NK, Viraktamath BC, Sivaramakrishnan S, Gangashetti MG, Khera P, Sundaram RM (2009) Validation of molecular markers linked to fertility restorer gene(s) for WA-CMS lines of rice. Euphytica 167:217–227 Singh SP, Singh SP, Pandey T, Singh RK, Sawant SV (2015) A novel male sterility-fertility restoration system in plants for hybrid seed production. Sci Rep 5:11274 Sisco PH (1991) Duplications complicate genetic mapping of Rf4, a restorer gene for cms-C cytoplasmic male sterility in corn. Crop Sci 31:1263–1266 Skibbe DS, Schnable PS (2005) Male sterility in maize. Maydica 50:367–376 Snyder RJ, Duvick DN (1969) Chromosomal location of Rf2, a male sterility 375 restorer gene for cytoplasmic pollen sterile maize. Crop Sci 9:156–157 Suresh PB, Srikanth B, Kishore VH et al (2012) Fine mapping of Rf3 and Rf4 fertility restorer loci of WA-CMS of rice (Oryza sativa L.) and validation of the developed marker system for identification of restorer lines. Euphytica 187:421–435 Wang K, Peng X, Ji Y, Yang P, Zhu Y, Li S (2013) Gene, protein and network of male sterility in rice. Front Plant Sci 92:1–10 Wei F, Jie H, Zhang X, Cheng L-m, Juan D, Si Y-h, Cao G, Tian B-m (2012) Male sterility induced by chemical SQ-1, as an effective male specific gametocide in maize (Zea mays). Maydica 57:244–248 Wise RP, Schnable PS (1994) Mapping complementary genes in maize: positioning the rf1 and rf2 nuclear-fertility restorer loci of Texas (T) cytoplasm relative to RFLP and visible markers. Theor Appl Genet 88:785–795 Wise RP, Bronson CR, Schnable PS, Horner HT (1999) The genetics, pathology, and molecular biology of T-cytoplasm male sterility in maize. Adv Agron 65:79–130

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Xie F, He Z, Esguerra MQ, Qiu F, Ramanathan V (2013) Determination of heterotic groups for tropical Indica hybrid rice germplasm. Theor Appl Genet 127:407–417 Yamagishi H, Bhat SR (2014) Cytoplasmic male sterility in Brassicaceae crops. Breed Sci 64:38– 47 Zhang ZF, Wang Y, Zheng YL (2006) AFLP and PCR based markers linked to RF3 , a fertility restorer gene for S cytoplasmic male sterility in maize. Mol Genet Genomics 2769:162–169 Zhang QY, Liu YG, Zhang GQ, Mei MT (2002) Molecular mapping of the fertility restorer gene Rf4 for WA cytoplasmic male sterility in rice. Yi Chuan Xue Bao 29:1001–1004

4

Male Sterility Technologies to Boost Heterosis Breeding in Pearl Millet K. B. Choudhary, H. R. Mahala, and Vikas Khandelwal

Abstract

A thriving pliability towards drought, low soil fertility, high salinity and high temperatures brands pearl millet (Pennisetum glaucum (L)) as an imperative cereal crop in the semi-arid zones of the biosphere, which demands significant contribution of researchers towards its hybrid breeding programmes owing to demonstration of marked heterosis both in seed yield and forage production of hybrids. Cytoplasmic male sterility (CMS), a more prominent genetic approach for F1 hybrid seed production enabling the development of effective systems for hybrid seed production by providing a sizeable amount of hybrid varieties to contemporary food production systems, remains a gifted approach to sustain improved productivity in pearl millet. Moreover, intuitive modern approaches are the need of the time likewise, a need of proper mapping of factors responsible for male sterility and fertility restoration in pearl millet ensuring the discovery of novel orfs as well as the development of DNA markers armoury ensure hybrid genetic purity, fast recovery of desirable candidate genes. This invites researchers not only to identification of male sterility-inducing factors and fertility-restorer factor but also to conduct deep innovation in molecular breeding of male sterility in pearl millet. Further, by using modern transcriptomic and proteomic tools in analysis of CMS or fertility restoration mechanisms in pearl millet, hybrid breeding can be oversimplified in terms of time and cost-effectiveness owing to fast incitement of DNA sequencing technology which provides a huge dataset of factors responsible of these plant attributes.

K. B. Choudhary (*) · H. R. Mahala ICAR—Central Arid Zone Research Institute (CAZRI), Jodhpur, Rajasthan, India V. Khandelwal ICAR—All India Coordinated Research Project on Pearl Millet, Jodhpur, Rajasthan, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_4

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Keywords

Cytoplasm · Fertility restoration · Genome · Heterosis · Hybrid · Male sterility · Pollen

4.1

Introduction

A thriving pliability towards drought, low soil fertility, high salinity and high temperatures brands pearl millet (Pennisetum glaucum [L]) as an imperative cereal crop in the semi-arid zones of the biosphere. In areas, where other cereal crops such as maize and sorghum cannot even survive, pearl millet, owing to its tolerance for difficult growing environments, is fruitfully enriching the farming community. Whereas, pearl millet is grown in almost 31 million ha in 30 countries, extended across the continents Asia, Africa, America and Australia (Yadav et al. 2012), in India, it occupies 6.93 Mha with an average production of 8.61 million tons and productivity of 1243 kg/ha during 2018–2019 (ICAR-AICRP on Pearl millet, 2020). By contributing to more than 90% of the production, the north-western parts of India are the major pearl millet-producing regions. Thus, for good sake of mainstream marginal landholders, it is staple food for human beings and fodder for livestock markedly in rainfed regions of India. Owing to its C4 nature, it has high carbonfixing properties, which is primarily climate-change compliant in addition to being an ample source of fibres and minerals, particularly iron, calcium, zinc among other cereals. Thus, it has conclusive remarkability of providing the majority of nutrients at the least cost equated with wheat and rice. Hence, due to its remarkable adaptiveness in the whole world, particularly in rainfed areas, it demands significant contribution from researchers towards hybrid breeding programmes of pearl millet owing to its demonstration of marked heterosis both in seed yield and forage production of hybrids. Moreover, developing commercial single cross hybrids in pearl millet has remained a remarkably successful breeding story among the witnessed significant advances in productivity of major crops. However, due to the cherished association of male and female reproductive organs in the same flower, the crossing of plants with one another is challenging in many agriculturally imperative crops. Thus, the anthers must be removed either mechanically or manually to prevent self-fertilization; if not, the plant fails to produce pollen considered as a genetic defect. Hence, a more prominent genetic approach for F1 hybrid seed production is exploited by many plant breeders labelled as cytoplasmic male sterility (CMS). The discovery of cytoplasmic male sterility has enabled the development of effective systems for hybrid seed production by providing a sizeable amount of hybrid varieties to contemporary food production systems, and remains a gifted approach to sustain improved productivity in majority of crops (Bohra et al. 2016; Mishra and Bohra 2018; Horn et al. 2014; Wang et al. 2013; Whitford et al. 2013). Practically, it deceives the need for removal of anthers, by this means, ambling the generation of dramatically superior F1 hybrids and gearing up the hybrid technology. These F1 progenies exhibit significant superiority

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for yield, biotic and abiotic stress, quality aspects and stability over its parents and predominant cultivars in the breeding chains. (Saxena et al. 2013; Reddy et al. 2006). Notably, by serving as decisive breeding armoury to strap up heterosis, male sterile plants are also used in providing important material to further dig up the pollen and stamen development systems as well as to study the various interactions between cytoplasm and nuclear genomes. Consequently, genetic and molecular mechanisms of restoration of male sterility and male fertility were the prime interest for researchers for a while. The pervasiveness of multiple sources of cms, the observation of high level of instability and the incidence of fertility restorer genes in the pearl millet germplasm make it a particularly intriguing system for the investigation of cms, fertility restoration and stochiometric shifting processes in plants. Whereas the majority of plant species generally exhibit one or two CMS types, millets provide a collection of at least eight independently identified forms. Here, we respectively review the CMS and its relevance to hybrid breeding in pearl millet with budding insights on cytoplasmic–nuclear genomic interactions as well as modern MAS technology.

4.2

Hybrid Development and Heterosis in Pearl Millet

Heterosis, whose biological basis is still mysterious, helped in transforming the production picture of numerous agricultural crops, contributing significantly not only to feed burgeoning global population but also in slowing down the continuous degradation of benign natural preserves. Owing to its distinctive ability to create male sterile gametophyte in the plant without disturbing its agronomic performance, the cytoplasmic male sterility has emerged as a godsend to exploit hybrid breeding in crop species (Touzet and Meyer 2014). After the mainstream intrusion of male sterile lines Tift 23A and Tift 18A, hybrid development in pearl millet was initiated. This intrusion markedly increased the yield potentials as well as tolerance to biotic and abiotic stress in F1 hybrids over the predominant open-pollinated varieties at that time. In India, HB 1 was the first pearl millet hybrid released in 1965. Later, greater efforts have been made by many breeders on the cytoplasmic and nuclear genome diversification in the parental genotypes of hybrids released. Identification, involvement and commercialization of male sterile lines has been proven to be one of the major milestones over the last decade in pearl millet breeding. In pearl millet, many researchers have reported higher level of heterosis remarkably for grain yield (Ugale et al. 1989). Rachie et al. (1967) demonstrated a large quantity of CMS-based hybrids (superior to its parental lines as well as contemporary open pollinated varieties) for various agro-climatic conditions in India. Among these hybrids, some out-yielded the controls as well by a margin of 75–200%. Further, maximum hetero-beltiosis of 105% and economic heterosis of 11.30% (Over standard check GHB-719) for grain yield was reported by Vagadiya et al. (2010). His findings were akin to many earlier researchers who reported higher extent of grain yield heterosis in pearl millet. Additionally, Amiribehzadi et al.

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Table 4.1 Studies in pearl millet hybrids on estimation of heterosis centred on male sterility

Hybrids X1, X2 Gahi 1 (Heterosis for dry matter yield)

JMSA-20072  J-2290 JMSA-20073  H-77/833-2 ICMA-98444  J-2498

Stress zone hybrids (for grain yield) ICMA 88004  PPMI 741 ICMA 89111  D 23

59 hybrids from different ecology of India (for Fe density) 45 hybrids (for Zn density)

Heterosis estimates 45% to local types 52% to “Common” 35% to “Starr” 11.30% 9% 9% (Over Standard check) 88% 14.02% (over better parent) 36.36% (for plant height) – Significant Heterosis

Male sterility source – –

References Rao et al. (1951) Burton (1965)

A1

Vagadiya et al. (2010)

843 A A1 A1

Yadav et al. (2012) Amiribehzadi et al. (2012)

– –

Kanatti et al. (2014)

(2012) reported 14.02% of heterobeltiosis in the cross ICMA 88004  PPMI 741 as well as economic heterosis of 36.36% in the cross ICMA 89111  D 23 (both belong to A1 cytoplasm) for plant height among the 26 hybrids. Recently, significant heterosis was observed in all sources of cytoplasm for mostly yield-attributing traits (Rafiq et al. 2016). Even for zinc and iron density, significant average heterosis was reported by Kanatti et al. (2014) as well as 88% level of heterosis for grain yield was reported in the stress zone hybrids in terminal drought conditions (developed by using early maturing ms line 843A) by Yadav et al. (2012). Additionally, Vetriventhan et al. (2008) found highly significant standard heterosis over standard hybrid (MBH163) for grain yield in seven hybrids developed using male sterile lines. Up to 50% level of heterosis has been reported in pearl millet using male sterility trait (Table 4.1). Even though the total acreage covered by the pearl millet has remained constant, the use of hybrid varieties has increased since the mid-1960s (Table 4.2), enhancing its production by about 50% in India. The productivity increase witnessed due to large scale adoption of hybrids ranges from 194 to 225% in various states of India.

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Table 4.2 A timeline of CMS-based hybrids developed in pearl millet in India Timeline of hybrid development in India 1965–75 1975–84

1986–95

1995–2010 After 2010

4.3

Male sterile source Tift 23 A 5141 A 5054 A 5054 A (mainly) 81 A 834 A 732 A Diverse ms lines Predominantly A1 cytoplasm

Hybrids HB series (HB1, HB2, HB3) BJ 104, BK 560, BD11, GHB 27, HHB 45 CJ 104,CM 46, PHB 47, X 5 40 hybrids JCMH 451 ICMH 501 MH 182

Reference links www.icrisat.org; www.icar. org; www.aicpmip.res.in; www.nandiseed.com; http:// smis.dacnet.nic.in/

62 hybrids, HHB 67-2 (2005), MH 1234 (2006) MH 1578 (2012), Nandi 52 (2008), Kaveri super Boss (2013), MPMH 17 (2014), Pratap (2014)

Diversity of Male Sterility-Inducing Cytoplasm

Albeit CMS is a cost-effective approach and available widely in plant breeding programmes, it is not without its drawbacks for commercial use in hybrid seed production. One of the major drawbacks is the presence of cytoplasmic uniformity in the hybrid owing to dependency on a single CMS source. The famous disastrous epidemic caused by fungal pathogen, Bipolaris maydis in maize was the epic example of genetic vulnerability associated with cytoplasmic uniformity due to consistent use of single T-cytoplasm in the 1970s (Levings 1993). To counter this problem, there should be a broader base of male sterile cytoplasm in a particular crop (Bohra et al. 2016, 2022). The discovery of Tift 23A was (A1 cytoplasmic male sterile cytoplasm) the major landmark success in pearl millet improvement programme (Athwal 1965; Burton 1965). Notably, for most of the pearl millet commercial grain yield hybrids, Tift 23-A was exploited as the major source of A1 cytoplasm. Hence, to counter this basic monopolistic approach, researchers made various efforts to develop alternate cytoplasmic male sterility sources in pearl millet which further advanced to identification of few distinct CMS systems in this crop, viz., A2, A3, Violaceum (Av), ex-borne (gero), A4 and A5 (Burton and Athwal 1967; Aken'Ova and Cbheda 1981; Marchais and Pernes 1985; Hanna 1989; Rai 1995). Owing to lesser stability in expression of male sterility in comparison to A1 cytoplasm, A2 and A3 cytoplasm were found to be not much suitable for pearl millet hybrid breeding (Rai et al. 1996). While, A4 and A5 were the most recent CMS sources (Rai 1995).

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Table 4.3 List of diverse male sterility-inducing cytoplasm/lines established in pearl millet along with the cytoplasm donors Cytoplasm A1 A2 and A3 A4 A5

Source of male sterility Tift 23A – Senegalese accession subspecies “Monodii” Male sterile plant of large seeded Gene Pool

Citation Burton (1965) Burton and Athwal (1967) Hanna (1989) Rai (1995)

As shown in Table 4.3, efficacy of alien cytoplasmic diversity as source of cytoplasmic male sterility (Violaceum) was also exemplified through distant hybridization between a wild relative of pearl millet (P.americanum subsp. Monodii) and a land race (Tiotande) from Senegal (Marchais and Pernes 1985) as well as from half sib progeny of early gene pool (ICMA 90111).

4.4

Mapping of Fertility Restoration Factors

Molecular interactions like discords between mitochondrial and nuclear genomes, candidate genes (for fertility restoration) can be estimated by a comprehensive analysis on fertility restoration. Additionally, it provides a detailed illustration on genetic tools that control traits responsible for inchoative fertility restoration process which can be relied markedly upon the Mendelian genetics as well as by use of simple monogenic and digenic models coupled with traditional allelic tests. Moreover, a strong selection pressure is created on the nuclear genome owing to altered sex ratios by reversal of male sterile plants to balance phenotype. On the other hand, there was a need to resolve the lost genomic harmony between two genomes lost by the alteration of sex ratios. These two phenomena were responsible for the emergence of fertility restorer- rf genes (Bohra et al. 2016). Involvement of single dominant gene in fertility restoration in A4 cytoplasm of pearl millet was reported by Du et al. (1996). Similarly, single dominant gene control for A1 cytoplasm was reported by Yadav et al. (2010). These studies illustrated the simulation pattern between sterility systems in pearl millet and Flor’s “gene-forgene” hypothesis developed for reviewing the growth of host plant resistance. Hence, to diversify the cytoplasmic base of pearl millet hybrids, restoration of breeding procedures using these single genes will be easy approach to trail. Moreover, to understand the molecular mechanisms behind cytoplasmic male sterility and fertility restoration cloning of these rf genes may be useful. (Chen and Liu 2014; Tan et al. 2015). Further, identification and rearrangements of cloning fragments owing to fertility restoration was reported by Smith et al. (1987). In another study, four Pst1 fragments, viz., 4.7 kbp (in A1 cytoplasm), 9.7 kbp (in 81B cytoplasm), 13.6 kbp (in 81A1 cytoplasm) and 10.9 kbp (in all cytoplasm) were cloned and presence of three genes cox1, rrn 18 and rrn 5 genes were depicted by using known mitochondrial gene probes (Smith and Chowdhury 1991).

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Table 4.4 Mapping of male sterility trait in pearl millet using mitochondrial probesa

a

Probe Cytochrome oxidase (subunit 1) Cytochrome oxidase (subunit 2) Cytochrome oxidase (subunit 3) Cytochrome reductase

Gene Cox1

Source Maize

F1-ATPase (subunit 1)

Atp1

F0-ATPase (subunit 9)

Atp9

F0-ATPase (subunit 6)

Atp6

Sorghum

NADH dehydrogenase (subunit 2) Ribsomal protein

Nad2

Oenothera berteriana

Cox2 Cox3 Cob

Rps13

Restriction sites EcoRV/ HaeIII Sau3A/ HindIII Sau3A/ Sau3A HindIII/ EcoRI SalI/BamHI EcoRI/ BamHI EcoRI/ HindIII EcoRI/ EcoRV EcoRIHindIII

Citation Isaac et al. (1985) Fox and Leaver (1981) Muise and Hauswirth (1992) Muise and Hauswirth (1992) Muise and Hauswirth (1992) Salazar et al. (1991) Dewey et al. (1985) Binder et al. (1992) Wissinger et al. (1990)

This table is adopted from Delorme et al. (1997)

In A1 cytoplasm, recombination events due to amplification of a sub-genomic molecule cox1-1-2 was reported as foremost determinant for spontaneous fertility restoration by Feng and Jan (2008). Moreover, it was hypothesized that owing to its detrimental nature for nuclear genome transmission, male sterility creates selection pressure that suppresses the mitochondrial mutations and hence fertility restoration occurs (Castandet and Araya 2011). Further, the association between RNA editing and rf genes were time-honoured due to post-transcriptional expression of sterility factors by these fertility restorer rf genes. This association was supported by assumptions for the rf genes nature to encoding the responsible targeted proteins responsible for the restoration, these specific targeted proteins could be having nuclear derived RNA binders as well as members of pentatricopeptide repeat (PPR) proteins. Several studies have reported possible involvement of mitochondrial sequences in conferring male sterility (Table 4.4).

4.5

Progress Towards Deciphering Male Sterility Determinants

Cytoplasmic male sterility in plants is firmly depicted as constructor of genomic bridge between the mitochondrial and nuclear genomes by many renowned researchers which further resulted in the production of non-functional pollen grains owing to these distorted cytoplasmic nuclear communications (Bohra et al. 2016; Touzet and Budar 2004; Fujii and Toriyama 2008; Touzet and Meyer 2014; Hanson and Bentolila 2004). Though genetic control of cytoplasmic male sterility is clearly

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far from much understanding. Whereas, few findings reported it as a result of interaction of cytoplasmic sterility-inducing factor and nuclear major genes. On the contrary, it was depicted either as a result of impression delivered by environmental factors, such as temperature and relative humidity (Rai et al. 2006) or by induced mutations, distant hybridization, protoplasmic fusion and genetic engineering (Yamagishi and Bhat 2014; Wang et al. 2013; Singh et al. 2015). In some cases, a less understood phenomenon of production of ORFs (open reading frames) made up by combination of different portions of various genes owing to the generation of chimeric genes formed by mitochondrial DMA recombination events denoted as the leading instigate of male sterility (Hanson and Bentolila 2004; Chase 2007). Notably, many reports suggesting the crucial involvement of tapetal cells (highly biosynthetic tissue) in pollen formation, as well as the requirement of fully functional mitochondria for the development of these tapetal cells which can be impaired by the production of mutant genes. Hence, resulting mitochondrial dysfunction can impair the pollen viability (Sykes et al. 2017; Pacini et al. 1985). Moreover, many reports suggested CMS gene-triggered programmed cell death as the prime cause of the degradation of these tapetal cells resulting in the impairment of anther development (Papini et al. 1999; Balk and Leaver 2001; Ku et al. 2003). The physiology of sterility in cytoplasmic male sterility lines can be further elucidated for proper understanding the fertility restoration process. Many reports suggested the two mechanisms firstly, repair; which may be involved in the removal or inactivation of sterility factors. Notably, inactivation of these factors may be either at the transcriptional or translational level (Inhibition of these expressions). This fertility restoration through a repair mechanism is very common in many cases of CMS. This was further exemplified as the involvement of rf2 restorer of maize CMS T cytoplasm in repair mechanism by Touzet and Meyer (2014). Secondly, compensation; which would involve in the counteracting of the sterilizing factors owing to modification of the cellular metabolism. Furthermore, involvement of disruption in the activities of ATPase and cytochrome oxidase enzymes towards male sterility was also reported by Kale and Munjal (2005) and considered as an example of energy deficiency model explained by Chen and Liu (2014). This further aligned by some researchers by establishment of a link between the male sterile phenotype and the lack of RNA editing (Castandet and Araya 2011), viz., the impaired mitochondrial functions caused by lack of RNA editing which further reduced the translated protein activity. This correction mechanism was further exemplified in sorghum by Howad and Kempken (1997) and Howad et al. (1999) by suggesting the pollen abortion caused by the editing defect in the mitochondrial complex F0 F1-ATPase. Notably, the candidate genes responsible for the expression of male sterility may be ideally located on the fragments which are rearranged in a fashion of loss of the one fragment of 4.7 kbp size with the gain of another fragment of size 9.7 kbp. This phenomenon was suggested on the basis of correlation between the fragment rearrangement and retravel of fertility in A1 cytoplasm. This rearrangement involving the cox1 gene may be achieved by using Pst1 mtDNA fragment present in A1,

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A5 and A7 cytoplasm (Delorme et al. 1997). This study was further exemplified by the various reports, including Rajeshwari et al. (1994), suggesting the rearrangement involving a cox gene might be the prime cause for male sterility in A1, A5 and A7 cytoplasm, whereas the rearrangement involving the atp6 or cox 3 cluster might be responsible for cytoplasmic male sterility in A4 cytoplasm. Similarly, it was reported by Feng and Jan (2008) that sub-stoichiometric shifting and mtDNA rearrangements caused by RNAi mediated knockdown of Msh1 nuclear genes are responsible for the naturally prevalent cytoplasmic male sterile lines, primarily, in pearl millet and sorghum. However, unlikely to other cereals, the genetic basis of most male sterile lines used for the development of hybrid varieties in pearl millet is still an area to be elucidated.

4.6

Marker-Assisted Selection (MAS) and Heterosis Breeding

Constant supply of genetically pure parental seed is the basic component in the success of any CMS-hybrid breeding programme (Sattari et al. 2007; Sundaram et al. 2008; Saxena et al. 2010; Qi et al. 2012; Bohra et al. 2012, 2015). Notably, many studies emphasizing gigantic significance of marker-assisted selection (MAS) either in tracking the transferred desirable genomic segment or to enable fast-track recovery of potential R-lines for diverse cytoplasm using the array of prevalent diverse set of DNA markers. Furthermore, crucial steps in male sterility-based hybrid breeding, viz., fasten searching of potential restorers from a vast diverse array of genotypes, perfect intrusion of rf genes to diverse genetic backgrounds, differentiation among parental lines and genetic purity assurance are the delivered products of MAS. Additionally, marker-assisted backcrossing helps in incorporation of desired dominant genes into putative parental lines for combining the multiple resistance into derived F1 hybrids (Reddy et al. 2006; Cheng et al. 2007). Employment of diverse marker technologies has served to determine genomic location of Rf genes. In conclusion, CMS circumvents the need of background selection owing to its maternal inheritance (Herzog and Frisch 2013). Therefore, through recurring backcross using DNA markers, diversity of CMS lines can be achieved by transferring these traits to the existing maintainer lines. Recently, for genetic improvement of pearl millet, use of molecular marker technology has made some headway, and owing to its stock capacity of genetic and genomic resources with various DNA-based molecular markers, including RFLP, STS, AFLP, SSRs, diversity array technologies, SNP, mapping populations, DNA markers based linkage maps, it has been devoted to the status of molecular crop through a series of research efforts (Liu et al. 1994; Allouis et al. 2001; Qi et al. 2004; Supriya et al. 2011; Sehgal et al. 2012). These genetic stocks or genomic resources are valuable for detection and breeding of promising QTLs for various desired traits and further helps in increase the economic lifespan of the elite hybrid parental lines (Jones et al. 2002; Morgan et al. 1998; Senthilvel et al. 2008).

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In pearl millet, despite these extensive availability of molecular tools and intensive exploration of MAS for other traits, there is no research attributed towards CMS platforms, whereas MAS is extensively explored for transferring male sterility in putative maintainers, transferring Rf genes and faster recovery of male sterility in other cereal crops like wheat, maize, rice and sorghum on a large scale. This invites researchers not only to identification of male sterility-inducing factors as well as fertility-restorer factor but also to conduct deep innovation in molecular breeding of male sterility in pearl millet.

4.7

Scope for Adoption of Modern Technologies

By using modern omic tools in analysis of cytoplasmic male sterility or fertility restoration mechanisms in pearl millet, hybrid breeding can be oversimplified in terms of time and cost-effectiveness owing to fast incitement of DNA sequencing technology, which provides a huge dataset of factors responsible for these plant attributes. Among them, transcriptome profiling, genomic tools as well as study of proteomic platforms contain a huge participation. Owing to a lack in reports on pearl millet transcriptomics (Mishra et al. 2007), there is ample scope of digging up the casual genetic factors responsible for induction of male sterility or fertility retrieval using whole transcriptome profiling. In other crops, to this end, to unveil the expression patterns of putative candidate genes or orfs, techniques like cDNA library screening, subtractive hybridization and microarray were predominantly used by Huang et al. (2011). Recently, these conventional methods of transcriptome profiling have been increasingly replaced by modern NGS techniques like RNA-seq, given its high-throughput nature to pinpoint critical gene(s) (Mantione et al. 2014). Notably, identification of differential expressive genes by whole transcriptome analysis using isogenic lines of male sterility and maintainers can further lead to determination of male sterility-causing factors in pearl millet owing to its huge diversity in NILS of different male sterility cytoplasm. Furthermore, the analysis of RNA sequence which deciphers the transcription details of factors could be co-understood with the energy conservation model of Chen and Liu (2014). Owing to the role of mitochondria in cytoplasmic male sterility, whole genome sequencing of these mitochondrial genomes provides the uncovering of several novel open reading frames (orfs) that will help greatly to illuminate the hitherto elusive mechanism of sterility and fertility restoration in pearl millet, likewise in rice (Igarashi et al. 2013), in B.juncea (Heng et al. 2014), in Sorghum (Klein et al. 2015). In area of proteomics, there is a comprehensive need of investigation of gene expression and protein profiling of crops, particularly in pearl millet, on account of the findings by Castandet and Araya (2011), indicating the role of PPR proteins in fertility reversion in this crop. Moreover, the abundance of transcripts may not significantly differentiate the fertile and sterile plants, but in few instances, at the translational level, differential expression patterns occurred indicating the role of gene expression and transcriptional profiling (Hu et al. 2013).

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Another approach may be the combination platform of omic tools and cellular data which will provide the gateway to an extensive landscape of gene network, for example, in rice (Suwabe et al. 2008). Moreover, the use of omic technology with DNA markers can profusely decrease the diversified network of genes by reducing the number of candidate genes responsible for male sterility and fertility restoration in pearl millet (Shemesh-Mayer et al. 2015).

4.8

Major Challenges and Potential Opportunities

In our review, there is clear-cut evidence that there are as yet very few reports with reference to CMS in pearl millet in comparison with other cereal crops despite its economic importance in hybrid breeding of this crop. Even though a less explained but fully available repository regarding linkage maps of this crop are available, but no evident discovery of novel sterility inducing orfs as well as fertility reversion factors has been made. This shows the lack of profoundly explainable model of molecular basis of CMS in this orphan crop. Yet there is ample scope of finding novel open reading frames (orfs) which induce sterility as well as fine mapping, facilitating the positional cloning and functional characterization of the responsible candidate rf genes in pearl millet. Notably, the monopolistic approach behind the use of single cytoplasmic source for the production of commercial hybrids in pearl millet is the greater concern that predisposes this crop to various biotic strains causing disease epidemics. Thus, proper genetic diversification of male sterile lines as well as their hybrids is indeed a prime necessity for today’s breeders to combat the potential hazards of vulnerability. This diversification can be achieved by systematic studies on the highly stable prevalent A4 and A5 cytoplasm, whose studies are predominantly restricted by their less known fertility restoration process. Hence a proper knowledge fertility restoration and systematic use of these cytoplasm can make a successful array in pearl millet hybrid development programmes (Rai et al. 2006). Moreover, by supplementing the traditional grow out test (GOT) for these male sterility derived hybrids, molecular markers befitted as the substitute breeding approach to ensure genetic purity by discriminating between the parental lines and their hybrids. These markers may also facilitate in fastening the incorporation of desired novel traits into parents and through them, in hybrids, ultimately. Hence, development of a wide range of these markers as well as mitochondrial DNA markers (owing to critical role of mitochondrial genome in male sterility) has received considerable attention among breeders engaged in CMS-related research (Rajendrakumar et al. 2007; Saxena et al. 2010; Zhou et al. 2003; Zhan et al. 2012; Jiang et al. 2012). In conclusion, the ultimate aim of pearl millet breeders are to develop varieties having high heterozygosity and higher genetic heterogeneity, providing the needful adaptiveness for better self and population buffering by aiming on the traits which potentially enhance the yield stability, phenotypic plasticity, abiotic and biotic stress tolerance, nutritional efficiency in general. In achieving these goals, requirement for

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the detailed studies on the cytoplasmic male sterility or fertility restoration is likely to enrich with the increasing applications of modern omic technologies. Greater relevance may be provided by further studies on mitochondrial genome at transcriptional and translational levels as well as the digging up of the extensive omics data repository using bioinformatics. Besides this, equally important will be the efficiently extracted potential restoration sources from a wider germplasm pool to combat the large scale phenotyping protocols as well as identification of the desired novel traits for pearl millet improvement (Langer et al. 2014).

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5

Sorghum Improvement: Male Sterility and Hybrid Breeding Approaches K. B. Choudhary, Vikas Khandelwal, and Sheetal Raj Sharma

Abstract

Sorghum (Sorghum bicolor L.) is the fifth among the world’s most important crops, mostly grown in arid and semiarid regions of the world. In sorghum, mounting commercial single cross hybrids, owing to their morphological homogeny, has been more preferred by growers. Hence, cytoplasmic male sterility (CMS) has emerged as a more prominent gifted approach to achieve sustainability in crop productivity systems due to its substantial contribution in the production of crop hybrids by practically deceiving the need of removal of anthers, resulting in generation of superior F1 hybrids. Owing to the ordinariness of multiple sources of CMS, the observation of shrewd level of instability and the frequency of fertility restorer genes in the germplasm make sorghum a particularly fascinating system for the investigation of male sterility and fertility restoration. Moreover, rapid isolation of potential restorers, precise introgression of rf genes to diverse genetic backgrounds, genetic purity assurance among parents and hybrids and swift prejudice among parental lines constitute crucial steps in cytoplasmic male sterility-based heterosis breeding. Here, we prospectively review the cytoplasmic male sterility (CMS) and its relevance to hybrid breeding in sorghum with potential impending on cytoplasmic–nuclear genomic interactions, marker-assisted selection (MAS) ensuring the eventual combination of multiple resistance with yield stability, omic technologies ensuring elaborative

K. B. Choudhary (*) ICAR—Central Arid Zone Research Institute (CAZRI), Jodhpur, Rajasthan, India V. Khandelwal ICAR—All India Coordinated Research Project on Pearl Millet, Jodhpur, Rajasthan, India S. R. Sharma Division of Plant Breeding and Genetics, RARI, Jaipur, Rajasthan, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_5

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analysis of sterility and fertility retrieval functionaries like introduction of novel sterility inducing orfs. Keywords

Cytoplasm · Fertility restoration · Genome · Heterosis · Hybrid · Male sterility · Pollen

5.1

Introduction

Sorghum (Sorghum bicolor L.) is the fifth among the world’s most important crops, mostly grown in arid and semiarid regions of the world. The area covered by sorghum crop in India is 4.48 million ha, whereas in India, sorghum production was 4.38 million tons (AICRP on Sorghum and Small millets: Project Coordinator report—2019–2020). The productivity of sorghum in India in 2019–2020 was 1051 kg/ha (AICRP on Sorghum and Small millets: Project Coordinator report— 2019–2020). A well communication is established between the adoption of hybrids and the increase in productivity by 47% in India from 1960 to 1990 (FAO, 1988). Further, it is exemplified that hybrids had a contribution of 85% in kharif season sorghum in India with compares to 95% contribution in the USA, Australia and China, depicting the role of hybrids in sorghum production (Reddy et al. 2006). Developing countries in Asian and African regions mostly have water- and nutrientscant soil, provide more than 70% of the sorghum production in comparison to other regions of the world. In the arid and semiarid areas, where other crops fall short to produce economic yield, owing to its heat tolerance, salt tolerance and ability to grow in water logging conditions, sorghum is denoted an ideal crop as well as industrial crop for its use in fuel production besides its uses in food and fodder utilities. Notably, its C4 nature provides higher photosynthetic efficiency, higher dry matter production capacity and carbon sequestration, suggesting it as an ecofriendly crop. In sorghum, mounting commercial single cross hybrids have remained a noteworthy triumphant breeding story among the witnessed significant advances in productivity of major crops. Owing to their morphological homogeny, single cross hybrids are more preferred in this crop by growers further exemplified by the commercial grain hybrids. Nevertheless, on account of the treasured association of male and female reproductive organs in the same flower, the crossing of plants with one another is challenging in many agriculturally domineering crops. Thus, the anthers must be removed either mechanically or manually to prevent selffertilization, if not, the plant fails to produce pollen considered as a genetic defect. Hence, a more prominent genetic approach for F1 hybrid seed production is exploited by many plant breeders labelled as cytoplasmic male sterility (CMS). It has emerged as a gifted approach to achieve sustainability in crop productivity systems due to its sizeable contribution in production of crop hybrids by practically deceiving the need of removal of anthers, resulting in generation of superior F1

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hybrids that exhibit significant advantage over their parents and existing popular cultivars in terms of yield, stress tolerance, adaptability, etc. (Wang et al. 2013; Whitford et al. 2013; Horn et al. 2014; Saxena et al. 2013; Reddy et al. 2006). Over time, researchers have shown keen interest in the genetic and molecular mechanisms of male sterility and fertility restoration processes because of its aptitude to strap up heterosis, ability to provide decisive breeding tools and to provide material regarding plant reproductive functionaries as well as interactions between cytoplasmic and nuclear genomes (Bohra et al. 2016; Mishra and Bohra 2018). In sorghum, the ordinariness of multiple sources of cytoplasmic male sterility, the observation of shrewd level of instability and the incidence of fertility restorer genes in the germplasm make it a particularly fascinating system for the investigation of male sterility and fertility restoration. While the mainstream plant species generally reveal one or two CMS types, millets provide a collection of at least eight independently identified forms. Here, we review the cytoplasmic male sterility (CMS) and its relevance to hybrid breeding in sorghum. We conclude with a discussion on the potential of harnessing cytoplasmic–nuclear genomic interactions as well as modern marker-assisted selection (MAS) technology for hybrid breeding in sorghum.

5.2

Hybrid Development and Heterosis in Sorghum

The basic concept of heterosis breeding lies in detection of better combiners, utilization of additive genetic variance, exploitation of panicle yield components and physiological criteria, incorporation of grain quality, insect and disease resistance in parental lines. Since heterosis has been successfully used in corn breeding, many workers had focused their attention on the findings of male sterile lines in the many different crop species in the hope of discovering the possibilities for utilizing heterosis. Notably, Conner and Karper (1927) pioneered the research that recorded occurrence of heterosis in sorghum. The role of hand emasculation in enhancing yield potential, yield stability and reduced maturity of sorghum hybrids was discussed by Stephens and Quinby (1952). At that point, the preventive factor to hybrid seed production was the lack of economic feasibility method to produce hybrid seed. Thus, by developing CMS systems, Stephens and Holland (1954) eliminated this gigantic problem and sorghum hybrids were adopted instantly as well as mass production of F1 hybrids and the practical exploitation of hybrid vigor in sorghum was made possible. The use of a CMS system ultimately determined by the existence of commercially viable heterosis even if all the requirements are met in a CMS system. Keeping this point in view, Quinby (1980) and Doggelt (1969) reported the heterobeltiosis of 58% and 22% under drought stresses and irrigated conditions, respectively. Additionally, Miller and Kebede (1984) reported 40% heterosis in new hybrids over commercial hybrids of the late 1950s. Ever since, a large number of researchers have reported recurrent occurrence of heterosis for yield and component traits over the better parent in this orphan crop. Basic reasons behind this remarkable heterosis over the years using CMS in sorghum are recombination of panicle

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Table 5.1 History of CMS-based sorghum hybrids in India Predominant cytoplasm A1

Hybrids CSH-1 CSH 25 CSH-13, CSH-16, CSH-18, CSH-22 CSH-27 CSH-21, SPH-1629, CSH-28 CSH-23, SPH-1567 CSH-24MF, CSH-19R

Table 5.2 Number of national- and state-released sorghum hybrids in India by the public sector

Year of release 1964 2008 2009 2011 2012 2013 2014

Phase 1961–1970 1971–1980 1981–1990 1991–2000 2001–2010

Source Singh and Lohithaswa (2006), Hariprasanna and Patil (2015), Reddy et al. (2012)

Number of hybrids 3 11 10 19 10

Source Kumara et al. (2011)

components and various types of genic interactions (Rana and Murty 1978). Moreover, 18–31% average heterosis for grain yield using A1 cytoplasm was reported by Indian national program testing in the years 1999 and 2000. By using A1 cytoplasm, Reddy et al. 2003 reported the 15–26% heterobeltiosis for the grain yield in sorghum. By comparing cytoplasm-based hybrid variability, Gangakishan and Borikar (1989) observed that A2-CMS-based hybrids gave higher yield and productivity followed by A4 cytoplasm-based hybrids and A1 cytoplasm-based hybrids. In India, attempts were first time made in the early 1960s to exploit heterosis using male sterile line CK 60A and Indian local or exotic dwarf lines as pollinators. It was an exotic  exotic combination between CK 60A  IS 84 which, released as CSH-1, denoted as the first hybrid in sorghum and further released as commercial variety in 1964 (Table 5.1). Moreover, the boost in sorghum productivity was further amplified by the spread of high yielding varieties in forms of hybrids likewise CSH-5, CSH-6 and CSH-9 from 1970 to 1980, and later till CSH-23 (Table 5.2); these varieties are the prime testimony of successful breeding programs in sorghum in terms of yield enhancement, parental lines diversifications and biotic stress resistance in this crop as well (Reddy et al. 2008). Notably, this progress was achieved in different phases; in the early phase (until 1975), CSH-1 predominantly replaced the pre-existing cultivars in India, in medial phase (1976–1986) CSH-5 and CSH-6 replaced the predominant CSH-1, CSH-2 and CSH-4 and in the later phase (after 1986) CSH-9, MSH-51 and JKSH-22 and many private sector hybrids replaced all the initial hybrid cultivars (Deb et al. 2004). The process of sorghum hybrid production includes crossing between A (male sterile) line and R (restorer) line, further harvesting seeds from male sterile line

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Table 5.3 Genotype and phenotype of A, B and R lines in cytoplasmic male sterility system Line A (male sterile) line B (maintainer) line R (restorer) line Hybrid

Cytoplasm A (sterility inducing cytoplasm)

Genotype mscmsc

Normal cytoplasm

mscmsc

A (sterility inducing cytoplasm) or Normal cytoplasm A (sterility inducing cytoplasm)

MscMsc

Phenotype Male sterile

Male fertile

Mscmsc

Source: Sleper and Poehlman (2006)

denoted as hybrid. Moreover, maintenance of A line is achieved by backcrossing of male sterile plants to B (maintainer) line. Enhance in seed production of B line and R line is achieved by selfing and isolation growing (Table 5.3).

5.3

Diversity of Male Sterility-inducing Cytoplasm

The first male sterility-inducing cytoplasm in sorghum was milo (denoted as A1), transferred into kafir nuclear background (Stephens and Holland 1954). Afterward, alternative cytoplasm exploited from the prevalent diversity present in different sorghum races and species related to different geographical locations have been reported in sorghum by several workers (Mital et al. 1958; Rao 1962; Hussaini and Rao 1964; Appathurai 1964; Rao and Gouda 1966; Nagur 1971; Nagur and Menon 1974; Rao et al. 1971; Tripathi 1979; Schertz 1977; Schertz and Ritchey 1977; Quinby 1980; Tripathi et al. 1980; Schertz and Pring 1982; Worstell et al. 1984). These different CMS sources have been classified and summarized in Table 5.4. Stephens and Holland (1954) assigned the designation A1 to milo cytoplasm. The designation A2 was given by Schertz (1977) ton the Ethiopian variety IS 12662C (TAM 428). Quinby 1980 assigned the A3 symbol to male sterility cytoplasm in Nilwa and IS 1112C and sources of A4 cytoplasm were obtained from IS-7920C, Guuntur, VZM and Maldandi (Worstell et al. 1984; Quinby 1982; Senthil et al. 1994; Reddy et al. 2005). On the other hand, a male sterile plant was discovered from 9E (a selection made in 9E) from Ghana (Webster and Singh 1964). A series of ms lines in Kansas with kafir nucleus and cytoplasm of wild grassy sorghum were developed and designated as KS 34–KS 39 (Ross 1965; Ross and Hackerott 1972).

5.4

Stability of Diverse CMS Systems

Ideally, stability of male sterile lines may be demarcated as the non-pollen shedding behavior as well as inability to produce seed when selfing is done in them regardless of the environmental factors, likewise in different locations or in different seasons. Many researchers had elucidated the stability of cytoplasmic–nuclear male-sterility systems in sorghum (Schertz and Pring 1982; Pedersen et al. 1998; Senthil et al.

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Table 5.4 Potential sources of cytoplasmic–nuclear male sterility in sorghum Cytoplasm A1

A2

A3

A4 9E A5 A6

KS 34 KS 35 KS 36 KS 37 KS 38 KS 39

Male sterile line 3-DwaRf Milo IS-1116C, IS-6771C, IS-8232C IS-2266C, IS-7007C, IS-3579C IS 6705C IS 7502C IS 12662C IS 2573C IS 2816C IS 1112C IS 12565C IS 6882C IS 7920C IS 112603C IS 7218 IS 7506C IS 1056C IS 2801C IS 3063C PI 208190/SA 1741 PI 258806 PI 208190 PI 247722 PI 155140 Kenya 53262

Race/Species Durra Guinea and Caudatum Guinea and Caudatum Guinea Guinea Guinea Caudatum Caudatum Durra-(Durra-Bicolor) Caudatum Kafir-Caudatum Guinea Guinea N/A Bicolor Durra Durra Durra S. arundinaceum S. arundinaceum S. verticilliflorum S. sudananse S. conspicnum S. niloticum

Place of origin Unknown India Sudan Burkina Faso Nigeria Nigeria Sudan Zimbabwe India Sudan USA Nigeria

India Zimbabwe Ethiopia Kansas, USA

Source: Schertz and Pring (1982); Schertz et al. (1989); Schertz (1994); Senthil et al. (1994); Xu et al. (1995); Pedersen et al. (1998); Reddy et al. (2005); Kumar et al. (2008)

1994; Reddy et al. 2003, 2005; Kumar et al. 2008). Occurrence of the restorer genes and commercially viable heterosis levels are the main factors of stability of cytoplasmic male sterility systems (Reddy et al. 2003). Instability increases the pollen shedding problems from seed production plots, which consequently enhances the seed production costs and reduces breeding efficiency owing to their degradation of sterility in the subsequent generations shown by the backcross progenies, which ultimately become the prime cause of their rejection in the breeding programs. Notably, in sorghum, almost all the commercially exploited hybrids are centered on A1 CMS system till date, depicting the low frequency of pollen shedders (72 diverse CMS sources that have been identified in 16 different Helianthus species (Serieys 2002), but have not been exploited in the production of hybrid seed (Serieys 1999a, b; Echeverria et al. 2003). CMS-89 and its derivatives were used to develop all commercial sunflower hybrids due to their stability across a wide range of environmental conditions and seasons, as well as the easy availability of fertility restorer gene(s) (Friedt 1992). In order to improve the sunflower hybrid yield and the introgression of resistance to various pathogens from wild Helianthus species into the cultivated sunflower, novel inbred lines have to be continually developed. To minimize the threat of pathogenic vulnerability, it is extremely important to enhance the cytoplasmic genetic diversity, and fertility restorers under agronomically superior genetic backgrounds will further enhance yields.

6.9

Desirable Characteristics of Female (CMS) Lines

The additional criteria for the cytoplasmic male sterile (female) line, are (1) high seed yield; (2) high oil content; (3) should be resistant/tolerant to major biotic and abiotic stresses; (4) should be less affected by environmental conditions; (5) should be a good general combiner; (6) should have perfect synchrony with the pollen parent (R) to facilitate nicking; (7) good seed characteristics; (8) maximum diversity from the R line chosen; and (9) appropriate height and capitulum diameter.

6.10

Methods for Developing New CMS Sources

There are different ways to develop new sources of CMS in sunflower, like intraspecific hybridization, interspecific hybridization, spontaneous mutation, chemicalinduced male sterility, mutagenesis and hormonal-induced male sterility. Table 6.3 provides detailed information on the origin, FAO code, and wild species of various cytoplasmic male sterilities in sunflower. (a) CMS sources obtain through interspecific hybridization: CMS frequently arises from interspecific crosses. The maximum numbers of CMS sources have been evolved by interspecific hybridization. There are a few successful examples of CMS developed by such crosses in sunflower. RIG-1 was discovered by Vulpe (1972) as a result of an interspecific hybrid between H. rigidus and the cultivated sunflower. Leclercq (1974) discovered a CMS source in a cross between diploid annual wild species H. petiolaris x H. annuus. This newly identified CMS source was different from the classic CMS PET-1 for pollen

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

1 2 3 4 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(continued)

S. no.

Designation Originated from FAO code HA-89 H. annuus MUT-10 HA-89 H. annuus MUT-11 HA-89 H. annuus MUT-12 CMS sources originated through Interspecific hybridization CMG-3 H. maximiliani MAX 1 – H. maximiliani MAX 2 ANOMALUS H. anomalus ANO-1 ARGOPHYLLUS H. argophyllus ARG-1 ARGOPHYLLUS H. argophyllus ARG-2 ARGOPHYLLUS H. argophyllus ARG-3 ARG3-MI H. argophyllus ARG-3-M1 ARGOPHYLLUS H. argophyllus ARG-4 BOLANDERI H. bolanderi BOL-1 DV-10 H. debilis DEB-1 EXI-1 EXILIS H. exilis EXI-2 H. exilis EXI-2 MOLLIS H. mollis MOL-1 NEGLECTUS H. neglectus NEG-1 CANESCENS H. niveus ssp canescens NIC-1 FALLAX H. petiolaris ssp fallax PEF-1 PET/PET H. petiolaris ssp petiolaris PEP-1 CMG-2 H. giganteus GIG-1 CLASSICAL CMS H. petiolaris Nutt PET-1 CMG-1 H. petiolaris Nutt PET-2 PETIOLARIS BIS H. petiolaris Nutt PET-3 PET-34 H. petiolaris PET-4

Table 6.3 Various CMS sources of sunflower S. no. 36 37 38

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Designation Originated from FAO code CMS sources originated through intraspecific crosses KOUBAN H. annuus ssp. lenticularis ANL-1 INDIANA-1 H. annuus ssp. lenticularis ANL-2 VIR 126 H. annuus ssp. lenticularis ANL-3 FUNDELEA-1 H. annuus ssp texanus ANT-1 Spontaneously occurring CMS sources H. annuus-367 wild H. annuus ANN-1 H. annuus-517 wild H. annuus ANN-2 H. annuus-519 wild H. annuus ANN-3 H. annuus-521 wild H. annuus ANN-4 NS-ANN-81 wild H. annuus ANN-5 NS-ANN-2 wild H. annuus ANN-6 – wild H. annuus ANN-7 – wild H. annuus ANN-8 – wild H. annuus ANN-9 AN-67 H. annuus ANN-10 AN-58 H. annuus ANN-11 AN-2-91 H. annuus ANN-12 AN-2-92 H. annuus ANN-13 – H. annuus ANN-14 CMS-G H. annuus ANN-15 CMS-Dp H. annuus ANN-16 CMS-VL H. annuus ANN-17 – H. annuus ANN-18 – H. annuus ANN-19 – H. annuus ANN-20

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Designation Originated from – H. annuus – H. annuus CMS induced by mutagenesis HEMUS H. annuus PEREDOVICK H. annuus STADION H. annuus PEREDOVICK H. annuus H. annuus PEREDOVICK VORONEJSKII H. annuus H. annuus HA 89 HA 89 H. annuus H. annuus HA 89

Source: Serieys (2005)

27 28 29 30 31 32 33 34 35

S. no. 25 26

Table 6.3 (continued)

MUT-1 MUT-2 MUT-3 MUT-4 MUT-5 MUT 6 MUT 7 MUT 8 MUT 9

FAO code ANN-21 ANN-22

S. no. 61 62 63 64 65 66 67 68 69 70 71 72

Designation – PRAECOX PHIR-27 PRAECOX PPR-28 RUN-29 RESINOSUS-243 VULPE RIG-M-28 STRUMOSUS – CMG-3

Originated from H. petiolaris H. praecox ssp praecox H. praecox ssp hirsutus H. praecox ssp praecox H. praecox ssp praecox H. praecox H. resinosus H. rigidus H. rigidus H. strumosus H. giganteus H. maximiliani

PRP-1 PRR-1 RES-1 RIG-1 RIG-2 STR-1 CMS-GIG3 MAX-1

FAO code PET-5 PRA-1

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fertility restoration. Through interspecific substitution of the nucleus of cultivated sunflower cv. Saturn into H. petiolaris cytoplasm, Whelan (1980) reported a source of CMS. Leclercq (1983) discovered another CMS source PET-3 again, by using diploid species H. petiolaris as the female parent. By crossing annual wild species with cultivated sunflowers, Serieys (1984) identified a large number of CMS sources. The first CMS source CMS PF (PEF-1) came from H. petiolaris ssp. fallax. The second CMS source CMS BOL (BOL-1) came from wild Helianthus species H. bolanderi. Serieys and Vincourt (1987) identified two sources, viz., EXI-1 and NIC-1 developed by crossing H. exilis, H. niveus ssp. canescens, and cultivated sunflower, respectively. Another CMS source CMS-KU-70 was discovered by Anashchenko (1974). Two CMS sources (CMG-3 and CMG-2) were also discovered by Whelan and Dorell (1980) and Whelan (1981). Christov (1990a, b, 1991, 1992) and Christov et al. (1993) identified various CMS sources such as ARG-1, ARG-2, ARG-3, RIG-2, PET-4, PRH-1, PRR-1, and DEB-1 using various diploid annual wild species such as H. argophyllus, H. petiolaris, H. rigidus, H. praecox, and H. debilis in interspecific hybridization. Some restorers of PET-1 seemed to be restorers for ARG-1 and ARG-2, indicating that these could be used in heterosis breeding programs. In an experiment conducted by Serieys (1994), it was found that CMS sources such as ANL-1, EXI-2, NEG-1, PEP-1, and PRP-1 were all distinct from each other as well as from the classical CMS PET-1. (b) CMS sources developed through intraspecific hybridization: A new CMS CMS KI-70 was originated by Anaschenko et al. (1974) by intraspecific hybridization between H. lenticularis and H. annuus ssp. annuus. CMS KI-70 was later given the name ANL-1 and is known as the Kuban source of CMS in the literature. Another CMS source, ANL-3, was discovered in the same year. A new CMS source was discovered by Heiser (1982) in a cross between H. lenticularis and H. annuus. A male sterility character was incorporated into RHA-265, which is a restorer of Classical CMS PET-1, resulting in a new source, Indiana 1 (CMS I), which was given the designation ANL-2. A CMS source designated as ANT-1 or FUNDULEA-1 was identified in a cross of H. annuus ssp. texanus x H. annuus by Vranceanu et al. (1986). (c) Spontaneously occurring cytoplasmic male sterility: Some novel CMS sources were discovered to have originated directly in wild H. annuus ecotypes, contributing to the CMS source diversity. Four CMS sources designated ANN-1, ANN-2, ANN-3, and ANN-4 were found directly in ecotypes of wild H. annuus by Serieys (1984). Jan (1995) discovered two more novel CMS sources in the wild H. annuus, ANN-7 and ANN-8, which appeared as spontaneous mutations. Marinkovic and Miller (1995) identified ANN-5 as a new CMS source from the wild sunflower population that was highly stable under various environmental situations. (d) CMS sources obtained through mutagenesis: In cultivated sunflowers, Jan and Rutger (1988) investigated the effectiveness of mitomycin C and streptomycin in inducing CMS. Streptomycin was found to be more effective than

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mitomycin C for inducing CMS mutations, and the induced CMS mutants might be used for hybrid seed production without requiring any changes to the current fertility restoration system. By treating inbred HA-89 seeds with streptomycin, they discovered a new CMS source designated STRP-555. Christov et al. (1993) described two more CMS mutants in two sunflower populations, viz., Peredovik and Hemus, which were originated by sonification and irradiation, respectively. Anashchenko (1977) summarized that the most important effects of chemical as well as physical mutagenesis are the creation of a substantial number of recessive genes and cytoplasmic mutations. (e) CMS sources induced by chemicals: Tripathi and Singh (2013) reported the effectiveness demonstrated by Benzotriazole (C6H5N3) in inducing perfect male sterility in sunflower. On a commercial scale, the double treatment with 1% benzotriazole can be used for hybrid seed production. Except for a single treatment of 1% benzotriazole, which resulted in 97% pollen sterility, 1.5 and 2% concentration treatments induced 100% male sterility. The benzotriazoleinduced male sterility was permanent and remained throughout the flowering period. It inhibits the growth of microspores, causing male sterility in plants. Benzotriazole was also found to be a suitable chemical hybridizing agent for other crops such as Indian mustard (Chauhan and Kinoshita 1982), faba bean (Shivana and Sawahney 1997), Datura (Chauhan and Agnihotri 2005), chilli peppers, tree cotton, and radish (Singh and Chauhan 2001). (f) Hormone induced male sterility: Piquemal (1970) and Miller and Fick (1978) also proposed that Gibberellic acid (C19H22O6) can be used to induce male sterility in sunflower. Chemical emasculation with gibberellic acid was first demonstrated by Schuster (1961) to be effective in inducing male sterility in sunflower, and it was widely utilized in the development of test crosses between inbreds and different testers (Anashchencko 1972; Skoric 1988). Piquemal (1970) reported that different inbreds may express various responses to gibberellic acid and require different concentrations (higher or lower) and different application times (earlier or later). Miller (1987) observed different negative effects of gibberellic acid on plants based on various concentrations and timing of application, such as reduced female sterility, partial male sterility, stem elongation, small head size, etc.

6.11

Molecular Characterization of Male Sterility

The existence of CMS in sunflower has been demonstrated. However, due to the current state of scientific understanding in the field of CMS and the available molecular discrepancies between the existing CMS fertility restoration systems, its application is limited to a small number of them. The reasons for these differences as well as the appearance of the new CMS are yet unknown (Christov 2003). As a result, molecular characterization of all CMS systems will be extremely beneficial in understanding the molecular mechanism.

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6.11.1 Open Reading Frames are Identified as a Cause of Male Sterility Brown et al. (1986) started a molecular investigation into the Classical CMS system developed by Leclercq. They investigated the differences in mitochondrial and chloroplast DNA, as well as double-stranded RNA, in male sterile and male fertile lines that were chromosomally isogenic. There were no differences observed in restriction fragment sizes of the chloroplast DNA or the double-stranded RNA. But the differences were observed in the lengths of DNA fragments from the mitochondrial genome between male sterile and male fertile lines. However, this was the first evidence that the sunflower CMS character may be caused by a mutation in the mitochondrial DNA. A small mitochondrial plasmid in the male fertile line that was absent from the male sterile line was detected by Brown et al. (1986). By studying RFLPs of mtDNA from isonuclear male fertile and male sterile lines, Siculella and Palmer (1988) focused attention on the mitochondrial genome. They found that the genomes were almost identical, except for a 12,000 base pair (12-kb) inversion and a 5-kb insertion in the male-sterile mtDNA. These changes occurred between the a-subunit of the F1-adenosine triphosphate (ATP) synthase (atpA) and the apocytochrome b (cob) genes. In CMS PET 1, the CMS-linked open reading frame orfH522, which is found in the 30 region of the atpA gene, consists of the first 57 bp of orfB (atp8) and unknown sequences for the rest of the part (Kohler et al. 1991). The open reading frame orfH522 is co-transcribed with the atpA gene on an additional larger transcript (Kohler et al. 1991). De la Canal et al. (2001) discovered a 0.5-kb insertion of unknown sequences in the 30 region of the atp9 gene in the PEF1 cytoplasm as the cause of male sterility. The orf linked with generating male sterility in higher plants appears to be the result of many recombination events involving known mitochondrial genes as well as sequences of unknown origin. Molecular characterization of another CMS line, CMS-3, has shown that genome sequences of at least five mitochondrial loci are altered. However, the typical 15 kDa protein is absent in the new CMS line. Some evidence has been provided for potential recombination events in COX-II and ATP-6 genes in the CMS phenotype (Spassova et al. 1994). Sunflower has joined the list of plants in which the CMS trait is known to be linked with a mutation in the mitochondrial genome. The mutation events involved are a 12-kb inversion and a 5-kb insertion/deletion in the vicinity of the ATP locus, resulting in the creation of a novel open reading frame (ORF522) located 30 to the α-subunit of the ATP-A gene in the sterile line (Siculella and Palmer 1988; Laver et al. 1991a, b; Horn et al. 1996). Translation experiments with mitochondria purified from seedlings of male fertile as well as male-sterile lines showed that a new polypeptide of about 15 kDa is synthesized by the mitochondria of the malesterile line, which, however, is not detectable in the male fertile line (Kohler et al. 1991; Laver et al. 1991a, b). The CMS gene, orf 522, for example, lies 30 to atpA sequences on the di-cistronic transcript (Monéger et al. 1994). CMS in sunflower has been shown to be linked with recombination events in the mitochondrial genome which involve an inversion or insertion rearrangement resulting in the creation of a

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novel ORF in the male sterile line (Kohler et al. 1991; Laver et al. 1991a, b). This ORF called orf 522 contains the first 18 codons of a mitochondrial gene of unknown function called orfB, while the remainder of the gene is of unknown origin. This chimeric gene is located 30 to the atpA gene and potentially encodes a polypeptide of 19 kDa or 14.6 kDa, depending on which two methionine codons are used for translation initiation (Laver et al. 1991a, b). In organelle translation experiments, a polypeptide of around 15 kDa was shown to be synthesized specifically by mitochondria from the sterile line (Horn et al. 1991, 1996; Laver et al. 1991a, b). This 15 kDa protein was immune-precipitated by antisera directed against orf 522 synthetic oligopeptides (Monéger et al. 1994). Furthermore, in situ hybridization experiments showed that orf 522 transcripts are decreased in abundance in meiotic cells in the presence of the fertility restorer gene and that the restorer gene might act cell-specifically (Smart et al. 1994). Spassova et al. (1991) studied mitochondrial DNA from one male fertile and six new CMS sunflower genotypes. CMS associated RFLPs were found, generated by various restriction enzymes in the vicinity of the atp A locus. CMS3 is another source of male sterility in sunflowers that have been previously described (Spassova et al. 1992). Male sterility is caused by orfB-cox III locus rearrangements, which alters the expression of these genes (Spassova et al. 1994). The genetic determination in sunflower male sterility types was established according to the location of the genetic factors that induce pollen degeneration and stamen atrophy (Vranceanu 1967a, b). CMS is a maternally inherited character that is frequently linked with unusual ORFs found in mitochondrial genomes (Hanson and Bentolila 2004). Numerous studies have exhibited that CMS in plants is associated with improper recombination events in the mitochondrial genome, resulting in the generation of chimeric ORFs that are expressed as novel polypeptides (Schnable and Wise 1998). The sequences that contribute to the generation of these chimeric ORFs are frequently derived from coding and non-coding regions of existing genes, but they can also come from somewhere else. Hahn and Friedt (1991) found that the mtDNA of the CMS cytoplasm MAX1 differs from fertile lines in the region of the atp A gene, as does the PET1 cytoplasm. Additionally, the former cytoplasm also displays differences in the region of the atp6 gene when compared to fertile sunflower lines. Gerlach et al. (1991) came to some important conclusions as well, by finding that the CMS discovered by Leclercq (1969) is linked to mtDNA rearrangements. The rearrangements consist of an 11 kb inversion and a 5 kb insertion near the atp A locus. Belhassen’s (1991) mitochondrial DNA analysis had allowed interesting characterizations of the 20 CMS sources. While the modification of some sequences seems to be correlated with CMS, the presence of the two mitochondrial plasmids P1 and P2 was described to be unrelated to male sterility. Laver et al. (1991a, b), emphasizing mitochondrial genome organization and expression linked with CMS, claimed that a region of mitochondrial genome variation between fertile and CMS phenotypes has been located in the three flanking regions of the gene encoding the alpha subunit of the F1 ATPase (atpA). The same researchers discovered that a 15 kDa protein is generated by sterile sunflower

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mitochondria, but not by male fertile sunflower mitochondria, in organelle labeling of mitochondrial translation products from sterile and fertile sunflowers. The ORFc sequence could encode this 15 kDa protein, which could be causally related to the CMS trait. Horn et al. (1991) reported, on the other hand, that the 16 kDa polypeptide is not expressed in the fertile lines of cultivated sunflower or H. petiolaris. The 16 kDa polypeptide, which is membrane-associated, might be the product of the new open reading frame orfH522, which is co-transcribed with the atpA gene and also seems to be correlated with CMS. On the basis of the findings achieved by some French authors (Crouzillat et al. 1991), Serieys (1991) stated that RFLP of mitochondrial DNA exhibits particular differences between the cytotypes investigated. Thirteen cytotypes were distinguished using three restriction enzymes and 12 probes. CMS cytotypes have no relation to the species from which they were derived. Phenograms were constructed based on the similarity indices between the cytotypes for genetical and mitochondrial RFLP investigations. The majority of the CMS is characterized by restoration patterns related to a mitochondrial DNA restriction fragment pattern. Perez et al. (1988) found that the presence of the P1T plasmid in the B lines has been established both by the ethidium bromide staining technique of Leroy et al. (1985) and by hybridization with the cloned plasmid used as a probe from CANP 3 B lines, Perez et al. (1988), and from the HA89 B line, Crouzillat et al. (1987). Nevertheless, some total DNA preparations of the A-line gave a sporadic hybridization signal depending upon the samples. So it is a question of whether the presence of P1T or any sequence homologous to P1T can be present in total DNA from the A-lines. Perez et al. (1988) claimed that the circular plasmid called P1T found in the sunflower mitochondria has been utilized as a probe on sunflower lines used by breeders and on wild forms or wild form derived CMS. P1T crosshybridized various sized plasmid, but not all of the H. petiolaris species. The Classical cytoplasm causes premature programed cell death (PCD) of the tapetal cells, which extends to anther tissues (Balk and Leaver 2001). PET1’s mitochondrial genome alterations are limited to a 17-kb region and consist of two mutations: a 12-kb inversion and a 5-kb insertion or deletion, both of which result in a change in the atpA gene’s transcription pattern (Siculella and Palmer 1988). The entire 5 kb insertion, observed in PET1, is also found in all other PET1 like CMS sources (Horn and Friedt 1999). In PET1 CMS source, the expression of a new orfH522, in the 30 flanking region of the atpA gene is linked with the CMS trait (Kohler et al. 1991), which encodes a 16 kDa polypeptide (Horn et al. 1991; Laver et al. 1991a, b). In nine more male-sterile cytoplasms, 16 kDa protein was also detected by organello translation (Horn et al. 1996). The adoption of novel diverse CMS sources would allow widening of the genetic base of the cytoplasm since molecular investigations on the relationships between CMS sources have disclosed polymorphisms in the mitochondrial DNA (Crouzillat et al. 1994; Hahn and Friedt 1994). Although MAX1’s mitochondrial DNA indicates homology to orfH522, the detailed molecular study revealed that MAX1 has a distinct CMS mechanism than PET1. This orf, and its 16 kDa translation product, have been associated with CMS in PET1 (Horn et al. 1991; Kohler et al. 1991; Laver et al. 1991a, b). Molecular investigations, on

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the other hand, demonstrate that distinct CMS source origins in sunflower do not always imply various CMS mechanisms (Korell et al. 1996). Only two orfs, orf288 and orf231, were found to be specific to the PET2 cytoplasm, which was also confirmed by transcript analyses (Reddemann and Horn 2010). In the presence of PET2 cytoplasm, the two novel orfs, the edited orf231 (8.4 kDa) and the orf288 (10.6 kDa) may be responsible for male sterility. Makarenko et al. (2018) discovered that two atp9 chimeric ORFs, orf228 and orf285, may be responsible for the PET2 CMS phenotype, which was verified using NGS sequencing. Makarenko et al. (2019) assumed that orf1197 is involved in the development of male sterility phenotype in ANN2.

6.11.2 Proteins Associated with Cytoplasmic Male Sterility Sabar et al. (2003) recorded the low level of ATPase activity in the PET CMS of sunflower. In sterile sunflowers, the ORF522 protein has not been shown to be involved in reducing ATPase activity. The Classical PET1 CMS protein is associated with the expression of orf522, a novel mitochondrial gene located downstream of the atp1 gene in sunflower. Open reading frame Orf 522 is co-transcribed with the atp1 gene and is expressed as a mitochondrial polypeptide of 15 kDa in all the tissues (Horn et al. 1991; Kohler et al. 1991; Laver et al. 1991a, b). The PET-1 cytoplasm leads tapetum cells to die prematurely due to programed cell death (PCD) (Balk and Leaver 2001). ORF522, the chimeric protein that causes the CMS phenotype in sunflower, has an N-terminal amino acid sequence, polypeptide domain structure, and molecular weight that are identical to ORFB. The N-terminal 19 amino acids of ORFB and ORF522, in combination with a specific carboxy-terminal domain, may cause competition between the two proteins, resulting in impaired biogenesis and uncoupling, as well as diminished phosphorylation activity of the F1FO-ATP synthase complex (Balk and Leaver 2001). The male-fertile phenotype can be restored by introducing nuclear fertility restorer Rf genes in a cross, which leads to a decrease in the levels of the atp1–orf522 co-transcript and of the ORF522 protein in the male florets (Monéger et al. 1994; Smart et al. 1994). A few studies of CMS lines point toward a role of mitochondrial reactive oxygen species (ROS) in sterility. The tissue-specific rise in the level of polyadenylated atpA-orfH522 transcripts was linked with the tissue-specific instability of atpA-orfH522 mRNA in the anther of the fertile hybrids (Gagliardi and Leaver 1999). In the anthers of the PET1-CMS of sunflowers, a ROS burst has been seen (Balk and Leaver 2001). Monéger et al. (1994) showed that this protein was produced by the extended orf and that its levels were decreased in the male florets of the restored hybrid. The orf522 transcript and protein are ample in the young meiocytes of male-sterile and fertile plants. At later stages of anther development, the levels of orf522 transcript and protein were decreased in the meiocytes of the restored hybrid (Smart et al. 1994). The actual roles of orf522, as well as the fertility restorer gene(s) in the disruption of pollen development and subsequent restoration, are unknown. Detailed

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information about mitochondrial genes, function, and aligned sequences to generate the consensus is presented (Table 6.4).

6.12

Limitations of the Diverse CMS Sources

The stability of female lines in relation to their sterility nature across a wide range of agro-climatic situations and seasons is one of the important prerequisites to its wider effective implications. However, available diverse CMS sources have not been exploited commercially in the heterosis breeding program due to various reasons, including CMS sources instability over a wide range of environments and seasons (Seetharam and Satyanarayana 1980; Seetharam and Virupakshappa 1993; Kumar et al. 1993; Rajanna et al. 1998), majority of the fertility restorers of the PET-1 CMS failed to restore fertility in the other novel CMS sources (Rukminidevi et al. 2006; Sujatha and Vishnuvardhan Reddy 2008; Reddy et al. 2008; Meena et al. 2013a, 2017), and fertility restorer genes have not been reported for the other CMS sources either due to the CMS having not yet been thoroughly studied or because restorer genes are extremely rare in Helianthus genetic resources (Serieys 1994). Lack of fertility restorers is also observed in the novel CMS sources, such as CMS I (H. lenticularis) and CMS PF (H. fallax) by Havekes et al. (1991), Virupakshappa et al. (1991, 1992), Ravi Kumar et al. (1994), Meena and Sujatha (2013), Meena and Prabakaran (2016), and Virupakshappa and Gowda (1996). Petrov (1992), Serieys (1992), Havekes et al. (1991) recorded negative effects on achene yield, and other plant and seed traits in several new CMS sources. Previous sunflower research has shown that wild cytoplasmic sources have a considerable impact on both qualitative and quantitative features (Nooryazdan et al. 2010). Jan et al. (2014) suggested that cytoplasm of perennial H. angustifolius increased lodging, while perennial cytoplasm of H. mollis, H. grosseserratus, and H. divaricatus reduced capitulum size and yield. Very recently, Tyagi et al. (2020) observed negative heterosis for oil content in the CMS source H. debilis in the regular water regime.

6.13

Opportunities

Many studies (Fleming 1972; Gracen and Grogan 1974; Kumar et al. 1983; Kruleves et al. 1988; Davidenko et al. 1988; Gill 1993) have emphasized the need for CMS diversification not only for excellent economically important characteristics but also to reduce the vulnerability of hybrids to major biotic stresses. Rajanna (1995) examined hybrids of three diverse CMS sources, namely, CMS F, CMS PF, and CMS I to downy mildew and found that the disease reaction of three CMS sources differs significantly. The hybrids of CMS PF were resistant to downy mildew, whereas hybrids with CMS F showed a susceptible reaction. In another study, CMS I was found to be tolerant to Alternaria leaf spot by Puttaranga Swamy (1997). In terms of achene yield, oil yield, and oil content, hybrids developed from the cytoplasmic background of CMS I ranked first, followed by CMS F and

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1 2 3 4

S. no.

Designation Originated from FAO code CMS sources originated through intraspecific crosses KOUBAN H. annuus ssp. lenticularis ANL-1 INDIANA-1 H. annuus ssp. lenticularis ANL-2 VIR 126 H. annuus ssp. lenticularis ANL-3 FUNDELEA-1 H. annuus ssp texanus ANT-1 Spontaneously occurring CMS sources H. annuus-367 wild H. annuus ANN-1 H. annuus-517 wild H. annuus ANN-2 H. annuus-519 wild H. annuus ANN-3 H. annuus-521 wild H. annuus ANN-4 NS-ANN-81 wild H. annuus ANN-5 NS-ANN-2 wild H. annuus ANN-6 – wild H. annuus ANN-7 – wild H. annuus ANN-8 – wild H. annuus ANN-9 AN-67 H. annuus ANN-10 AN-58 H. annuus ANN-11 AN-2-91 H. annuus ANN-12 AN-2-92 H. annuus ANN-13 – H. annuus ANN-14 CMS-G H. annuus ANN-15 CMS-Dp H. annuus ANN-16 CMS-VL H. annuus ANN-17 – H. annuus ANN-18 – H. annuus ANN-19

Table 6.4 Various CMS sources of sunflower

39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

S. no. 36 37 38

Designation Originated from FAO code HA-89 H. annuus MUT-10 HA-89 H. annuus MUT-11 HA-89 H. annuus MUT-12 CMS sources originated through Interspecific hybridization CMG-3 H. maximiliani MAX 1 – H. maximiliani MAX 2 ANOMALUS H. anomalus ANO-1 ARGOPHYLLUS H. argophyllus ARG-1 ARGOPHYLLUS H. argophyllus ARG-2 ARGOPHYLLUS H. argophyllus ARG-3 ARG3-MI H. argophyllus ARG-3-M1 ARGOPHYLLUS H. argophyllus ARG-4 BOLANDERI H. bolanderi BOL-1 DV-10 H. debilis DEB-1 EXI-1 EXILIS H. exilis EXI-2 H. exilis EXI-2 MOLLIS H. mollis MOL-1 NEGLECTUS H. neglectus NEG-1 CANESCENS H. niveus ssp canescens NIC-1 FALLAX H. petiolaris ssp fallax PEF-1 PET/PET H. petiolaris ssp petiolaris PEP-1 CMG-2 H. giganteus GIG-1 CLASSICAL CMS H. petiolaris Nutt PET-1 CMG-1 H. petiolaris Nutt PET-2 PETIOLARIS BIS H. petiolaris Nutt PET-3

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– H. annuus – H. annuus – H. annuus CMS induced by mutagenesis HEMUS H. annuus PEREDOVICK H. annuus STADION H. annuus PEREDOVICK H. annuus PEREDOVICK H. annuus VORONEJSKII H. annuus HA 89 H. annuus HA 89 H. annuus HA 89 H. annuus

Source: Serieys (2005)

27 28 29 30 31 32 33 34 35

24 25 26 MUT-1 MUT-2 MUT-3 MUT-4 MUT-5 MUT 6 MUT 7 MUT 8 MUT 9

ANN-20 ANN-21 ANN-22

60 61 62 63 64 65 66 67 68 69 70 71 72

PET-34 – PRAECOX PHIR-27 PRAECOX PPR-28 RUN-29 RESINOSUS-243 VULPE RIG-M-28 STRUMOSUS – CMG-3

H. petiolaris H. petiolaris H. praecox ssp praecox H. praecox ssp hirsutus H. praecox ssp praecox H. praecox ssp praecox H. praecox H. resinosus H. rigidus H. rigidus H. strumosus H. giganteus H. maximiliani PRP-1 PRR-1 RES-1 RIG-1 RIG-2 STR-1 CMS-GIG3 MAX-1

PET-4 PET-5 PRA-1

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CMS PF. Abdul Gafoor (1997) reported that oil content hybrids generated using CMS PF showed superiority over CMS F hybrids. The positive or negative influence of cytoplasm type has been confirmed by many researchers (Matvienko 1989; Baldini et al. 1991). Serieys (1992) recorded the positive effect on oil content. The hybrids, based on ANL1, ANL2, MAX1, PEF1, PET2, and ANN4, exhibited good agronomic performance for various traits, including days to 50% flowering, days to maturity, plant height, and oil content (Horn and Friedt 1997). However, in terms of achene oil content, the CMS source from H. lenticularis outperformed the Classical cytoplasm by producing hybrids with significantly higher oil content. Meena et al. (2013b) reported that sunflower hybrids developed using two novel CMS sources, viz., FMS and IMS, displayed much higher oil content compared to the PET-1 CMS based hybrid. Tyagi and Dhillon (2016) observed that CMS analogues E002-91A, ARG-2A (H. argophyllus) and ARG-3A (H. argophyllus) showed very good combining ability for seed yield both under normal and stress conditions. Therefore, these novel, diverse male sterility sources can replace the traditional source while offering additional benefits for various desirable agronomical traits.

6.14

Fertility Restorer (Rf) in Sunflower

A restorer line is used to restore the fertility of hybrid after crossing with a malesterile (A) line in a hybrid production plot. The CMS source was commercially utilized in the production of F1 fertile hybrid seeds due to the availability of fertility restorer sources in sunflower. The discovery and development of new fertilityrestoring inbreds was a critical step in the success of sunflower hybrid breeding programs. The American breeder Kinman (1970) was the first to discover the male fertility restorer gene Rf1, which was found in the T66006-2-1-B line, that was selected from a composite cross involving the annual sunflower wild H. annuus. Velkov and Stoyanova (1974) discovered that the Rf1 gene(s) tracing back to Kinman’s T660066-2 source were extremely stable in a variety of environments. Since then, many restoration lines from USDA-ARS, such as RHA lines, RHA-271, RHA-272, RHA-273, RHA-274, RHA-275, RHA-276, RHA-278, RHA-279, and RHA-296 carrying the Rf1 gene derived from T66006-2-1-B were reported (Korell et al. 1992; Jan et al. 2002). The development of the first fertility restorer line with the recessive nature of branching coupled with resistance to downy mildew by Fick and Zimmer (1974) was significant for commercially exploiting heterosis in sunflower breeding. In Texas, Kinman, in 1970, developed three restorer lines, viz., RHA-271, RHA-273, and RHA-274 by crossing CMS PI-343765 with single plant selections from the cross T-660066-2. Tarpomanova et al. (2010) isolated two restorer lines, viz., 2530-R and 2534-R from the F8 generation of the cross between H. bolanderi
H. annuus.

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Sources for Fertility Restoration Genes

Sources of genetic fertility restoration that restore fertility to the hybrid seed that the farmer actually grows as the crop are more common. It has been reported that the occurrence of genes for perfect fertility restoration of CMS is rare in cultivated sunflowers. Although genes for fertility restoration have been found in cultivated sunflowers (Vranceanu and Stoenescu 1973; Fernandez et al. 1974; Fick and Zimmer 1974), they are more common in wild species (Fick et al. 1976; Jan 1990), which provide most of the sources of genetic fertility restoration in current breeding programs. Twenty wild perennial and six annual Helianthus species were proven to carry fertility restoration factors (Christov 1992). At the IFVCNS in Serbia, five new fertility restorer inbreds (RHA-D-2, RHA-D-5, RHA-D-6, RHA-D-7, RHA-D-8) were obtained from interspecific populations derived from H. deserticola (Hladni et al. 2009). Marinkovic et al. (1996) also reported fertility restoration genes from 75 populations of wild H. annuus L., H. petiolaris.

6.16

Desirable Characteristics of a Good Male Line

The additional criteria for parental choice, particularly for the restorer line, are (1) good fertility restoration against the majority of the CMS lines with diverse sterility systems; (2) less affected by changes in environmental conditions; (3) should be a good general combiner (capability of producing high yielding hybrids); (4) possesses desirable agronomic characteristics like high yielding inbred with desirable features for outcrossing, for example, more pollen production, good anther dehiscence, long shedding period, more attractiveness to insects, particularly bees and high pollen load, etc.; (5) good pollen shedding (large anthers with many pollen grains) in hybrid seed production fields in various environment conditions; (6) reasonable synchrony with the CMS line to facilitate nicking; (7) high SCA of the cross between A-line and the R line; (8) maximum diversity from the A-line chosen; and (9) appropriate height.

6.17

The Use of Molecular Markers

Molecular markers have found significant application in hybrid production by giving a better knowledge of the mechanisms underlying heterosis and by proliferating the overall hybrid development system. Some of the salient applications of molecular markers relevant to hybrid breeding are highlighted (Table 6.5).

6.17.1 Germplasm Characterization DNA-based molecular markers have also been utilized to characterize unique sunflower collections. For instance, Dong et al. (2007) characterized a non-oil type

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Table 6.5 Applications of molecular markers in heterosis breeding Characters Markers Genetic diversity analysis Isozymes

Population/material

References

Germplasm

Isozymes

Germplasm

Isozymes

Germplasm

RAPD markers

RAPD markers

Commercial cultivars (5), breeding lines (7) & wild sunflower (2) Domesticated and wild relatives of sunflower –

Carrera and Poverene (1995) Cronn et al. (1997) Tersac et al. (1993) Lawson et al. (1994)

RAPD markers

24 sunflower inbreds

RAPD markers

–

RFLP markers

25 elite inbred lines

RAPD markers

30 inbred sunflower lines

RAPD markers

8 sunflower lines

RAPD, ISSR & SRAP markers

13 sunflower genotypes

RFLP markers RFLP markers

A diverse set of 24 inbred lines 17 sunflower inbreds

RFLP markers

26 sunflower inbred lines

AFLP markers

24 public inbred lines

AFLP markers

F2 population

AFLP markers

25 sunflower inbred lines

AFLP markers

Populations of Helianthus argophyllus 70 germplasm accessions of confectionary sunflower 2 German inbreds and 8 North American lines

RAPD markers

AFLP markers SSR markers

Arias and Reisberg (1995) Sivolap and Solodenko (1998) Gopalan and Sassi Kumar (2004) Sivolap et al. (1998) Carrera et al. (2002) Popov et al. (2002) Iqbal et al. (2008) Mahmoud and Abdel-Fatah (2012) Berry et al. (1994) Gentzbittel et al. (1994) Zhang et al. (1995) Hongtrakul et al. (1997) Gedil et al. (2001) Ronicke et al. (2005) Quagliaro et al. (2001) Dong et al. (2007) Dehmer and Friedt (1998) (continued)

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Table 6.5 (continued) Characters

Markers SSR markers

Population/material 16 elite inbred lines

SSR markers

374 F3 lines

SSR markers

94 RILs populations

SSR markers

94 RILs populations

SSR markers

42 sunflower genotypes

SSR markers

U.S. sunflower inbreds and hybrids 20 sunflower inbred lines

SSR markers SSR markers

78 SSR markers

21 species & subspecies of Helianthus, and 6 Ukrainian inbred lines 47 domesticated and wild germplasm accessions 177 USDA-ARS inbreds 250 back-cross derived inbred lines 67 maintainers and 57 restorer lines 6 inbred lines

SSR markers

15 confectionary sunflowers

SSR markers

433 cultivated accessions from North America & Europe and Wild Helianthus annuus 28 sunflower genotypes

122 SSR markers SSR markers SSR markers 78 SSR markers

102 SSR markers 10 SSR & 12 retrotransposon markers 11 SSR markers SNP markers

TRAP markers Minisatellite SCAR markers

Among confectionery sunflower (50 populations) 17 CMS lines and 12 paternal lines 16 cultivated sunflower lines and 16 wild H. annuus populations 177 sunflower inbreds 2 German inbreds and 8 North American lines 500 sunflower genotypes representing VIR World Collection

References Yu et al. (2002a, b) Burke et al. (2002) Tang et al. (2002) Tang et al. (2003) Duca et al. (2015) Smith et al. (2009) Pankovic et al. (2004) Solodenko and Sivolap (2005) Tang and Knapp (2003) Yue et al. (2009) Sujatha et al. (2008) Zhang et al. (2005) Hvarleva et al. (2007) Kholghi et al. (2011) Mandel et al. (2011)

Darvishzadeh et al. (2010) Jannatdoust et al. (2016) Markin et al. (2016) Liu and Burke (2006) Yue et al. (2009) Dehmer and Friedt (1998) Anisimova et al. (2010) (continued)

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Table 6.5 (continued) Characters

Markers Interretrotransposon amplified polymorphism (IRAP) 96 SSR markers 8 AFLP markers

10 SSR and 12 retrotransposon markers 42 SSR markers 13 SSR markers

64 ISSR & 29 SSR markers 78 SSR markers Hybridity confirmation and genetic purity SSR markers Seed proteins and isozymes markers SSR markers SSR markers

SSR markers SSR markers RAPD and SSR markers SCAR and RAPD markers Isozymes, proteinbased markers and seed storage proteins Simple and repetitive sequences as hybridization probes, isozyme and random primers for PCR

Population/material 26 varieties and 36 wild accessions of sunflower

References Vukich et al. (2009)

40 sunflower lines 70 confectionery sunflower germplasm representing 12 provinces of China 50 confectionery sunflower germplasm

Zia et al. (2014) Dong et al. (2007)

170 sunflower accessions 10 sunflower parental lines, including 5 restorers and 5 CMS lines 10 natural populations & 6 inbreds 124 sunflower inbred lines (67 B lines and 57 R lines) 17 sunflower inbreds and 2 hybrids 5 sunflower hybrids 5 hybrids, 4 female and 2 male lines 8 parents, 6 populations and 7 F1 hybrids 6 public sector hybrids and 1 variety 8 parents and their 16 hybrids 3 sunflower hybrids and their parents DRSH-1 16 F1 hybrids

Sunflower lines & F1 hybrids

Kholghi et al. (2012) Filippi et al. (2015) Duca et al. (2013) Garayalde et al. (2011) Zhang et al. (2005) Antonova et al. (2006) Pallavi et al. (2010) Pallavi et al. (2011) Nandini and Chikkadevaiah (2005) Kulkarni et al. (2015) Iqbal et al. (2010) Bhosle et al. (2015) Kumar et al. (2009) Nikolić et al. (2008)

Mosges and Friedt (1994)

(continued)

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Table 6.5 (continued) Characters

Markers SSR markers

Population/material F1 plants

RAPD markers

Interspecific cross between cultivated sunflower
H. petioiaris Interspecific cross cultivated sunflower
H. argophyllus Interspecific cross cultivated sunflower
H. argophyllus

SSR markers SSR markers QTLs for agronomic traits Seed weight (F3 generation) and oil content (F2, F3 & F4 generation) Flowering, seed weight per plant, 1000 achene weight, oil content in kernel, plant height, head diameter, and stem girth Time to 50% germination, percentage of germinated seeds, root and shoot length, fresh weight of root shoot and dry weight of shoot, and root and percentage of normal seedlings Flowering date (F3 generation), oil content (F2 generation), seed weight (F2 generation), plant height (F3 generation), plant lodging, maturity dates, and days from flowering to maturity

References Terzic et al. (2006) Rieseberg et al. (1995) Meena et al. (2017) Meena et al. (2020)

RFLP/isoezymes markers

F2:F3 (GH
PAC-2)

Mestries et al. (1998)

AFLP and SSR markers

RIL (PAC-2
RHA-266)

Rachid Al-Chaarani et al. (2004)

AFLP and SSR markers

RIL (PAC-2
RHA-266)

Rachid Al-Chaarani et al. (2005)

RFLP and AFLP markers

XRQ
PSC-8

Bert et al. (2003)

(continued)

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Table 6.5 (continued) Characters Oil content in wellwatered and waterstressed condition under greenhouse, oil content in wellwatered and waterstressed under field condition Seed oil percentage Physiological traits (leaf chlorophyll content, net photosynthesis, and internal CO2 concentration) and water status (stomatal conductance, transpiration, relative water content) Days to flowering Growing degree days to flowering Seed oil concentration Seed weight per plant, 1000 achene weight, percent oil content in kernel, and flowering date Sowing to flowering date, Leaf area at flowering and seed yield per plant Seed oil concentration and 100-seed weight Seedling vigor, seed germination, seedling growth, and developmental traits

Markers SSR markers

Population/material RIL (PAC-2
RHA-266)

References Ebrahimi et al. (2008)

RFLP markers

F2/F3 (ZENB-8
HA-89)

AFLP markers

RIL (PAC-2
RHA-266)

Leon et al. (1995a) Harve et al. (2001)

RFLP markers

F2/F3 (ZENB-8
HA-89)

RFLP markers

F3 and F4 (ZENB8
HA89) 235 F2:F3 populations

RFLP markers

Leon et al. (2000) Leon et al. (2001) Leon et al. (2003) Mokrani et al. (2002)

AFLP and SSR markers

244 F3 families

AFLP and SSR markers

RIL (PAC2
RHA266)

Poormohammad Kiani et al. (2009)

SSR and INDEL markers

RILs (RHA280
RHA801)

Tang et al. (2006)

AFLP and SSR markers

RIL (PAC2
RHA266)

Davar et al. (2011)

(continued)

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Table 6.5 (continued) Characters Markers AFLP and SSR Days from sowing markers to flowering, achene yield per plant, 1000 seed weight, head diameter, and leaf area at flowering Leaf traits (leaf SSR and SNP number, length, markers width), plant height, stem girth, and head diameter Germination AFLP and SSRs parameters markers Stripes on seed SSRs markers margin, between margin, test weight, and achene yield per plant Prediction of heterosis and F1 performance AFLP markers

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References Haddadi et al. (2011)

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Eyvaznejad and Darvishzadeh (2014)

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Ebrahimi and Sarrafi (2014) Vanitha et al. (2014)

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86 SNPs & Interretrotransposonamplified polymorphism (IRAP) protocol 160 AFLP markers 44 SSR markers

Hybridization and introgression RAPD markers

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Hongtrakul et al. (1997) Cheres et al. (2000) Darvishzadeh (2012) Gvozdenović et al. (2009) Tersac et al. (1994) Buti et al. (2013)

5 sunflower parental lines of diallel population 49 sunflower hybrids along with parents (7CMS + 7 R lines)

Darvishzadeh (2012) Kulkarni et al. (2016)

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exotic Helianthus petiolaris populations H. argophyllus
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Gutiérrez et al. (2012)

Vischi et al. (2002)

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(confectionary) sunflower collection of 70 germplasm accessions from China with Amplified Fragment Length Polymorphism (AFLP) markers. They observed that there were no duplicate entries and that the material was not properly classified according to its geographical origin. Various sources of cytoplasmic male sterility have also been characterized using molecular markers. Proper understanding of the molecular mechanisms of the distinct CMS systems helps in the classification of different types and the identification of new ones.

6.17.2 Prediction of Heterosis In crop plants, genetic distance has been extensively investigated as a determinant of heterosis and hybrid performance. Tersac et al. (1994) measured the genetic distance from isozyme data in sunflower and observed that it was unrelated to heterosis. Cheres et al. (2000) revealed that genetic distance alone was a poor determinant of hybrid performance in sunflowers employing AFLP markers and co-ancestries. Genetic distance and F1 heterozygosity determined from randomly chosen molecular markers are often not connected with heterosis in other crops, such as soybean or maize (Melchinger et al. 1990; Gizlice et al. 1993). To improve this prediction, methods such as screening for heterosis-related markers (Melchinger et al. 1990), employing specific heterozygosity (Zhang et al. 1994), and obtaining desirable combinations of allele and heterotic patterns (Liu and Wu 1998) could be used.

6.17.3 Gene Introgression Different QTLs underlying oil content (per cent of kernel and kernel oil concentration) have been recognized, and some of them have been found to be co-located with the phenotypic locus B, hyp, and P (Leon et al. 1996, 2003; Tang et al. 2006). Tang et al. (2006) explained this as a pleiotropic influence of such phenotypic loci on oil content, which were apparently targeted by selection in the transition from boldseeded, low-oil to small-seeded, high-oil cultivars. In addition to marker-assisted background selection, Leon et al. (1995b) allowed for both combined marker- and phenotypic- (based on the hyp locus underlying a QTL for the percentage of the kernel) assisted selection for high oil content in the backcross process.

6.17.4 Characterization and Verification of Interspecific Hybrids DNA-based markers have also proven effective for identifying introgressed DNA fragments from wild species in interspecific progenies and for molecular characterization and verification of interspecific hybrids (Natali et al. 1998; Binsfeld et al. 2001). Some of these introgressed DNA fragments have been related to increased levels of resistance to diseases such as head and stalk rots (Rönicke et al. 2004),

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Phomopsis (Besnard et al. 1997), or downy mildew (Wieckhorst et al. 2008) in sunflower.

6.17.5 Identification of Maintainers and Restorers Marker-assisted selection is not only being employed for gene introgression but also as a testcross and progeny testing alternative. In sunflower, hybrid seed production depends on a good CMS system by mitochondrial genes combined with pollen fertility restoration by nuclear genes. The development of novel fertility restorer and maintainer pools for the sterile cytoplasm requires a lot of test-crossing and progeny testing, which can be substituted by MAS using molecular markers that are closely linked to fertility restoration genes. Yue et al. (2007) proved the efficacy of a target region amplification polymorphism (TRAP) marker generated from a sunflower expressed sequence tag (EST) that exhibited homology to a Petunia fertility restorer gene in progeny tests for the non-oil type of maintainer lines to recover rf1/rf1 genotypes with greater efficiency. These markers will be helpful in the rapid development of novel restorer as well as maintainer pools for new CMS sources, as well as for the identification of fertility-restored plants in breeding programs.

6.17.6 Testing for Genetic Purity of Parental Lines and Hybrids The commercial potential of hybrid technology mainly depends on the good quality of hybrid seed supplied, especially the genetic purity. Traditionally, the grow-outtest (GOT) was used to determine the genetic purity of a hybrid. On the other hand, GOT takes one full season, thus preventing the quick cultivation of hybrid seeds produced. The cost of hybrid seed increases when capital spent on hybrid seed production is locked up and additional expenses for hybrid seed storage are incurred. This constraint, and the environmental dependence of the whole procedure, can be handled efficiently by the use of molecular markers.

6.17.7 Protection of Parental Lines and Hybrids In hybrid seed production, the availability of tools for the identification of F1 seeds and for the legal protection of the parental lines is of utmost importance. Molecular markers are not yet accepted as a means for registering and protecting plant varieties. However, they have all the characteristics to become one of the preferred tools in the future for this purpose. Several researchers highlighted the utility of DNA markers in effective protection of parental lines and hybrids as they satisfy the three essential criteria for cultivar identification, namely, maximum inter-varietal polymorphism, minimum intra-varietal polymorphism, and temporal and environmental stability of the marker. Further, molecular fingerprinting of hybrids can be utilized to check spurious seeds in the market by labeling them with the hybrid’s graphical fingerprint.

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Hybrid Seed Production

Commercial sunflower hybrid seed production: The CMS restoration system is used to produce the commercial sunflower hybrid seed. The following are the steps involved in hybrid seed production: Maintenance of parental lines: The male sterile or female line (A-line) carries cytoplasmic genetic male sterility, the maintainer line (B line) is male fertile and carries non-pollen restoring genes, and the restorer or male line (R line) carries fertility restoring genes to be used for the purpose of producing hybrid seed. The male fertile or pollen restoring line has to be maintained in an isolated condition.

6.18.1 Production of Hybrid Seed This involves crossing a female line that is a male-sterile line (A-line) with a male line that is a restorer line (R line) (Fig. 6.2). The first stage of increased, i.e., parental line maintenance, is referred to as foundation seed production, and hybrid seed production is referred to as certified seed production.

Fig. 6.2 Procedure for parental lines maintenance and hybrid seed production

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6.18.2 Maintenance of the Male Sterile Line (A-Line) The A-line carries male sterility due to cytoplasmic genetic factors. It is maintained by crossing with a male fertile, non-pollen restoring strain (line B), which is a sister strain of line A in an isolated plot. Line B is essentially similar to line A in all respects except that line B is pollen fertile, whereas line A is pollen sterile. In a crossing block, the usual planting ratio of lines (male sterile and maintainer) is 3:1. Four border rows with line B seeds are planted all around the seed plot. The seed harvested from line A is male sterile and is used for commercial hybrid seed production and further multiplication of line A. The seed harvested from line B is pollen fertile and could be used in further multiplication of line A in subsequent years. Five to six hand pollinations are done to increase seed filling. Any rogues, pollen shedders, genetic variants (off-types), and diseased plants are uprooted before flowering. In recent years, separate block planting of A and B lines has been done to avoid the problem of the mechanical mixture.

6.18.3 Maintenance of Pollen Parent (R Line) In order to multiply the male parent seed of a hybrid, all the rows of the seed production plots are planted with the pollen parent under isolation. Rouging is done to eliminate non-branching or any off-type plants or diseased plants in the seed plot. Nipping of side branches ensures high test weight and reduces loss in seed processing.

6.18.4 Hybrid Seed Production (A 3 R Crossing Block) There are two methods of hybrid seed production in sunflowers. To realize high productivity levels on a commercial scale, the supply of quality hybrid seeds assumes importance. Seed producers must plan and manage their operations in order to produce high-quality seed from parental lines and certified seed from hybrids. The practices that are to be followed for efficient seed production in different situations are detailed below. 1. Row method: The male-to-female ratio is 1:3, i.e., one row of males to three rows of females (Fig. 6.3). However, four rows of restorer line seeds are planted all around the seed plot to supply enough pollen. To increase the seed set and yield of hybrid seeds, five-to-six-times supplementary pollinations are necessary. Roguing will be performed 2–3 times before flowering in both the lines. The seeds harvested from the A line are hybrid seeds and can be used for commercial cultivation. There are some disadvantages to this method, as the chances of mixing R line seed into hybrid seed. To avoid the disadvantages of this method, a new method has been proposed by the researchers.

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Method I: Row method Border rows

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2. Block method: Seed production agencies have recently experienced major seed quality issues as a result of large amounts of male line plants contaminated during harvesting and drying, as well as the various stages of post-harvest operations followed in the above 1:3 method. As a result, the new method of planting in blocks is currently being used (Fig. 6.4). The A and R lines are planted in a 75:25 ratio, utilizing different blocks in the prescribed block system. In order to produce certified seed, pollen from the R lines is collected separately and pollinated on the A-line at the time of anthesis. This method assures high-quality hybrid seed production that meets the genetic standards.

6.19

General Principles of Seed Production

6.19.1 Selection of Land A field in which seed is produced should be properly isolated from the source of contaminating pollen, including other cultivars and related species. A hybrid sunflower seed crop shall not be certified if planted on land where the same kind of crop was grown the previous year, unless the crop grown the previous year was of the same variety and of an equivalent or higher class of certified seed and was certified.

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The land selected for hybrid seed production should be free from perennial weeds and volunteer plants and should be well-drained, deep, fertile, productive, and level.

6.19.2 Isolation Requirement Sunflower is a partially self- and predominantly cross-pollinated crop, the degree of cross-pollination ranges from 17 to 62% due to insect activity. The provision of a suitable isolation distance for seed production is a prerequisite for minimizing genetic contamination of a seed crop. The ideal distance differs from crop to crop and is dependent on various factors, including mode of reproduction, stage of multiplication, varietal mass, windbreaks and barriers, geographic location of the seed plot, and prevalence of wild plants that can cross with seed crop (Bateman 1952). So, seed fields must be isolated from the contaminants using appropriate isolation distance. Changes in the isolation distance are not permitted for a hybrid seed production program.

6.19.3 Seed Source The use of seed of a suitable class and from approved sources is mandatory for raising a seed crop. During the submission of the application, its scrutiny, and/or the first inspection of the seed crop, the individual intending to produce seed under certification shall submit to the certification Agency one or more relevant evidence such as certification tags, seals, labels, seed containers, purchase records, sale records, and other documents as may be requested by the certification Agency during the submission of the application, its scrutiny, and/or during the first inspection of the seed crop in order to confirm that the seed used for raising the crop was obtained from the source approved by it. This condition also pertained to both parents in seed production involving two parental lines. To produce the foundation seed class, it is necessary to obtain breeder seed from the SAUs and ICAR schemes of concerning crop and, for certified seed production, it is better to use foundation seed from the foundation seed production agencies like SAU Forms, SSCs, NSC and Central State Farms, etc.

6.19.4 Field Inspection The field examination work, which requires technically trained workers, must be performed by individuals who have been authorized by the certification entity. A field inspection meant to check those factors that can cause irreversible damage to the genetic purity of seed health shall be conducted without advance notice to the seed producer. At least a minimum of four inspections should be performed. The initial inspection should be done at the stage of 6–7 leaf pairs in order to verify isolation, outcrosses, volunteer plants, planting ratio, planting errors, mentioned

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diseases, and other relevant factors. The second and third inspections should be done during the flowering stage to verify for isolation, pollen shedders, off-types, and other issues. The fourth and last inspection should be done at crop maturity and prior to harvesting to assess the variety’s specified disease, true to type plant and head, seeds traits, and other imperative factors.

6.19.5 Roguing of Seed Fields The occurrence of off-types is a potent source of genetic contamination. The presence of some recessive genes in heterozygous conditions at the time of the variety’s release may result in off-type plants. The recessive genes may also arise due to mutation. In later production cycles, heterozygous plants segregate for the characters disrupted by the specific gene(s) resulting in off-types. Sometimes, off-types may arise from volunteer plants, i.e., plants arising from intermittently planted seeds or from seeds produced by previous crops. Before pollination arises, off-type plants should be removed from the seed production fields.

6.19.6 Seed Certification Seed certification is frequently used to ensure the genetic purity of commercial hybrid seed production. The basic goal of seed certification is to maintain and make true-to-type seeds available. To accomplish this purpose, qualified and wellexperienced personnel from Seed Certification Agency perform field inspections at proper stages of crop growth and inspections at post-harvest stages as to variety of the requisite genetic purity and quality. It will also specify the field and seed standards that must be met by the seed crop and seed in order to be certified as certified seed.

6.19.7 Grow-Out Test (GOT) Grow-out tests should be performed on hybrids or varieties raised for seed production on a regular basis to ensure that they are maintained in their original form.

6.20

Problems in Hybrid Seed Production

(a) Major Causes of Genetic Purity Loss in Parental Lines 1. A & B Lines: The female line, or A-line, loses purity because of the presence of the following types of plants in varying proportions. – Pollen shedders and – Off-types

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Pollen shedders: There are two main reasons for the appearance of pollen shedders in the female or A-line. 1. Seeds from the maintainer (B) line are mixed into the female (A) line and 2. Outcrossing of the female or A-line with lines that carry restorer genes is more often because of the contamination of the maintainer or B line. 3. Occasionally, due to male sterility breakdown. How maintainer line seeds are mixed with female (A) line seeds: It is recommended to sow ‘A’ and ‘B’ lines in a 3:1 row proportion during breeder and foundation seed production of the ‘A’-line. Because the ‘A’ and ‘B’ lines are identical (isogenic line), there is a chance that ‘B’ seeds will be mixed with ‘A’ seeds at various phases such as harvesting, processing, packing, and storage. When the harvesting of plants from the maintainer (B) line is delayed, they are then harvested along with the female (A) line, leading to the mixing of ‘A’ and ‘B’ seeds. Lack of required isolation distance: Outcrossing of a line with any other line occurs due to a lack of adequate isolation distance. In the seed production program, the chance of crossing a line with another line having restorer genes results in the creeping of that gene into a line. This results in pollen shedders in ‘A’-lines. When this occurs, it becomes difficult to eliminate the restorer gene from the ‘A’-line and this issue is dealt with at length later in this paper. Off-types: An off-type is any plant that is genetically or morphologically different from the respective line. Variation may be with respect to traits like plant height, days to flowering, branching, presence or absence of pigmentation on various parts, which is due to mechanical mixtures and or outcrossing with any other sunflower line, population, or hybrids. Rarely do variants occur due to mutations. For the reasons explained above, off-types may occur both in ‘A’ and ‘B’ lines. The occurrence of male-sterile plants in B lines is also off-types and therefore needs to be rogued out (Fig. 6.5). R line (restorer line): Lack of purity in the R line arises because of the presence of off-types and male-sterile plants. In many cases, the restorer lines of sunflower hybrids are branching types. In such cases, the non-branching types and the male sterile plants are the off-types. Variations in branching pattern differences, pigmentation in various parts of the plant and other off-types in the R lines due to the mechanical mixture outcrossing, and rare mutations.

6.21

Approaches to Overcome Problems in Hybrid Seed Production

The following measures are suggested to produce the parental lines in pure form and for successful sunflower hybrid seed production:

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(a) General measures: Follow precisely the appropriate field as well as seed standards prescribed for seed certification. 1. Follow absolute isolation distance: Although isolation of 400–600 m is recommended depending on the class of seed, this distance is sometimes inadequate because the bees can travel up to a distance of 3–4 km. It is therefore often recommended that the seed production be taken up in non-traditional areas of the crop so that a safe isolation distance is followed. 2. Take up rigorous rouging: This is the most important step in seed production and rigorous rouging of pollen shedders and other off-types should be done before harvest. 3. Avoid mechanical mixtures at various stages: Care should be taken to see that ‘A’ line seeds do not get mixed with ‘B’ seeds since this has been the major reason for the recurring pollen shedder problems. Since plants and seeds of ‘A’ and ‘B’ plants look alike, it will be impossible to separate them once the flowering is over. Therefore, the identification of ‘A’ and ‘B’ lines in the field should be well demarked so that there is no confusion. While harvesting, care should also be taken at every stage to avoid contamination at all stages of harvest, threshing, processing, packing, and storage. 4. Special measures: Seed production of hybrids has been crippled in the recent past, mainly because of the high percentage of pollen shedders in the female lines. As indicated, the two main reasons for the occurrence of pollen shedders are: • Mixing of B line seeds with A-line seeds, and • Outcrossing of ‘A’-line with other lines, particularly the lines carrying restorer genes.

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Avoid mixing B line with A-line seeds: It is recommended to grow ‘A’ and ‘B’ lines in a 3:1 row proportion. While this is convenient and technically correct, some problems have cropped up with this practice. In certain fields, it is difficult to differentiate ‘A’ and ‘B’ lines because the lines are not straight, especially in the border lines where the lines are out off in between. This may be because of the lack of symmetry in the field. Sometimes, ‘A’ and ‘B’ lines merge because of the imperfect marking of the lines. As a result of all these issues, plants of ‘A’ and ‘B’ lines get mixed up, resulting in the pollen shedders in the ‘A’-line in the subsequent crop. The problem is further complicated if the harvesting is delayed, resulting in the fall of ‘B’ plants in ‘A’-lines and vice versa. To overcome the above problem, it is suggested to sow the ‘A’ and ‘B’ lines separately in adjacent blocks (side by side) instead of row planting. The pollen has to be collected from the adjacent ‘B’ plot and pollinated on the ‘A’-line. Although this method is comparatively more laborconsuming (for pollination work), it will avoid mixing of ‘A’ and ‘B’ seeds and therefore minimizes the problem of pollen shedders. The other approach suggested is to cut off ‘B’ lines (after completion of the flowering period and collecting seed from ‘A’ lines). In this case, the ‘B’ line is multiplied separately in adjacent blocks elsewhere. In the commercial hybrid seed plots, problems with pollen shedders occur, along with multi-headed (R) plants, leading to variations in plant height, days to flowering, and days to maturity.

6.22

Future Prospects of Heterosis Breeding in Sunflower

1. Search for novel cytoplasm donors to avoid the cytoplasmic vulnerability to economically important diseases and insect pests. 2. Need for the development of heterotic pools: There is an urgent need to synthesize breeding programs to develop and improve heterotic pools for use as source populations for hybrid breeding. The development and improvement of maintainer and restorer heterotic pools through reciprocal recurrent selection requires extensive intermating. Whatever approaches have been used at IRRI to develop maintainer and restorer pools in rice can also be applied to sunflower. 3. Recycling of B and R lines: Development and improvement of gene pools is a long-term project. To meet immediate needs, B
B and R
R crosses should be attempted by using carefully selected lines that complement each other. The desired types may be selected in segregating generations. For recycling, pedigree as well as backcross methods may be used, depending upon the objectives and materials. However, there is a need for the utmost caution not to cross maintainers and restorers except when specific genes from a special source have to be incorporated, presumably through backcrossing. 4. Strong maintenance program: The goal of plant breeding research is to see that high-quality seed, generally identical to the variety released by the breeder, is produced in successive years. Seetharam and Virupakshappa (1993) emphasized the need for proper maintenance breeding of the parental lines. Maintenance of genetic purity of the parental lines of the released hybrids is one of the most

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difficult aspects, and this has not received adequate attention till now. Earlier, it was reported that the hybrids after their identification (and sometimes even before release) show rapid genetic deterioration, thereby defeating the purpose of plant breeding research. Many reasons could be attributed to genetic deterioration. As mentioned above, the maintenance of genetic purity of the parental lines of hybrids is not being followed meticulously. Today, DNA marker technology offers an efficient alternative to this approach, which is based on sequence variation of specific genomic regions and provides powerful tools for cultivar identification and seed quality control in different crops with the advantages of time and labor saving and more efficiency (Liu et al. 2007). 5. Conversion of good combiner inbreds into diverse CMS backgrounds: The scarcity of good combiner inbred lines continues to be a major impediment to the large-scale production of high-yielding hybrids. Inbred lines derived from the same narrow gene pool are used to develop sunflower hybrids. Inbreds must be examined for their breeding value in more than one environment before being converted to CMS lines for utilization in heterosis breeding programs in order to generate hybrids with higher heterosis and resistance or tolerance to major diseases and insect pests. It should be ensured that newly converted inbreds into CMS should not have any adverse effects on agronomic or seed oil traits.

6.23

Concluding Remarks

During the past decade, sunflower production in several countries has migrated from regions of adequate rainfall and fertile soil to less productive areas where water and fertility restrict yields. Since the discovery and use of CMS and pollen fertility restoration, sunflower heterosis breeding has progressed significantly. These spectacular achievements account for the rapid extension of sunflower hybrids across the globe, with a significant and positive impact on the achene and oil yield levels. Unfortunately, all currently available F1 hybrids are based on the PET-1 CMS source, and much more effort is needed to diversify the CMS sources that can be exploited for enhancing sunflower yields. Therefore, the breeding of efficient and stable fertility restorer lines and stable CMS lines is the need of the hour. As a result, using various breeding methods, the development of diverse fertility restorer lines should be continued in order to reduce the genetic vulnerability of sunflower hybrids to ever-changing climatic conditions and major diseases. The main current challenges in sunflower hybrid breeding include enhancing productivity by increasing seed and oil yields as well as minimizing genetic vulnerability to diseases and water stress in various ecological zones. Modern technologies of biotechnology and genetic engineering, which will allow the development of a novel, more productive type of sunflower, will expedite the genetic progress already achieved. Conventional breeding for developing restorer lines is laborious and time-consuming, since selected plants must be tested for their ability to restore fertility. As a result, identification of suitable gene-based markers for fertility restoration character would be extremely helpful in distinguishing restorers from non-restorers. Thus,

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the use of molecular markers linked to pollen fertility restorer genes would save both time and money, besides adding accuracy in the restorer identification and evaluating these restorer populations across locations to know the stability of these newly developed restorer populations. The utility of the DNA-based molecular markers linked to fertility restorer genes is to increase the selection efficiency, save time, and avoid the complexities linked with phenotype-based screening. Sunflower heterosis breeding cannot progress with the rapid pace of modern molecular genetics development without the combined efforts of scientists and research institutes involved.

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Discovery and Application of Male Sterility Systems in Pigeonpea Abhishek Bohra, S. J. Satheesh Naik, Abha Tiwari, Alok Kumar Maurya, Shefali Tyagi, and Vivekanand Yadav

Abstract

Pigeonpea is an important food legume crop of the semi-arid tropics. Often crosspollinating nature of pigeonpea offers a unique opportunity for exploitation of hybrid vigour. In this context, discovery of male sterility and its application in hybrid breeding has yielded promising results in pigeonpea. The latest breakthrough in the hybrid pigeonpea research is the discovery of CMS (cytoplasmic male sterility) system. Of the total nine CMS sources reported so far, cytoplasms from Cajanus cajanifolius and Cajanus scarabaeoides represent the two key sources of male sterility that gave a strong impetus to hybrid pigeonpea research. Several pigeonpea hybrids based on CMS technology have been released for cultivation across different agro-ecologies in India. Accelerating the efficiency of hybrid breeding program demands deployment of new tools and methodologies in order to relieve the conventional bottlenecks. Understanding of the genetic architectures and regulatory mechanisms underlying CMS/fertility restoration (Rf) system of pigeonpea has been substantially improved in recent times. Genomic technologies have been proven useful in this regard. For instance, the role of epigenetic changes in the development of hybrid vigour and fertility restoration has been demonstrated in pigeonpea. Similarly, DNA markers associated with fertility restoration trait in pigeonpea will facilitate rapid development of restorer lines. Cost-efficient SNP assays have been developed in pigeonpea to test the genetic purity of hybrids and the parental lines. The enormous potential of novel breeding methods based on genome-wide marker information such as genomic prediction could be harnessed to construct heterotic pools and identification of heterotic patterns. Development of a hybrid breeding

A. Bohra (*) · S. J. Satheesh Naik · A. Tiwari · A. K. Maurya · S. Tyagi · V. Yadav ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_7

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pipeline informed from heterotic pools and heterotic patterns will be crucial for long-term gains from hybrid breeding of pigeonpea. Keywords

Cytoplasm · Hybrid · Heterosis · Male sterility · Restoration · DNA marker · GoT

7.1

Introduction

Pigeonpea (Cajanus cajan (L.) Millsp.), also known as red gram, tur or arhar, is an annual to perennial shrub that is traditionally cultivated in the Indian sub-continent as integrated with different cropping systems (Bohra et al. 2014, Saxena et al. 2021a, b, Singh et al. 2016). Pigeonpea is considered to be an excellent and affordable source of protein, fibres, minerals and vitamins. Globally, 5.96 million tons (mt) of pigeonpea is being harvested from 6.99 million hectares (mha) area, making it the sixth most important legume food crop (FAOSTAT 2019). India is the largest producer (3.83 mt) and consumer of pigeonpea (source: http:// agricoop.gov.in) sharing the world’s 76.69% area and 71.54% production. The major pigeonpea-growing states in India are Karnataka (1.3 mha), Maharashtra (1.12 mha), Telangana (0.29 mha), Uttar Pradesh (0.28 mha), Madhya Pradesh (0.25 mha), Andhra Pradesh (0.24 mha) and Gujarat (0.21 mha), all together contributes around 85.75% of total area (https://agricoop.nic.in/en/annual-report). However, productivity-wise few states which cultivate long-duration pigeonpea such as Bihar (1802 kg/ha) and West Bengal (1500 kg/ha) have high productivity compared to early- and mid-early-duration pigeonpea-cultivating states, viz., Haryana (1133 kg/ha), Gujarat (1123 kg/ha), Punjab (920 kg/ha) and Uttar Pradesh (902 kg/ha) (Project coordinator’s report, 2019). Despite the higher productivity in Bihar, West Bengal, Haryana, Punjab and Uttar Pradesh, pigeonpea-growing area declined while remarkably increased in Karnataka, Maharashtra, Gujarat and Andhra Pradesh at a faster rate from a decade (Fig. 7.1). During this transition phase of geographical shifting, though, high productivity area had to sacrifice but compensated from Karnataka, Maharashtra, Gujarat and Andhra Pradesh with the availability of additional area coupled with high-yielding varieties and favourable management practices. Pigeonpea was domesticated about 3500 years ago in India (Vavilov 1951; De 1974; Royes 1976), and it remains the chief source of proteins for more than a billion people worldwide. The grains harvested from pigeonpea and other byproducts support the livelihoods of millions of marginal farmers in developing countries (Mula and Saxena 2010; Naik et al. 2020). The per capita protein availability in the developing world is often less than one-third of minimum dietary requirements (Latham 1997); this scenario is likely to worsen due to ever-increasing population and crop yield saturation. Therefore, from a nutritional security perspective, legumes help in balancing the cereal-based diets to meet the calories and protein requirements especially in the developing world.

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Discovery and Application of Male Sterility Systems in Pigeonpea

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Bihar Haryana

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Fig. 7.1 Shift in pigeonpea-cultivated area over five decades

The gradual increase in pigeonpea production in India over the last five decades could be attributable to increase in area, particularly in the less fertile, non-traditional area in southern and central zones at the cost of traditionally fertile alluvial soil of north Indian states. Nearly 166 high-yielding and disease-resistant varieties of different maturity groups have been released for commercial cultivation in India (Naik et al. 2020). Notwithstanding the progress in pigeonpea genetic improvement, a considerable yield gap still exists between the potential and realized yields at farmers’ field. Unlike other pulses, unique floral morphology of pigeonpea allows considerable proportion of out-crossing mainly aided by insects. Therefore, it is considered as an often cross-pollinated crop (Saxena et al. 1990). The extent of out-crossing in pigeonpea varies from 20 to 70% and from one place to another (Saxena et al. 2010a, b, c). The often cross-pollination nature offers an excellent system to exploit the hybrid vigour for enhanced crop yield. The first case of hybrid vigour in pigeonpea was reported nearly 25% heterosis over the better parent for grain yield (Solomon et al. 1957). Subsequently, various reports have been published on hybrid vigour for yield and yield components in pigeonpea (Saxena and Sharma 1990). The cost of hybrid seed production presents the major challenge to effective and rapid deployment of hybrid technology; for instance, manual emasculation contributes to enhanced seed production cost. The phenomenon of male sterility is one such event that raises special interest among the breeding community to harness the genetic gain in pigeonpea. Male sterility has played a vital role to accelerate the progress of hybrid breeding in different crops species (Bohra et al. 2016). Male sterility trait refers to the formation of non-functional pollen (Figs. 7.2 and 7.3). In recent years, availability of stable male sterility systems in combination with efficient fertility restoration has paved the way for utilization of cytoplasmic male sterility (CMS) for hybrid development in pigeonpea (Bohra et al. 2017a, 2020). Maintaining the genetic purity of

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Fig. 7.2 Visual difference in anthers of CMS line (left) and fertile maintainer line (right)

seeds of improved pigeonpea cultivars or hybrids is crucial for realizing their productivity and adaptability of the genotypes at different agro-ecological niches. To this end, a definitive hybrid breeding technology could facilitate an efficient seed production that provides adequate quantity of quality seeds at economically viable costs. The immense potential of hybrid technology cannot be fully realized until quality seeds of the released hybrids are produced at commercially scale and made available at affordable prices to the farmers. Hybrid technology opens new avenues for improving pigeonpea productivity. However, the prerequisites for efficient hybrid seed production include (1) male sterility systems showing stability over years and locations, (2) identification of efficient fertility restorers, (3) substantially higher and exploitable levels of heterosis and (4) the large-scale seed production of hybrids for commercialization.

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Discovery and Application of Male Sterility Systems in Pigeonpea

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Fig. 7.3 Pollen grains in CMS and cognate male fertile lines as differentiated by 1% acetocarmine solution

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Male Sterility Systems and Hybrid Breeding in Pigeonpea

Discovery of genetic male sterility (GMS) as spontaneous mutant in pigeonpea provided pigeonpea breeders with the opportunity to use male sterility trait for hybrid development (Reddy et al. 1978). Considerable research efforts have been dedicated towards this end, which paved the way for the development of the first GMS-based pigeonpea hybrid ICPH 8 in 1991 in India. Saxena et al. have reported up to 35% yield advantage of the hybrid ICPH 8 over the best control in the farmer’s field. Other GMS-based hybrids released for commercial cultivation in India include PPH 4 (1994), CoH 1 (1994), CoH 2 (1997), AKPH 4104 (1997) and AKPH 2022 (1998) (Saxena et al. 2010a). These GMS hybrids provided 14–64% superiority over the popular pigeonpea varieties. However, GMS proved to be an expensive system with immense technical difficulties to maintain the genetic purity of hybrid seeds, a fact that greatly obstructed the cultivation of GMS hybrids at commercial levels in the farmers’ fields. The difficulties encountered in GMS hybrid system led to the discovery of an improved hybrid seed production technology that harnesses the non-functionality of pollens resulting from the impaired harmony between the nuclear and cytoplasmic genomes. Also known as three-line hybrid breeding, the CMS system requires CMS line (A), maintainer (B) and restorer (R) lines. Mitochondrial genes have been reported in different crops that confer CMS trait (Bohra et al. 2016). The fertility in CMS system is rescued because of fertility-restoring elements located in the nucleus. Interspecific hybridization resulting in male-sterile progenies helped in achieving this breakthrough of discovering CMS system in pigeonpea. The pioneer CMS line GT 288A and its maintainer B line was developed by Pulse Research Station, SDAU, GAU, SK Nagar, Gujarat. The first CMS hybrid GTH 1 was developed by combining the CMS line GT 288A with the restorer line GTR 11, and the hybrid was subsequently released by the Central Variety Release Committee (CVRC) for cultivation in central zone of India. Currently, nearly 40 CMS lines of pigeonpea have been registered with ICAR-National Bureau of Plant Genetic Resources (NBPGR), and six CMS-based hybrids have been released in India for cultivation in diverse agro-ecologies (Bohra et al. 2020). The pigeonpea hybrids IPH 15-03 and IPH 09-5 were developed by the ICAR-Indian Institute of Pulses Research (IIPR), Kanpur, using A2-CMS and released and notified for cultivation in the north western plain zone (NWPZ). The hybrids offer 28% (IPH 09-5) and 30% (IPH 15-03) yield superiorities over the check varieties (Bohra et al. 2020). The field image of the pigeonpea hybrid IPH 09-5 at podding stage is shown in Fig. 7.4. Recent advances in genomics have greatly improved our understanding of the molecular mechanisms of male sterility and fertility restoration in different crops (Bohra et al. 2016). In pigeonpea, a mitochondrial gene (nad 7a) has been proposed as responsible for CMS occurrence in A-4 cytoplasm (Sinha et al. 2015). Similarly, nucleus-coding Rf loci that rescue male fertility in the hybrids derived from A4 cytoplasm have been mapped in the pigeonpea genome (Bohra et al. 2012; Saxena et al. 2018).

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Fig. 7.4 Field photograph of the pigeonpea hybrid IPH 09-5

Once a stable CMS system is established, the sterilizing cytoplasm may be combined with different nuclear backgrounds using standard backcross protocol. Several popular variety lines such as UPAS 120, PA 163, PDA 89-2E, Hy 4 and H 28B have been converted into CMS lines using backcross techniques (Singh et al., 2009). More recent CMS lines developed at ICAR-IIPR, Kanpur, include Pusa 992A, ICPL 88039A and DPP 3-2A (Bohra et al. 2017a, b, c) using both A2 and A4 cytoplasms. Over the last decade, several CMS-based hybrids were tested in multiple locations under ICAR-All India Coordinated Research Project (AICRP) on pigeonpea across different locations.

7.2.1

Scope of Pigeonpea Hybrids

The heterosis manifested in pigeonpea hybrids offers many advantages over varieties (Saxena et al. 2019a). The notable remarks are as given below: 1. Enhanced grain yield: A notable increase in grain yield of hybrid pigeonpea possibly results from an efficient use of inputs including water and nutrients that enables hybrid pigeonpea to manifest greater biomass while their partitioning reaming comparable with the most popular varieties. 2. High initial vigour: A vigorous growth has been reported in the hybrid pigeonpea seedlings in comparison to the pure line cultivars (Saxena et al. 2013a, b), rendering them competitive advantage over the weeds that otherwise poses

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considerable challenge to the initial crop growth. Also, the initial vigour helps overcome the problem of waterlogging and oxygen deprivation that the pigeonpea crop often encounters during onset of monsoon. 3. Reduced seed rate: Spreading-type growth habit/a wider canopy with a higher number of primary and secondary branches enables hybrid pigeonpea to be grown at reduced seed rate by 40–50% compared to the popular varieties. 4. Ability to tolerate biotic and abiotic stresses: Hybrids may acquire extra degree of genotypic plasticity compared to varieties, which helps them tolerate and produce higher yields under biotic stress such as fusarium wilt and sterility mosaic disease. Comparatively higher root depth and root system biomass of pigeonpea hybrids than that of varieties greatly enhance the formers’ ability for water intake from deeper soil profiles, thus sustaining the drought conditions.

7.3

Diversity of Male Sterility-Inducing Cytoplasms

In 1974, researchers at ICRISAT first started the hybrid breeding using natural out-crossing phenomenon of pigeonpea. Subsequently, Reddy et al. (1978) identified the genetic male sterility (GMS) system, controlled by a single recessive gene (ms1ms1). This GMS system led to develop hybrid technology to assess the extent of hybrid vigour and ability of out-crossing in seed production on male-sterile plants. Later on the difficulties encountered in GMS system were further provoked to identify an alternate and cost-effective source of male sterility; as a result, today, we have a reliable cytoplasmic male-sterile system in pigeonpea. The male sterility events resulting from interspecific hybridization remain the most promising. The brief details about the different male-sterile systems available in pigeonpea are discussed in the following section.

7.3.1

Genetic Male Sterility (GMS) System in Pigeonpea

The first case of genetic male sterility in pigeonpea was observed as spontaneous mutants from two different sources (Reddy et al. 1978). The male-sterile MS 3A was reported in ICP 1555, a collection from Andhra Pradesh, whereas MS 4A arose in ICP 1596 collected from Maharashtra. Further genetic analysis of the segregating progenies elucidated the monogenic recessive nature of the male sterility gene, with the authors proposing the symbol ms1 for the gene. The occurrence of GMS might be credited to a mutation in the male-fertility controlling dominant (Fr) nuclear gene under the influence of some natural forces. Male-sterile offsprings ( frfr) appear in the next generation by natural selfing of heterozygotes (Frfr). In the absence of cross-pollination by fertile pollen, these genotypes are subject to gradually elimination from its parental population. Therefore, rate of natural out-crossing in a given population influences elimination processes. Alternatively, mate sterility trait has also been induced in populations following mutagenesis. However, an associated occurrence of female sterility or reproductive abnormalities in such male-sterile

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mutants has been reported to plague their further maintenance for use in breeding (Dundas 1990). The most spontaneous male-sterile mutants reported so far are recessive. Abundance of recessive male sterility genes results in high frequency of such natural mutations, causing a slow removal from the parental populations (Kaul 1988). Among legumes, male sterility trait controlled by recessive genes was reported in broad bean [Vicia faba (L.)], grass pea [Lathyrus sativus (L.)], groundnut [Arachis hypogea (L.)], soybean [Glycine max (L.) Merr.], pea [Pisum sativum (L.)], common bean [Phaseolus vulgaris (L.)], etc., while dominant control of male sterility was reported in white clover [Trifolium repens (L.)]. Similarly, many reports are available on genetic male sterility systems in pigeonpea. The progenies of the cross msms  Msms segregate for male-sterile trait, with the two types of genetic constitution [male sterile (msms) and male fertile (Msms)] obtained in equal proportions (1:1). Hence, maintenance of male-sterile line presents a formidable task in the GMS system. Hybrid seed production using GMS system suffered a major bottleneck as removal of 50% plants in each generation led to the increase in the seed production cost. Most importantly, the operation of discarding the fertile lot must be carried out before flowering. Therefore, the presence of morphological and molecular markers tightly linked with the ms locus is of great value in selection and removal of the fertile individuals, or else it may lead to contamination of whole seed lot (Colombo and Galmarini 2017).

7.3.2

Cytoplasmic Male Sterility (CMS) Systems in Pigeonpea

Wild relatives and landraces serve as a rich source of male sterility trait in several crops (Bohra et al. 2022). Nine sterilizing cytoplasms have been reported so far in pigeonpea (Bohra et al. 2010, 2016). Of these, seven have been developed utilizing wild relatives from secondary gene pool of pigeonpea, while two CMSs carry cytoplasm from Cajanus cajan, resulting from crossing cultivated pigeonpea with the Cajanus acutifolius as the pollen donor (Mallikarjuna and Saxena 2005). More recently, the cross between Cajanus lanceolatus (ICP 15639) and Cajanus cajan (ICPL 85010) led to CMS occurrence when Cajanus lanceolatus was used as a pollen parent (Srikanth et al. 2015) and the CMS was designated as A9.

7.3.3

Cytoplasmic-Nuclear Male Sterility (CMS) System in Pigeonpea

Reproductive abnormality leading to male sterility involves a complex interplay between the specific genetic elements residing in the nucleus and cytoplasm (Bohra et al. 2016). The phenomenon of CMS results from a competition in the cytoplasmic and nuclear genes that differ in their inheritance, i.e. paternal and maternal. The sterility is induced by the factors contained in the cytoplasm (Chen and Liu 2014). On the other hand, the genes that counteract the effect of sterility, thus enabling CMS

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Table 7.1 Genomic loci/QTL associated with fertility restoration trait in pigeonpea

a

S. no. 1.

Population ICPA 2039  ICPR 2447 ICPA 2043  ICPR 3467 ICPA 2043  ICPR 2671

2.

ICPA 2039  ICPL 87119

3.

ICPA 2039  ICPL 87119

QTL QTL-RF1 QTL-RF2 QTL-RF3 QTL-RF4 S8_7664779 and S8_6474381 on CcLG08 qRf8.1

Markers SSR

Phenotypic variations (R2)a % 24.17

SNP

28.5

Saxena et al. (2018)

SNP

45.06

Saxena et al. (2020)

References Bohra et al. (2012)

Only the highest R2 values are shown

to restore fertility, reside in the nucleus. Isogenic lines (B lines) are required to retain the male sterility trait of CMS line that perpetuates production of non-functional pollen grains. Further, restoration of fertility in the hybrids is facilitated through fertile parent that carries fertility-restoring genes (Table 7.1). Involving three different parents, this system is also known as the A, B, and R system or three-line breeding. The CMS trait may result from spontaneous mutation, intra-specific crosses or wide hybridization (interspecific/inter-generic crosses).

7.3.4

Environment-Sensitive Genetic Male Sterility

The two cases of EGMS include male fertility status modulated by temperature (TGMS) and photoperiod (PGMS). Chen and Liu (2014) have described the high temperature and long photoperiod as “restrictive condition (RC)” that creates pollen sterility. The same line, however, behaves as male fertile under “permissive condition (PC)”, represented by low temperature and short photoperiod in case of TGMS and PGMS, respectively. This is also known as the two-line system where the EGMS line is propagated under PC and hybrid seed is produced under RC. TGMS system in pigeonpea was reported by Saxena (2014) based on four selections, viz. EnvsSel 1, EnvsSel 2, EnvsSel 3 and EnvsSel 5, that were completely male sterile at temperature above 25  C (RC), while the same selections had fertile pollen at temperature below 24  C (PC). The temperature-sensitive selections were made in advanced generations of the populations derived between C. cajan (ICPA 85010) as female parent and Cajanus sericeus as the pollen donor.

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Molecular Understanding of the CMS/Restoration and Genomic Tools to Assist Hybrid Breeding

A compromised crosstalk between cytoplasm and nucleus is reported to cause CMS, and several mitochondrial CMS-associated genes (MCAGs) have been discovered in plants (Mishra and Bohra 2018). Resulting from the extensive rearrangement events in plant mitochondrial genomes, these MCAGs show a chimeric structure comprising fragments of a mitochondrial gene and alien (unknown) sequences and “translate into peptides with membrane spanning domains” (Bohra et al. 2016). The high-throughput sequencing methods have facilitated generation of valuable genome resources in different flowering plants, especially those with CMS cytoplasm (Heng et al. 2014). Analysis of the mitochondrial sequences between CMS line and cognate maintainer line established the role of these MCAGs in CMS induction (Tables 7.2 and 7.3). A high-quality mitochondrial (mt) genome (545.7 kb) was assembled for A4-CMS line ICPA 2039 with Roche/454 FLX and Sanger platforms (Tuteja et al. 2013). Further comparison of this mt genome with cognate fertile line (ICPB 2039), hybrid (ICPH 2433) and C. cajanifolius (ICPW 29) identified a set of 13 chimeric MCAGs for CMS in pigeonpea. Further research on expression and structural variation in 34 mitochondrial genes between ICPA 2039 and ICPB 2039 led authors to associate a 10-bp deletion in the nad7 gene with A4-CMS. Subsequently, a gel-based marker was developed and successfully validated in a set of nine A4-CMS lines and cognate fertile lines. Analysis of the other CMS cytoplasms with the gene-based marker system alludes towards the occurrence of diverse mechanisms underlying different CMS cytoplasms of pigeonpea (Bohra et al. 2017a). More recently, differential RNA editing patterns in mitochondrial transcripts of male fertile and CMS line causing no major change in the protein structure were elucidated in pigeonpea (Kaila et al. 2019). Research on various CMS systems has revealed that the CMS phenotypes are rescued by the action of nuclear genes called restoration-of-fertility (Rf) genes. Pollen fertility is restored through “reconciliation” of the “lost” harmony between the two genomes with different inheritance. Researchers suggest a metaphorical Table 7.2 Candidate genes for male sterility in pigeonpea Genes atp4, atp6, nad3, nad6, nad9, cox1, cox3, atp1, nad4, rps4, nad5 and atp9 nad 4L (129–130;SNP) nad 7a (180–189;InDel)

References Tuteja et al. (2013) Sinha et al. (2015) Sinha et al. (2015)

Table 7.3 Organellar genome sequencing Organelle Mitochondria

Highlights 51 genes, 34 protein coding genes and 17 rRNA

Chloroplast

116 genes, 30 tRNAs, 4 rRNAs, 78 protein coding genes and 5 pseudogenes

References Tuteja et al. (2013) Kaila et al. (2016)

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“molecular arms race” between the newly evolving mitochondrial orfs and the corresponding Rf genes (Touzet and Budar 2004). Genomic locations of Rf genes responsible for CMS restoration have been determined in a wide range of plant species. In pigeonpea, gene(s)/QTL controlling fertility restoration were mapped which could enable fast-track identification and introgression of fertility-controlling gene(s)/QTL. For example, Saxena et al. (2018) recorded pollen fertility data in F2 population (ICPA 2039  ICPL 87119) and assayed the population with genotyping-by-sequencing (GBS) technology. QTL analysis identified one major QTL on CcLG08 that explained up to 28.5% phenotypic variance (PV) for A4 fertility restoration. Earlier, Bohra et al. (2012) identified four QTLs, viz. QTL-RF-1, QTL-RF-2, QTL-RF-3 and QTL-RF-4 for A4-CMS restoration from three different F2 populations. The two studies share a common candidate genomic region on CcLG8 that harbours one major QTL for fertility restoration. Recent findings on deep sequencing of flower bud transcriptomes in pigeonpea have associated anther/pollen development processes with differential expression of several genes/transcripts (Bohra et al. 2021a, b). Also, methylation profiles of CMS, restorer and the hybrid suggest that fertility/hybrid vigour phenomenon in pigeonpea might involve epigenetic changes such as DNA methylation (Junaid et al. 2018; Sinha et al. 2020). Epigenetic changes cover modifications beyond DNA sequence such as methylation, histone modification and non-coding (nc) RNAs (small RNA and long ncRNA). Accumulating literature suggests involvement of ncRNA molecules in CMS occurrence across several crop species (Mishra and Bohra 2018). Following sequencing of small RNAs from floral buds, the differential expression of miRNAs between CMS line and maintainer line has been demonstrated in different crops like rice (Yan et al. 2015), maize (Yu et al. 2013), soybean (Ding et al. 2016), cotton (Yu et al. 2020), etc. In pigeonpea, Bohra et al. (2021c) recently sequenced small RNA and degradome libraries from floral buds of CMS line UPAS 120A and maintainer line UPAS 120B using Illumina platform. The analysis of the small RNAome dataset identified a total of 316 miRNAs, of which 248 miRNAs were known (belonging to 46 miRNA families) and 68 miRNAs were of novel type. Target prediction using the web tool psRNATarget (https:// plantgrn.noble.org/psRNATarget/home) enabled identification of a total of 637 and 324 genes as targets for known and novel miRNAs, respectively. Efforts were also made to reveal the molecular mechanism explaining the fertility transitions in the TGMS pigeonpea. Pazhamala et al. (2020) applied multi-omics approach integrating information generated through transcriptomic, proteomic and metabolomics platforms. The study demonstrated impairments in auxin biosynthesis as a prime cause explaining fertility transitions below and above critical temperature, i.e. 24  C. DNA marker-based technology is applied for testing the genetic purity of the hybrid seed lots and their parental lines, bolstering hybrid breeding programs in several crops (see Bohra et al. 2016). To support grow-out test (GoT) of hybrid pigeonpea, genetic purity testing kits based on DNA markers have also been developed (Table 7.4). Saxena et al. (2010b) reported two diagnostic SSR markers

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Discovery and Application of Male Sterility Systems in Pigeonpea

Table 7.4 DNA markers for genetic purity testing in hybrid pigeonpea programs

Marker CCB4 and CCttc006 42 markers CcGM 08896

Type SSR SSR SSR

161

References Saxena et al. (2010a, b, c) Bohra et al. (2011) Bohra et al. (2017a, b, c)

CCB4 and CCttc006 for purity testing of the pigeonpea hybrid ICPH 2438, with purity index of CCB4 being 94.2 and 95.6, while CCttc006 showing index of 98.7 and 97.8 for two different seed lots. Similarly, a set of 42 SSRs was identified each for two hybrids (ICPH 2671 and ICPH 2438) for their genetic purity assessment, and importantly, multiplexing of eight SSRs to a single PCR assay was shown. More recently, Bohra et al. (2017b) demonstrated the utility of hyper-variable SSRs for DNA profiling and seed purity testing of pigeonpea hybrids and their inbred lines. These genomic tools and technologies would contribute to improve efficiency of hybrid breeding in pigeonpea.

7.5

Scope for Adoption of Modern Technologies

The success of pigeonpea hybrid breeding would be estimated in terms of improvements in farmers’ livelihood. Nevertheless, to achieve the stated objective, hybrid breeding in pigeonpea has to go a long way. It is an opportune time in hybrid pigeonpea breeding to learn key lessons from the crops where hybrid breeding has been established as a successful entrepreneurship and efforts should be done towards developing new tools and strategies to optimize and fully leverage the worth of sequencing data in pigeonpea (Bohra et al. 2017d). As mentioned in the previous section, a variety of genomic resources have been made available in pigeonpea to enhance the operational efficiency of the hybrid breeding program in pigeonpea. In parallel, knowing the molecular mechanisms of heterosis, defining heterotic groups (Saxena et al. 2021c), diversification of cytoplasm sources, etc. present future researchable areas.

7.6

Major Challenges and Potential Opportunities

Even today the considerable chunk of pigeonpea cultivable area is under sustainable crop model despite yield gaps in cultivars belonging to all maturity groups (early, medium and long duration). The current gap between potential and realized yields of pigeonpea offers substantial opportunities for mass dissemination of modern technological interventions that could boost cultivation of this crop at the commercial level. To enhance the area under pigeonpea cultivation, new production niches with extra early and early-maturing cultivars have a huge demand in the near future (Saxena et al. 2019b). Pigeonpea-wheat rotation is prevalent in the north western plain zone (NWPZ) in India including states of Punjab, Haryana and western

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Uttar Pradesh. Breeding new cultivars and hybrids for aforementioned states will have high practical utility. It has been realized that, at majority of the pigeonpea-growing region, the average grain yield is stagnated over the years. Developing pigeonpea hybrids with shortmaturity duration offers the best alternative to enhance the productivity per unit area, and the recent results from hybrid pigeonpea experimentations show a great promise (Saxena et al. 2021a; Saxena et al. 2013a, b). Hybrid pigeonpea would increase the per se crop productivity in the existing and potential new niches. There is an immense scope for the CMS conversion with respect to incorporate beneficial traits including photo-thermal insensitivity, early maturity and resistance to major biotic and abiotic stresses. To this end, the male sterility traits were transferred to 11 diverse short-duration pigeonpea genotypes through standard backcrossing scheme (Saxena et al. 2005a, b; Dalvi et al. 2008; Sawargaonkar et al. 2012). The resulting CMS lines showed a considerable range in important plant traits including flowering, maturity and grain weight. At ICAR-IIPR, Kanpur, scientists have demonstrated successful transfer of male sterility-inducing cytoplasm (A2 and A4) into the background of different early-maturing popular pigeonpea cultivars such as PA 163, UPAS 120, ICPL 88039 and Pusa 992 (Bohra et al. 2017a). The lines are being deployed in hybrid breeding programs. For instance, the CMS line PA 163A is the female parent of the two pigeonpea hybrids IPH 15-03 and IPH 09-5 that have been released and notified for cultivation in the north western plain zone (NWPZ). The often cross-pollinating nature of pigeonpea crop demands strict adherence to the isolation distance (minimum of 1000 m) to allow large-scale generation of pure seeds of hybrid and parental lines in a cost-effective manner. Relying solely on the insect (pollinator) activities, female line (A) and maintainer line (B) are planted in 4: 1 or 6:1 (female-to-male) ratio. Similar ratio is adopted in case of hybrid seed production in which female line (A) and restorer line (R) are planted in 4:1 or 6:1 ratio. The extent of seed harvest from 6:1 planting is subjected to the higher activity of pollinators, while 4:1 ratio is known to provide optimum harvest in general. To ensure the genetic purity, the seeds are harvested first from fertile line(s), i.e. B and R lines. Subsequently, A-line is also harvested. Experiments were conducted at IIPR, Kanpur, for two seasons to standardize the technique that enables cost-effective seed production of A-line. For example, the relative efficiencies of planting at different ratios were examined for UPAS 120 A and its cognate maintainer (UPAS 120B) using three ratios, viz. 4A:1B, 6A:1B and 8A:1B. Notably, the 6:1 ratio witnessed the highest seed yield of A-line. At ICRISAT, good yield was harvested from both A  B and A  R seed production plots using a planting ratio of 4:1. On an average, yields of 1135 kg/ha and 975 kg/ha were recorded for A-line seed (A  B) and hybrid seed (A  R), respectively. Regular monitoring of the seed production plot and removal of the off-types (roguing) are essentially needed in order to maintain genetic purity, and roguing is performed at seedling or pre-flowering stages. Keeping this in view, development of breeder friendly molecular markers for assessment of genetic purity at the early stage has an immense potential in saving the resources and increasing the productivity of hybrid and its parental yield.

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Future Outlook

Significantly higher performance of hybrids over the popular pigeonpea cultivars has become evident from multi-location evaluation of pigeonpea hybrids (Saxena et al. 2015). However, a growing demand for nutritious food will require more productive pigeonpea hybrids in combination with cost-efficient large-scale production of hybrid seeds. Following areas need to be emphasized for planning and implementing of hybrid pigeonpea breeding: • Developing high-yielding hybrids for specific agro-ecologies • Diversifying cytoplasmic base of new hybrids to minimize the risk of disease outbreaks • Applying molecular diagnostic tools for rapid improvement of disease and pest resistance • Identification of key seed parameters and development of cost-effective practices for high-quality seed production • Optimizing agronomy practices to realize potential productivity gains of hybrid pigeonpea • Sequence-based characterization of hybrids and their parents • Construction of high heterotic groups and identification of heterotic patterns • Implementing modern breeding techniques like genomic selection in hybrid breeding.

References Bohra A, Mallikarjuna N, Saxena K, Upadhyaya H, Vales MI, Varshney RK (2010) Harnessing the potential of crop wild relatives through genomics tools for pigeonpea improvement. J Plant Biol 37:1–16 Bohra A, Dubey A, Saxena RK, Penmetsa RV, Poornima KN, Kumar N, Farmer AD, Srivani G, Upadhyaya HD, Gothalwal R, Ramesh R, Singh D, Saxena KB, Kavi Kishor PB, Singh NK, Town CD, May GD, Cook DR, Varshney RK (2011) Analysis of BAC-end sequences (BESs) and development of BES-SSR markers for genetic mapping and hybrid purity assessment in pigeonpea. BMC Plant Biol 11:56 Bohra A, Saxena RK, Gnanesh BN, Saxena KB, Byregowda M, Rathore A, KaviKishor PB, Cook DR, Varshney RK (2012) An intra-specific consensus genetic map of pigeonpea [Cajanus cajan (L.) Millspaugh] derived from six mapping populations. Theor Appl Genet 125:1325–1338 Bohra A, Saxena RK, Saxena KB, Sameerkumar CV, Varshney RK (2014) Advances in pigeonpea genomics. In: Gupta S, Nadarajan N, Sen Gupta D (eds) Legumes in the Omic Era. Springer, New York, pp 95–110 Bohra A, Jha UC, Adhimoolam P, Bisht D, Singh NP (2016) Cytoplasmic male sterility (CMS) in hybrid breeding in field crops. Plant Cell Rep 35:967–993 Bohra A, Jha A, Singh IP, Pandey G, Pareek S, Basu PS, Chaturvedi SK, Singh NP (2017a) Novel CMS lines in pigeonpea [Cajanus cajan (L.) Millspaugh] derived from cytoplasmic substitutions, their effective restoration and deployment in hybrid breeding. Crop J 5:89–94 Bohra A, Jha R, Pandey G, Patil PG, Saxena RK, Singh IP, Singh D, Mishra RK, Mishra A, Singh F, Varshney RK, Singh NP (2017b) New hypervariable SSR markers for diversity

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analysis, hybrid purity testing and trait mapping in pigeonpea [Cajanus cajan (L.) Millspaugh]. Front Plant Sci 8:377 Bohra A, Jha R, Pandey G, Patil PG, Saxena RK, Singh IP, Singh D, Mishra RK, Mishra A, Singh F, Varshney RK, Singh NP (2017c) New hypervariable SSR markers for diversity analysis, hybrid purity testing and trait mapping in pigeonpea [Cajanus cajan (L.) Millspaugh]. Front Plant Sci 8:1–15 Bohra A, Pareek S, Jha R, Saxena RK, Singh IP, Pandey G et al (2017d) Modern genomic tools for pigeonpea improvement: status and prospects. In: Varshney RK, Saxena RK, Scott J (eds) The pigeonpea genome. Springer, Cham, pp 41–54 Bohra A, Saxena KB, Varshney RK, Saxena RK (2020) Genomics assisted breeding for pigeonpea improvement. Theor Appl Genet 133:1721–1737 Bohra A, Prasad G, Rathore A, Saxena RK, Satheesh Naik SJ, Pareek S, Jha R, Pazhamala L, Datta D, Pandey G, Tiwari A, Maurya AK, Soren KR, Akram M, Varshney RK, Singh NP (2021a) Global gene expression analysis of pigeonpea with male sterility conditioned by A2 cytoplasm. Plant Genome 14:e20132 Bohra A, Rathore A, Gandham P, Saxena RK, Satheesh Naik SJ, Dutta D, Singh IP, Singh F, Rathore M, Varshney RK, Singh NP (2021b) Genome-wide comparative transcriptome analysis of the A4-CMS line ICPA 2043 and its maintainer ICPB 2043 during the floral bud development of pigeonpea. Funct Integr Genomics 21:251–263 Bohra A, Gandham P, Rathore A, Thakur V, Saxena RK, SatheeshNaik SJ, Varshney RK, Singh NP (2021c) Identification of microRNAs and their gene targets in cytoplasmic male sterile and fertile maintainer lines of pigeonpea. Planta 253(2):59. https://doi.org/10.1007/s00425-02103568-6 Bohra A, Kilian B, Sivasankar S, Caccamo M, Mba C, McCouch SR, Varshney RK (2022) Reap the crop wild relatives for breeding future crops. Trends Biotechnol 40(4):412–431. https://doi. org/10.1016/j.tibtech.2021.08.009 Chen L, Liu YG (2014) Male sterility and fertility restoration in crops. Annu Rev Plant Biol 65: 579–606 Colombo N, Galmarini CR (2017) The use of genetic, manual and chemical methods to control pollination in vegetable hybrid seed production: a review. Plant Breed 136:287–299 Dalvi V, Saxena K, Madrap I, Ravikoti V (2008) Cytogenetic studies in A(4) cytoplasmic-nuclear male-sterility system of Pigeonpea. J Hered 99:667–670 De DN (1974) Pigeonpea. In: Hutchinson J (ed) Evolutionary studies in world crops: diversity and change in the Indian subcontinent. Cambridge University Press, London, pp 79–87 Ding X, Li J, Zhang H, He T, Han S, Li Y, Yang S, Gai J (2016) Identification of miRNAs and their targets by high-throughput sequencing and degradome analysis in cytoplasmic male-sterile line NJCMS1A and its maintainer NJCMS1B of soybean. BMC Genomics 17:24 Dundas IS (1990) Pigeonpea: cytology and cytogenetics—perspectives and prospects. In: Nene YL, Hall SD, Sheila VK (eds) The pigeonpea. CAB International, Wallington, pp 117–136 FAOSTAT (2019) Food and Agriculture Organization of United Nations. http://faostat.fao.org Heng S, Wei C, Jing B et al (2014) Comparative analysis of mitochondrial genomes between the hau cytoplasmic male sterility (CMS) line and its iso-nuclear maintainer line in Brassica juncea to reveal the origin of the CMS-associated gene orf288. BMC Genomics 15:322 Junaid H, Kumar AR, Rao AN, Patil NK, Singh K, Gaikwad K (2018) Unravelling the epigenomic interactions between parental inbreds resulting in an altered hybrid methylome in pigeonpea. DNA Res 25:361–373 Kaila T, Chaduvla PK, Saxena S, Bahadur K, Gahukar SJ, Chaudhury A, Sharma TR, Singh NK, Gaikwad K (2016) Chloroplast genome sequence of pigeonpea (Cajanus cajan (L.) Millspaugh) and Cajanus scarabaeoides (L.) Thouars: genome organization and comparison with other legumes. Front Plant Sci 7:1847 Kaila T, Saxena S, Ramakrishna G, Tyagi A, Tribhuvan KU, Srivastava H, Chaudhury A, Singh NK, Gaikwad K (2019) Comparative RNA editing profile of mitochondrial transcripts in

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cytoplasmic male sterile and fertile pigeonpea reveal significant changes at the protein level. Mol Biol Rep 46(2):2067–2084 Kaul MLH (1988) Male-sterility in higher plants. In: Frankel R, Grassman M, Maliga, Riley R (eds) Monograph, vol. 10. Springer, Berlin Latham MC (1997) Human nutrition in the developing world. Food and nutrition series—no. 29. Food and Agriculture Organization of the United Nations, Rome Mallikarjuna N, Saxena KB (2005) A new cytoplasmic nuclear malesterility system derived from cultivated pigeonpea cytoplasm. Euphytica 142:143–148 Mishra A, Bohra A (2018) Non-coding RNAs and plant male sterility: current knowledge and future prospects. Plant Cell Rep 37(2):177–191 Mula MG, Saxena KB (2010) Lifting the level of awareness on Pigeonpea—a global perspective. International Crops Research Institute for the Semi-Arid Tropics, Patancheru, 540 pp Naik SJS, Singh I, Bohra A, Singh F, Datta D, Mishra R, Tyagi S, Maurya AK, Singh NP (2020) Analyzing the genetic relatedness of pigeonpea varieties released over last 58 years in India. Indian J Genet Plant Breed 80:70–76 Pazhamala LT, Chaturvedi P, Bajaj P, Srikanth S, Ghatak A, Chitikineni A, Varshney RK (2020) Multiomics approach unravels fertility transition in a pigeonpea line for a two-line hybrid system. Plant Genome 13:e20028 Reddy BVS, Green JM, Bisen SS (1978) Genetic male-sterility in pigeonpea. Crop Sci 18:362–364 Royes WV (1976) Pigeon pea Cajanus cajan (Leguminosuae-Papilionatae). In: Simmonds NW (ed) Evolution of crop plants. Longman, London Sawargaonkar SL, Madrap IA, Saxena KB (2012) Study of inheritance of fertility restoration in pigeonpea lines derived from Cajanus cajanifolius. Plant Breed 131:312–314 Saxena KB (2014) Temperature-sensitive male sterility system in pigeonpea. Curr Sci 107:277–281 Saxena KB, Sharma D (1990) Pigeonpea genetics. In: Nene YL, Hall SD, Sheila VK (eds) The pigeonpea. CAB International, Wallingford, pp 137–158 Saxena KB, Singh L, Gupta MD (1990) Variation for natural out-crossing in pigeonpea. Euphytica 46:143–146 Saxena KB, Kumar RV, Srivastava N, Bao S (2005a) A cytoplasmic-nuclear male-sterility system derived from a cross between Cajanus cajanifolius and Cajanus cajan. Euphytica 145:289–294 Saxena KB, Kumar RV, Srivastava N, Shiying B (2005b) A cytoplasmic-genic male-sterility system derived from a crosses between Cajanus cajanifolius and Cajanus cajan. Euphytica 145:291–296 Saxena KB, Sultana R, Mallikarjuna N, Saxena RK, Kumar RV, Sawargaonkar KL (2010a) Malesterility systems in pigeonpea and their role in enhancing yield. Plant Breed 129:125–134 Saxena KB, Sultana R, Mallikarjuna N, Saxena RK, Kumar RV, Sawargaonkar SL, Varshney RK (2010b) Male sterility systems in pigeonpea and their role in enhancing yield. Plant Breed 129: 125–134 Saxena RK, Saxena K, Varshney RK (2010c) Application of SSR markers for molecular characterization of hybrid parents and purity assessment of ICPH 2438 hybrid of pigeonpea [Cajanuscajan (L.) Millspaugh]. Mol Breed 26:371–380 Saxena KB, Kumar RV, Tikle AN, Saxena MK, Gautam VS, Rao SK, Khare DK, Chauhan YS, Saxena RK, Reddy BVS, Sharma D, Reddy LJ, Green JM, Faris DG, Nene YL, Mula M, Sultana R, Srivastava RK, Gowda CLL, Sawargaonkar SL, Varshney RK (2013a) ICPH 2671— the world’s first commercial food legume hybrid. Plant Breed 132:479–485 Saxena KB, Kumar RV, Tikle AN et al (2013b) ICPH2671—the world’s first commercial food legume hybrid. Plant Breed 132:479–485 Saxena KB, Singh IP, Bohra A, Singh BB (2015) Strategies for breeding, production, and promotion of pigeonpea hybrids in India. J Food Legumes 28:190–198 Saxena RK, Patel K, Sameer Kumar CV, Tyagi K, Saxena KB, Varshney RK (2018) Molecular mapping and inheritance of restoration of fertility (Rf) in A4 hybrid system in pigeonpea (Cajanus cajan (L.) Millsp.). Theor Appl Genet 131:1605–1614

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Saxena KB, Sharma D, Vales MI (2019a) Development and commercialization of CMS pigeonpea hybrids. Plant Breed Rev 41:103–167 Saxena KB, Choudhary A, Srivastava R, Bohra A, Saxena R, Varshney RK (2019b) Origin of early maturing pigeonpea germplasm and its impact on adaptation and cropping systems. Plant Breed 138:243–251 Saxena RK, Molla J, Yadav P, Varshney RK (2020) High resolution mapping of restoration of fertility (Rf) by combining large population and high density genetic map in pigeonpea [Cajanus cajan (L.) Millsp]. BMC Genomics 21:460 Saxena KB, Bohra A, Choudhary AK, Sultana R, Pazhamala L, Saxena RK (2021a) The alternative breeding approaches for improving yield gains and stress response in pigeonpea [Cajanus cajan (L.) Millsp.]. Plant Breed 140:76–86 Saxena RK, Hake A, Bohra A, Khan A, Hingane A, Sultana R, Singh IP, Satheen Naik SJ, Varshney RK (2021b) A diagnostic marker kit for fusarium wilt and sterility mosaic diseases resistance in pigeonpea. Theor Appl Genet 134:367–379 Saxena RK, Jiang Y, Khan A, Zhao Y, Singh VK, Bohra A, Sonappa M, Rathore A, Sameerkumar CV, Saxena CV, Reif J, Varshney RK (2021c) Characterization of heterosis and genomic prediction based establishment of heterotic pattern for developing better hybrids in pigeonpea. Plant Genome 14(3):e20125. https://doi.org/10.1002/tpg2.20125 Singh IP, Singh BB, Ali I, Kumar S (2009) Diversification and evaluation of cytoplasmic nuclear male sterility system in pigeonpea (Cajanuscajan L. Millsp.). Indian J Agric Sci 79:291–294 Singh IP, Bohra A, Singh F (2016) An overview of varietal development programme of pigeonpea in India. Legume Perspect 11:37–40 Sinha P, Saxena K B, Saxena R K, Singh VK, Suryanarayana V, Sameer Kumar CV, Katta M, Khan AW, Varshney RK (2015) Association of nad7a gene with cytoplasmic male sterility in Pigeonpea. The plant genome 8(2)eplantgenome2014.11.0084 Sinha P, Singh VK, Saxena RK, Kale SM, Li Y, Garg V, Meifang T, Khan AW, Kim KD, Chitikineni A, Saxena KB, Sameer Kumar CV, Liu X, Xu X, Jackson S, Powell W, Nevo E, Searle IR, Lodha M, Varshney RK (2020) Genome-wide analysis of epigenetic and transcriptional changes associated with heterosis in pigeonpea. Plant Biotechnol J 18:1697–1710 Solomon S, Argikar GP, Solanki HS, Morbad JR (1957)A study of heterosis in Cajancajan L. Millsp. Indian J Genet Plant Breed 17: 90–95 Srikanth S, Saxena RK, Rao MV, Varshney RK, Mallikarjuna N (2015) Development of a new CMS system in pigeonpea utilizing crosses with Cajanuslanceolatus (WV Fitgz) van der Maesen. Euphytica 204(2):289–302 Touzet P, Budar F (2004) Unveiling the molecular arms race between two conflicting genomes in cytoplasmic male sterility? Trends Plant Sci 9:568–570 Tuteja R, Saxena RK, Davila J, Shah T, Chen W, Xiao YL, Fan G, Saxena KB, Alverson AJ, Spilliane C, Town C, Varshney RK( 2013) Cytoplasmic male sterility-associated chimeric open reading frames identified by mitochondrial genome sequencing of four Cajanus genotypes. DNA Res 20:485–495 Vavilov NI (1951) The origin, variation, immunity and breeding of cultivated plants. Chron Bot 13: 1–366 Yan J, Zhang H, Zheng Y, Ding Y (2015) Comparative expression profiling of miRNAs between the cytoplasmic male sterile line MeixiangA and its maintainer line MeixiangB during rice anther development. Planta 241:109–123 Yu JH, Zhao YX, Qin YT, Yue B, Zheng YL, Xiao HL (2013) Discovery of microRNAs associated with the S type cytoplasmic male sterility in maize. J IntegrAgric 12:229–238 Yu D, Li L, Wei H et al (2020) Identification and profiling of microRNAs and differentially expressed genes during anther development between a genetic male-sterile mutant and its wild type cotton via high-throughput RNA sequencing. Mol Gen Genomics 295:645–660

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Achievements, Challenges and Prospects of Hybrid Soybean Subhash Chandra, Shivakumar Maranna, Manisha Saini, G. Kumawat, V. Nataraj, G. K. Satpute, V. Rajesh, R. K. Verma, M. B. Ratnaparkhe, Sanjay Gupta, and Akshay Talukdar

Abstract

Soybean is the number one oilseed crop in India as well as in the world. Globally, most of the soybean cultivars have been released through hybridization followed by pedigree selection approach. In general, papilionaceous flower of soybean makes outcrossing difficult through wind pollination or pollination via insects/ bees. Soybean exhibits sufficient heterosis, so improving the yield and other agronomical traits through hybrid vigour is the reliable possibility. Being a selfpollinated crop, cross-pollination in natural fields is challenging due to highly dependence on pollinator’s population and environmental factors. However, around 50 cytoplasmic-genetic male sterility systems, ~30 genetic male sterility systems and ~11 male-sterile mutants have been identified/recognized in this crop. Utilizing ‘three-line breeding’ approach, few hybrids also have been commercially released by China. There are several reports showing mapping of male sterility/restoration genes in soybean, so marker-assisted breeding may be utilized to develop stable and diverse restorers and male-sterile lines for efficient hybrid seed production. Wide hybridization, functional genomics and genome editing also may pave the ways forward to develop more stable and improved hybrid seed techniques in soybean.

Subhash Chandra, Shivakumar Maranna and Manisha Saini contributed equally with all other contributors. S. Chandra (*) · S. Maranna · G. Kumawat · V. Nataraj · G. K. Satpute · V. Rajesh · R. K. Verma · M. B. Ratnaparkhe · S. Gupta ICAR—Indian Institute of Soybean Research, Indore, India M. Saini · A. Talukdar Division of Genetics, ICAR—Indian Agricultural Research Institute, New Delhi, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_8

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Keywords

Fertility restoration · Genomics · Heterosis · Hybrid vigour · Male sterility · Pollen

8.1

Introduction

Soybean (Glycine max L. Merrill) is the most significant oilseed crop in India as well as in the world. This oilseed is an imperative source of protein (40–45%) and oil (18–22%). It is considered to be the nature’s highest-yielding source of plant origin usable protein (Chandra 2017). Soybean is widely used in preparation of diversified food and food ingredients including soy flour, milk, cheese, curd, ice cream, dietary fibre, single-cell protein, citric acid, sprouted and roasted snacks, soy fortified bakery, soy protein concentrate, margarine, etc. (Kumar 2005). Soybean has a large share (around 25%) in global edible oil production; simultaneously it also contributes for two thirds of the world’s protein essence for livestock feeding (Maranna et al. 2021). This bean has a wide spectrum of industrial and pharmaceutical applications in manufacturing of soap, cosmetics, plastics, inks, resins, crayons, solvents, clothing, etc. (Chandra 2017). Soybean also helps in enhancement of soil fertility/soil health by fixing atmospheric N2 (nitrogen) via symbiotic association with soil bacteria Bradyrhizobium japonicum. Owing to its diverse and quintessential utilities, this ‘golden bean’ also has been termed as ‘miracle bean’ (Orf 2010). Soybean was originated in Chinese continent but now cultivated worldwide (Hymowitz and Singh 1987). The USA, Argentina, Brazil, China and India are the leading countries for soybean production (AMIS, FAO website). Globally, soybean occupies an area of 122.6 million hectares (mha) with an annual average production of 336.6 million tonnes (mt) (USDA 2020). India occupies the fifth place in production and fourth in terms of area among soybean-producing countries of the world (AMIS, FAO website). During 2020–2021, soybean was raised in an area of 12.06 million hectare with grain production of 13.58 million tonnes and average yield of 1126 kg/ha (SOPA website). Soy revolution in India is a milestone success. In the past, an extraordinary growth in soybean cultivation has been recorded; acreage of soybean has been enlarged from 0.03 mha (1970) to 13.58 mha (2021), and average yield has increased from 0.43 t/ha (1970) to 1.12 t/ha (2021). The Indian soybean productivity (~1.2 t/ha) is poor in comparison of world soybean productivity (~2.9 t/ha), and the USA and Brazil have the highest soybean productivity (>3 t/ha) (AMIS, FAO website). The contribution of India in the global soybean production is about 4% with 10% area in globe, representing the poor status of productivity of the crop in the country (Agarwal et al. 2013). There are several factors affecting soybean productivity likewise (1) narrow genetic base of the released commercial cultivars; (2) rainfed environment and short maturity duration (90–100 days) of the crop in India; (3) emergence of biotic stresses, i.e. anthracnose, Rhizoctonia aerial blight, charcoal rot, rust, mosaic viruses, defoliators, tobacco

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caterpillar, stem fly, etc.; and (4) occurrence of abiotic stress, i.e. moisture deficit, water logging, low solar radiation, high temperature, etc. (Agarwal et al. 2013; Maranna et al. 2021). Seed of improved varieties is one of the essential agricultural inputs for achieving higher productivity in any crop. Improved varieties are developed for better yielding traits along with tolerance for biotic and abiotic stress and quality traits. Maturity duration is another important aspect on which breeders focus during cultivar development in any crop, especially in soybean. Soybean is highly sensitive to day length as it is a short-day plant. The history of varietal development for soybean in India is not too old. Soybean’s introduction in the country was started with field trials using varieties from the USA at GBPUAT, Pantnagar, and JNKV, Jabalpur, during 1963. On the basis of development strategies, Indian soybean varieties may be grouped broadly in five categories. (1) Indigenous varieties are land races or selection from them during research trails. Some of them are black-seeded indigenous cultivars such as ‘Bhat’ or ‘Bhatmash’ and habitual flora of northern hills of India; others are yellow seeded, representing from Tehri-Garhwal regions of northern hills. Overall these land races have provided release of three varieties, viz. Type 49 (yellowseeded), Kalitur (black-seeded) and JS-2 (yellow-seeded) (Agarwal et al. 2013). (2) The second group represents direct introduction of soybean varieties or selection in them. This category comprises varieties, i.e. Bragg, Lee, Improved Pelican, Hardee, Davis, Monetta, Clark 63, KM-1, etc. (Tiwari 2014). (3) The third group consists of varieties which were developed through mutation breeding. Mutation breeding resulted in development of many varieties, i.e. NRC 2, Birsa Soya, NRC 12, Aarti, VLSoya 1, Pusa 97-12, TAMS 98-21, MACS 450, etc. (Tiwari 2014). (4) The fourth group contains varieties developed through hybridization and pedigree selection. Most of the varieties in India have been developed through pedigree method. Even mega varieties, viz. JS 335, JS 93-05, NRC 7, JS 95-60 and JS 20-34, have been developed through this conventional breeding methodology. (5) The fifth group encompasses recent varieties, which were developed through marker-assisted breeding methodologies. It includes recently released varieties, i.e. NRC 127, NRC 142, NRC-SL 2, etc. If the genetic potential of varieties developed in the Indian history compared, then varieties developed before year 1990 produced four times higher grain productivity than indigenous black-seeded cultivar Kalitur by advantage of more number of pods plant
1, short duration, seed weight, etc. (Agarwal et al. 2013). The varieties developed after 1990 depicted 18% better yield than varieties developed before year 1990, due to improvement in harvest index and rapid seed-filling. Over the years, genetic improvement and yield stability were the main focus with concentration of short maturity duration. The ideotype of soybean plant for high yield consists of determinate/semi-determinate growth habit, erect and non-lodging, suitability for mechanical harvesting (first reproductive node should be above from the ground with at least 7–10-cm distance), long juvenile period, rapid seed fill duration and maturity duration of 95–100 days (Agarwal et al. 2013; Maranna et al. 2021). The most important yielding attributes in soybean are pods per plant, seeds per pod and seed size. Worldwide, basically soybean yield has been increased through hybridization and selection. Specth et al. (1999) reported that

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soybean yields are increasing with a rate of 23 kg/ha/annum, while Wilcox (2001) assessed a rise of 60% in grain yields during the last 60 years through public sector soybean programs in the USA. In India, this annual genetic gain for soybean seed yield has been estimated around 22 kg/ha/annum based on data collected between years 1969 and 1993 (Karmakar and Bhatnagar 1996). Most of Indian soybean varieties have yield potential of 2.5–3 t/ha, while some of them produce up to 5 t/ha. The conventional breeding techniques along with newly developed molecular techniques have increased yield by around 60% in the last 70 years, and around 4000 varieties of soybean have been released globally for commercial cultivation. The availability of genome sequence, gene mapping, QTL analysis, the utilization of functional genomics and transgenic development has accelerated soybean improvement in recent past. Soybean, being a self-pollinated crop, is not too much researched for possibilities of utilization of hybrid vigour for enhancing yield and other characteristics. However, more than 20% heterosis for yield and other traits have been documented in soybean (Tiwari 2014; Li et al. 2019). In this chapter, we discussed the botany, floral morphology, pollination, hybridization in soybean, recent achievement in the perspective of development of male-sterile lines and hybrids in soybean along with current challenges and future prospects.

8.2

Botany, Pollination and Hybridization in Soybean

The present-day cultivated soybean [Glycine max (L.) Merr.] belongs to the family Leguminosae, the subfamily Papilionoideae, the tribe Phaseoleae and the genus Glycine. Further, Glycine is divided in to Soja and Glycine as two sub-genera. Cultivated soybean (Glycine max L. Merrill) and its wild ancestor Glycine soja are annuals and belong to the sub-genus Glycine. The sub-genus Soja possesses >25 wild perennial species in its pool (Hymowitz and Singh 1987). It has been suggested through morphological, cytological and molecular evidence that G. soja is the direct predecessor of G. max and Glycine gracilis is believed to be the semi-wild form of G. max (Hymowitz 1970). Wild perennial species of soybean includes G. tabacina, G. tomentella, G. clandestine, G. latifolia, G. latrobeana, G. canescens, G. falcate, etc. (Hymowitz and Newell 1981; Grant 1984; Tindale 1986). The gene transfer from these wild to cultivated soybean needs special techniques like protoplast fusion and embryo rescue. However, Glycine tomentella and Glycine tobacina has been used for gene transfer to cultivated soybean for a number of useful traits (Singh 2019). Cultivated soybean contains 20 chromosomes and (2n ¼ 40) cytologically diploid in nature. It is a bushy herbaceous erect annual that can attain height up to 150 cm and having three types of growth habits, viz. determinate, semi-determinate and indeterminate (Bernard and Weiss 1973). Determinate and semi-determinate types of genotypes are preferred by Indian farmers due to their early maturity characters. In soybean, the root system contains a taproot with strong nodulation system for N2 fixation that suits for intercropping system with cereal crop.

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Soybean contains papilionaceous flower, and it has four parts, viz. calyx, corolla, androecium and gynoecium, which are present in a single flower. The five petals— standard (01), wings (02) and keels (02)—enclose the pistil and 10 (9 + 1) stamens. Two keel petals enclosed the sexual parts stamens and stigma for ensuring selffertilization and less than 1% natural cross-pollination. In flower, the 10 stamens (male parts) are closely situated near the pistil (female structure) so that pollen grains produced in the anthers (part of stamen) are deposited directly onto the stigma (part of the pistil). More than 98% of pods formed on single plants are a result of selffertilization (Gupta et al. 2019). Soybean flowers are protogynous in nature wherein the stigma matures 24 h before anthesis and remains receptive for about 48 h. Therefore, emasculation procedure is not necessary to produce hybrid plant/seed, if the correct flower bud is selected for the pollination. Although size and colour of the bud vary according to genotype, by experience one can choose the flower bud which is going to open the next day, and receptive stigma is ready for pollination. Selection of male flower is also equally important in making true hybrids, and fresh and same-day opened flowers need to be used as male parents for achieving more number of true crosses (Talukdar and Shivakumar 2012). Fertilization of soybean flowers is nearly 100% because of the following reasons: many pollen grains are produced in the 10 stamens; no pollen grains are lost by wind; pollen tubes need to travel a short distance from the stigma to the ovary; and the flower petals cover the pistil, which reduces dehydration. In soybean flowers, inflorescences called racemes in botanical terms and it takes 4–10 days for all flowers to open on a single raceme. Artificial hybridization in soybean is very difficult using the emasculation method. Soybean flower is very small and delicate; it drops even with minor injuries to the pistil. Walker et al. (1979) reported soybean flowers are protogynous in nature. To exploit the protogyny condition, pollination without emasculation (PWE) method (Talukdar and Shivakumar 2012) is suggested to get more true F1s in soybean. Soybean seed number per plant is determined by the number of flowers produced, the number of pods retained on the plant and the number of seeds per pod. Because flowers can be produced on all stem and branch nodes, flower number is highly influenced by the amount of branching. So overall, pollination behaviour in soybean crop highly influences the possibility of utilization of heterosis phenomena. There is no secondary gene pool (GP-2) in soybean. There are possibilities of development of hybrids through interspecific and intergeneric crosses with related species. Interspecific, fertile hybrids between G. max. and G. soja (Sieb and Zucc.) have been observed successfully by Ahmad et al. (1977), Hadley and Hymowitz (1973), Broich (1978), Yashpal et al. (2015) and Chandra et al. (2020a) and between G. max and G. gracilis by Karasawa (1952). Intergeneric hybrids can also be obtained with the help of embryo rescue and other newly developed techniques. Intersub-generic hybrids were obtained between G. max and G. clandestina Wendl, G. max and G. tomentella Hayata (Singh and Hymowitz 1985; Singh et al. 1987) and G. max and G. canescens (Broue et al. 1982).

172

8.3

S. Chandra et al.

Heterosis in Soybean and Its Scope

Heterosis is defined as a superior performance of heterozygous F1 hybrid plants in terms of increased grain yield, biomass, size and speed of growth/development, fertility, resistance/tolerance to disease/insect pest or any kind of climatic stress in comparison to the mean of their homozygous parents/parental lines (Falconer and Mackay 1981). It can be mean heterosis or better parent heterosis, depending upon the comparison with the average of both parents and better parent accordingly. In soybean, heterosis for many traits has been reported. Furthermore, it was evaluated and tested mainly for grain yield. Chaudhary and Singh (1974) studied 17 F1s of 8 promising Indian varieties in soybean. They reported about better parent heterosis for grain yield, and it was ranging from
30.3 to 67.8% with a mean (heterosis) of 26.1%. They also reported about positive heterosis for pods per plant and seeds per plant along with negative heterosis for seeds per pod and seed size (Chaudhary and Singh 1974). Burton (1987) observed that 85% of the F1 soybean crosses displayed mid-parent heterosis for seed yield and 62% showed high parent heterosis. Burton and Brownie (2006) worked out about 16% and 5% higher yield in F1s derived from crosses, viz. Holladay/Hutcheson and Brim/Boggs, respectively. Shivakumar et al. (2019) estimated higher heterosis for yield traits in the crosses during development of NAM populations, whereas negative heterosis is also observed in few crosses studied for grain yield and days to maturity. Recently, Yamgar et al. (2021) reported about negative heterosis for flowering and maturity duration, while positive heterosis for plant height, number of pods per plant, seed yield and seed size. The differential appearance of heterosis in soybean is also reported by other studies (Gadag and Upadhyaya 1995; Perez et al. 2009). Cregan et al. (1989) reported about positive heterosis for N2 fixation traits; they showed high parent heterosis for nodule mass (34%), N2 accumulation (28%) and dry matter accumulation (28%) in crosses derived from Glycine max  G. soja. The preliminary and small-scale evaluation of F1 hybrids in soybean indicated the presence of significant amount of heterosis for seed protein and oil content (Sharma and Maloo 2009). Several reasons could be there for explaining possible genetic basis of heterosis in soybean. It can happen due to gene complementation/interaction of duplicate favourable loci in repulsion, more number of dominant alleles in the hybrid than either parent separately, linked dominant alleles that advance in groups, multiple quanta-dependant regulatory genes, and/or overdominance nature. The existence of heterosis may be one of the evidence of possibility of combination of superior genes. The advantages of cultivation of soybean hybrid vs variety are discussed below: 1. Increased grain yield: Generally, the hybrid produces greater biomass by utilizing inputs more efficiently. The hybrids will produce more number of branches (primary as well as secondary) coupled with more number of pods per plant and larger seed size that help towards better yield. 2. Increased seedling vigour: Seedling vigour is also one of the major yield attributing traits. The hybrid soybean is more competitive than the weeds during the initial crop growth stages. Thus it also helps in reduction of weeds, and thus it will enhance the grain yield under sole crop as well as intercrop.

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3. Reduced seed rate: Soybean varieties require high seed rate (60–70 kg/ha). However, by the introduction of hybrids, it is possible to reduce the seed requirements as hybrids are known to produce more number of primary and secondary branches, number of pods, etc. 4. Root biomass and N2 fixation: Soybean hybrids show greater root mass and deep root system, which provide more ability to draw water from deeper soil profiles during drought conditions. In India, soybean crop is grown under rainfed condition, crop experiences more dry spells and the cultivation of hybrids with deep roots will help in mitigating the drought phenomenon. Similarly, soybean hybrids enable plants for N2 fixation due to more nodules and more dry matter accumulation. 5. Oil and protein content: Some of the scientific reports indicated that hybrids are superior in oil and protein content than the soybean varieties.

8.4

Male Sterility Systems in Soybean

In sexually reproducing plant, male sterility is a state where male reproductive organs (anthers, pollen mother cells, pollens, pollen tubes, male gametes) develop abnormally or failure of active involvement of these organs resulting in nonfunctional pollen grains without interrupting the development of female reproductive organs and plant behaves as gynoecious or female plant. Generally, male sterility (MS) is governed by the nuclear gene and cytoplasm genes; particularly when MS is controlled by the nuclear gene termed as genetic or nuclear male sterility (GMS/NMS), by the cytoplasmic genes as cytoplasmic male sterility (CMS). Whereas occurrence of male sterility due to the complementary effects of cytoplasmic male sterility and nuclear genes is known as cytoplasmic-genetic male sterility (CGMS) in which both nuclear gene and cytoplasmic gene play a significant role (Chen and Liu 2014). In the era of hybrids, choosing a male sterility system in hybrid seed production avoids artificial emasculation and cross-pollination which represent an efficient tool in commercial hybrid seed production (Horn et al. 2014). In many crops, GMS and CMS encourage the production of hybrid seeds and fasten the yield gain with heterosis and hybrid vigour (Chen and Liu 2014). Presently >200 plant species CMS has been known/expressed (Hu et al. 2012). MS not only provides an essential basis of breeding for heterosis in crops, but it also provides opportunities to understand the regulation mechanisms of sterile genes in floral organs (Chen and Liu 2014). Firstly, genetic male sterility in soybean is observed by Owen in 1928, while CMS is reported by Davis in 1985. Further, with the help of the continuous studies on MS, around 50 CMS systems (NJCMS1A-NJCMS5A, OA, YA, JLCMS9A, W931A, etc.) and ~30 NMS lines/mutants (St1, ms1-ms9, msMOS, msp, etc.) have been recognized. Moreover, immense studies had been conducted on various characteristics of soybean MS (CMS and GMS) based on conventional breeding along with the development of modern molecular technologies which majorly includes hybrid seed production systems and population improvement programs

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(Ding et al. 2002), three-line breeding (Dai et al. 2017) and recurrent selection (Zhao et al. 2007). In addition, inheritance, cytology and structural characteristics, tissue structures (chloroplasts and mitochondria), epigenetics, omics level, localization and utility of fertility and restoring gene traits were also studied in regard to male sterility in soybean (Zhang et al. 2018). The brief details about the different male-sterile systems available in soybean are discussed hereunder.

8.4.1

Genetic Male Sterility System in Soybean

In the twentieth-century, research on GMS system has been started, and the first soybean GMS mutant (St1, male and female sterile) was reported by Owen (1928). Further numerous soybean GMS systems were identified which includes msp (Stelly and Palmer 1980), fs1fs2 (Johns and Palmer 1982), St2–St5 (Palmer and Kaul 1983), 88-428-BY (Wei 1991), NJ89-1 (Ma et al. 1993), D8804-7 (Zhao et al. 1995), msMOS (Jin et al. 1997), Wh921 (Zhang et al. 1999), ms1-ms9 (Palmer 2000), N7241S (Zhao et al. 2005) and NJS-1H (Li et al. 2010a, b) (Table 8.1). Up to now, over 30 GMS systems in soybean have been reported which are structural MS ( fs1fs2), partially MS (msp), MS and female fertile (ms1-ms9), male and female sterility (St1), photosensitive MS (88-428-BY), MS controlled by a single-dominant gene (N7241S), etc. In commercial hybrid seed production, the use of the GMS is limited by the inefficient maintenance of the pure MS line, although this bottleneck is broken down with the exploitation of the advanced molecular techniques, viz. molecular gene cloning, plant transformation and recombinant DNA approaches, gene editing, etc. (Zhang et al. 2018). Although the main lacunae for the adoption of three-line breeding system with CMS is the limited number of the maintainer line of GMS, while for CMS, more number of restorers are available.

8.4.2

Cytoplasmic-Genetic Male Sterility System in Soybean

Davis (1985) first discovered soybean CMS line in the USA with patent no. 4545146; moreover afterwards research on breeding of CMS lines in soybean has extensively been carried out. Sun et al. (1994) reported the first CMS line in China (OA) from a cross between ‘Runantianedan’ (Glycine max) and 5090035 (Glycine soja), and RN-CMS line was the one of the first CMS line which developed from RNTED (Sun et al. 2003). Another new CMS cytoplasm, ZD-CMS line, was derived from ‘Zhongdou 19’ (ZD8319), which was differentiated from earlier ones (Li et al. 1995; Sun et al. 2003). Subsequently, it is further reported that the MS in both RN-CMS and ZD-CMS lines were controlled by a single-genic system, and their maintainer and restorer lines can mutually exchange for breeding (Sun et al. 2003; Zhao et al. 2012). Gai et al. (1995) reported NJCMS type as a new sterile cytoplasm (N8855). Furthermore based on above and some other MS cytoplasm,

St6, St7

St8

St6, St7

w4-mutable line D8804
7

ms2

ms3

ms4

ms5

ms6

ms7

ms2

ms3

ms4

ms5

ms6

ms7

ms1

St2–St5

St2–St5

Introducing exogenous DNA ms1

Gene St1

GMS name St1

Male sterile, female fertile Male sterile, female fertile Male sterile, female fertile

Male sterile, female fertile Male sterile, female fertile Male sterile, female fertile Male sterile, female fertile

Male and female sterile Male and female sterile

Male and female sterile

MS type Male and female sterile Male and female sterile

Satt595 and AW186493 –

–

–

BARCSOYSSR_02_1477

–

Sat_190, Satt153, Sat_274

13

13

2

2

10

Satt595 and AW186493

–

–

13

Satt132, Sct_065

BARCSOYSSR_11_137, BARCSOYSSR_11_122, Satt030, Satt146, Satt436, Satt468 BARCSOYSSR_14_109, BARCSOYSSR_14_84, Satg001

Marker type –

16

14, 2

11, 1, 13

Ch. no. –

Table 8.1 Details of GMS lines developed and reported in soybean

Single-recessive gene Single-recessive gene Single-recessive gene

Single-recessive gene Single-recessive gene Single-recessive gene Single-recessive gene

Duplicate-factor inheritance gene mutation Single-recessive gene Single-recessive gene

Genetic characteristics Single-recessive gene Single-recessive gene

Achievements, Challenges and Prospects of Hybrid Soybean (continued)

Skorupska and Palmer (1989), Palmer et al. (1998) Palmer (2000), Palmer et al. (2001)

Brim and Young (1971), Palmer (1979a, b), Palmer et al. (1978) Graybosch et al. (1984), Cervantes-Martinez et al. (2007) Palmer (1979a), Palmer et al. (1980), Buss (1983) Palmer (1979b), Graybosch and Palmer (1987), Delannay and Palmer (1982) Buss (1983)

Palmer and Horner (2000), Kato and Palmer (2003) Zhao et al. (1995)

Hadley and Starnes (1964), Palmer (1974), Palmer and Kaul (1983) Palmer and Horner (2000)

References Owen (1928)

8 175

Male sterile, female fertile –

Male sterile, female fertile Partial male sterile Structural male sterile Photosensitive male sterile Male sterile and partially female sterile Female partial sterile Sterility mutant

ms9

–

–

–

msp

fs1fs2

–

–

–

mst-M

ms0

ms9

ms MOS

Wh921 – – – –

N7241S

Msp

fs1fs2

88-428-BY

NJS-1H

L67-3483

St-M

NJ89
1

–

W1, dCAPS-1

13 –

Satt266, Satt157

–

–

–

–

–

– –

Satt172

–

–

Satt542, Satt157

Marker type BARCSOYSSR_16_0430, BARCSOYSSR_16_0428 Satt237, Satt521

2

–

–

2

2

Ch. no. 7

Note: This table is adopted from Li et al. (2019) with minor modifications

Male sterile, female fertile

MS type Male sterile, female fertile Male sterile, female fertile

Gene ms8

GMS name ms8

Table 8.1 (continued)

Single-recessive gene Single-recessive gene

–

Single-recessive gene Single-recessive gene Single-dominant gene Single-recessive gene Duplicaterecessive genes Short-day sterile, long-day fertile A pair of recessive

Genetic characteristics Single-recessive gene Single-recessive gene

Ma et al. (1993), Yang et al. (2003)

Zhao et al. (2019)

Kato and Palmer (2003)

Li et al. (2010a)

Wei (1991), Wang et al. (2004)

Stelly and Palmer (1980), Frasch et al. (2010) Johns and Palmer (1982)

Zhao et al. (2005)

References Palmer (2000), Palmer et al. (2001), Frasch et al. (2010) Palmer (2000), Palmer et al. (2001), Cervantes-Martinez et al. (2007) Jin et al. (1997), CervantesMartinez et al. (2009) Zhang et al. (1999)

176 S. Chandra et al.

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numerous soybean CMS lines were evolved and registered likewise as YA lines (Sun et al. 1997), ZA lines (Zhao et al. 1998), NJCMS lines (Li et al. 2017), FuCMS lines (Li 2007, 2015) and M-type CMS lines (W931A) (Zhang et al. 1999) (Table 8.2). In a nutshell, there were five cytoplasmic sources, viz. RNTED, Zhongdou-19, N8855, N21566 and N23661, which were further utilized as cytoplasm donors and many CMS identified (Dai et al. 2017). Ding et al. (2002) and Bai and Gai (2006) developed two CMS lines, i.e. NJCMS1A and NJCMS2A, with utilization of N8855 as the cytoplasm provider. Zhao et al. (2006) developed the CMS line NJCMS3A with the use of N21566 as the cytoplasm donor. Recently, with utilization of N23661, Nie et al. (2017) developed the CMS line (NJCMS4A). So, many restorer lines and their maintainer lines based on diverse CMS lines have been identified and established the ‘three-line breeding’ system for utilization of heterosis and improvement in soybean production via hybrid soybean.

8.4.3

Inheritance of Male Sterility and Fertility Restoration System in Soybean

The nucleus genotype and their cytoplasm decide the genetics of the male sterility (Bohra et al. 2016). In some crops, male fertility restoration mechanism is controlled by the single gene, whereas in others same genetic makeup of nucleus interacts with a diverse range of cytoplasm that generates polygenic genetics (Schertz 1993). Additionally, fertility restoration is also affected by the intra- and inter-allelic complementation and their interactions. For the development of new hybrids in soybean, the information regarding the genetic mechanism of fertility restoration and male sterility of these CMS system can enhance options to choose maintainers and restorer lines for hybrids (Guha et al. 2002). However, there are limited reports available in regard to iso-nuclear male-sterile lines with different sterility-inducing cytoplasms and common restorer lines. Zhao and Gai (2006) reported about the presence of a single restorer-of-fertility (Rf or rf) gene, showing gametophytic restoration regulation, whereas Bai and Gai (2005) reported about sporophytic control over for restoration, possessing two dominant Rf-genes which were identified for N8855 cytoplasm. Some evidence provide more clarity with facts that a dominant Rf-gene restoring ZDCMS was mapped on chromosome 16 (Dong et al. 2012), and the same genomic region was previously reported for fertility restoration against RN-CMS (Zhao et al. 2007). Assuming that a common chromosomal fragment among the two gametophytically restored CMSs (RN and ZD), which means either a common CMS-inducing cytoplasm or a similar regulatory system describes the fertility restoration (Dong et al. 2012). This type of common fertility restoration mechanism with multiple CMS in soybean is similar to other crops, viz. cotton (Zhang and Stewart 2001), sorghum (Jordan et al. 2011; Praveen et al. 2015) and wheat (Tsunewaki 2015).

N21249

N23658 Wandou 28 W206

W207

W203

NJCMS3A

NJCMS4A NJCMS5A

W933A

W936A

W931A

N1628

NJCMS2A

NJCMS1A

CMS name Cms

Male (nuclear) Bedford and Braxton variety N2899

Zhongyou 89B

Zhongyou 89B

Zhongyou 89B

N23661 N8855

N21566

N8855

N8855

Female (cytoplasmic) Elf variety

Recessive gametophyte sterility gene Recessive gametophyte sterility gene Recessive gametophyte sterility gene

Two pairs of sporophyte sterility overlapped genes Two pairs of sporophyte sterility overlapped genes Each pair of gametophyte, sporophyte and inhibitor genes – –

Nuclear sterile gene r1r1r2r2

Table 8.2 Details of CMS line developed and reported in soybean

Nie et al. (2017) Li et al. (2017) Zhang et al. (1999), Tang et al. (2009), Wang et al. (2016) – –

Satt477, Satt331, CSSRl33

– – Satt276, Satt684, Satt545, PPR proteins – –

– – A, J

– –

Ding et al. (1999, 2002), Gai et al. (1995), Yang et al. (2007), Jiang et al. (2011b) Bai and Gai (2002, 2006), Han et al. (2010), Dong et al. (2008), Jiang et al. (2011a) Li et al. (2010b), Zhao and Gai (2006)

References Davis (1985)

O

Atp9, Satt135, MADS-box

GmMF1, Satt300, Satt626

M, A1

D2

MS/restorer gene/QTL –

Linkage group (LG) –

178 S. Chandra et al.

ZD8319

Zhongdou 19 FuCMS4A

Zhongdou 19 FuCMS5A FuCMS5A Zhongdou Zhongdou Zhongdou FuCMS5A Zhongdou 19

167 (RNTED)

– – SG01

JX03

PI004

– Fubao 5

– – – – – – – JD24

035

W951A W952A FuCMS1A

FuCMS2A

FuCMS3A

FuCMS4A FuCMS5A

FuCMS6A FuCMS7A FuCMS8A FuCMS9A FuCMS10A FuCMS11A FuCMS12A SXCMSlA

OA

ZD8319

W212

W948A

Zhongyou 89B Zhongyou 89B – – ZD8319

W210

W945A

– – – – – – – Single gametophyte sterility gene

Single-recessive nuclear sterility gene Single-recessive nuclear sterility gene – – Single-dominant sporophyte sterility gene Single-dominant sporophyte sterility gene Multigene control sporophyte sterility – Single-dominant gametophytic gene – – – – – – Sctt011, BARCSOYSSR-161076, BARCSOYSSR-16-1070, BARCSOYSSR-16-1077 – – – – – – – Satt267, Sattl84 –

– – – J

– – – – – – – D1a –

–

– – – –

–

–

(continued)

Achievements, Challenges and Prospects of Hybrid Soybean

Sun et al. (1994, 2003)

Li (2007) – – – – Li et al. (2015) – Lian et al. (2016)

Li (2007) Li (2007, 2015), Dong et al. (2012)

Li (2007), Xu et al. (1999)

–

Dai et al. (2002) – Xu et al. (1999)

–

–

8 179

– 167 (RNTED) 167 (RNTED) 167 (RNTED)

ZB

– –

–

–

YA

ZA

XXT JLCMS9A

JLCMS82A

JLCMS89A

–

–

J –

– –

Satt215

– –

–

–

– –

MS/restorer gene/QTL

–

Nuclear sterile gene Recessive gametophyte sterility gene Single gametophyte sterility gene Single gametophyte sterility gene – –

Linkage group (LG)

Note: This table is adopted from Li et al. (2019) with minor modifications

167 (RNTED) ZD8319

YB

CMS name

Female (cytoplasmic)

Male (nuclear)

Table 8.2 (continued)

Zhao et al. (2009), Wang et al. (2010) –

Zhao et al. (1998) Zhao et al. (2004, 2009)

Zhao et al. (1998, 2005)

Zhao et al. (1998)

References

180 S. Chandra et al.

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Achievements, Challenges and Prospects of Hybrid Soybean

8.4.4

181

Mutation for Male Sterility System in Soybean

In many crops, mutation in genes tangled with microsporogenesis and/or microgametogenesis results in male sterility (Bohra et al. 2016). Mainly genetic alteration in genomic regions tangled in synapsis governs for modifications in phenotype, viz. male-sterile female-sterile, male-sterile female-fertile, or male-fertile female-sterile, in plants. In soybean, a total of 11 male-sterile, female-fertile (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, msMOS and msp) mutants have been discovered, registered and mapped onto different 20 chromosomes. Based on genetic analysis and test of allelism, all 11 mutants were found to be genetically independent to each other. Additionally cytological reports on these mutants explain about diverse mechanism behind the development of the male-sterile phenotype, which mainly includes cytokinesis failure during telophase II, failure of tetrad formation during microsporogenesis, abnormality/lack of vacuoles, microspore and pollen degeneration and low callose level. Fine mapping and map-based cloning of ms4 mutant revealed that the continuous spontaneous mutation at ms4 locus is accountable for the male sterility, whereas MS4 gene is involved in the production of normal stamens, successful tetrad formation, fertile pollens and viable seeds (Zhang et al. 2017). Thus, genetic and molecular characterization of male sterility genes may assist to develop stable male-sterile parental lines highly required for the efficient hybrid seed production in soybean.

8.5

Hybrid Development in Soybean

Soybean is a highly self-pollinated crop, so production of true F1s/hybrid seeds is tedious in general. Manual cross-pollination to develop F1 seeds for hybrid soybean seed is very difficult and time-consuming; however pollination without emasculation (Talukdar and Shivakumar 2012) technique may be useful to produce F1 seeds in small quantity or identify the best cross combination during genetic tests. In selfpollinated crops, flower morphology, flowering timings, pollinator insects, etc. decide the fate of cross-pollination in natural field conditions. Likewise in rice, short fluorescence and concentrated flowering time within a week favours for outcrossing, so through enhanced wind pollination, hybrid rice may be developed easily than other some self-pollinated crops (Li et al. 2003). However, soybean is having natural outcrossing rate lower than 1% (Ray et al. 2003). Soybean plants have dispersed flowers throughout at flora with around 15–30-day-long flowering period. It has particular concentrated flowering time in few morning hours with morning dew which reduce the possibility for emergence of viable pollens for outcrossing (Dai et al. 2017). Therefore, economic and efficient hybrid seed production technique is impatiently required for hybrid soybeans. There are two basic needs for hybrid seed production in soybean: first is a stable male-sterile line with adequate exogenous pollen source; second is that effective pollinators are required for transferring pollens from male anthers to the male-sterile gynoecium (Palmer et al. 2001). Effective and viable pollens are pivot for hybrid

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seed production in soybean because the stigma of the flowers on male-sterile lines is not exposing in nature and the pollen liveliness period is comparatively very less under the natural field conditions, which cause poor feasibility for wind pollination, so fertilization on male-sterile flowers in soybean has to rely on insects in the field. Luckily, complete nectarium system available in soybean can invite bees and other insects for outcrossing (Erickson 1984). Ray et al. (2003) reported about 6.32% outcrossing as highest, under natural conditions in field, suggesting a role of insect in hybrid soybean. Several workers investigated outcrossing in soybean with different types of bees, i.e. bees (Apis mellifera), alfalfa cutting bees (Megachile rotundata), etc., and utilized in studies of male-sterile lines and recurrent selections (Ortiz-Perez et al. 2006, 2008; Dai et al. 2017). There are several biological factors and environmental factors which affect insect diversity and outcrossing via insect pollinators in soybean. Dai et al. (2017) reported that nectar amount and nectarium structure in flower may be the important indicators in the breeding programs for hybrid soybeans. In their studies, they found that planting season without morning dew may improve the outcrossing in soybeans (Dai et al. 2017). Another important aspect for hybrid soybean is male-sterile lines with companion pollen source. Both male-sterile line systems have been utilized for hybrid seed in soybean. In two-line systems, male sterility is propagated only by the heterozygotes with male-sterile plants, so both types of plants (male-sterile and male-fertile) will be equal in numbers in the next generation. So identification of male fertile in population is challenging. In ‘traditional method’ (Specht and Graef 1992), it is identified at flowering by anther observation and identification of a pollen source. In this method, rogueing plants by inspecting anthers are time-consuming. Male-sterile plants often got fertilized with pollen grain from fertile plants in same plot before the fertile siblings can be removed. In another method ‘dilution method’, the desired pollen parent is mixed more times than fertile siblings expected in the pod-parent line, so it enhances the possibility of outcrossing, but same time this method needs more land and pollen-parent seed is required. But rogueing is not required in this method; however possible difficulty to identifying male-sterile plants among male-fertile plants still is there. A third method, ‘cosegregation’ method, of F1 seed production avoids most of the difficulties of the traditional and dilution methods. This method takes advantage of the close genetic linkage in between the W1 and Ms6 loci (Lewers et al. 1996). The W1 locus is involved in anthocyanin pigment production, so malefertile plants could be identified by hypocotyl colour in initial phase. The segregation method produced higher seed yield with good efficiency and better seed quality than both previous methods (Lewers et al. 1996). The cosegregation technique may be useful for male-sterile-enabled selections and marker-assisted recurrent selection (Lewers and Palmer 1997) for variety development. The two-line system/GMS may play an important role in hybrid seed CMS system, and the three-line system is generally considered a better choice for large-scale seed production (Dalvi et al. 2010). This male-sterile system has been utilized in different legumes, especially in pigeon pea and soybeans (Saxena et al. 2018). For soybean, China emerged as the world’s leader like hybrid rice. China has released its first soybean hybrid ‘HybSoy 1’ in the world during year 2002 through continuous backcrossing

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Table 8.3 List of soybean hybrids released in China Female Yield advantage (%) parent over check genotype Name Male parent Hybrids with disease resistance as the main characteristics HybSoy 1 Jihui 1 JLCMS9 A 20.8 Zayoudou No. 1 WR016 W931 A 19.14 HybSoy 2 JLR2 JLCMS47 A 22.7 HybSoy 3 JLR9 JLCMS8 A 19.2 Jiyu 606 JLR100 JLCMS47 A 16.9 Jiyu 607 JLR83 JLCMS14 A 11.2 Hybrids with grain quality traits as the main characteristics FuHybSoy 1 Fuhui 6 FuCMS5 A 13.33 Zayoudou No. 2 WR99071 W931 A 0.79 HybSoy 5 JLR1 JLCMS84 A 19.7

References Zhao et al. (2004) Zhang et al. (2007) Peng et al. (2008) Peng et al. (2010) Peng et al. (2013a) Peng et al. (2013b) Li and Zhou (2014) Huang et al. (2013) Peng et al. (2011)

Note: This table is adopted from Li et al. (2019) with minor modifications

(JLCMS9A  Jihui 1) (Zhao et al. 2004). ‘HybSoy 1’ depicted about 20% higher yield than control (‘Jilin 30’) with strong disease resistance (SMV). Zhang et al. (2007) reported ‘Zayoudou No. 1’ (world’s first summer soybean hybrid) by crossing between W931A and WR016. Subsequently, many hybrid cultivars of soybean such as HybSoy 2, HybSoy 3, Jiyu 606 and Jiyu 607 were developed with disease resistance and released for commercial purposes (Table 8.3) (Peng et al. 2008, 2010, 2013a, b). Many soybean hybrids with better grain quality, viz. HybSoy 5, Zayoudou No. 2 and FuHybSoy 1, have been released in China (Table 8.3) (Peng et al. 2011; Huang et al. 2013; Li and Zhou 2014). In India, no hybrid has been developed and released so far. For this, a dedicated long-term project is necessary to get advantage on a quid pro quo (something for something in return) basis (Tiwari 2014).

8.6

Genomic Tools for Molecular Understanding of the CMS/Restoration and Assisting Hybrid Breeding

Molecular markers are a potential tool for different aspects of marker-assisted breeding (Bohra et al. 2020). For heterosis breeding and hybrid development, development of stable male-sterile line and restorer line using diverse sources is a routine process. Identification of markers associated with male sterility and fertility restorer genes fastens the introgression of MS/restoration in the desired diverse genetic background (Bohra et al. 2016, 2017). There are several molecular markers (AFLP, SSR, SNP, etc.) identified which are associated with different GMS and CMS genes in soybean (Tables 8.1 and 8.2). It has been also suggested that molecular marker-based genetic diversity may be used to select diverse parents for hybridization (Chandra et al. 2020b). Several studies have been reported for undertone between molecular genetic distance and heterosis expression (Melchinger et al. 1990). Significant correlations between RFLP-based genetic diversity and heterosis

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expression of yield were reported in maize (Smith et al. 1990). High-yielding lines had been developed in soybean by hybridizing genotypes possessing significantly more genetic distances (Kiang and Gorman 1983). Appropriate parental combinations and an economical method of producing hybrid seed are a must for the success of soybean hybrid technology (Perez et al. 2009), where molecular markers assist with different means. Molecular understanding of male sterility and heterosis is important to breed new and improved hybrid varieties (Mishra and Bohra 2018). Further, identification of genes and metabolites conditioning male sterility will help in the development of the desired male-sterile lines using new genome editing tools. Recently, Nadeem et al. (2021) cloned and characterized GmMS1 gene from a male-sterile soybean line SYMS01. GmMS1 (Glyma.13G114200) has homology with a gene which encodes a kinesin-like protein NACK2 in Arabidopsis. NACK2 encodes TETRASPORE, which is involved in meiotic cytokinesis (Spielman et al. 1997). CRISPR/Cas9mediated GmMS1 knockout lines displayed male-sterile phenotype (Nadeem et al. 2021). Metabolic profiling of male-sterile and male-fertile individuals showed that there were significant differences in phenylpropanoids, plant hormones, starch and sucrose metabolite level. Male-fertile individuals had more of trans-zeatin-7-glucoside and N6-isopentene adenine, while sterile plants had more zeatin nucleoside and jasmonate isoleucine. The identification of GmMS1 should facilitate heterosis breeding in soybean. Transcriptome analysis can be used to identify differential gene expression and their network involved in controlling male sterility (Bohra et al. 2021a, b). The comparative gene expression analysis between CMS line NJCMS1A and its maintainer NJCMS1B identified that 339 genes were downregulated and 26 were upregulated in NJCMS1A, as compared to NJCMS1B (Li et al. 2015). These differentially expressed genes (DEGs) were predicted to be involved in carbohydrate and energy metabolism, regulation of pollen development, cellular signal transduction, transcription factors, elimination of reactive oxygen species, programmed cell death, etc. Among identified DEGs, 38 were found to be involved in regulation of pollen development. Of 38 DEGs, 34 genes were predicted to participate in the regulation of pollen wall development, and all have shown lower expression in NJCMS1A compared to NJCMS1B. CMS in plants is associated with mitochondrial genome rearrangement, and fertility restorer (Rf) genes could suppress or activate many mitochondrial genes restoring fertility (Bentolila and Stefanov 2012; Chen et al. 2017). Restriction endonuclease analysis of mitochondrial genome between CMS line NJCMS1A and its maintainer NJCMS1B identified significant differences in three restriction digestion maps (Ding et al. 1999). Jiang et al. (2011a, b) compared atp9 gene sequence of CMS line NJCMS2A and its maintainer NJCMS2B and found that identical sequence was obtained from the genomic DNA of NJCMS2A and NJCMS2B, but differences were observed when coding DNA sequences were amplified from cDNAs of NJCMS2A and NJCMS2B. RNA editing was detected at two nucleotide sites (C to U) in the conserved region of atp9 gene of the maintainer NJCMS2B; this in turn leads to conversion of serine (hydrophilic amino acid) into

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leucine (hydrophobic amino acid), whereas no RNA editing was detected in cDNA sequence of atp9 in the male-sterile line NJCMS2A. To understand the molecular basis of heterosis, Taliercio et al. (2017) performed comparative transcriptome analysis between F1 hybrid and its parents. Hypothetically the likely basis of many of inherited phenotypes should be the recombinant additive gene expression pattern. However, differential gene expression analysis identified between the F1 hybrids, and both parents was not additive (Taliercio et al. 2017). These genes were expressed at a lower (6–31 genes) or higher level (20–25 genes) in the F1 hybrid than both parents. Zhang et al. (2017) also studied transcriptome of two hybrids HYBSOY-1 and HYBSOY-5 and their parental lines. A total of 146 and 193 DEGs were identified showing dominance of parental expression in HYBSOY-1 and HYBSOY-5, respectively. Among all DEGs, 169 DEGs showed additive, transgressive downregulation and transgressive upregulation expression, in the two F1 hybrids, and were identified as potential heterosis genes. But they could not find a common set of parental expression-level dominance genes for HYBSOY-1 and HYBSOY-5. Invention and advancements in genome editing techniques allowed targeted modification in plant architecture. Particularly CRISPR/Cas9 system is now commonly used for genome editing in crop plants. Chen et al. (2021) used CRISPR/Cas9 editing technology for mutating ABORTED MICROSPORES (AMS) gene. AMS gene affects tapetal development, and aborted microspores (ams) mutant showed male-sterile phenotype in Arabidopsis (Sorensen et al. 2003). Soybean genome contains two AMS genes. Targeted editing of GmAMS1 gene generated the male-sterile phenotype but not by GmAMS2. Histological studies showed that GmAMS1 controls development of pollen wall and degradation of tapetum (Chen et al. 2021).

8.7

Challenges of Hybrid Soybean

Soybean is a self-pollinated crop, and its floral biology favours complete selffertilization. Looking at the success of hybrids in self-pollinated crop like rice, soybean crop needs male sterility system for production of hybrid seeds and efficient pollination mechanism. However, many GMS and CGSM lines have been identified in soybean to exploit heterosis phenomena through two- and three-line breeding strategies, respectively. Utilizing three-line breeding approaches, few hybrids of soybean also has been released in China (Table 8.3). In India, CGMS/GMS have not utilized yet to develop F1s, hybrids, etc. In India, artificial hand pollinations have been attempted to develop F1 seeds which are very limited in terms of the number of true F1s produced. A lot of opportunities are available for hybrid soybean, but there are many challenges also which are as follows: • • • •

Low level of heterosis has been found in soybean in comparison to other crops. Natural outcrossing is very poor (0–1%) in soybean. Genetic base of male-sterile lines and restorers is narrow in general in soybean. Very few strong dominant restorer lines are available in soybean.

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• Lack of availability of stable CMS types in soybean. • Pollination of male-sterile lines with exogenous pollens of restorers in fields highly depends on weather conditions, i.e. temperature, humidity, etc. • Outcrossing in natural fields highly depends on diversity and populations of insects/bees. • Seed setting in outcrossed pod in soybean is less than other crops. • Soybean is a high-volume crop and requires high seed rate, so commercial production of hybrid/F1s seed in large quantity is difficult than other crops. • Unlike cereals, one pollination may yield maximum of four F1 seeds which again limits the fulfilment of high volume of hybrid seed demand.

8.8

Future Prospects

Productivity enhancement of the oilseeds particularly in soybean is remaining a challenge for the breeders. In this direction, development and popularization of hybrid soybean could be an option to break the yield barrier. Development of male sterility lines for two-/three-line breeding and efficient seed production is essential for producing large quantity of hybrid seed. New opportunities of molecular biology and gene editing-based approaches which can change soybean flower biology to favour the cross-pollination by hand or insects may be helpful. In case of soybean varieties, the recommended seed requirement per ha is 60 kg which is a very high volume. For hybrid soybean, suitable agronomic practices including plant geometry need to be worked out to exploit the full potential of hybrids. Presently very less information is available on the success of hybrid soybean cultivation, to establish its superiority in yield advantage over the varieties. Large area testing of hybrid soybean comparative analysis will guide the future path for attaining benefit of hybrid soybean. The following areas need to be emphasized for planning and implementing hybrid soybean breeding: • Desired alternation in soybean flower biology to favour outcrossing through pre-breeding/molecular biology/genome editing approaches. • Development of male-sterile and restorer lines with abundant nectarium to attract insect/bee for pollination in fields. • Development of high-yielding hybrids specific to agro-ecological regions is desired. • Diversification of parental lines to suit a variety of target environments. • Incorporation of resistances to biotic stresses especially anthracnose, charcoal rot, Rhizoctonia rot, etc. • Development of cost-effective hybrid seed production technology with increased efficiency. • Conduction of basic agronomical research for improved productivity of hybrid soybean. • Genomic characterization of hybrids and their parents in soybean.

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• Construction of high heterotic groups using molecular techniques and identification of heterotic patterns. • Exploring the potential of modern breeding techniques like genome editing/ genomic selection in hybrid breeding.

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Zhang JF, Stewart JM (2001) CMS-D8 restoration in cotton is conditioned by one dominant gene. Crop Sci 41:283–288 Zhang L, Dai OH, Huang ZP, Li JK (1999) Selection of soybean male sterile line of nucleocytoplasmic interaction and its fertility. Sci Agric Sin 32:34–38 Zhang L, Dai OH, Huang ZP, Li JK, Zhang LY, Hu C (2007) Breeding of hybrid soybean Zayoudou No. 1. Soybean Bull 2:14–16 Zhang C, Lin C, Fu F, Zhong X, Peng B, Yan H et al (2017) Comparative transcriptome analysis of flower heterosis in two soybean F1 hybrids by RNA-seq. PLoS One 12(7):e0181061 Zhang DF, Wu SW, An XL, Xie K, Dong ZY, Zhou Y et al (2018) Construction of a multicontrol sterility system for a maize male-sterile line and hybrid seed production based on the ZmMs7 gene encoding a PHD-finger transcription factor. Plant Biotechnol J 16:459–471 Zhao TJ, Gai JY (2006) Discovery of new male-sterile cytoplasm sources and development of a new cytoplasmic- nuclear male-sterile line NJCMS3A in soybean. Euphytica 152:387–396 Zhao LM, Liu DP, Sun H (1995) A sterile material of soybean gained by introducing exogenous DNA. Soybean Science 14:83–87 Zhao LM, Sun H, Huang M (1998) The development and preliminary studies on cytoplasmic malesterile soybean line ZA. Soybean Science 17:268–270 Zhao LM, Sun H, Wang SM, Wang YQ, Huang M, Li JP (2004) Breeding of hybrid soybean HybSoy 1. Chin J Oil Crop Sci 26:15–17 Zhao TJ, Yang SP, Gai JY (2005) Discovery of a dominant nuclear male sterile mutant N7241S in soybean and analysis of its inheritance. Sci Agric Sin 38:22–26 Zhao SJ, Zhang MC, Jiang CZ, Yang CY, Liu BQ et al (2006) Studies on quality improvement effect and separate character of soybean male sterile (MS1) recurrent selection population. Sci Agric Sin 39:2422–2427 Zhao SJ, Zhang MC, Jiang CZ, Yang CY et al (2007) Study on quality improvement effect and separate character of soybean male sterile (MS1) recurrent selection population. Agric Sci China 6:545–551 Zhao L, Sun H, Peng B, Li J, Wang S, Li M et al (2009) Pollinator effects on genotypically distinct soybean cytoplasmic male sterile lines. Crop Sci 49:2080–2086 Zhao LM, Sun H, Peng B, Zhang CB, Zhang WL, Zhang JY et al (2012) Genetic modes analysis of the RN and ZD-type cytoplasmic male sterility lines in soybean. In: International conference on utilization of Heterosis in crops, pp 105–106 Zhao QS, Tong Y, Yang CY, Yang YQ et al (2019) Identification and mapping of a new soybean male-sterile gene, mst- M. Front Plant Sci 10:94

9

Recent Progress in Brassica Hybrid Breeding Javed Akhatar, Hitesh Kumar, and Harjeevan Kaur

Abstract

Rapeseed-mustard is a second important edible oilseed crop globally. India is the biggest importer of edible oilseeds despite ranked as second in cultivated area and fourth in production of rapeseed-mustard. The low productivity along with biotic and abiotic stresses is a limiting factor for cultivation on a larger scale. The required genetic gain could not be achieved alone by current varietal development programmes. Therefore, it should be accompanying with hybrid development programme. The heterosis breeding is a vital option to break the yield barrier to increase productivity which could meet our current and future edible oil’s requirement. Several efficient stable cytoplasmic male sterility systems with fertility restorer, understanding genetic basis of heterosis and established heterotic gene pool have laid the foundation of hybrid breeding. As a result, six hybrids of rapeseed-mustard have been released with improved genetic potential for commercial cultivation in India. Further, to meet the country’s edible oil requirement, hybrid breeding has to be exploited fully to achieve yield potential in rapeseed-mustard. Keywords

Alloplasmic · Epigenetics · Hybrid · Molecular markers · Pollen viability

J. Akhatar · H. Kaur (*) Punjab Agricultural University, Ludhiana, Punjab, India H. Kumar Banda University of Agriculture and Technology, Banda, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_9

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Introduction

Oilseed brassicas, also referred to as rapeseed-mustard, represent an important group of edible oilseed crop. Rapeseed-mustard is a group of three diploid progenitor species Brassica rapa (AA genome, 2n ¼ 20), B. nigra (BB genome, 2n ¼ 16) and B. oleracea (CC genome, 2n ¼ 18), and three amphidiploid species B. juncea (AABB genome, 2n ¼ 4x ¼ 36), B. napus (AACC genome, 2n ¼ 4x ¼ 38) and B. carinata (BBCC genome, 2n ¼ 4x ¼ 34) are evolved by natural hybridization of three diploid species (U, 1935). Among six spp. four leading cultivated brassica species are B. rapa, B. juncea, B. napus and B. carinata which contribute to major oilseed production globally. In trade B. rapa (Toria, brown sarson, yellow sarson) and B. napus (gobhi sarson, canola) are categorized as ‘rapeseed’, and B. juncea (Indian mustard) and B. carinata (African sarson) are known as ‘mustard’. Indian mustard is largely grown in Asia; however, B. napus L. and B. rapa L. are cultivated in Europe, Canada and China under winter and summer planting season (Rai et al. 2007). Among nine major oilseed crops, Brassica ranks among the second largest crop group of edible oilseeds with a global production of 73,209 thousand metric tonnes after soybean (366,231 thousand metric tonnes) which was cultivated in diverse geographical regions of the world during 2020–2021 (USDA 2021). The highest rapeseed-mustard was produced by Canada 19.48 MMT by the European Union (16.57 MMT), China (14.00 MMT) and India (9.12 MMT) during 2019–2020 (OECD/FAO 2020). In India, rapeseed-mustard is a major oilseed crop occupying an area of 23.5% with productivity of 24.2% out of total oilseeds. Despite the third largest producer, India ranked seventh in importing edible oils and fulfilled nearly 57% domestic requirement (Jat et al. 2019). Since the inception of organized breeding effort in the past, significant increase in productivity has been achieved by Brassica varietal development programme for commercial cultivation along with improved quality oil traits. Despite the long breeding effort, the average productivity levels are far from genetic yield potential. In addition, Brassica crop productivity is hampered by several biotic and abiotic stress challenges. A total of 248 varieties including six hybrids of rapeseed-mustard have been released with improved genetic potential. These hybrids are tolerant to biotic stresses such as white rust, Alternaria blight and powdery mildew and to abiotic stresses such as salinity and high temperature. Moreover, oil and meal quality traits like erucic acid and glucosinolate have been improved in these varieties and hybrids (DRMR 2021). Availability of various varieties and improved technologies are not sufficient to meet our current and future demands. The projected edible oil demand will be 25 million tonnes by 2030 for increasing population under declining natural resources which cannot be met by the available varieties. The extent of heterosis ranged 10–15% in Brassica which is the key indicator of the yield enhancement in mustard crop. Thus, heterosis breeding is a conceivable alternative approach to improve crop productivity, production and edible oil availability. To ease hybrid breeding, several highly efficient pollination control

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mechanisms cytoplasmic male sterility (CMS) systems and fertility-restoration systems have been exploited commercially in mustard crop for hybrid seed production. A number of CMS systems such as Ogura (ogu), Polima (pol), oxyrrhina (oxy), tournefortii (tour), Moricandia (mori), Enarthrocarpus lyratus (lyr) and B. fruticulosa ( fruit) have been discovered in Brassica spp. (Ogura 1968; Fu 1981; Banga and Banga 1997; Prakash et al. 1998; Rana et al. 2019). For production of commercial hybrids in B. juncea and B. napus, stable CMS-RF systems (Mori and Ogu) are widely used. This chapter will discuss heterosis breeding, pollination control mechanism, genetics of heterosis and development of heterotic gene pool and present the status of hybrid development to meet the productivity enhancement in rapeseed-mustard.

9.2

Status of Hybrid Breeding

Heterosis in oilseed brassica has been reported by Sun (1943), and subsequently numerous studies estimated a significant level of yield heterosis in brassicas (B. rapa and B. juncea) (Singh 1973; Schuster and Michael 1976; Lefort-Buson et al. 1982; Sernyk and Stefansson 1983; Brandle and McVetty 1990; Larik and Hussain 1990; Schuler et al. 1992; Pradhan et al. 1993a). Later on, in 1968, Ogura system has upturned the assurance of hybrid breeding in Brassica. The Ogura cytoplasmic male sterility (CMS) system of radish (Raphanus sativus L.) was introgressed in Indian mustard (Banga and Labana 1984). In India, the significant level of heterosis for yield—11 to 82% in B. juncea (Banga and Labana 1984; Kumar et al. 1990; Thakur and Bhateria 1993; Verma et al. 1998), 10–72% in B. napus (Rai 1995; Thakur and Sagwal 1997) and 20–107% in B. campestris (Patnaik and Murty 1978; Verma et al. 1989; Srivastava and Rai 1993; Yada et al. 1998)—were studied. A sufficient level of heterosis has to be identified for crossing between genetically distant groups and to use it for exploitation in hybrid breeding in brassicas. In most cultivated Brassica crops, there is 14–30% natural outcrossing which is adequate to develop CMS and fertility restorer lines for hybrid seed production. Principal components of yield heterosis include traits such as the number of branches, pods number and seed size which have lower heritability, are also affected by population density and have to be improved by different methods (Labana et al. 1978; Thakur and Sagwal 1997). Lower expression of heterosis is observed for harvest index and oil percent due to some inherent physiological barriers. Majority of studies involving heterosis reports the estimation of lower heterosis over mid-parental value, heterobeltiosis and small population size as compared to commercial heterosis and CMS-based studies.

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Table 9.1 Development of alloplasmic male sterility systems for brassicas CMS Raphanus/ ogu siifolia Trachystoma Moricandia catholica lyratus oxyrrhina canariense erucoides berthautii

Cytoplasm donor Raphanus sativus

Discovered by Ogura (1968)

Fertility restoration Restorer gene in B. juncea NRA Single dominant gene Single dominant gene RNIU RNIU

Diplotaxis siifolia Trachystoma ballii Moricandia arvensis D. catholica Enarthrocarpus lyratus B. oxyrrhina Erucastrum canariense D. erucoides D. berthautii

Rao et al. (1994) Kirti et al. (1995a, b, c) Prakash et al. (1995) Kirti et al. (1995a, b, c) Banga and Banga (1997) Prakash et al. (2001) Prakash et al. (2001)

NRA RNIU

Bhat et al. (2006) Bhat et al. (2008)

RNIU RNIU

NRA no restoration available, RNIU reported but not in use

9.3

Pollination Control System in Brassica

A large number of male sterile forms have been investigated for hybrid development in oilseed brassicas (Stiewe and Röbbelen 1994; Prakash et al. 1995). Out of these, CMS systems have been rapidly used by breeders worldwide for the production of hybrids (Bohra et al. 2016, 2017). However, other male sterility systems have been only developed for research purposes. Most of the CMS sources have been studied intensively, and some are under development stage. A number of CMS systems listed in Table 9.1. In western countries, Raphanus-based Ogura CMS and Polima (pol) CMS are used, whereas in India most breeders are using B. tournefortii CMS, B. juncea CMS, Polima CMS and siifolia CMS systems. Fortunately, Brassica breeders from Punjab Agricultural University, Ludhiana (India), have recommended the release of the first CMS-based B. napus (PGSH-51) and B. juncea (RCH-1) hybrid for cultivation in Punjab (India). In B. juncea, alien introgression programme employs the exploitation of alloplasmic variation for the development of cytoplasmic male sterility-fertility restoration. Wild crucifers backcross substitution with Brassica genomes (B. rapa and B. juncea) could be used for development of CMS lines. Wild crop species also serve as bridging species to facilitate the somatic hybrids or sexually synthesized allopolyploids. For such crosses, wild species was used a female parent. As expected, maternal inheritance plays an exclusive role by unaltered organelle genomes in sexual hybridizations for CMS systems. Earlier, for CMS line development, various combinations of mitochondrial, chloroplast and somatic hybrids have been studied, and thus cytoplasmic genomic constitution varies from these (Prakash et al. 2009). Successful establishment of hybrid seed production is based on threeline hybrid breeding system, i.e. CMS line, maintainer line (same nucleus from CMS

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line) and fertility restorer line encompassing the Rf gene(s) (Chen and Liu 2014; Yamagishi and Bhat 2014; Bohra et al. 2016; Mishra and Bohra 2018).

9.4

Male Sterility and Fertility Restoration

When alien species is used as cytoplasmic donor with genetic background of other species, it is called as cytoplasmic male sterility. A male sterile line is considered to be of immense importance in facilitating the efficient production of hybrid seeds in oilseed brassicas by the use of wild relatives. Several CMS systems have been successfully developed from intergeneric and interspecific hybridizations. In general, alloplasmic CMS lines would show normal plant development and morphological characteristics as euplasmic lines. Many of them exhibit abnormal plant development like flowering abnormalities, poor or absent nectarines, chlorosis, lower seed fertility, crooked style and thick pistil as a consequence of altered nucleo-cytoplasmic interactions. Fertility restoration has been introduced and developed from cytoplasmic wild species used as a donor by introgressing gene(s) for catholica, Ogura, erucoides, Moricandia and lyratus CMS system (Banga and Banga 2009; Prakash et al. 2009). At present, mori and Ogura CMS systems are widely used for production of hybrid seeds at a commercial scale. Most widely and adoptive Ogura CMS system used in B. oleracea, B. juncea and B. napus describes use in Japanese radish (R. sativus) (Ogura 1968). This is one of the CMS systems which has been studied in terms of molecular mechanism and practised in Brassica breeding. Some European radishes have an Rf gene, whereas no such gene has been observed in Ogura CMS (Bannerot et al. 1977; Bonnet 1977). Intergeneric hybridization with repeated backcrossing of Ogura CMS has introduced and showed male sterility in alloplasmic B. napus lines. These lines show chlorotic leaves (yellows at temperatures 15  C) due to functional incompatibility between R. sativus chloroplasts and B. napus nucleus. To overcome chlorosis, cell fusion approaches were applied in Ogura CMS in B. napus and B. juncea (Pelletier et al. 1983; Jarl and Bornman 1988; Kirti et al. 1993, 1995c). Recombination events were observed in mitochondrial genome of B. napus and Ogura CMS, and causal genes were identified for male sterility (Bonhomme et al. 1991, 1992; Grelon et al. 1994). CMS types are defined based on their origins, which are based on the combination of mitochondrial genome and nuclear genome that lack Rf genes. It is caused by intraspecific variation, interspecific/intergeneric hybridization and cell fusion.

9.5

Intraspecific Variation-Induced CMS

Polima (pol) CMS is well-known studied at the molecular level in B. napus. This system is temperature sensitive with limited practical usage in hybrid breeding (Liu et al. 1978; Fu 1981). Pol plants contain a causal CMS gene—orf224—in mitochondrial genome and is transcribed together with atp6 which specifically alters the transcripts (L’Homme and Brown 1993; Singh and Brown 1993). In addition,

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introduction of orf224 in B. oleracea by somatic cell fusion induced male sterility (Wang et al. 1995). Line 681A in B. napus and hau in B. juncea had been identified as another spontaneous CMS variants and are suggested as same maintainer and restorer for pol (Wan et al. 2008). pol CMS might be a variant of 681A lines because of different mitochondrial genome organizations. Another novel CMS gene orf288 expressed together with atp6 induced male sterility in A. thaliana with expression from nuclear genome and mitochondrial presequences and also causal gene of hau CMS.

9.6

Alloplasmic CMS Caused by Interspecific or Intergeneric Hybridizations

In B. rapa, several male sterility lines were developed by establishment of CMS lines with cytoplasm of Diplotaxis muralis (Hinata and Konno 1979). In Southern Asia, some most important species has been inducing CMS in B. juncea used as cytoplasm (Prakash and Chopra 1990; Rao et al. 1994; Malik et al. 1999; Banga et al. 2003; Kumar et al. 2012). The cytoplasm of D. muralis had been used to introduce male sterility in B. rapa and B. oleracea (Deol et al. 2003; Shinada et al. 2006; Tsutsui et al. 2011) (Table 9.2). Interspecific hybridization between B. rapa and B. juncea induced CMS in B. juncea, and later on, CMS was transferred to leaf mustard (var. multiceps) (Yang et al. 2005) and stem mustard (Yang et al. 2009). Table 9.2 Combination of recipient and cytoplasmic donor used to produce CMS plant in alloplasmic and cell fusion Recipient B. rapa

B. oleracea B. juncea

B. napus

Raphanus sativus a

Cytoplasm donor In alloplasmic CMS B. oxyrrhinaa, Eruca sativa, Diplotaxis muralisa, Enarthrocarpus lyratusa, Moricandia arvensisa D. muralisa, M. arvensisa, Erucastrum canariensea B. oxyrrhinaa, B. tournefortiia, E. lyratusa, E. canariensea, D. berthautii, D. siifoliaa, D. catholicab, D. erucoides B. tournefortiia, D. muralisa, E. lyratusa, D. siifoliaa B. maurorum

CMS in more than one Brassicaceae species

By cell fusion Raphanus sativus

Arabidopsis thaliana Diplotaxis catholica, Trachystoma ballii Moricandia arvensis B. tournefortii, A. thaliana, Orychophragmus violaceus, Sinapis arvensis –

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CMS as a Result of Cell Fusion

Cell fusion doesn’t require fertilization like sexual hybridization, and thus there is a wider range of availability of crossing species. As depicted in Table 9.2, somatic cell fusion approach has been used to transfer cytoplasm from A. thaliana, Trachystoma ballii, Orychophragmus violaceus and Sinapis arvensis into B. napus, B. juncea or B. oleracea to develop CMS (Kirti et al. 1993; Forsberg et al. 1994, 1998a, b; Kirti et al. 1995b; Prakash et al. 1995; Yamagishi et al. 2002; Yamagishi and Nakagawa 2004). Identification of CMS causal gene is more complicated in somatic hybridization as compared to sexual hybridization for development of alloplasmic CMS lines. Gene orf108 upstream of atpA as CMS casual gene was observed in the somatic hybrid lines made by cross-pollinating B. juncea and M. arvensis, similarly found in alloplasmic CMS plants (Prakash et al. 1998). Another genes such as ofr193 and orf263 have been identified as causal CMS genes in progeny of a somatic hybrids like observed in alloplasmic CMS plants (Stiewe and Röbbelen 1994; Dieterich et al. 2003).

9.8

Restorer Fertility (Rf) Genes

Seed must be male fertile for producing F1 hybrid seeds by CMS system in oilseed hybrid breeding. Therefore, critical exploitation of CMS system to discover Rf genes is required which are insuring male fertility to CMS lines. Rf genes were usually found among the germplasm lines used for intraspecific cytoplasmic variants. By introgression of Rf genes from cytoplasm donor species into recipient species, many cultivated Brassica lines were successfully introgressed for various CMS systems such as T. ballii (Kirti et al. 1997), E. canariense (Prakash et al. 2001) and E. lyratus (Banga et al. 2003). In Brassica CMS plants, majority of fertility restoration is sporophytic, and some of other CMS lines (M. arvensis, D. berthautii and D. erucoides) show gametophytic fertility restoration (Bhat et al. 2005), i.e. F1 plants are 50% male fertile if they carry Rf gene. A very interesting relationship has been observed between co-evolution of nuclear and mitochondrial genomes and has been also studied for alloplasmic CMS lines of B. juncea, pol and nap CMS of B. napus and Ogura CMS. Rf gene has two alleles—Rfp and Rfn for pol and nap. They remove the sequences from 50 -transcripts end and alter the mRNA processing of CMS genes (Li et al. 1998; Menassa et al. 1999). In Ogura CMS, causal Rf gene—orf138—has evolved a different molecular mechanism in radish to suppress this gene’s expression. Some Rf gene does not affect the transcription but regulates orf138 mRNA translation, and others regulate orf138 mRNA processing (Brown et al. 2003; Koizuka et al. 2003; Yasumoto et al. 2008, 2009).

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Genetically Engineered Male Sterility

The hybrid seed production in Brassica CMS systems is used worldwide with certain constraints such as partial or unstable or inefficient fertility restoration systems (Jagannath et al. 2002; Bisht et al. 2004a, b; Martinez et al. 2009). Genetic engineering ensures to overcome these limitations and develop techniques for fully functional male sterility systems. In B. juncea, a complete male sterility/restoration system has been developed in a study by misfunction of the tapetum cell layers of pollens which render them non-functional (Jagannath et al. 2001). This system is known as barnase-barstar system which works by constructing a spacer DNA sequence between barnase gene and CaMV35S promoter-driven BAR gene. Highfrequency male sterility/restoration for hybrid seed production can be identified by this system in Indian mustard. Different combinations were evaluated by crosspollinating wild-type and modified barstar lines. Out of these, only one combination was observed in F1 plant with effective restoration of male fertility. Another success story was reported based on a two gene-two promoter system approach for improving fertility restoration in B. juncea with enhanced expression of barstar gene (Bisht et al. 2004a, b). They retransformed a transgenic male sterility by using two different barstar constructs; resultant 66–90% plants had fertility restoration, up to 30% contained single gene copy and 90% transgenic plants demonstrated high pollen viability in Indian mustard. Segregated T1 progeny (male sterile (barnase) and restorer (barstar)) could be diversified into suitable hybrid development.

9.10

Genetic Basis of Heterosis

Heterosis or hybrid vigour is the phenomenon where hybrids formed are more vigorous and robust than their parents. In plants, heterosis results in increase in traits such as growth rate, size, fertility, yield, etc. in the hybrids. The first progeny shows the desired characteristic in higher measure than the subsequent progenies, and thus the purity of parental lines should be maintained. Heterosis is a complicated biological process caused by the combination of various genes/factors. Numerous studies till date elucidate the genetic basis of heterosis. Genetically, heterosis has been explained by various mechanisms such as dominance, overdominance, epistasis, etc. These models are based on the allelic and non-allelic interactions. Dominance and overdominance hypotheses were first proposed in 1908. With the advent of various markers such as restriction fragment length polymorphism (RFLP), allozymes and high-density molecular linkage maps, the genetic mechanism of hybrid vigour had been validated in crops such as maize, rice, wheat, etc.

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Dominance Hypothesis

In 1908, dominance hypothesis was proposed (Davenport 1908) and was explained later on in the 1910s (Bruce 1910; Jones 1917). According to it, desirable genes are dominant or superior over undesirable genes which are recessive or inferior, and during the hybrid generation, the desirable alleles are expressed in the hybrids by inhibiting the recessive ones. This leads to the phenomenon of heterobeltiosis which means hybrids perform better than the best parent through genetic complementation. Various genetic studies in rice and maize have favoured the dominance hypothesis (Xiao et al. 1995; Swanson-Wagner et al. 2009). In some cases, closely linked alleles behave in an overdominance state, and this process is termed as pseudo-dominance.

9.12

Overdominance Hypothesis

In contrary, overdominance hypothesis relies on principal opposite to the dominance hypothesis. According to it, heterogeneous alleles received from both parents interact with each other which lead to heterosis. Heterogeneous combination is superior to homozygous combinations, and moreover, there is no dominant-recessive relationship between the alleles. Thus, heterobeltiosis is caused by strong interaction of heterogeneous alleles (Shull 1908; East 1936). There are numerous experimental evidences to validate this hypothesis in crops such as maize (Stuber et al. 1992; Hollick and Chandler 1998), rice (Zhu et al. 2016), cotton (Ma et al. 2019) and tomato (Krieger et al. 2010). Overdominance results from allelic interactions of a single gene. It can also result from linked loci in dominant and recessive state in repulsion, called as pseudo-overdominance. The dominant and recessive alleles are observed on the opposite gene homologs, and it is validated in tomato hybrids (Semel et al. 2006). Due to genetic recombination, pseudo-overdominance heterosis can disperse in the self-pollinated generation (Chen 2007).

9.13

Epistasis

Both dominance and overdominance hypotheses assume that heterosis is caused by individual locus. In contrary, quantitative traits such as yield have polygenic inheritance. The concept of epistasis for heterosis was given by Sheridan in 1981 (Sheridan 1981). He proposed that heterosis occurs due to interaction of non-alleles. Various studies have validated these results in eggplant (Sao and Mehta 2010), maize (Tang et al. 2010) and rice (Yu et al. 1997; Zhou et al. 2012).

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Active Gene Effect

Active gene effect hypothesis (Zhong 1994) compares the relationship between the genomic imprinting (a form of epigenetic inheritance where expression of a genomic region depends on its parental origin) and heterosis. According to it, the cause of heterosis is the additive effect of the active genes. In homozygous state, only one allele is active, while in the heterozygous form, both alleles are active showing an overall effect. The latter is the case with hybrids showing heterosis. One of the examples of active gene effect and genomic imprinting includes red1 (r1) involved in anthocyanin pigmentation in maize; when this gene is inherited from both parents, it results in expression of different colours of kernels (Kermicle 1970; Kermicle and Alleman 1990). In genomic imprinting, maternal and paternal genes are expressed differentially due to histone modifications and DNA methylation (Huh et al. 2008).

9.15

Gene Network Effect

In an organism, genes form a sequential expression network, and thus activity of one gene is related to another one. The entire gene network may be affected by alteration of a single gene. Thus, in F1 hybrids, two different gene networks from both the parents will form a new gene network that will interact and lead to heterosis (Yu et al. 2021). Various heterosis studies to increase seed yield have been conducted in B. napus (Quijada et al. 2004; Udall et al. 2004). B. rapa was shown to be a valuable source of heterosis for seed yield and biomass in B. napus through introgression (Qian et al. 2003, 2005). Heterosis has also been observed by crossing spring and semi-winter B. napus genotypes (Qian et al. 2007, 2009). In rapeseed (B. napus), a study on heterosis for yield and its components on a doubled haploid and test cross populations was conducted (Radoev et al. 2008). They summarized that in rapeseed, epistasis together with partial to overdominance is responsible for heterosis. A study in B. napus used two doubled haploid populations and two backcross hybrids and observed that epistasis is the major cause of heterosis expression (Basunanda et al. 2010). Another study to broaden the genetic base of B. napus involved lines derived from interspecific crosses between B. napus and B. oleracea. They observed seed yield heterosis in hybrids having C-genome introgression (Nikzad et al. 2020). Aakanksha et al. 2021 used a doubled haploid population derived from F1 between one parent from Indian gene pool and another from east European gene pool studied heterosis in B. juncea. They also used two corresponding backcross hybrids. Overdominance was observed to be the main heterosis mechanism in B. juncea. Various epistatic loci were also identified where the main effect was not detectable. In the same crop, genetic background, genetic complexity, trait variability and maternal-paternal effects are present which leads to different heterosis mechanisms. In addition, multiple genetic loci affect heterosis. Thus, these genetic models have their limitations. Multiple models of heterosis such as dominance, overdominance and epistatic mechanisms of heterosis were observed in various studies for the same

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crop. Heterosis is unexplained or has limited explanation in polyploids through the described genetic models as allelic and genomic dosage comes to an effect (Birchler et al. 2003; Chen 2010). Polyploidy in various crops causes genomic dosage effects which are known as progressive heterosis. Allopolyploids such as tetraploid brassicas exhibit more genomic dosage effects than homologous polyploids (Chen 2010).

9.16

Understanding of Molecular Basis of Heterosis

Numerous genetic models have been rigorously tested for heterosis, but its molecular basis still remains elusive. Advances in molecular genetics and genome sequencing technologies have expedited the exploration of the molecular basis of heterosis in crops. Various genetic factors associated with heterosis have been unravelled in recent years through genomic, transcriptomic, proteomic, metabolic, epigenomic and system biology studies. They have offered new understandings into regulatory and network changes in hybrids. Brassica tetraploids—B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC)—are allopolyploids which can be called as ‘doubled interspecific hybrids’ as these are formed by spontaneous hybridization between B. rapa (AA), B. nigra (BB) and B. oleracea (CC) as indicated in the famous ‘U triangle’ (UN 1935). In these tetraploids, heterozygosity and hybrid vigour have been fixed permanently. Various models of molecular basis of heterosis studied in various crops are discussed below.

9.17

Epigenetic and Transcriptional Regulation

At the molecular level, overdominance model suggests that gene expression in hybrids is the result of non-additive expression of alleles from parents. Non-additive gene expression pertains to deviation in expression levels from parental additivity. Epigenetic regulation suggests that heterosis is caused by non-additive expression of key regulators in hybrids which mediates expression of other regulatory network genes involved in the developmental and metabolic processes (Chen 2010). In a study, three possible modes of non-additive expression are proposed— high and low parent dominance, underdominance and overdominance (SwansonWagner et al. 2006). Through transcriptome and functional analysis, non-additive gene expression has been studied in maize, citrus and oil palm (Auger et al. 2004; Bassene et al. 2009; Jin et al. 2017). There is gene activation if the effects in hybrids are greater than the parents or gene repression if the effects in hybrids are lower than the parents. Moreover, in hybrids, due to allelic interactions, there is genetic and epigenetic reprogramming of genes and regulators resulting in their enhanced growth and productivity as compared to parents (Chen 2013). In B. rapa (Chinese cabbage), differentially expressed genes were studied in hybrids using transcriptome analysis (Kong et al. 2020). They observed variation in various loci involved in glycolysis, photosynthesis and phytohormone synthesis in hybrids. Whole genome

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SNP markers have been used for heterosis prediction and parental selection for hybrid generation in Chinese cabbage (Yue et al. 2022).

9.18

Small RNAs and MicroRNAs

Post-transcriptional regulations such as RNA-mediated pathways such as microRNAs (miRNAs), small RNAs, small-interfering RNAs, etc. are known to control non-additive gene expression. MicroRNAs (miRNAs) were discovered in the 1990s (Lee et al. 1993) and mediate RNA degradation followed by translational repression. Thus, there is negative gene regulation. These are non-coding small RNA molecules which either repress the mRNA (messenger RNA) translation or degrade mRNA. In hybrids, combination of miRNAs from different parental origin reprograms their expression which is non-additive in nature. miRNA expression variation is controlled by epigenetic mechanisms that may result in accumulation of miRNAs and thus differential expression (Bartel 2004; Brown et al. 2007; Chen 2007; Ha et al. 2009).

9.19

Molecular Circadian Clock Model

Circadian-mediated molecular clock model had also explained the molecular basis of heterosis (Chen 2010; Ko et al. 2016). In plants, circadian clock is controlled by many feedback loops—one of them is a major loop having two negative regulators named CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) and LHY (LATE ELONGATED HYPOCOTYL) and their reciprocal positive regulators TOC1 (TIMING OF CAB EXPRESSION 1) or PRR1 (PSEUDO RESPONSE REGULATOR 1), CCA1 HIKING EXPEDITION (CHE) and GIGANTEA (GI). Input signals such as light and temperature are received by the clock, and thus output signals such as photosynthesis and other metabolic activities are controlled through expression of certain genes (McClung 2006). When hybrids are formed by the combination of two parents, allelic interactions lead to epigenetic repression of CCA1 and LHY while upregulating TOC1, CCA1 and GI expression in hybrids in comparison to parents. Thus, the clock periodicity and rhythm remain the same to maintain plant growth (Jin et al. 2017). As CCA1 amounts are reduced during the days, this leads to expression of various circadian clock-associated genes in the pathways that control the vegetative growth and vigour. Superior performance in the physiological pathways pertains to allelic interactions of hybrids. Overdominance is the result of epigenetic repression of key regulators in the clock which mediates downstream genes in physiological processes. Circadian clock-mediated heterosis is considered a universal phenomenon of heterosis. This molecular mechanism has been validated for heterosis in Arabidopsis and maize (Green et al. 2002; Chen 2010; Ko et al. 2016).

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Chromatin Architecture

In B. napus, three-dimensional (3D) chromatin architecture was studied as a mechanism of heterosis using genetic, transcriptomic and three-dimensional genomic approaches (Hu et al. 2022). In 3D genome architecture, two compartments are present—A compartments (transcriptionally active regions of euchromatin) and B compartments (transcriptionally non-active regions of heterochromatin) (Doğan and Liu 2018). In the study, it was observed that A compartments were higher in superior hybrids as compared to inferior hybrids (Hu et al. 2022). Higher A compartments result in higher levels of expression-level dominance in hybrids, and key genes involved in the cell cycle are upregulated in hybrids.

9.21

Hydrogen Sulphide Mechanism

Hydrogen sulphide (H2S) is an important universal signalling molecule and is synthesized by desulphurization of cysteine (Corpas et al. 2019). This endogenous signal is linked with plant growth and development (Xuan et al. 2020). A study in B. napus F1 hybrids showed the role of H2S in heterosis during salt stress. Under saline conditions, the increased levels of H2S in the hybrids as compared to parents restore ion homeostasis and redox balance (Cheng et al. 2022).

9.22

Development of Heterotic Groups

Heterotic group discovery dates back to 1942 when Sprague and Tatum gave the principle of general combining ability (GCA) and specific combining ability (SCA) (Sprague and Tatum 1942). When the genetic diversity in a germplasm is increased, it doesn’t mean that the heterosis level would also increase. Heterosis depends on the combining ability of the lines, and thus in heterotic groups, germplasm is subdivided into divergent populations based on their combining ability and heterotic response. Usually, the crosses within heterotic groups will lead to lower heterosis as compared to crosses between groups. Identification of heterotic groups has a major role in prediction of heterosis in early stages. Specific pair of heterotic groups of related or unrelated genotypes that possess high heterosis and similar combining ability and thus good hybrid performance exhibit heterotic pattern (Melchinger and Gumber 1998). There are various methods to develop heterotic pools such as pedigree analysis, measurement of combining ability, geographical isolation inference, use of molecular markers, etc. Heterotic patterns and development of heterotic group are advantageous as it does not allow the development and evaluation of hybrids that have to be rejected later on. It also allows the maximum levels of heterosis by cross-pollinating lines belonging to different heterotic groups (Tracy and Chandler 2005). Various methods can be used for evaluation of heterotic groups such as parental pedigree analysis, SCA method based on evaluation of specific combining ability

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effects (Fan et al. 2004), HSGCA method based on the evaluation of general combining ability and group specific combining ability (Fan et al. 2009), HGCAMT method based on general combining ability of many traits (Badu-Apraku et al. 2013), SCA-Y method based on evaluating SCA effect (Yang 1983), etc. In the 1990s application of molecular markers to study heterosis was employed. Heterotic groups have been evaluated using molecular markers by measuring the genetic diversity at the genomic level. Representative genotypes are selected from each group and are cross-pollinated, followed by their field evaluation. Thus, heterotic groups are identified based on their performance. But studies have showed the low reliability of molecular markers to predict heterotic pools for genetically similar germplasm (Barata and Carena 2006). Another study has shown no correlation between heterosis and genetic distance in Chinese cabbage based on SSR (simple sequence repeats) and CAPS (cleaved amplified polymorphic sequence) markers (Kawamura et al. 2016). Various heterotic groups were predicted in B. napus using different methods including molecular markers (Tian et al. 2015). Genome-wide haplotype analysis has been used in B. napus for predicting heterosis using 24,403 genome-wide SNP sites (Jan et al. 2019). In B. juncea, there are two main distinct gene pools—Indian and east European— identified by various molecular markers (Pradhan et al. 1993b; Srivastava et al. 2001). Yield heterosis has been observed in hybrids from these heterotic gene pools in Indian mustard. DMH-1 was developed in B. juncea by crossing Pusa Bold (Indian gene pool) and EH-2 (east European gene pool). DMH-1 is the first commercial hybrid and showed ~30% higher yield than controls (Pradhan et al. 1993b; Sodhi et al. 2006). In B. rapa, Asian and European heterotic gene pools are present (Zhao et al. 2005). Rapeseed has been divided into four heterotic groups – Asian, Canadian spring type, European winter type and European spring type. The crosses of genotypes between these groups resulted in high heterosis for yield (Qian et al. 2007, 2009). Another study involved the heterotic groups evaluation in Chinese cabbage (B. rapa L. ssp. pekinensis) and divided the inbred lines into five gene pools (Chen et al. 2021).

9.23

Present Status of Commercial Hybrids

Countries are expected to create a demand for hybrid seeds in order to provide profitable opportunities for the market growth. At Asia-Pacific hybrid seeds market (2016), India occupied the highest market grosser place due to an increased demand for fruit and vegetable crops. The top-notch players operating in this market include Bayer Crop Science, Kaveri Seeds, Advanta Limited, KWS AG, Biostadt India Limited, Monsanto, DOW Agrosciences LLC, Syngenta, EI DuPont De Nemours, Mahyco, Ajeet Seeds, Ankur Rallis India and Shriram Bioseed (https://www. alliedmarketresearch.com/hybrid-seeds-market). The current status of oilseed crop sector in India has estimated an upward projection of per capita consumption due to an increase in income and change in food habits.

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For hybrid development in oilseed brassicas, continuous efforts are made with CMS systems. Mostly, Ogura and tour system in B. juncea and Polima system in B. napus are used (Chauhan et al. 2011). Till 2008, CMS-based commercial F1 hybrids were available only in B. napus using Polima (China canola), tour (India) and Ogura (Europe/Canada) CMS systems (Basu et al. 2013). Further, fertility restorers for mori and trachy CMS systems were developed by ICAR-National Research Centre for Plant Biotechnology, New Delhi, India. In oilseed brassica, various other systems such as lyratus, canariense, 126-1, erucoides and berthautii CMS were also reported. A large number of rapeseed-mustard varieties have been apprised by the Indian government. These varieties play a pivotal role in enhancing the crop productivity, provided their selection is based on their fit for the concerned environment (Kumar 2012). The first commercial hybrid PGSH-51 in B. napus was released in 1994 by Punjab Agricultural University, Ludhiana, based on tour CMS. In this hybrid, there was 18% increase in yield over the best hybrid (Downey and Chopra 1996). The other hybrids are Hyola 401 hybrid (1997–2000) which was based on pol CMS system and developed by Advanta for north western plain zone, DMH-1 (2008) on 126-1 CMS, NRCHB-506 (2008) on mori cytoplasm and PAC-432 (2009) on ogu cytoplasm. Genetic engineering approaches led to the development of India’s first transgenic hybrid, DMH-11, instigated by Delhi University, but it could not be commercialized owing to resistance posed by environmental activist in thought of its harm to the environment (Chand et al. 2018, 2021), even after approval from Genetic Engineering Appraisal Committee (GEAC) of India. The Centre for Genetic Manipulation of Crop Plants (CGMCP) and Delhi University South Campus submitted an application to GEAC in 2015 for the environmental release of GM mustard (B. juncea) hybrid Dhara Mustard Hybrid-11 (DMH-11) and the use of parental events (bn 3.6 and modbs 2.99 with barnase, barstar and bar genes) for the development of new hybrids (https://www.ISAAA.org). The first B. napus hybrid, PGSH-51, has already been released commercially, while in B. campestris, the hybrid is under the final phase of evaluation. The technology to develop GM-based hybrids (YSMS 2, YSMS 6 and YSMS 8163) in B. rapa was refined (Anonymous 1997), and seed production technique has also been improved for B. napus hybrid seed production (Rai 1995; Anonymous 1997). Private seed companies like Mahyco, Hindustan Lever Ltd., ProAgro, etc. are also working towards the development of commercial Brassica hybrids (Poojitha et al. 2021). Attempts for commercialization of CMS-based hybrids have been successful in Indian mustard. Hybrids, namely, NRCHB-506, Coral-432 (PAC-432) and DMH-1, have been recently identified for release by CVRC (Central Variety Release Committee). In 2008, the first CMS-based B. juncea hybrids—NRCHB-506 and DMH-1—were released by ICAR-DRMR, Bharatpur. NRCHB-506 has been developed by the National Research Centre on Rapeseed Mustard, Bharatpur (Rajasthan), while Coral-432 (PAC-432) and DMH-1 have been evolved by private sector, 26% and 15% in zone-II and zone-III, respectively. Later on, three more hybrids (Table 9.3) were also released in 1997 by Mahyco through the central subcommittee

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Table 9.3 B. juncea and B. napus hybrids released in India Genome B. juncea

B. napus

Hybrid name NRCHB506 DMH-1 CORAL432 (PAC-432) CORAL437 (PAC 437) PGSH-51 HYOLA401 (PAC-401) DMH-11 (transgenic)

Developing institution DRMR, BHARATPUR DUSC, DELHI Advanta India, Bangalore

Release location Rajasthan, UP

Year 2008

CMS source mori

Punjab, Haryana, Delhi, J & K, Rajasthan, UP, MP Punjab, Haryana, Delhi, J & K, Rajasthan

2008

126-1

2010

ogu

Advanta India, Bangalore

Punjab, Haryana, Delhi, J & K, Rajasthan

2011

PAU, Ludhiana Advanta India, Bangalore

Punjab

1996

tour

Punjab, Haryana, Delhi, J & K, Rajasthan

1997

ogu

Delhi University

–

2015

barnase, barstar

on crop standards, notification and released of varieties for agricultural crops. All these released hybrids demonstrated more than 10% higher productivity than the best inbred lines (Zehr et al. 1997). Ogura CMS is now the most widely used pollination control system in this crop. For improving oil and seed meal quality in rapeseedmustard varieties, three canola quality varieties of gobhi sarson (GSC-5, GSC-6 and TERI Uttam Jawahar) and two low erucic acid varieties of Indian mustard (Pusa Karishma and Pusa Mustard-21) were also recommended (Kumar 2012).

9.24

Genetic Resource for Hybrid Breeding

The National Bureau of Plant Genetic Resources (NBPGR), New Delhi, has registered many genetic stocks of oilseed brassica with novel traits for use in future breeding programme such as dwarf, earliness, low erucic acid (TERI-Swarna and PRQ-2005-1 registered in B. juncea), high oil content, low erucic acid and low glucosinolates (Heera, TERI-GZ-05 and NUDHYJ- 5 registered in B. juncea; TERIPhaguni, TERI-Shyamali, TERI-Gaurav, TERI-Garima, NUDB-38, NUDB-42 and TERI-Uttam in B. napus), high oleic acid and low linolenic acid, tetralocular siliquae, long main shoot, bold seed, yellow seed, high temperature tolerance, high salinity tolerance, white rust resistance, high-water use efficiency, etc. Moreover, under AICRP-RM (All India Coordinated Research Project on Rapeseed Mustard) quality improvement programmes, canola quality double low Australian (JR042, JN010, JN033, JN031, JN049, JN009, JN004, JM016 and JM006) and Chinese (CBJ001, CBJ002, CBJ003 CBJ004 and XINYOU5) double low lines were used as donors.

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Hybrid development in Indian mustard has witnessed a good progress as every year under AICRP-RM 67; new CMS-based hybrids are tested and attempted at developing hybrids with higher heterosis than the presently released hybrids. DRMR, Bharatpur; IARI, New Delhi; NRCPN, New Delhi; PAU, Ludhiana; HAU, Hisar; University of Delhi, South Campus; and many private seed organizations have actively participated in the hybrid development programme so far (Basu et al. 2013).

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Conclusion

Among oilseed crops rapeseed-mustard is a potential crop that could reduce India’s dependence on edible oil imports. Rapeseed-mustard crop is a crucial oilseed crop for marginal farmers under low input grown in diverse ecosystems of the country. A long breeding effort has delivered a series of improved varieties with increased productivity. However, the genetic yield potential is far away from realised yield through varietal cultivation. The development of more hybrids would depend on stable and efficient cytoplasmic male sterility-fertility restorer and identification of diverse parental lines. Implementation of heterosis breeding, using new cytoplasmic male sterility system with strong fertility restorer genes, broadening of diverse heterotic gene pool and introgression of quality trait, will play a greater role to achieve genetic yield potential in rapeseed-mustard crop.

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Cytoplasmic Male Sterility: A Robust and Well-Proven Arsenal for Hybrid Breeding in Vegetable Crops

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Pradip Karmakar, B. K. Singh, Vidya Sagar, P. M. Singh, Jagdish Singh, and T. K. Behera

Abstract

Vegetables are the integral part of our daily diet as they are loaded with various phytonutrients and considered as an important component for nutritional security and alleviating malnutrition. There is a huge demand of vegetables to fulfil the requirement of ever-growing population. This can be achieved only through vertical expansion by enhancing productivity. In this respect exploitation of heterosis and hybrid breeding will be a feasible and sustainable intervention. Vegetable crops exhibit robust and durable hybrid vigour, and exploitation of heterosis is the important way forward in their genetic improvement. Production of hybrid seeds in a commercial scale and cost-effective way, pollination control mechanism like male sterility system is considered as robust and proven arsenal in vegetables. Among the various types of male sterility mechanism available in plant species, cytoplasmic male sterility is the most extensively utilized in different vegetable crops. In this chapter, we thoroughly documented the various available male sterility mechanisms; their potential utilization in F1 hybrid seed production; sterilizing cytoplasm, their sources and diversification; fertility restoration mechanism, inheritance, molecular mapping and cloning of Rf elements; recent advances in deciphering male sterility determinants; role of markerassisted selection in hybrid breeding utilizing CMS system; and adoption of modern omics technology to elucidate the CMS systems in vegetables. Moreover, this chapter also summarizes the current status and future strategies of utilization and improvement of the available CMS systems in various vegetables. Keywords

Vegetables · Hybrid seed production · CMS system · Fertility restoration P. Karmakar (*) · B. K. Singh · V. Sagar · P. M. Singh · J. Singh · T. K. Behera ICAR—Indian Institute of Vegetable Research (IIVR), Varanasi, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_10

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Introduction

India ranked second in vegetable production after China with an acreage of 10.35 million ha and production of 191.76 million tonnes (http://www.nhb.gov.in/) in the world. The demands of vegetables increase with the ever-increasing population in the country. The possibility of enhancing vegetable production by expanding the area under vegetable cultivation is quite impossible as arable shrinking day by day in India. The only way to increase the vegetable production is through enhanced productivity. In this regard exploitation of heterosis and hybrid breeding will be a feasible and sustainable contrivance. Exploitation of heterosis and development of F1 hybrids become popular in crop plants including vegetables after the successful demonstration of the revolutionary work related to heterosis by G. H. Shull in maize. In vegetables the first F1 hybrid for commercial cultivation was developed in eggplant during 1924 in Japan (Nishi 1967). After that, F1 hybrids were also developed for watermelon in 1930, cucumber in 1933, radish during 1935, tomato in 1940 and cabbage during 1942 (Liedle and Anderson 1993). Coyne (1980) has emphasized the predominance of hybrid cultivars of sweet corn and squash in the USA and the increasing demands of hybrid seed production in cucumber, summer squash, cabbage, broccoli, onion and carrot. In the USA commercial cultivation of hybrid vegetable started during 1945, but in some European nations, viz. the Netherlands, France and Denmark, it come into market for commercial exploitation after 1940. Since then the popularity of hybrid vegetables and hybrid seed industries in those countries augmented with immense pace. In recent time more than 90% of cabbage, tomato, onion, cucumber, muskmelon, sweet pepper, lettuce, etc. cultivated are hybrids in Japan, the Netherlands, Denmark, France Australia, the United Kingdom and the USA. In India though the exploitation of the first F1 hybrid at the experimental level reported from ICAR-Indian Agricultural Research Institute in 1933, the very first commercial hybrid Pusa Meghdut was developed in bottle gourd in 1971 by the same Institute. Subsequently in 1973 two F1 hybrids Pusa Alankar and Pusa Sanjog were developed in summer squash and cucumber, respectively. Since then a number of F1 hybrid varieties of tomato, brinjal, chilli, sweet pepper, muskmelon, watermelon, bottle gourd, cucumber, summer squash, pumpkin, bitter gourd, cauliflower and cabbage were developed and released by various ICAR institutes, SAUs and other public sector organizations in India. Vegetable crops are well suited for exploitation of heterosis and the development of F1 hybrids. Vegetable breeders select hybrid breeding as it is comparatively simple to integrate the genes conferring resistance to biotic and abiotic stresses in the F1 hybrids. F1 hybrid seed production in vegetables such as onion and carrot, those having numerous small hermaphrodite flowers where executing hand emasculation and pollination is very difficult, where male sterility proved to be an important pollination control tool. Hybrid seed production in a huge extent occasionally becomes handicapped due to soaring cost of labour, scarcity of trained manpower during flowering stage and unpredictable weather conditions such as continuous rains, hail storm, etc. Hybrid seeds produced by utilizing natural crossing through

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competitive fertilization among self- and cross-pollen in various vegetables contained only 40–80% of true hybrid (Liedle and Anderson 1993) that was far away from recommended acceptable limit of impurity in hybrid seeds. Consequently, search for techniques for production of uncontaminated hybrid seeds at a profitable scale was an utmost important task. In vegetables, three basic genetic mechanisms of pollination control systems have been used to exploit the expression of heterosis. These systems are cytoplasmic male sterility (CMS), selfincompatibility (SI) and the manipulation of sex forms. Pearson (1933) utilizing SI system in cabbage and Jones and Clarke (1943) exploiting CMS system in Allium cepa suggested the approaches of producing true hybrid seeds for commercial purpose. Afterwards, male sterility was identified and utilized in an array of crop plants as well as in vegetable crops. Male sterility in plants was either isolated as a spontaneous mutant in natural gene pool or was artificially developed by mutagenesis (Kaul 1988). In this chapter, efforts have been made to illustrate various aspects of male sterility with a special emphasis to CMS and its exploitation in hybrid development in vegetables.

10.2

Hybrid Development and Heterosis

The tendency for using F1 hybrid seed in vegetables is going up worldwide day by day not only in terms crop plants but also the quantity of seed required. In developed nation hybrid varieties were developed, and generally hybrid seeds were sown in tomato; capsicum; brinjal; cucumber; squash; pumpkin; muskmelon; watermelon; cole crops like cabbage, cauliflower and broccoli; Chinese cabbage; Raphanus; and Allium. The F1 hybrid varieties are more widespread owing to their uniformity, vigour, resistance to diseases, tolerance to environmental stress and expression of superior horticultural characters like earliness, long shelf-life and consistent high productivity. Development of F1 hybrid is a quick and handy means to combine desirable traits, viz. size and colour of fruits, plant type and reaction to disease collectively in vegetable crops. At present only about 15% area is under hybrid varieties of vegetables, of which 36% and 30% crop area in tomato and cabbage occupied by F1 hybrid.

10.2.1 Development of Hand-Pollinated F1 Hybrids This is a very simple method that involves either hand emasculation of male reproductive parts in bisexual flowers, followed by manual pollination by the pollen of the male parental line in tomato, brinjal, sweet pepper, etc., or pinching of staminate flower plus hand pollination with the pollen of desirable male parent as in cucurbits like bitter gourd, bottle gourd, etc. or pinching of staminate flower with natural pollination used in cucurbitaceous crops including bitter gourd and summer squash. It is a labour-intensive method; requires skilful growers and skilful labour having good eye health, calm hands, ample of perseverance and dedication; and is

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capable of following instructions precisely. This system is cost-effective only in crop plants where a single pollinated female flower is able to produce lots of seeds. This is true in the case of solanaceous crops like tomato, brinjal and sweet pepper and cucurbitaceous crops where more number of seeds are produced/pollinated. On the one hand in legumes and Brassica vegetables, a few number of seeds per pollination or pod make hand pollination a non-profitable proposition and not recommended for these crops. The F1 hybrids of vegetables produced through hand pollination are presented in Table 10.1.

10.2.2 Development of Male Sterility-Based F1 Hybrids In India male sterility was successfully utilized for the exploitation of development in muskmelon and chilli (Kalloo et al. 1998). Hybrid seed production in the crops like carrot, sweet corn and onion is based on the use of male sterility mechanism. Genetically male sterility is either governed by clear-cut nuclear genes or by the interaction of nuclear gene with a cytoplasmic gene. In modern time, vegetable breeders are attempting to exploit male sterility mechanism as an alternative to the self-incompatibility (SI) system for developing F1 hybrids in brassica vegetables. Due to insufficient production of nectar, male sterile plants were unable to attract pollinator. Thus for proper seed set, necessary arrangement should be made to attract pollinator. In India F1 hybrids of vegetables were developed utilizing male sterility system in India (Table 10.2).

10.2.3 Heterosis Estimates in Vegetable Crops Though heterosis does not express universally in all vegetable crops, still a number of the pioneering work on heterosis breeding were reported in the vegetables. The identification of CMS in onion is regarded as the foundation for making hybrid seed production economical. However, the heterotic effect of hybrid varieties had not been utilized commercially in self-fertilized vegetables as compared to crosspollinated vegetables. Self-pollinated vegetables do not exhibit both in breeding depression and hybrid vigour. Vegetables are classified into self-pollinated vegetables with few to many seeds produced/pollination and cross-pollinated crops having low to high rate of natural cross-pollination (Wehner 1999). Extensive research was carried out to find out the extent of heterosis in both self-pollinated (bean, pea, brinjal, pepper, tomato and lettuce) and cross-pollinated (cucumber, melon, squash, cabbage, cauliflower, broccoli, radish, carrot, onion, spinach, etc.) vegetables. The estimate on heterosis for different traits including yield varies considerably in different vegetables, and the estimate of heterosis is presented in Table 10.3.

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Table 10.1 F1 hybrids of vegetables produced through hand pollination

Crop Bitter gourd

Bottle gourd

Brinjal

Capsicum Chilli

Cauliflower

Name of the hybrids Pusa Hybrid-1 Pusa Hybrid-2 CO Bgo H1 Pusa Hybrid-3 Kashi Bahar Pant Sankar Lauki-1 Pant Sankar Lauki-2 NBGH-4

Source IARI, New Delhi IARI, New Delhi

Pusa Hybrid-5

TNAU, Coimbatore IARI, New Delhi IIVR, Varanasi GBPUA & T, Pantnagar GBPUA & T, Pantnagar NDUA & T, Faizabad IARI, New Delhi

Pusa Hybrid-6

IARI, New Delhi

Pusa Hybrid-9 Arka Anand

IARI, New Delhi IIHR, Bengaluru

Kashi Sandesh Pusa Deepti Chilli hybrid-1 COCH-1

IIVR, Varanasi IARI, New Delhi AAU, Anand TNAU, Coimbatore IARI, New Delhi IARI, New Delhi

Pusa Hybrid-2

Recommended area NCR Uttar Pradesh, Punjab, Odisha, Bihar, Jharkhand, Chhattisgarh, Rajasthan, Gujarat, Haryana, Delhi and Andhra Pradesh Tamil Nadu All over India Punjab, Uttar Pradesh, Jharkhand and Bihar Uttarakhand and Uttar Pradesh

Uttarakhand and Uttar Pradesh

Punjab, Bihar, Uttar Pradesh, Haryana and Rajasthan Uttar Pradesh, Punjab, Bihar, Rajasthan, Delhi, West Bengal, Jammu and Kashmir, Haryana, Madhya Pradesh, Himachal Pradesh, Chhattisgarh, Karnataka, Tamil Nadu and Kerala Punjab, Bihar, Rajasthan, West Bengal, Uttar Pradesh, Delhi, Himachal Pradesh, Haryana, Jammu and Kashmir, Chhattisgarh, Madhya Pradesh Tamil Nadu, Karnataka, Kerala and Andhra Pradesh Maharashtra and Gujarat Haryana, Rajasthan, Gujarat, Madhya Pradesh, Delhi, Maharashtra, Kerala, Karnataka and Tamil Nadu Uttar Pradesh All over India Gujarat Tamil Nadu Assam, West Bengal, Punjab, Bihar, Uttar Pradesh and Delhi Assam, West Bengal, Punjab, Bihar, Jharkhand and Uttar Pradesh (continued)

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Table 10.1 (continued)

Crop

Cucumber

Pumpkin

Summer squash Tomato

Watermelon Okra NHB (2012)

Name of the hybrids Pusa Kartik Sankar Pant Sankar Khira-1 Kashi Nutan Pusa Hybrid-1 Kashi Shishir Pusa Alankar Pusa Hybrid-2

Source

Recommended area

GBPUA & T, Pantnagar IIVR, Varanasi IARI, New Delhi IIVR, Varanasi IARI, New Delhi IARI, New Delhi

Uttarakhand and Uttar Pradesh

Pusa Hybrid-4 Pusa Hybrid-8 Arka Ananya

IARI, New Delhi IARI, New Delhi IIHR, Bengaluru

Arka Vardhan

IIHR, Bengaluru

Arka Vishal Arka Abhijit

IIHR, Bengaluru IIHR, Bengaluru

Kashi Abhiman Arka Jyothi Kashi Shristi

IIVR, Varanasi IIHR, Bengaluru IIVR, Varanasi

Uttar Pradesh NCR Uttar Pradesh All over India Chhattisgarh, Jharkhand, Andhra Pradesh, Madhya Pradesh, Odisha, Maharashtra, Bihar, Karnataka, Rajasthan, Punjab, Gujarat and Tamil Nadu Uttar Pradesh, Bihar, Jharkhand and Punjab Uttar Pradesh, Bihar and Punjab Uttar Pradesh, Punjab, Bihar, Jharkhand, Odisha, Chhattisgarh, Arunachal Pradesh, Rajasthan, Haryana, Gujarat, Delhi, Madhya Pradesh and Maharashtra Himachal Pradesh, Jammu and Kashmir, Uttar Pradesh, Punjab, Bihar, Jharkhand, Karnataka, Kerala and Tamil Nadu Uttar Pradesh, Bihar, Jharkhand, Punjab, Tamil Nadu, Karnataka and Kerala Odisha, Chhattisgarh, Rajasthan, Arunachal Pradesh, Delhi, Gujarat, Haryana, Karnataka, Tamil Nadu and Kerala Punjab, UP, Bihar and Jharkhand Karnataka, Tamil Nadu and Kerala Uttar Pradesh

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Table 10.2 F1 hybrids of vegetables developed utilizing male sterility system Crop Chilli

Muskmelon Carrot

Cabbage Cauliflower

Radish

10.3

Hybrid CH-1 CH-3 Kashi Surkh Arka Meghana Arka Sweta Arka Harita Punjab Hybrid Punjab Anmol Pusa Nayanjyoti Arka Kirthiman Arka Lalima Hybrid-63 Hybrid-35 KCH-5 KTCBH-81 KTH-27 KTH-52 KTH-51 Kashi Rituraj (VRRAD-201
VRRAD-200)

MS system used GMS GMS CGMS CGMS CGMS CGMS GMS GMS CMS CMS CMS CMS CMS CMS CMS CMS CMS CMS CMS

References Hundal and Khurana (1993a, b) Hundal and Khurana (2001) www.iivr.org.in Prasanth and Kumary (2014) Prasanth and Kumary (2014) Prasanth and Kumary (2014) Nandpuri et al. (1982) Lal et al. (2007) Dhall (2010) Dhall (2010) Dhall (2010) Dhall (2010) Dhall (2010) Dhall (2010) Parkash et al. (2015) www.iari.res.in www.iari.res.in www.iari.res.in Singh and Singh (2020)

An Overview of Male Sterility in Vegetable Crops

Male sterility system can be grouped into two broad categories, viz. genetic where the sterility is governed by genes and non-genetic which can momentarily be induced through stresses (Kaul 1988). In vegetable crops first one use extensively for the development of F1 hybrids, but induced male sterility had not been exploited commercially. On the basis of phenotypic expression, there are three classes of genetic male sterility, i.e., sporogenous, structural and functional. Likewise, induced male sterility is classified into chemical-, physiological- and ecological-induced male sterility. On the basis of the position of factor (gene/genes) accountable for the male sterility, which may be either isolated spontaneously or induced artificially using mutagenesis or artificially developed using protoplast fusion or genetically engineered male sterility mechanism through transgenic approach (inherited type) is grouped into genic male sterility (GMS) also known as nuclear gene (s) induced male sterility and cytoplasmic male sterility or CMS (also known as nuclearcytoplasmic male sterility).

10.3.1 Genic or Genetic Male Sterility (GMS) in Vegetables When pollen sterility is governed by nuclear gene(s), then such type of male sterility is defined as genic or genetic male sterility (GMS). GMS is reported to have occurred

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Table 10.3 Estimate on Heterosis for Different Traits in Vegetables Crop Bean Brinjal

Trait Dry seed yield Yield Yield Yield

Estimated heterosis % over parents/check 28 33 97 60

Pepper

Yield

9

Export quality yield Yield Yield

75

Tomato

Early yield Late yield Yield

50 4 60

Cucumber Melon

Yield Yield

5 8

Earliness

3

Yield

11–84

References Gritton (1975) Tiwari (1966) Dharme (1977) Mulualem and Abate (2016) Shifriss and Rylski (1973) Shifriss and Rylski (1973) Dikil et al. (1973) Mulualem and Abate (2016) Yordanov (1983) Yordanov (1983) Mulualem and Abate (2016) Wehner (1999) Lippert and Legg (1972) Lippert and Legg (1972) Wehner (1999)

28–47 35

Summer squash Pumpkin Watermelon Tropical cauliflower Cabbage Broccoli Carrot

Yield Yield Yield

40 10 10

Wehner (1999) Wehner (1999) Pearson (1983)

Yield Yield Yield

12–15 40–90 25–30

Onion

Yield

14–67

Pearson (1983) Morelock et al. (1972) Bonnet and Pecaut (1978) Dowker and Gordon (1983)

in approximately 175 plant species including several important vegetables (Kaul 1988). Though in most of the cases GMS is generally inherited through recessive nuclear gene(s), there are some exceptional cases like in cabbage, broccoli and genetically engineered male sterility where dominant genes are also involved in male sterility (Kaul 1988; Williams et al. 1997). In majority of cases, sterility is caused by a single gene. However there are few cases, where two or more genes control the process of male sterility. GMS may be resulted from mutation in any gene (s) that either involves in stamen development or pollen formation (microporogenesis) or micro-gametogenesis. There are certain mutants reported,

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which even though produce viable pollen, unable to self-fertilize either due to lack of pollen dehiscence or special flower phenotype. These types of mutants are frequently termed as functional male sterile, such as genotypes having exerted stigma or positional sterility in tomato (Georgiev 1991; Atanassova 1999), non-dehiscence of pollen grain in brinjal and some other vegetables (Kaul 1988). Environmentally influenced male-sterile mutants were reported various in vegetables. Thermo-sensitive genic male sterility (TGMS) was reported in cabbage, Brussels sprout, broccoli, pepper, tomato and carrot (Rundfeldt 1961; Nieuwhof 1968; Dickson 1970; Daskalov 1972; Rick 1948; Sawhney 1983; Kaul 1988), and PGMS or photoperiod-influenced genic male sterility was reported in cabbage by Rundfeldt (1961). Monogenic recessive gene-controlled male sterility was exploited for the development of cost-effective experimental hybrids in tomato (Sawhney 1997; Kumar et al. 2001). In tomato more than 55 genes of recessive nature have been documented (Kaul 1988; Georgiev 1991), and temperature sensitivity is reported for sl-2, ms-13 and ms-15 (Sawhney 1997), but only ps-2 gene has been utilized for commercial hybrid seed production (Atanassova 1999). Monogenic recessive functional sterility was reported in brinjal (Phatak and Jaworski 1989). Male sterility in the case of Capsicum spp. reported to be caused by more than 12 recessive genes (Shifriss 1997). MS-12 (ms-509/ms-10) and ms-3 genes were exploited commercially in India and Hungry, respectively, for hybrid seed production of chilli (Kumar et al. 2000). In chilli ms-10 gene is linked with morphological traits such as taller plant height, erect growth and dark purple anther (Dash et al. 2001). In cauliflower both recessive and dominant inheritance are reported for male sterility (Kaul 1988; Kumar et al. 2000). While Nieuwhof (1961) reported recessive gene, Fang et al. (1997) identified dominant genes in cabbage. This dominant gene-controlled male-sterile mutant can be used for F1 hybrid development in cabbage (Fang et al. 1997), and they proposed the multiplication of male-sterile line through tissue culture. Zhang et al. (1994) proposed the used of monogenic recessive male sterile mutant for hybrid development in watermelon. Dutta (1983) identified genetic male-sterile plant in bottle gourd and subsequently used for the development of experimental crosses. In muskmelon five non-allelic genes of recessive nature have been identified for male sterility (McCreight et al. 1993), and ms-1 has been gene exploited commercially in India (Kumar et al. 2000). Some morphological markers had been reported to have linked with male sterility in different vegetables. Bright green hypocotyl is linked with ms gene in broccoli (Sampson 1966). Green stem colour, potato leaf shape and parthenocarpic fruit development in tomato are some identified morphological markers reported to have linked with ms gene (Kaul 1988; Soressi and Salamini 1975). Zhang et al. (1996) reported delayed green seedling marker in watermelon.

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10.3.2 Cytoplasmic Male Sterility (CMS) in Vegetable Crops CMS is a situation, in which a plant species is incapable of developing viable pollens and is inherited maternally as the mitochondrial genome is accountable for the manifestation of male sterility and the mitochondria are generally barred from the pollen grain at the time of fertilization (Bohra et al. 2016; Mishra and Bohra 2018). In CMS plants male sterility arose because of the incompatibility interaction of recessive nuclear gene and genome from specific male-sterile cytoplasm. It is reported to occur in several crop plants and is frequently linked with mitochondrial open reading frames (ORFs) with chimeric nature. CMS was reported to have occurred in more than 150 plant species (Laser and Lersten 1972). CMS system is an outstanding model to describe the relation among nuclear and cytoplasmic genes, as restoration of fertility depends on nuclear factors that overturn cytoplasmic abnormalities (Schnable and Wise 1998). On the other hand, when the dominant (Rf) gene for fertility reinstatement which is present in the nucleus and accountable for fertile pollen production in a CMS line is recognized, then it is called as CGMS or cytoplasmic genetic male sterility. Thus the production of sterile pollen in cytoplasmic genetic male-sterile genotype is expressed when sterile-type mt-genome situated in cytoplasm and recessive allele for fertility restoration (rf) is positioned in the nuclear genome that is present together, but none of them alone can produce sterile phenotype. Cytoplasmic male sterility in crop plants may develop in nature through distance hybridization (interspecific/intergeneric), but it can be also induced artificially using mutagenesis or antibiotic effect (Kaul 1988). Because of their commercial value in F1 hybrid development and their seed production, cytoplasmic malesterile systems have been reported and thoroughly studied in many crop species including vegetables like cole crop, radish, Phaseolus vulgaris, beet, carrot, onion, etc. (Kück et al. 1995; Singh et al. 2021; Singh and Karmakar 2021). For commercial hybrid seed production, cytoplasmic male sterility is the most commonly exploited genetic emasculation system in crop plants. This system is used for three-line hybrid developments in many crops comprising A line or male-sterile line (S rfrf), B line or maintainer line (N rfrf) and C or R (restorer) line (S/N RfRf). A line or male-sterile line can be developed through back cross-breeding. Back crossing of a designated maintainer line (B line) with an existed male-sterile line or ‘A’ line for six to seven generations is required to develop a new CMS line. This creates a couple of male-sterile and maintainer lines in the novel and desired genetic background. Fertility restoration gene or (Rf) is moreover introgressed into recognized pollen parent, or it can be straightway utilized for hybrid seed production using CMS line, if the Rf gene of homozygous type is present in pollen parent. It is impossible to use CMS in fruit vegetables like tomato, chilli and melon without restorer gene, but it may be exploited in vegetables where vegetative parts are of economic importance like in cole crop, carrot, onion, radish, beet and leafy vegetables even without restorer gene.

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Sterility-Inducing Cytoplasm in Vegetables

Cytoplasmic male sterility system is commonly known as maternally inherited defect in a plant system which makes it to produce nonviable/sterile pollen and facilitate cross-pollination. In most of the cases, the mitochondria are accountable for CMS, and generally mutation in the mitochondrial genome damages the appropriate functioning of this cytoplasmic organelle, leading to male sterility (Budar and Pelletier 2001). CMS may arise from spontaneous mutation or from intraspecific, interspecific or intergeneric hybridization (Kaul 1988). Till date, the mitochondrial mutations in the case of CMS are composed of sequence chimeras, usually melding detectable mitochondrial gene sequences with fragments of unidentified origin (Schnable and Wise 1998; Hanson and Bentolila 2004).

10.4.1 Source of Sterile Cytoplasm in Vegetables According to the source of the S cytoplasm and nuclear gene which constitute the cells of the male-sterile individual, it is possible to categorize various cases of cytoplasmic male sterility into different classes. Edwardson (1956) distinguished the CMS system arose through intergeneric/inter- or intraspecific hybridization from the spontaneously originated cytoplasmic male sterility in a given crop plant. Following a similar principle, cytoplasmic genetic male sterility can be categorized into autoplasmic male sterility, homoplasmic male sterility and alloplasmic male sterility. In autoplasmic male sterility, the plants whose cells are established by nucleus and cytoplasm as a result of an extensive nucleus- cytoplasm variation and are precise to the genetic population to which the male-sterile individual belong (Lacadena 1968). Cytoplasmic genetic male sterility arose spontaneously in maize, beet root, onion and chilli pepper (Duvick 1965; Owen 1945; Jones and Clarke 1943; Peterson 1958) which is the example of this type of CGMS. But homoplasmic male sterility originated because of the interaction between S cytoplasm and nuclear gene from different genetic backgrounds belonging to the same taxonomic species. It is mostly suited to the case of CMS arisen by intraspecific combination as in rice and sorghum (Sampath and Mohanty 1954; Stephens and Holland 1954). Obviously the autoplasmic male sterility might have evolved from an ancestral homoplasmy. On the other hand, alloplasmic male sterility originated from the interaction of cytoplasm and chromosome from different species. Alloplasmic male sterility which derived from substitution back crosses can be classified into interspecific and intergeneric alloplasmy based on the species involved that belong or not to the same taxonomic genus, respectively (Kihara 1951). Alloplasmic male sterility is reported in carrot and onion (Nothnagel et al. 2008; Vu et al. 2011). Cytoplasmic male sterility are reported in the number of vegetables including chilli, cole crops, radish, carrot, beet root, onion, etc., and the source of sterilityinducing cytoplasm in these vegetables is either spontaneous or intraspecifically/ interspecifically/intergenerically derived which is enlisted in Table 10.4.

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Table 10.4 Source of sterility-inducing cytoplasm in vegetables Crop Chilli

Tomato Brinjal

French bean Cole crop

Source USDA P.I. 164835 1005 and 1006 C. frutescens Seungchon and Suwon CMS-pennellii S. gilo S. kurzii S. violaceum S. virginianum S. aethiopicum Aculeatum Group S. anguivi S. grandifolium CV. Morton Accession line G08063 P. coccineus Cultivar ‘Kurodane Kinugasa’ Japanese radish (Raphanus sativus)

B. nigra

Radish

Carrot

Beet root

Onion

B. tournefortii D. muralis Erucastrum canariense and Moricandia arvensis Ogura cytoplasm from Japanese radish NWB cytoplasm from Korean radish Radish from Uzbekistan B. maurorum Japanese radish cultivar Kosena Wisconsin or Cornell cytoplasm from wild carrots Daucus carota carota Petaloid cytoplasm ‘Guelph’ from wild carrot population Cytoplasm of Daucus carota gummifer, D. carota maritimus and D. carota gadecaei Owen cytoplasm from cultivar ‘US1’ New source of CMS I-12CMS (3) from wild beets collected from Pakistan BMC-CMS cytoplasm of the wild beet Beta maritime S cytoplasm from cultivar Italian Red T cytoplasm from cultivar Jaune Paille des Vertus

References Peterson (1958) Shifriss and Frankel (1971) Csillery (1983), Woong Yu (1990) Anon (2006) Petrova et al. (1999), Radkova (2002) Fang et al. (1985) Khan and Isshiki (2009) Isshiki and Kawajiri (2002) Khan and Isshiki (2008) Khan and Isshiki (2010) Khan and Isshiki (2011) Hasnunnahar et al. (2012), Saito et al. (2009) Singh et al. (1980) Bannerot and Charbonnier (1987) Bannerot et al. (1974), McCollum (1981), Dickson (1975), Hoser-Krauze (1987) Pearson (1972), Kaminski and Dyki (2007) Pradhan et al. (1991) Shinada et al. (2006) Chamola et al. (2013) Ogura (1968) Nahm et al. (2005) Kim et al. (2007), Lee et al. 2008, 2009) Bang et al. (2011) Ikegaya (1986) Thompson (1961), McCollum (1966) Wolyn and Chahal (1998) Nothnagel et al. (2000)

Owen (1945) Mikami et al. (1985) Mann et al. (1989) Jones and Clarke (1943) Berninger (1965) (continued)

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Table 10.4 (continued) Crop

Source Cytoplasm from Allium roylei Cytoplasm from Nasik White Globe

References Vu et al. (2011) Pathak (1997)

10.4.2 Diversity of Male Sterility-Inducing Cytoplasm in Vegetables Heterosis is a well-proven genetic tool that has contributed significantly to enhance productivity in crops including vegetables, and CMS is one of the most important mechanisms for commercial exploitation heterosis and hybrid development in chilli, carrot, onion, French bean, cole crops, etc. Genetic diversity in the source of sterile cytoplasm is essential for exploitation significant amount of heterosis and to safeguard the hybrids from outbreak of devastating disease. The use of a solitary source of sterile cytoplasm makes hybrid varieties more susceptible to contamination of cytoplasm-inherited diseases, results in devastating yield reduction and is considered as a restrictive element for hybrid cultivar development (Cao and Rong 1997). Utilization of male-sterile lines having same source of sterilizing cytoplasm will certainly lead to F1 hybrid cultivars carrying a single source of cytoplasm because of maternal inheritance of cytoplasm. The race of a particular cytoplasm-related pathogen may rapidly develop, may become the principal race under high selection intensity and result in epidemics of the disease that can cause yield losses to a great extent in hybrid cultivar (Liu et al. 2014). Therefore exploitation of the new and novel sterilizing cytoplasm to develop male-sterile lines is quite essential to diversify the CMS lines which are very basic for the detection and utilization of novel classes of cytoplasm in breeding programme of vegetable crops. In chilli Peterson cytoplasm is most commonly used for the production of the seeds of F1 hybrid (Duvick 1959; Shifriss 1997). Utilizing the sterile cytoplasm of ‘Seungchon’ and ‘Suwon’, AVRDC—the World Vegetable Center diversified the CMS lines of chilli (Anon 2006) and transferred the CMS into 14 chilli and 6 bell pepper maintainer lines (Gniffke et al. 2009). In brinjal CMS system can be diversified by using different sources of sterilizing cytoplasm and S. kurzii, S. gilo, S. virginianum, S. violaceum, S. aethiopicum Aculeatum Group, S. grandifolium CV. Morton and S. anguivi are some potential sources for this purpose. Based on the types of cytoplasmic male sterility and genetic background during the process of transferring sterility, there was a complex variation observed in the morphologies of CMS floral organs (Zhu et al. 1998). Variation in the floral organ of CMS lines may perhaps substantially influence the efficiency in pollination during the production of hybrid seeds (Shu et al. 2015). Brassica crops are reported to have an array of sources of various types of cytoplasmic male sterility, which includes ogu CMS, pol CMS, nap CMS, nig CMS and hau CMS (Ogura 1968; Fu et al. 1995; Thompson 1972; Pearson 1981; Wan et al. 2008) which can be used for diversification of CMS line in cole crop. In broccoli, Shu et al. (2016) detected the diversity of cytoplasmic male sterility sources using 39 accessions and mitochondrial marker. In

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radish ogura, NBW and Kosena cytoplasm are well characterized and can be used for diversification of CMS line in radish. In onion two different sources of sterile cytoplasm, i.e., S and T cytoplasms, were genetically well characterized. In T and S cytoplasmic line, different alleles or loci restore male fertility. Supplementary sources of cytoplasmic male sterility in onion are reported in European countries, Japan and India and their relationships with S and T cytoplasms established. Putative CMS lines derived from the Indian variety Nasik White Globe were similar to S cytoplasm. On the other hand, T cytoplasm and two different sources of CMS from Japan were analogous. These sterility-inducing cytoplasms isolated independently were of similar type (Havey 2000). In carrot two CMS systems, i.e., brown anther type which become deformed brown colour and petaloid type which is categorized by altered petal like stamens, lack pollen production present for exploiting in hybrid breeding. The brown anther type is present in many cultivars of carrot with a varying degree of expression and also reported in wild carrot genotypes (Morelock 1974; Ronfort et al. 1995), but petaloid type was detected in wild carrots (Daucus carota var. carota) by Thompson (1961) and McCollum (1966). Various findings established the genetic uniqueness of the brown anther- and petaloid-type sterile cytoplasms (Pingitore et al. 1989; Ronfort et al. 1995). Six STS markers are used to classify CMS lines and male fertile lines. STS 2 marker classified five CMS lines into two groups, i.e., Wisconsin Wild and Cornell origins (Nakajima et al. 1999). Owen CMS is the only male sterility system in sugar beet on which the F1 hybrid development and production of hybrid seeds entirely depend up on in this crop (Bosemark 2006). Till date quite few additional sources of sterile cytoplasm had been identified in wild relatives of beet (Mikami et al. 1985; Hallden et al. 1988; Touzet et al. 2004) which provide enormous opportunity to broaden the genetic base of the CMS lines. In the natural populations of wild beet (Beta maritime), a new sterile cytoplasm (Smar) has been sought (Boutin et al. 1987; Hallden et al. 1988; Desplanque et al. 2000). Furthermore to the previously identified (Smar) sterile cytoplasm, a novel mitochondrial genotype (R type) of sterile plant has been described in wild beet from the Canche and Somme bay (Saumitou-Laprade et al. 1993). An accession of B. maritime which is collected from Brittany carried the lone sterile type of mt-DNA which resembles the R type as revealed by Eco-RI restriction pattern (Jassem 1985; Sadoch and Goc 1997). These novel CMS types identified in wild Beta species seem to be a potential source of male sterility which can be used for diversification of sugar beet genotypes. Three cytoplasmic male-sterile breeding lines reported to have male-sterile cytoplasm from B. maritima and Beta cicla were analysed. These breeding lines contained three distinctly different kinds of sterile mt-DNA (Hallden et al. 1990).

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Fertility Restoration in Cytoplasmic Male-Sterile Line

Though CMS is governed through extra nuclear genome, occasionally genes from the nucleus are capable of reinstating fertility in male-sterile lines. In CMS system once the nuclear genes for fertility restoration (Rf) are identified, then it is known as cytoplasmic genetic male sterility. In this system, pollen sterility is exhibited through the influence of both nuclear and extra nuclear or cytoplasmic genes. The fertility restoration or Rf genes are different from that of the genes responsible for genic male sterility, and Rf genes worked only in the presence of sterile cytoplasm ‘S’. The Rf genes are essential to bring back fertility in the background of sterile cytoplasm. Accordingly plants carrying normal or ‘N’ cytoplasm and ‘S’ cytoplasm along with Rf gene are always breed fertile phenotypes, while ‘S’ cytoplasm with ‘rfrf’ produces only male-sterile phenotypes. Additional distinctive attribute of these systems is that the Rf mutations are common. Thus ‘N’ cytoplasm with ‘Rfrf’ is preeminent for stable fertility reinstatement. CMS is maternally inherited and frequently related to rare open reading frames (ORFs) occurred in mitochondrial genomes (Hanson and Bentolila 2004; Chase and Babay-Laughnan 2004). In most of the cases of CMS, pollen fertility is reinstated by nuclear encrypted fertility restorer ‘Rf’ gene(s). Thus CMS/Rf mechanisms are of great value to study the interaction of nuclear and mitochondrial genomes. Rf genes are generally responsible for the synthesis of pentatricopeptide repeat (PPR) proteins which enable them to musk the effect of mitochondrial genes governing CMS (Schnable and Wise 1998; Castandet and Araya 2012; Fujii et al. 2011). The pollen sterility in CMS lines resulted from genes of mitochondrial origin responsible for cytoplasmic dysfunction, but restoration of fertility depends on nuclear genes that overturn cytoplasmic dysfunction (Eckardt 2006). The Rf genes are very important in the crops like chilli, French bean and brinjal where fruits/seeds are an economic part than the crops like cole crops, carrot, radish, etc. whose vegetative parts are of economic important as it is indispensable to produce fertile F1 hybrid in the former type of crop plant. Rf genes reported in various vegetables were documented in Table 10.5.

10.5.1 Inheritance of Rf Elements in Vegetables There are lots of reports available on the relationship between CMS and Rf genes responsible for fertility reinstatement in various plants and offer a better understanding of genetic variation and the interaction of mitochondrial and nuclear genomes in crop plants (Budar and Pelletier 2001). The Rf genes in the nucleus overpower development of the male-sterile phenotype and also permit utilization of CMS system for hybrid seed production at the commercial scale. Though in chilli restoration of fertility is governed by one major gene as suggested by the earlier studies (Gulyas et al. 2006; Peterson 1958), numerous studies revealed that the inheritance pattern of fertility restoration is very complex. Various reports also documented that in sweet pepper two complementary genes governed the fertility restoration mechanism (Novak and Betlach 1971). Still it is not quite clear about the number of Rf

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Table 10.5 Source of sterility-inducing cytoplasm in vegetables Rf gene Rf

CMS type Peterson CMS

French bean Brinjal

Fr, Fr2

CMS-Sprite

Rf

Beet root

Rf1 RfG1, RfG2 Rfk1

aethiopicum-, anguivi- and grandifolium-derived CMS Owen CMS CMS-G

Crop Chilli

Radish

Cole crop

Rfd1 Rfo Rfo2 Rfp

CMS-Kos CMS-Don Ogura-CMS 9802A1 CMS radish pol CMS

References Peterson (1958), Shifriss (1997), Yoo (1990) Abad et al. (1995), Mackenzie and McIntosh (1999) Khan et al. (2014) Yamamoto et al. (2005) Ducos et al. (2001) Iwabuchi et al. (1999), Koizuka et al. (2003) Park et al. (2013) Yasumoto et al. (2009) Wang et al. (2008) Liu et al. (2012)

genes that contribute towards the fertility reinstatement in chilli pepper. Wei et al. (2020) studied the genetics of Rf genes in the two different populations and reported that it is governed by two major additive-dominant epistatic genes and also observed that the two major genes also reported for high additive and dominant effects. Besides, substantial epistatic reaction among the two major genes is also reported. In brinjal segregation patterns for fertility restoration unravelled that two dominant Rf genes which are independent to each other are responsible for fertility restoration in available CMS system (Khan and Isshiki 2016). Hasnunnahar et al. (2012) also reported two independent dominant genes regulate the pollen development in brinjal with the cytoplasm of S. grandifolium. In Phaseolus vulgaris reinstatement of fertility in cytoplasmic male-sterile line was governed by a single gene Fr (Mackenzie and Bassett 1987).

10.5.2 Molecular Mapping and Cloning of Rf Elements in Vegetables Fertility restoration (Rf) genes have enormous importance from an economic point of view for efficient seed production of F1 hybrids and academic points of view for their logical significance as a prototypical system to explore the control of nuclear gene (s) on mitochondrial gene(s) expression and also for simultaneous development of the nuclear and mitochondrial genome (Hanson and Bentolila 2004; Chen and Liu 2014). In the past two decades, Rf genes have been effectively cloned in many crop plants including vegetables, viz. radish, sugar beet, Chinese cabbage and chilli (Koizuka et al. 2003; Matsuhira et al. 2012a, b; Zhang et al. 2017, 2020a, b; Ortega et al. 2020). In chilli pepper CA00g82510 is described as a strong candidate gene for

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CaRf032 which is an only dominant locus for fertility reinstatement in CMS system. Five KASP markers were also identified as the ones capable of detecting SNPs in CA00g82510 of 77013 and IVF2014032, were also jointly segregated with CaRf032 and exhibited 64.4% effective genotyping of the population of maintainer and restorer lines (Zhang et al. 2020a, b). Ortega et al. (2020) also demonstrated the efficiency of the previously identified CAPS marker Co1Mod1-CAPS and PPR derivative CaRf648 in predicting the Rf phenotype in a segregating population. The marker Co1Mod1-CAPS identified the genotype with Rf gene, while CaRf648 can rapidly and economically recognized the existence of Rf element in Capsicum annuum. A fertility restorer gene, BrRfp1, which is most likely similar to the B. napus restorer gene (Rfp) was cloned in Chinese cabbage for pol CMS (Zhang et al. 2017).

10.6

Progress Towards Deciphering Male Sterility Determinants

In crop plant inability to produce fertile pollen grain inherited maternally and mainly triggered through mitochondrial genes is the characteristic feature of CMS system. There is ample diversity reported in various crops for mitochondrial sterilizing genes, and this diversity can be realized in the intra- and interspecies level in maize, beet or rice (Kumar et al. 2020). In spite of this diversity, most of the sterile-inducing genes are of de novo origin. These genes perhaps are generated through recombination and characterized by their chimerical nature. Mitochondrial sterilizing genes are associated with male sterility, and the production of pollen grains is exclusively exaggerated by the appearance of mitochondrial sterilizing factors, which can be explained by two nonexclusive theories. The first assumption was known as ‘pollen hypothesis’ which adopts the fact that normal growth and development of male reproductive organ is disturbed due to interaction of an element present solely in anthers and organelles with changed structures (Flavell 1974). Phaseolus vulgaris CMS is an example of the ‘pollen hypothesis’ where the protein which is responsible for CMS was degenerated by a protease enzyme in the mitochondria of vegetative tissues (Sarria et al. 1998). The second hypothesis is regarded as ‘ATP hypothesis’ which describes that sterilizing factors responsible for the mitochondrial dysfunction resulted from pollen grain developmental phase which is extremely energy demanding as described by an upsurge of the number of mitochondria in each cell in the tapetum or sporogenous cells in maize (Warmke and Lee 1978). As per ‘ATP hypothesis’, genes responsible for sterilizing are frequently co-transcribed along with atp genes of the ATP synthase enzyme, and thus they disturb expression of sterilizing gene which could consequently affect ATP synthesis (Hanson and Bentolila 2004). Yamamoto et al. (2008) identified a unique 12-kDa polypeptide in I-12 CMS resulting from the cytoplasm of wild beet through an in organello protein translation study. A unique mitochondrial CMS-associated gene (MCAG) characteristic was reported in Owen CMS where an alternate 35-kDa polypeptide was translated in the mitochondria only in Owen CMS encoded by the 50 leader sequence of an ATP synthase subunit encrypting atp6 (Yamamoto et al.

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2005). Unique genes were also reported in the Ogura CMS and Kosena CMS in radish. In the case of Ogura CMS, ORF138 was associated with the translation of a 19-kDa protein (Grelon et al. 1994), while an orthologous sequence has been reported from Kosena CMS radish which carried 13 amino acid-condensed ORF125 (Iwabuchi et al. 1999).

10.7

Marker-Assisted Selection and Hybrid Breeding

Hybrid breeding utilizing cytoplasmic male sterility system comprises of several essential steps like quick identification of potential sources of sterile cytoplasm along with possible restorers for fruit vegetables like chilli and French bean from diverse and improved gene pool; transfer of sterile cytoplasm into the desirable background; precise introgression chromosomal segment containing Rf element into desirable genetic background; characterization of parental lines to differentiate them; and assuring genetic purity in parental lines and hybrids. In recent time several findings revealed the phenomenal application of DNA markers and marker-assisted breeding quick identification, characterization and introgression of CMS system in suitable background in many vegetables. In cabbage two sets of SSR markers, i.e., cpSSR and mtSSR primers, detected polymorphism in six different types of cabbage CMS system (Pol CMS, Nig CMS, Ogu CMSHY, Ogu CMSR1, Ogu CMSR2 and Ogu CMSR3), and three primers, namely, ACP43, ACP47 and mtSSR2, clearly differentiated these six cabbage CMS types. Precise analysis of either mitochondrial or chloroplast SSR might be a realistic choice for cabbage CMS-type identification (Wang et al. 2012). Eight useful mitochondrial molecular markers identified, which were capable of identifying ogu CMS in 39 diverse CMS broccoli genotypes, contained the CMS-related orf138 fragment (Shu et al. 2016). Yu et al. (2016) showed that the unique allele-specific marker might be useful for the marker-assisted breeding for Rfo gene in B. oleracea, and it will show the way forward towards the improvement of Ogu-CMS restorer stock in cabbage and allied subspecies. One primer pair at the 30 region of the atp6 gene and the 50 region of the nad3 gene that created a 2-kbp DNA fragment was specific to NWB CMS and absent in other radish CMS types. This 2-kbp DNA fragment might be utilized as a DNA marker to detect NWB CMS in a radish (Nahm et al. 2005). Bach et al. (2002) identified PCR-based molecular markers targeting atp1, atp6, atp9, orfB (atp8), nad6 and cob loci, which were capable of differentiating the mitochondrial genomes of petaloid CMS line and male fertile carrot. Santos et al. (2010) identified maintainer and male-sterile onion lines within Brazilian onion-derived progeny and S, T and N cytoplasms examined using PCR-based DNA markers. T cytoplasm associated with 180-bp and 473-bp DNA fragments was produced by 50 cob-marker and orfA501-marker, respectively, while 180-bp fragment produced by 50 cob-marker is linked with the maintainer line. Inverse PCR was carried out to distinguish the nucleotide sequences of the 50 and 30 adjoining to the mitochondrial atp6 and coxII from the cytoplasms of male fertile and CMS chilli plant to design a CMS-specific SCAR markers. Based on the sequence

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data, 607 and 708 bp long two CMS-specifics SCAR markers developed, which clearly distinguished N-c8.

10.8

Adoption of Modern Omics Technologies

The era of omics technologies evolve continuously in terms of quantity and excellence of output in crop plants. Omics technologies like genomics, transcriptomics and proteomics undergoing constant refinement with improved efficiency and precision for data mining in crop plants are becoming exceptionally abridged. Novel insights of various omics techniques are equally capable of decoding the information related to CMS/Rf mechanism in various vegetable crops where CMS system is exclusively exploited for hybrid development.

10.8.1 Mitochondrial Genome Sequencing In plants larger variation (100–11,000 kb) was detected for the mitochondria size as compared to animal mitochondria (~16 kb) and is considered as the power house of the cell (Tuteja et al. 2013). Mitochondria in the cell are also known for their widespread genomic restructurings in the non-coding regions which operate as the primary driving power that controls the plant mitochondrial genomes and are also responsible for the initiation of male-sterile phenotypes (Tan et al. 2015). In present time an inclusive appraisal with the reference mitochondrial genome has been made possible to discover several novel orfs which could elucidate the indefinable mechanism of sterility and fertility reinstatement in plants. In chilli whole mitochondrial genomes were sequenced and assembled for a CMS line (138A) and its maintainer line (138B), and the size of the mitochondrial genome was reported 504,210 bp for 138A and 512,828 bp for 138B. Besides, more than 214 and 215 open reading frames longer than 100 amino acids are also reported in 138A and 138B, respectively. Due to the presence of recombination and reorganization events, the mitochondrial genome structure of 138A was quite different from the maintainer 138B (Wang et al. 2019). Though in the mitochondrial genome a larger fraction of background sequence was common in the CMS and male-fertile chilli lines, the widespread genome reorganization was also spotted. Candidate genes for CMS were positioned on the edges of extremely reorganized DNA regions’ specific cytoplasmic male sterility which is close to repeat sequences. Wang et al. (2020) performed mitochondrial genome sequencing which revealed that orf463a might be accountable for the induction of male sterility in NWB cytoplasm of radish and also reported the most fascinating finding that orf463a was indistinguishable to open reading frame (orf463), which is accountable for sterility in Dongbu cytoplasm and genic male-sterile (DCGMS) radish. In Owen CMS system of beet root, the mitochondrial genomes of male-fertile and male-sterile cytoplasms were reported to be highly reorganized which were related to each other, and lots of inversional recombinations

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and other reorganization actions were essential to be hypothesized for interconverting the two genomes (Satoh et al. 2006).

10.8.2 Whole Transcriptome Profiling In whole transcriptome profiling of an organism, all RNA transcripts come under consideration regardless of whether the RNA is a coding or non-coding one, which provided an accurately inclusive picture of transcriptomes of that organism. The causal genetic factors which play a major role to induce male sterility as well as the reclaim can be deciphered in crop plant by investigating the plenty of transcripts in the male reproductive part or flower buds. Screening of the cDNA library or subtractive hybridization and microarray was commonly practised to reveal the appearance patterns of putative candidate genes/ORFs accountable for sterility/ fertility restoration. Liu et al. (2019) examined the complete mechanism of CMS and homeotic organ interchange at the time of flower development in carrot through transcriptome analysis between the petaloid CMS line and its maintainer line at various flower development stages and reported 2838 differentially expressed genes, out of which 1495 genes were expressively downregulated and 1343 genes were appreciably upregulated in the petaloid CMS line. In CMS broccoli differentially expressed genes related to sterility were unveiled through comparative transcriptomic analysis. A total of 39,694 and 44,513 unigenes were assembled from 24 and 53.5 million transcriptome reads in CMS line and its maintainer, respectively. These results also identified differentially expressed 218 unigenes which include 145 downregulated and 73 upregulated unigenes (Pei et al. 2017). In radish a comparative analysis of radish floral buds from a CMS line and its maintainer line was carried out. Digital gene expression (DGE) profiling had recognized 3504 expressively differentially expressed genes between CMS line and maintainer line, out of which 1910 and 1594 reported as upregulated and downregulated, respectively.

10.8.3 High-Throughput Sequencing and Degradome Sequencing A microRNA or miRNA can be defined as a small piece of non-coding RNA molecule which contains approximately only 22 nucleotides and reported to occur in plants, animals and some viruses; that role in RNA silencing and posttranscriptional regulation of the expression of a gene is known as microRNA or miRNA. Involvement of miRNAs in the growth and development of male and female reproductive tissues/organ has been increasingly given emphasis in plants in the modern time (Millar and Gubler 2005). Zhang et al. (2016) studied comparative small RNAome sequencing of the flower buds in CMS line and its maintainer using high-throughput sequencing in radish. They isolated a sum of 162 known miRNAs belonging to 25 conserved and 24 non-conserved miRNA families and also identified 27 potential unique miRNA families for the first time in flower buds in

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radish. Out of these miRNAs, 28 recognized and 14 potential unique miRNAs were differentially expressed at the time of anther development. For improved understanding of the role and regulatory mechanism of miRNAs for the expression of CMS, Zhang et al. (2020) created miRNA libraries from the male reproductive organ of CMS line (N816S) and its maintainer (Ning5m) by miRNAome sequencing in chilli. They detected 76 differentially expressed miRNAs; among them, 18 miRNAs were additionally confirmed by quantitative RT-PCR. Besides, degradome sequencing was used to identify miRNA targets which showed 1292 targets were potentially cleaved by 321 miRNAs.

10.8.4 Proteomic Approach for Greater Understanding Transcript abundance was not always able to reveal considerable variation between male-sterile and fertile lines in crop plants, but there was discrepancy in the expression patterns observed at the proteomic level (Hu et al. 2013). The decisive expression of any characters is because of the expression of proteins and metabolites determined by different genes, and these proteins were controlled through particular metabolic pathways and monitored the expression of a said character. Therefore, an inclusive investigation of proteome to have better understanding for initiation of sterility male reproductive organ and retrieval of fertility in CMS/Rf system may perhaps be handy in unraveling the molecular mechanism and discovery of unique proteins accountable for cytoplasmic male sterility and fertility reinstatement (Kumar et al. 2020). Wu et al. (2013) conducted proteomic analysis using two-dimensional gel electrophoresis of the male reproductive organ in a CMS line along with its maintainer to identify few precise proteins linked with CMS in chilli. On the basis of mass spectrometry, 27 spots were signifying the 23 distinct proteins in these identified 33 spots. Various proteins were reported to have downregulated in CMS anthers/buds. Proteomic analysis based on iTRAQ in ogura-CMS (CMS01-20) cabbage along with the maintainer revealed that there were 162 differential abundance protein species identified between these two lines, among them 92 and 70 were identified as down-accumulated and up-accumulated, respectively, in CMS01-20. Besides, eight differential abundance protein species responsible for the oxidative phosphorylation were reported to have down-accumulated in oguraCMS during energy metabolism in the mitochondrion (Han et al. 2018).

10.9

Conclusion and Future Perspectives

The male sterility or more specifically cytoplasmic male sterility as a matter of research is an evergreen and never-ending process in crop plants including vegetables due to speedy development in the molecular biology techniques and their implementation. Significant advancement has been carried out in the research and development front to understand the male sterility system in various vegetables. The extraordinary research accompanied by the detection of unique orfs responsible

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for nonviable pollen production as function of sterility in the male reproductive part and elucidation of genomic segment through fine mapping of the candidate genes paves the way for purposeful characterization vis-à-vis positional cloning of Rf elements in different vegetables. In the last few decades, different male sterility systems especially CMS and CGMS were exploited significantly at the global level for the development of yield potential hybrids. In the past decades, CMS lines were introduced from AVRDC, Taiwan, and were judiciously used for hybrid seed production in chilli. In Brassica vegetables GMS and CMS systems were utilized for F1 hybrid development, though CMS remains the most extensively exploited pollination control mechanism throughout the world because of its stability and inclusive accessibility. At the global level, CMS and CGMS are the most widely utilized in most vegetables. In the recent past, chilli CMS lines were introduced at IIVR from AVRDC, which are utilized directly or indirectly to produce CMS-based hybrids. Male sterility is an important pollination control system in Brassica crops used extensively in F1 hybrid development. Broadly, GMS and CMS are two male sterility systems exploited in Brassica crops for hybrid seed production, and CMS is the most used system worldwide. It is quite apparent that CMS system is an evidential proof of the conflict between two genomes, i.e., mitochondrial and nuclear genomes, and happens at various stages of reproductive developments. Novel orfs resulted from the mitochondrial genome reorganization are considered as the most common reason for arising CMS system in plant. It has been observed that there is a common molecular mechanism even though the sterile cytoplasm was identified in different genotypes. In contrast, different ways of masking CMS via the evolution of different fertility restoration (Rf) genes are also evident. The thorough analysis of cellular and molecular events related to the development and differentiation for male reproductive organs has been significantly important. Therefore a better knowledge of amphimixis and related gene regulation can simplify the applications of male sterility for hybrid development in vegetables. Consequently, diversification of sterile cytoplasm will be useful in improving the risk of outbreak of different diseases. Moreover, DNA markers as a tool to discriminate precisely the parental lines and hybrids may guarantee their genetic purity and bypassing the conventional grow-out test in a hybrid breeding programme.

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Male Sterility and Hybrid Breeding Strategies in Safflower

11

Vrijendra Singh, Nandini Nimbkar, and C. V. Sameer Kumar

Abstract

Safflower crop is an excellent oilseed crop owing to health benefits and quality attributes of its oil. Enhancing productivity assumes specific significance in the crop as it is cultivated by small and marginal farmers of rainfed agro-ecologies. The productivity of safflower has remained stagnated at around 600 kg/ha despite the availability of several high-yielding varieties with high yield potential (up to 2 t/ha). Exploitation of hybrid vigour to develop high-yielding hybrids to rainfed environments opens avenues to overcome the problem of yield stagnation. The safflower hybrids developed by ICAR and State Agricultural Universities have exhibited a yield potential of 2.5 t/ha in multilocation evaluations. The journey of hybrid technology in India began with the discovery of genic male sterility (GMS) system, resulting in the development of promising hybrids including DSH-129 and NARI-NH-1. These hybrids exhibited 20% heterosis over standard check varieties. Recent release of cytoplasmic male sterility (CMS)-based hybrids DSH-185 (2018) and ISH-402 (2020) has raised hope of successful exploitation of heterosis for breeding and development of heterotic pool. Several genomic resources are available in the crop for tagging of sterility and fertility and purity assessment of the hybrids. Success of CMS system in the crop aids for large-scale commercial hybrid seed production and will further enhance adoption of hybrid seed by the large number of farmers. Efforts are needed to utilize the available germplasm lines to develop new hybrid combinations having pest and disease resistance. There is an immediate need to standardize the seed production technology in varied ecologies of the country to tap the potential of hybrids.

V. Singh (*) · N. Nimbkar Nimbkar Agricultural Research Institute, Phaltan, Maharashtra, India C. V. Sameer Kumar Department of Genetics and Plant Breeding, College of Agriculture, PJTSAU, Hyderabad, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_11

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Keywords

Cytoplasm · Fertility restoration · Heterosis · Male sterility · Pollen · Thermosensitivity

11.1

Introduction

Safflower is an ancient oilseed crop with quality oil rich in poly-/mono-unsaturated fatty acids which help in reducing the cholesterol level in blood, and its petals possess several medicinal and pharmaceutical properties to cure many chronic diseases like hypertension, coronary artery disorders, arthritis, spondylosis, sterility in men and women, menstrual cycle disorders, etc. Safflower flowers also produce natural colours for food, fabrics and cosmetics. Safflower at an early stage of plant growth is consumed as a leafy vegetable in and around its area of production since ages in India and neighbouring countries (Knowles 1969). Safflower leaves are rich in protein, fat, vitamin C and phenolic compounds (Singh et al. 2017). India is the second largest producer of safflower in the world after Kazakhstan. In India, Maharashtra and Karnataka account for 90% of total safflower area and production. The other important safflower-growing states are Madhya Pradesh, Andhra Pradesh, Telangana, Chhattisgarh, Gujarat and Odisha. Safflower belongs to family Asteraceae and has a hermaphroditic nature. It is a self-pollinated crop, but the stigma extrusion before anther dehiscence and pollen liberation has put it in the category of an often cross-pollinated crop. The productivity of the crop is very low at not more than 600 kg/ha though several high-yielding varieties and hybrids are available. Biotic stresses like aphids, wilt and Alternaria leaf spots are major threats affecting the productivity. Success in exploitation of GMS and recently CMS in the crop provides an opportunity to the crop improvement group in breeding and development of parental lines, highyielding hybrids and standardization of seed production technology.

11.2

Exploitation of Heterosis and Hybrid Vigour in Safflower

Safflower is an often cross-pollinated crop, and exploitation of heterosis has always been a challenge to plant breeders for commercial production of hybrid seeds at a large scale. Safflower has been reported to have very high heterosis for seed yield and its components (Classen 1950; Smith and Classen 1963; Rubis 1969; Urie and Zimmer 1969; Yazdi-Samadi et al. 1975; Deokar and Patil 1979; Ramachandram and Goud 1982; Narkhede and Patil 1987; Kulkarni et al. 1992; and Pandya et al. 1992). Efforts to commercially exploit hybrid vigour in safflower were initiated as early as 1970 by Urie and Zimmer by using the structural male sterility associated with thin hull. Subsequently Karve et al. (1977) tried to produce commercial-scale hybrid seed by following the mass emasculation method which works on the principle of causing pollen grains of a flower to become unviable by artificially

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increasing the humidity around the flower. This was done by bagging the safflower capitula a day before flowering with polythene bags of low to medium density followed by hand pollination the next day with the pollen of the desired parent. Another method employed by Karve et al. in 1979 to produce hybrid seed on a commercial scale was the irradiation of safflower seed with high dosages of gamma rays to render the resultant plants male sterile and use them for hybrid seed production programme. However, these efforts did not lead to commercialization of hybrid safflower due to inherent problems associated with the methods developed.

11.3

History and Genesis of Hybrid Development

The hybrid development in safflower got an impetus with the availability of genetic male sterile lines UC-148 and UC-149 of recessive nature developed in the USA by Heaton and Knowles (1980). The preliminary studies with the hybrids developed with these lines indicated the existence of exploitable heterosis, i.e. 19% average increase over A-1 in multilocational coordinated trials (Deshmukh et al. 1989). But the hybrids based on derivatives of UC-148 and UC-149 male sterile lines could not be commercialized owing to certain deficiencies such as poor yielding ability, improper segregation ratio of sterile and fertile plants, segregation for different traits, poor architecture making plants prone to lodging, emergence of plants which exhibited a creeping nature, greater succulence making them vulnerable to insect attack, delayed flowering and maturity, etc. associated with them. In view of the above bottlenecks, it was envisaged to improve these GMS lines by transferring the male sterility into indigenous high-yielding, well-adapted cultivars by following backcrossing as well as to search the germplasm and early and advanced generation lines for new sources of male sterility. The latter resulted in identification and development of genetic male sterility systems which can be broadly categorized as below: 1. Recessive genetic male sterility 2. Dominant genetic male sterility

11.3.1 Recessive Genetic Male Sterility The genetic male sterility sources reported to be controlled by recessive genes in addition to the source reported by Heaton and Knowles (1980) are as follows: 1. GMS (DOR) male sterility reported by Ramachandram and Sujatha (1991) 2. MSN and MSV GMS lines developed by Singh (1996) 3. Genetic male sterility associated with dwarfness as a marker trait (DMS) reported by Singh (1997) 4. GMS derived from safflower cultivar HUS-305 reported by Ghorpade (1999)

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Due to the monogenic recessive control of male sterility in the sources listed above, they segregate into 50% male sterile (MS) and 50% male fertile (MF) plants during flowering of the crop. The male sterile sources UC-148, MSV and MSN exhibited allelic interaction among the male sterility-causing genes in them and non-allelic interaction with the sterility genes in DMS and GMS (DOR). Singh (1996, 1997) also observed the non-allelic interaction between the sterility-causing genes in DMS and GMS (DOR). Male sterile and male fertile plants are distinguished at flowering by the pinched, incomplete opening of the capitulum in case of male sterile plants and a normal spreading opening in case of male fertile plants of all the GMS sources mentioned above except the DMS. In DMS lines due to the linkage between the genes responsible for male sterility and dwarfness, the male sterile and fertile plants are identified at 30 to 40 days after sowing. At the age of 30 to 40 days, the male fertile (MF) plants attain a height of 20–25 cm; however, the male sterile (MS) plants remain dwarfs of 5–10 cm. The difference in height of MF and MS plants helps in easy identification and roguing of MF plants at this stage from the seed production plot, thus leaving a pure stand of dwarf MS plants.

11.3.2 Dominant Genetic Male Sterility Joshi et al. (1983) developed the dominant genetic male sterility in safflower. The identification of MS and MF plants in dominant gene-controlled MS lines is similar to that in male sterility sources governed by single recessive genes and is feasible only during flowering of the crop. Due to the dominant nature of the male sterility gene, hybrids and the male sterile line segregate into 50% MS and 50% MF plants. The occurrence of 50% MS plants in the hybrid population adversely affects the yielding ability of the hybrids if external pollinators are not sufficient to produce 100% seed setting in the male sterile plants. The use of dominant male sterility for hybrid development therefore remained only at the experimental stage. The advent of new genetic male sterility sources as mentioned above followed by development of agronomically promising male sterile lines of diverse nature provided the desired thrust to accelerate the hybrid development in safflower. The accelerated efforts in this direction resulted in development and release of hybrids DSH-129 and MKH-11 during 1997 giving an increase of 22% in seed yield and 30% in oil yield over the national check A-1 at all India level. This enhancement in safflower productivity due to exploitation of hybrid vigour in fact was a breakthrough by any measure, as the conventional methods of breeding did not result in any increase in the safflower productivity, and as a result, no safflower variety was released between 1990 and 2000. The development of non-spiny GMS-based hybrids NARI-NH-1 (Table 11.1; Fig. 11.1) in 2001 and NARI-H-15 (Table 11.2; Fig. 11.2) in 2006 further boosted the success of hybrid safflower production in India not only because of their greater yields but also because of an ease of hybrid seed production in them owing to non-spiny nature of their female parents. The non-spiny hybrid NARI-NH-1 in addition to giving high seed yield also provided an opportunity to harvest

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Table 11.1 Performance of NARI-NH-1 for seed and oil yield in coordinated trials

Mean seed yield (kg/ha)

Mean oil yield (kg/ha)

Year of testing 1998– 1999 1999– 2000 2000– 2001 Mean 3 years 2 years 1998– 1999 1999– 2000 2000– 2001 Mean 3 years 2 years

No. of trials 6

NARINH-1 2016

JSI-7 (non-spiny check) –

Percentage (%) increase over check –

7

1619

1369

18.26

5

2172

1668

30.22

18 (T) 12 (T)

1936 1895

– 1518

– 24.83

6

643

–

–

7

532

419

26.97

5

654

474

37.97

18 (T) 12 (T)

610 593

– 446

– 32.96

Fig. 11.1 GMS-based non-spiny hybrid NARI-NH-1

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Table 11.2 Performance of NARI-H-15 in coordinated trials

Mean seed yield (kg/ha)

Mean oil yield (kg/ha)

Year of testing 2002– 2003 2003– 2004 2004– 2005 Weighted mean 2002– 2003 2003– 2004 2004– 2005 Weighted mean

No. of trials 6

NARIH-15 2522

NARI-NH-1 (hybrid check) 2008

8

2214

2057

7.63

9

1975

1573

25.56

23 (T)

2201

1855

18.65

5

722

589

22.58

7

636

613

3.91

4

657

530

23.96

16 (T)

668.6

584.8

14.35

Fig. 11.2 GMS-based hybrid NARI-H-15

Percentage (%) increase over check 25.6

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high-quality flowers to the extent of 250 kg/ha. These flowers fetched high market price which made safflower highly remunerative.

11.4

Thermosensitive Genetic Male Sterility in Safflower

Thermosensitive genetic male sterility (TGMS) (Fig. 11.3) in safflower has been developed from the derivatives of a CMS-based hybrid of an exotic origin. The thermosensitive nature of male sterility was first noticed in sib-mated crosses of CMS-based hybrid derivatives during winter 2004–2005 when two to three sib-mated crosses and their respective male parents exhibited 100% male sterility in the sib-mated crosses and 75–84% in the pollinator parents. To confirm the thermosensitive nature of male sterility in them, the leftover seeds of the concerned crosses and the related pollinator parents were sown in summer 2005. The screening of the said crosses and their parents for male sterility during flowering of the crop revealed only 2.94% male sterility in the crosses and no sterility in the parents as compared to 84 and 75% male sterility, respectively, observed in them during previous winter. The contrasting nature of the crop in the two seasons thus confirmed the thermosensitive nature of male sterility in the crop. Fertile plants of the crosses as well as of pollinator parents were selfed before flowering to prevent any chance of outcrossing and were threshed individually to evaluate them in Rabi 2005–2006. The seeds of each individual plant were divided into two parts: one part was used for screening for male sterility during Rabi 2005–2006, and the other part was kept for multiplication during summer. Only the lines expressing 100% male sterility during Rabi 2005–2006 were selected for multiplication during summer season. This process was followed for 2–3 years to enable development of uniform and stable thermosensitive genetic male sterility for

Fig. 11.3 Capitulum of thermosensitive genetic male sterility

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Table 11.3 Performance of NARI-H-23 in coordinated trials

Mean seed yield (kg/ha)

Mean oil yield (kg/ha)

Year of testing 2019– 2010 2010– 2011 2011– 2012 W. mean 2019– 2010 2010– 2011 2011– 2012 W. mean

No. of trials 4

NARI-H-23 TGMS hybrid 1817

NARI-H-15 GMS hybrid 1839

5

1693

1655

+2.30

10

1677

1570

+6.81

19 (T) 3

1710.68

1649.0

+3.74

524

474

+10.55

4

477

445

+7.19

10

535

474

+12.87

17 (T)

519.65

467.23

Percentage increase/ decrease over NARI-H15 1.20

11.22

hybrid development. The thermosensitive male sterility shows 100% male sterility during winter when average daily minimum and maximum temperature is 21 and 39  C, respectively, in the period from capitula formation to completion of flowering. The thermosensitive male sterility in safflower was reported to be controlled by inhibitory genes (Singh et al. 2008). The advantage of thermosensitive male sterility over genetic male sterility in safflower is that it enables a 100% stand of male sterile plants to be obtained as in the case of cytoplasmic male sterile line, while only 50% plants in GMS line in a seed production plot are male sterile when grown under winter conditions. Similarly TGMS has another advantage over CMS line in that the seed of TGMS line can be used as female for hybrid seed production in winter and for multiplication in summer. There is no need to have a separate male sterility maintainer line as is required in the case of CMS system (Chen and Liu 2014). Another advantage of thermosensitive male sterility over CMS system is that any fertile genotype can act as a male parent for hybrid development, unlike CMS system in which only those genotypes which contain fertility restorer genes can be used as a male parent. The thermosensitive genetic male sterility-based hybrids recorded high heterosis of 20–40% over the genetic male sterility-based hybrid checks (Singh et al. 2008, 2014). The safflower hybrid NARI-H-23 (Table 11.3; Fig. 11.4) developed and released for commercial production by Nimbkar Agricultural Research Institute (NARI), Phaltan, in 2014 is based on thermosensitive genetic male sterility system.

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Fig. 11.4 TGMS-based hybrid NARI-H-23

11.5

Cytoplasmic-Genetic Male Sterility (CMS) in Safflower

In CMS plants, non-functional pollen grains are formed as a result of aberrations in cytoplasmic-nuclear communication (Bohra et al. 2016, 2017; Mishra and Bohra 2018). The work on development of cytoplasmic-genetic male sterility in safflower was initiated by A. B. Hill in 1972 by making interspecific crosses. Hill (1989) reported the development of cytoplasmic male sterility in safflower. The cytoplasmic male sterile plants of this source produced either rudimentary anthers or no anthers at all. This source of cytoplasmic male sterility had restoration of its fertility when sown 1 month later than the recommended date of sowing in April in the USA. Delayed sowing led the crop to flower under relatively higher temperatures than the temperatures which prevailed during flowering of the crop sown in April, thus indicating the thermosensitive nature of male sterility in it (Hill 2008). Development of cytoplasmic male sterility in safflower in India was first reported by Singh et al. (2001). In this case an interspecific cross between Carthamus palaestinus and C. glaucus showed an occurrence in F3 generation of a male sterile plant with rudimentary anthers but a fertile and fully grown stigma. Subsequent crossing of this male sterile plant with fertile sib-counterparts and C. tinctorius genotypes revealed the male sterility to be cytoplasmic in nature. The sterile cytoplasm in the present case is donated by C. palaestinus. The cytoplasmic male sterility in this source appeared to be under the control of more than one gene. Fertility restoration to the sterile cytoplasm was caused by many germplasm lines as

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Table 11.4 CMS hybrid released by Indian Institute of Oilseeds Research Hybrid DSH-185 ISH-402

Year of release 2018 2020

Pedigree A-133 X 1705 – P22 –

Area of adaptability All India All India

well as by fertile sib-counterparts. The cytoplasmic male sterility of this source was observed to be variable in nature as the expression of male sterility was less than 100% in some years. In order to have diverse cytoplasmic male sterility systems in safflower, mutagenesis with streptomycin was carried out in a highly adapted genotype NARI-2 at NARI, Phaltan, during 2001–2002. The male sterile mutants were identified in the plants treated with streptomycin dosage of 50 mg/L. Similar to C. palaestinus-based CMS system mentioned above, the streptomycin-induced male sterility also exhibited multigenic control of male sterility and the presence of fewer fertility restorer genotypes. The expression of male sterility in this system also was less than 100% in some years. Since the cytoplasmic male sterility was induced in a cultivated species, it is categorized as autoplasmic cytoplasmic male sterility (Singh 2005; Singh and Nimbkar 2018). Another attempt was made to develop cytoplasmic male sterility from an exotic CMS-based hybrid following selfing and crossing of the resultant male sterile plants with fertile sib-counterparts and diverse genotypes of cultivated species. This was followed by screening of the CMS-based crosses and further backcrossing of the male sterile plants of the lines giving >70% male sterility with the concerned male parents. This activity was repeated for 3–4 years and resulted in identification of a male sterility maintainer genotype which conferred 100% male sterility to the sterile cytoplasm. This male sterility maintainer genotype was identified from an advanced generation line, viz. NARI-66. This genotype with further refinement by carrying out pairwise crossing with the male sterile plants for 2–3 generations has resulted in development of a stable male sterility maintainer genotype giving 100% male sterility. The hybrids based on this source were found to be highly heterotic and productive (Anonymous 2017). Anjani (2005) also reported the development of cytoplasmic male sterility following interspecific crossing between C. oxyacantha and C. tinctorius. C. oxyacantha was indicated as the donor of sterile cytoplasm, and the cytoplasmic male sterility was reported to be controlled by a single recessive gene. Fertility restorer gene was indicated to be present in cultivated species C. tinctorius, and its inheritance was suggested to be monogenic dominant. The CMS-based safflower hybrid DSH-185 developed and notified for commercial production during 2018 was derived from this source of male sterility (Table 11.4). Deshmukh et al. (2014) reported the successful development of cytoplasmic male sterility in safflower at Dr. P. D. K. V. Akola. This was done by carrying out the selfing of a known CMS-based hybrid followed by rising of the F2 generation and crossing of resultant male sterile plants with the fertile plants of germplasm lines and of genetic male sterile lines. This was followed by evaluation of CMS-based crosses

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along with the concerned pollinator parents and subsequently backcrossing of the incumbent male sterile plants with the pollinator parents in pairwise fashion. This process was repeated for 3–4 years and consequently resulted in development of two CMS lines AKS CMS-2A and AKS CMS-3A giving 100% male sterility across the safflower-growing areas. However, this source of cytoplasmic male sterility also showed varied male sterility percentage if not planted on an appropriate date. The hybrids based on Akola CMS lines are also highly heterotic, and hence these lines are being exploited for hybrid development.

11.6

Major Challenges and Potential Opportunities

In the days to come, only those agricultural crops will survive which either are economically important or are required for food security. Safflower is one such crop which is fast disappearing from commercial cultivation due to its practically non-existent economic importance. Notwithstanding the significant achievements made in development of high oil and high-yielding varieties and hybrids based on GMS, TGMS and CMS systems, of spiny and non-spiny nature with built-in tolerance to wilt apart from the suitable technologies for raising the crop in rainfed and irrigated ecosystems, the area and production of the crop in India have witnessed drastic reduction. Safflower area has declined from 1.05 m ha in 1987–1988 to 0.08 m ha in 2017–2018. Thus the reduction in safflower area was 92.4% in a span of 30 years. The rate of decline in safflower area has worked out to be more than 3% per annum. If this trend of decline in area continues, the remaining safflower area may be lost in the near future. The major cause of reduction in safflower area in the country is its poor profitability owing to depressed market price as compared to competing crops like chickpea and Bt cotton. Therefore, in the future crops with high profitability will gain importance and will be preferred for hybrid development. The crops with average or low profitability will continue to depend upon conventional varieties for their production. In order to boost safflower production and to make it more remunerative than at present, the Govt. of India has raised the minimum support price of the safflower seed from Rs. 3700/q to 4950/q in years 2018–2019. This may augur well for increasing its area and production.

11.7

Future Thrust Areas and Prospects of Safflower Hybrid

The prospects of safflower hybrids are highly optimistic with the rise in MSP of the crop. Development and release of TGMS-based hybrid NARI-H-23 and CMS-based hybrid DSH-185 which possess simple and cost-effective technology of seed production make them highly suitable for commercial exploitation of hybrid vigour. Conversion of TGMS and CMS lines into high-yielding, high oleic and non-spiny background for development of high oleic, non-spiny hybrids will make them widely acceptable due to the high value of the oleic-rich oil which has a very high

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probability of becoming a quality substitute to imported olive oil in the country. Several germplasm lines are available in the gene banks, and there is a need for identification of maintainer and restorer lines and diversification of the CMS sources. Aphids and wilt are the major constraints in the crop, and there is a need to identify and develop the resistant parental lines and hybrid. Vast genomic resources are available, and there is an immediate need for deploying these for hastening the process of hybrid breeding through line conversions, for development of heterotic gene pools and for the purpose of testing purity of the parental lines and hybrids. Being an often cross-pollinated and entomophilous crop, large-scale efforts are needed for standardization of seed production technology to various agro-ecologies to harness the fruits of hybrid technology in safflower. Thus, the future of hybrid safflower looks quite bright and is expected to have the capability to become one of the most profitable crops grown under rainfed conditions in India.

References Anjani K (2005) Development of cytoplasmic-genic male sterility in safflower. Plant Breed 124: 310–312 Anonymous (2017) Annual progress report. All India Coordinated Research Project, Safflower, NARI, Phaltan, pp 107 Bohra A, Jha UC, Adhimoolam P, Bisht D, Singh NP (2016) Cytoplasmic male sterility (CMS) in hybrid breeding in field crops. Plant Cell Rep 35:967–993 Bohra A, Jha A, Singh IP, Pandey G, Pareek S, Basu PS, Chaturvedi SK, Singh NP (2017) Novel CMS lines in pigeonpea [Cajanus cajan (L.) Millspaugh] derived from cytoplasmic substitutions, their effective restoration and deployment in hybrid breeding. Crop J 5:89–94 Chen L, Liu YG (2014) Male sterility and fertility restoration in crops. Annu Rev Plant Biol 65: 579–606 Classen CE (1950) Natural and controlled crossing in safflower, Carthamus tinctorius L. Agron J 42:381–384 Deokar AB, Patil FB (1979) Heterosis studies in safflower. Indian J Agric Sci 49:82–86 Deshmukh AK, Patil RM, Nimbkar N (1989) Commercial scale exploitation of hybrid vigour in safflower using genetic male sterility systems. In: RangaRao V, Ramachandram M (eds) Proc second international safflower conference 9–13 January, Hyderabad, pp 163–167 Deshmukh SN, Wakode MM, Ratnaparakhi RD (2014) Cytoplasmic male sterility development in safflower. PKV Res J 38:1–3 Ghorpade PB (1999) Development of new genetic male sterile lines in safflower. Annual Research Report (Unpub) of Dept of Agril Botany College of Agriculture Nagpur Dr PDKV Akola Heaton TC, Knowles PF (1980) Registration of UC-148 and UC-149 male-sterile safflower germplasm. Crop Sci 20:554 Hill AB (1989) Hybrid safflower breeding. In: RangaRao V, Ramachandram M (eds) Proc second international safflower conference, 9–13 January, Hyderabad, pp 169–170 Hill AB (2008) Selection for cytoplasmic genetic male sterile lines with stable sterility in late season planting. In: 7th international safflower conference, Wagga Wagga, Australia Joshi BM, Nerkar YS, Jambhale ND (1983) Induced male sterility in safflower. J Maharashtra Agric Univ 8:194–196 Karve AD, Deshmukh AK, Nagvekar DV (1977) New methods for cross pollination in safflower. Oilseeds J 7:22

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Karve AD, Deshmukh AK, Nagvekar DV (1979) Hybrid safflower. In: International congress on oilseeds and oils abstracts of technical papers, pp 13–14 Knowles PF (1969) Centres of plant diversity and conservation of crop germplasm: safflower. Econ Bot 23:324–329 Kulkarni RR, Patil VD, Mahajan AR, Nerkar YS (1992) Heterosis for yield and yield contributing characters in safflower. J Maharashtra Agric Univ 17:72–75 Mishra A, Bohra A (2018) Non-coding RNAs and plant male sterility: current knowledge and future prospects. Plant Cell Rep 37:177–191 Narkhede BN, Patil AM (1987) Heterosis and inbreeding depression in safflower. J Maharashtra Agric Univ 12(3):337–340 Pandya HM, Patil VD, Nerkar YS (1992) Dominant genic male sterility in safflower. Heterosis for yield and yield components. J Maharashtra Agric Univ 17:472–473 Ramachandram M, Goud JV (1982) Heterosis for seed yield and oil content in safflower. Indian J Agric Sci 52:561–563 Ramachandram M, Sujatha M (1991) Development of genetic male sterile lines in safflower. Indian J Genet Plant Breed 51:268–269 Rubis DD (1969) Biological and technical basis for the production of hybrid safflower without using male sterility. Agron Abst 16 Singh V (1996) Inheritance of genetic male sterility in safflower. Indian J Genet Plant Breed 56: 490–494 Singh V (1997) Identification of genetic linkage between male sterility and dwarfness in safflower. Indian J Genet Plant Breed 57:327–332 Singh V (2005) Final report of adhoc project on “Identification of early plant growth male sterility marker in existing GMS systems and search for cytoplasmic genetic source of sterility in safflower”. Submitted to ICAR, New Delhi, pp 61 Singh V, Nimbkar N (2018) Safflower research at the Nimbkar Agricultural Research Institute (NARI), Phaltan, pp 49 Singh V, Galande MK, Deshmukh SR, Deshpande MB, Nimbkar N (2001) Identification of male sterile cytoplasm in safflower. In: Bergman, JW, Mundel HH (eds) Proc fifth international safflower conference, Williston, North Dakota and Sidney, pp 123–126 Singh V, Deshmukh SR, Deshpande MB, Nimbkar N (2008) Potential use of thermo-sensitive genetic male sterility for hybrid development in safflower. In: Proc seventh international safflower conference, Wagga Wagga New South Wales Australia 3rd–7th November 2008 Singh V, Ashwini C, Burangale SV, Deshpande MB, Nimbkar N (2014) Heterosis for yield and its components in thermosensitive genetic male sterility-based hybrids in safflower. J Agric Res Technol 39:320–323 Singh V, Jadhav RR, Atre GE, Kale RV, Karande PT, Kanbargi KD, Nimbkar N, Rajvanshi AK (2017) Safflower (Carthamus tinctorius L.)—an underutilized leafy vegetable. Curr Sci 113: 857–858 Smith PL, Classen CE (1963) Development and performance of F1 hybrid safflower (Carthamus tinctotius L.). Agron Abst 90 Urie AL, Zimmer DE (1969) The performance of hybrid safflower in competitive yield trials. In: Proc third safflower research conference, University of California, Davis, pp 54–56 Urie AL, Zimmer DE (1970) Yield reduction in safflower hybrids caused by female selfs. Crop Sci 10:419–422 Yazdi-Samadi B, Sarafi A, Zali AA (1975) Heterosis and inbreeding estimates in safflower. Crop Sci 15:81–83

Insect Pollinators and Hybrid Seed Production: Relevance to Climate Change and Sustainability

12

Anup Chandra, Gopalakrishnan Kesharivarmen Sujayanand, Revanasidda, Sanjay M. Bandi, Thejangulie Angami, and Manish Kanwat

Abstract

The role of pollination in supporting food production is more than significant. Insects are the most important means of natural cross pollination. Higher foraging preference for restorer lines enables efficient pollination from male to female lines in the process of hybrid seed production of fruits, vegetables, oilseeds, pulses, commercial and many other major food crops. These have a considerable impact not only on yield enhancement but also on quality of the produce. Deliberate deployment of beehives for the above-said purpose is a century-old practice now. The order Hymenoptera represents majority of the insect pollinators; however, the role of other insects and wild insect fauna cannot be underestimated. Mainly, the crop ecosystem and nature of climatic conditions amount to the occurrence and diversity of insect pollinators. A holistic view of the diverse group of insect pollinators and tending to a particular crop ecosystem is certainly an area needed to be explored. Seen in the perspective of changing climate, insect pollinators impart resilience to the plants in stress; however, these ecologically important creatures are also subjected to direct or indirect adverse impact. In this chapter, an effort has been made to spotlight the importance and diversity of insect pollinators. The impact of changing climate has also been discussed.

A. Chandra (*) · G. K. Sujayanand · Revanasidda · S. M. Bandi ICAR—Indian Institute of Pulses Research (IIPR), Kanpur, Uttar Pradesh, India T. Angami ICAR Research Complex for NEH Region, AP Centre, Basar, Arunachal Pradesh, India M. Kanwat Krishi Vigyan Kendra (ICAR-CAZRI), Bhuj, Gujarat, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. Bohra et al. (eds.), Plant Male Sterility Systems for Accelerating Crop Improvement, https://doi.org/10.1007/978-981-19-3808-5_12

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Keywords

Climate change · Diversity · Hybrid · Insects · Male sterile · Pollination · Restorer lines

12.1

Introduction

The need and craving for higher agricultural productivity and quality traits have created buoyancy in production of hybrids in many cultivated crops (Saxena et al. 2021). In nature, a number of higher plants are sexually reproduced and perpetuated through the natural phenomenon of cross pollination (McGregor 1976; Crane and Walker 1984; Free 1993; Williams 1994; Nabhan and Buchmann 1997; Westerkamp and Gottsberger 2000) where pollens from a flower are transferred to the stigma of another flower of a different plant through external agents like insects, bats, birds, winds, etc. Different types of plants rely on different pollinating agents based on the flower-pollinating agent suitability (Table 12.1). In some plants, pollens are transferred within the flower, referred to as automatic self-pollination where pollinators are not required in the process, whereas flowers of many cultivated crops create the conditions which necessitate cross pollination. Heterogamous (varied arrangements of sexual parts), dioecious (individual plants having either male or female flowers), monoecious (flowers of both sexes on same plant but not crossable), pistillate (female flowers), staminate (male flowers), polygamous (both unisexual and bisexual flowers on same plant), protandrous (stamens matures before the pistil) and protogynous (pistil matures before the stamen) are some of the flower conditions where the role of external pollinating agents becomes imperative. In order to achieve a good quantity and quality of seed setting under hybrid seed production system, factors impacting pollination find their own importance. Type of flower, compatibility of flower shape and structure with its pollinator, feeding preferences of insects to pollen over nectar, attractiveness of flower owing to its colour and scent, visiting frequency and stay of pollinators, duration of flower opening, vibrations produced by the insect pollinators, species and their abundance affect the pollination in a considerable way. Table 12.1 Different types of pollinating agents and associated form of pollination Pollinating agent Insects Bees Butterflies Beetles Moths Bats Birds Thrips

Form of pollination Entomophily Melittophily Psychophily Cantharophily Phalaenophily Chiropterophily Ornithophily Thripophily

Pollinating agent Ants Snails and slug Snake Humans (artificial pollination) Animals Wind Water

Form of pollination Myrmecophily Malacophily Ophiophily Anthropophily Zoophily Anemophily Hydrophily

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Table 12.2 Percent increase in yield attributes reported in crops due to the effect of bee pollination Crop Sunflower (Helianthus annuus) Mustard (Brassica sp.) Bittergourd (Momordica charantia) Buckwheat (Fagopyrum esculentum) Cotton

Apple Onion Small cardamom Sweet orange

Increase in yield (%) 34 43 1356

References Abbasi et al. (2021) Devkota et al. (2021)

169 12 (fibre weight) 17 (seed number) 11.1 (bolls harvested) 16.5 (mass of bolls) 15.8 (total lint mass) 19.7 (total seed mass) 16.5 (total number of seeds) 39.50–108.3 77.45 (honey bees) 87.68 (open pollination) 21–37 35.30

Pires et al. (2014) Rhodes (2002)

Sharma et al. (2003) Padamshali and Mandal (2018) Meena et al. (2015) Malerbo-Souza et al. (2004)

Generally, insects prefer flowers which are scented and have colourful attractive petals. The insects visit flowers for nectar and transfer pollens among different flowers. Flowers of many crops depend totally on insects for their pollen to be transferred, and in the absence of these pollinators, seeds would fail to set. Most of our food crops are exclusively pollinated by insects, and it could bring famine if these creatures are wiped out. The importance of insects in pollination service can be realized with the example of the Smyrna fig which being female does not produce pollens, but the transfer of pollens is accomplished from wild caprifig, Ficus carica var. sylvestris, through the female of Agaonidae wasp, Blastophaga psenes. It is considered that the pollination done by the bees is far more valuable than their products like honey and wax (David 2001). Boosting pollination by deployment of suitable insect pollinators is the most economical and eco-friendly way to attain quality seeds as well as increased yield (Free 1970). The practice of hiring bee colonies for augmented pollination was started well in the early twentieth century in western countries and continues today. Bee-boxes are brought on a rental basis at the time of peak flowering period which results in significant enhancement in the yield and quality of crop. Remarkable yield enhancement has been observed in many crops with deliberate introduction and establishment of insect pollinators. The contribution of bees to some crops, in terms of yield magnification, has been listed in Table 12.2. In order to make balance between the foraging capacity (distance) and cost involvement of beehives, the suitable number of beehives recommended per unit area of different crops has been listed in Table 12.3.

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Table 12.3 Recommended honey beehives per acre in different crops Crop Squashes, zucchini Cucumbers Watermelons Muskmelons, cantaloupes Pumpkins Blackberries Raspberries Strawberry Blueberries Cranberries Apples Pears Plums, prunes Peaches, nectarines, apricots Cherries

Recommended hives per acre: range (avg.) 0.04–3 (1.5) 0.1–4 (2.2) 0.2–5 (1.8) 0.2–5 (1.8) 0.04–3 (1.5) 1–4 (2.7) 0.2–1 (0.8) 0.5–10 (3.5) 0.5–10 (3) 0.2–10 (3) 0.25–5 (1.5) 0.4–2 (1.5) 1–2 (1.3) 0.08–2 (0.8) 0.5–5 (1)

Source: Benjamin (2019), Delaplane and Mayer (2000), Free (1970)

The practice of spraying harmful chemical insecticides has threatened to our beneficial insect pollinators to an excess. Any adverse effect on pollinator’s population may have a straight effect on the plants pollinated by them (Biesmeijer et al. 2006). Many insecticides are highly harmful to honey bees, the use of which should be done in a judicious way. Spraying against insect-pests must be avoided during flowering time and also during the peak foraging time of the pollinators. Apart from the hazard of chemical insecticides, the bee colonies in close proximity to cell phone towers could also be affected by the electromagnetic radiation (Taye et al. 2017), but further research is needed in this area with supportive data and technological evidences (Kumar 2018). Although different types of insects contribute to pollination service, among all, Hymenoptera consists of most of the insect pollinators. Other than bees and wasp, pollination in many crops is also achieved by ants, flies, beetles, moths and butterflies, thrips and many other insects. The study on biodiversity parameters like richness and abundance of insect pollinators present in a particular crop ecosystem has their own importance from both ecological and economical points of view. The most potential pollinators are considered to be honey bees which visit about 100 flowers during a field trip and make four million field trips in a year (David 2001). Pollen is an important source of protein for honey bees and other insects too. Honey bees have specialized morphological adaptation for efficient pollination. Hind legs of worker bees are comparatively larger than that of queen or drones and carry special modification, i.e. pollen basket or corbicula. Hybrid in crops is achieved through the cross of genetically different plants wherein the role of pollinating insects becomes quintessential. In this chapter, we discuss the criticality of pollination aid provided by the diverse group of insects,

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their role in improving quality and yield attributes and influence of changing climatic conditions on the process.

12.2

Diversity of Insect Pollinators and Their Economic Role

Fijen et al. (2020) describes insect pollination as the most sensitive factor, compared to other yield-boosting agronomic interventions, which can affect the production of a hybrid seed crop. In hybrid seed production system through the cytoplasmic male sterility (CMS) method, it becomes essential for the pollens of restorer lines to be transferred to the stigma of male sterile lines. The availability of CMS system circumvents the need of cumbersome manual emasculation procedure (Bohra et al. 2016, 2017). Zheng-Hong et al. (2012) observed 25 different insect species transferring pollens from restorer lines to the male sterile lines of pigeonpea, of which, Megachile velutina Smith, along with two other species, Xylocopa tenuiscapa Westwood and a species of Apinae, was observed as a major pollination contributor in hybrid seed production of ICPH 2671 in Yunnan province of China. The family Megachilidae was most abundant with 50.9% of total captured insects. The flowervisiting insects had a greater preference to CMS restorer lines with normal flowers (5.2 times per 10 min) as compared to the A-lines with sterile flowers (2.8 times per 10 min). More than 60% of the global population sees rice as their staple food. Although it is a self-pollinated crop, hybrid seed production mainly resorts to the mechanical way of pollen transfer, which in rice is generally known to be wind driven, but the role of insects is little explored. Pu et al. (2014) observed not less than 510 different species of insects involved in transferring pollens from genetically modified (GM) rice lines to their non-GM counterparts and realized the role of honey bees foraging up to a distance of 500 m with viable pollens boosting the efficiency of gene flow. Honey bees are significant contributors and relied on for transfer of pollen in hybrid seed production of sunflower. Sunflower capitulum attracts a range of insect pollinators in its boom. Diversity and abundance of insect pollinators and their role (especially honey bees) in seed production of sunflower cannot be sidelined (Kumar and Srivastava 2021). Not only seed setting percentage and yield enhance considerably when pollination is augmented with honey bees, but it also enhances seedling vigour, germination percentage, field emergence, oil content (Rajasri et al. 2012) and seed weight (Altayeb and Nagi 2015) of the sunflower seeds. Satyanarayana and Seetharam (1982) reported a diverse group of insect species on the capitula of sunflower from 21 different genera, which was mainly consisted of bees from the genus Apis, constituting more than 85%. Singh et al. (2000) noted different insects belonging to 41 genera from 23 families and 6 orders foraging on male sterile and restorer lines of sunflower, out of which A. mellifera, A. dorsata and A. florea made 42.2% of the total insects observed. Kumar and Srivastava (2021) observed 17 species of Apis and non-Apis bees during different stages of flowering in sunflower. The composition of different orders comprising of pollinator fauna was Hymenoptera

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(11), Diptera (2) and Lepidoptera (4). Among the Apis species, A. dorsata, A. mellifera and A. cerana indica, followed by non-Apis, Tetragonula iridipennis, were most abundant visitors in the variety Morden. It was also found that ‘R’ line was preferred more by honey bees than ‘A’ lines during hybrid seed production in sunflower (Singh et al. 2000; Rajasri et al. 2012). In this context, beehives must be kept around the fields for augmented pollination (Altayeb and Nagi 2015). In hybrid seed production of mustard, Apis sp., especially dorsata, plays a significant role in the transfer of pollens from male to female lines (Maity et al. 2014). Safflower is mainly a self-pollinated crop where the role of insect pollinators comes in focus when it has to be cross pollinated for hybrid seed production. Selected parent lines may be grown in alternate rows, and effective pollination is achieved in the presence of high population of insect pollinators (Abrol 2012). Among insects, the chief pollinating agents in this crop are honey bees (Langridge and Goodman 1980; Khalil et al. 1986). According to a survey conducted by Khalil et al. (1986), honey bees represented 58.15–63.36% of total pollinators. In that, 19 insect species of 5 orders (Hemiptera, Lepidoptera, Coleoptera, Diptera and Hymenoptera) were recorded as visitors to safflower in bloom apart from the native bees Ceratina tarsata Mor., Megachile sp., Nomia sp. and Prosopis klugi Fr. which too proved to be of great importance in safflower pollination. Kucera et al. (2006) showed that hybrid breeding in cauliflower can be achieved through self-incompatibility and cytoplasmic male sterility in isolation cages using insect pollinators like bumblebees. Bees are very important for pollination and fruit setting in eggplants (Patricio et al. 2012). Pollinators belonging to the genus Bombus, Xylocopa, Exomalopsis, Centris, Oxaea, Halictus sp., many species of family Halictidae, Apis sp., Pseudaugochloropsis graminea of Hymenoptera and Eristalinus sp. of Diptera (Patricio et al. 2012; Bodlah and Waqar 2013; Montemor and Malerbo Souza 2009; Mainali et al. 2015) are the main contributors in eggplants. Nunes-Silva et al. (2013) reported Melipona fasciculata as an efficient pollinator of eggplant in Brazil. Fruit set and seed set in eggplant, being a member of Solanaceae family, are efficiently enhanced by buzz pollination (Jayasinghe et al. 2017) which happens due to high-frequency vibration produced by the insect pollinators and occasions the pollens to come out of the anther. These pollinators could be deployed in hybrid seed production of eggplant. Syrphid flies and Apis spp. are the most common contributors in open-pollinated carrot among a large variety of flower visitors (Gaffney et al. 2011). The variation and diversity of insect pollinators may depend upon weather conditions and surrounding vegetation. For commercial seed production, artificial introduction and establishment of Apis mellifera are important. Hawthorn et al. (1960) stressed the importance of insect pollinators in gaining adequate yield and quality carrot seeds, which can save considerable time by using insect pollinators rather than hand pollination (Wilson et al. 1991). Hybrid in cotton too is achieved by cross pollination between different parental lines. It is mainly done by honey bees (Bozbek et al. 2008; Vaissière et al. 1984) and can be used in production of F2 hybrid lines with successful transfer of genetically useful traits (Zumba et al. 2013). The number of colonies deployed per ha area significantly influences the seed yield (Vaissière et al. 1984). Approximately, one

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honey bee per 100 flowers is considered well enough for effective seed setting. Apart from population density, ratio or arrangement of male sterile and male fertile lines also influences the effectiveness of the pollination. Wailer et al. (1985) observed that honey bees usually prefer a ratio of 2:1 for male sterile to male fertile lines in cotton. For almost all the seed spices, honey bees bear higher weightage over all other insects, as far as their pollination is concerned. Crops like small cardamom, onion, clove, coriander, fennel, vanilla, cumin, anise, celery, sesame, etc. are chiefly pollinated by bees. In small cardamom, nearly 21–37% increase in yield is observed on account of pollination (Meena et al. 2015). To this crop, A. cerana is the most abundant visitor followed by A. dorsata, A. florea and Tetragonula spp. In coriander, yield increase of up to 187% has been known due to augmented pollination by honey bees. A. florea constitutes 81% of the total insect pollinators visiting fennel flowers (Narayana et al. 1960). Pollination in vanilla is mainly achieved by small bees, Melipona sp. Ovinge and Hoover (2018) compared single (one brood chamber) and double (two brood chambers) honey bee colony units in hybrid seed production of canola and found that singles are sufficient to provide the level of pollination given by doubles and their use can lower the cost. Pollination in coconut is achieved by both wind and insects, later being the important mechanism (Thomas and Josephrajkumar 2013; Meléndez-Ramírez et al. 2004) and mainly contributed by honey bees (Free et al. 1975), especially A. mellifera (Meléndez-Ramírez et al. 2004). Sadakathulla (1991) observed three species of honey bees, viz. A. cerana indica F., A. florea F. and T. iridipennis in the inflorescence of coconut genotypes, whereas Sholdt (1966) observed 51 different insects. The population of Indian bees visiting the inflorescence remains higher than the dammer bees both in the varieties and hybrid palms and latter (hybrid palms) attracts insects more than the varieties. Ashburner et al. (2000) reported exclusively insect-pollinated palms (up to 96.3%), mainly by bees belonging to the family Halictidae (Hymenoptera). Nitidulid beetles are important pollinators in Annona sp. Soil temperature favours perpetuation of these beetles, whereas rainfall afflicts (George et al. 1989, 1991). Insect diversity is important for apple yield, but its influence varies with cultivars (Nunes-Silva et al. 2020). Contribution of insect pollinators is essential for achieving both yields and quality of apple across different regions of the world. Pardo and Borges (2020) realized that wild pollinators occur abundantly in apple orchards and they are frequently more effective pollinators than honey bees; however, Ramírez and Davenport (2013) reported that the most common insect pollinator of apple is honey bee. Insect diversity studies on apple flowers conducted by Kaundil and Thakur (2020) showed 34 different species from 11 families and 5 orders, and the most frequent visitors were Apis mellifera, A. cerana and Episyrphus balteatus. Garratt et al. (2016) reported solitary bees and bumblebees as more effective pollinators of apple as compared to the hoverflies. In case of hybrid citrus, sexual fertilization is needed for good yield. Bagging of fruits in citrus crop has a significant effect on fruit setting due to hindrance in pollination (Phartiyal et al. 2012). Malerbo-Souza et al. (2004) observed 35.30% higher sweet orange production in uncovered flowers due to open pollination. The

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quality of fruit in terms of fruit weight and acid content (low acid content) is achieved from uncovered flowers (open pollination) as compared to the covered one. Singh (2016) reported 12 different insect species belonging to Hymenoptera, Diptera, Lepidoptera and Coleoptera, as major pollinators during the peak flowering stage of sweet orange flowers. Tetragonula iridipennis, Nomia sp. and Xylocopa tenuiscapa are some of the efficient pollinators in this fruit. Insect pollinators play an irreplaceable role in cross pollination in a number of crops. Some of the commonly encountered species in different crop ecosystems have been listed in Table 12.4; Fig. 12.1).

12.3

Impact of Climate Change on Insect Pollinators and Pollination

Climate change is threatening the pollination ecology and so the food production system (Bhagawati et al. 2017). It has been estimated that more than one-third of the global food crop production is contributed exclusively by pollinators and it alone contributes to US$ 117 billion per year (Klein et al. 2007). The honey bees are the major source of pollinators, and the genus Apis has a total of 6 to 11 species and 44 subspecies (Engel 1999). The golden honey bee, Apis mellifera, has 25 subspecies or races, and it well adapts to climate change by virtue of its plasticity and genetic variability. Numerous factors such as habitat loss, fragmentation, chemical-intensive agriculture and climate change were contributing for honey bee population decline (Potts et al. 2010), however, the latter is the major contributor as reported by Hegland et al. (2009) and Schweiger et al. (2010). A. mellifera sahariensis is well adapted to oasis present in sub-Saharan deserts, but due to the impact of climate change, they cannot be able to migrate or swarm to long distance to the next suitable oasis in nearby deserts. The long-distance swarming or migration is dependent on many environmental factors like temperature, wind direction, El Niño, relative humidity and changes in rainfall pattern (Sujayanand and Karuppaiah 2016). A. dorsata responds to seasonal change, flowering pattern and flowering disruption by migrating up to 200 km to escape from starvation (Mattila and Otis 2006). The elevated temperature disrupts the bee foraging behaviour as reported in the case of A. florea in mango (Reddy et al. 2012) and weakens several characteristics relating to the nectar composition and floral volatiles (Broussard et al. 2017). The elevated CO2 is expected to modify the carbon-and-nitrogen ratio in the plant tissues, and it leads to change in nectar composition (Rusterholz and Erhardt 1998). The egg-laying by queen honey bee will start only at the onset of congenial temperature for brood development of worker bees. Further, the nectar availability will also be at same time by the onset of spring. Thus, climate change may influence the developmental cycle of honey bees drastically. The excessive drying in autumn leads to shortage of pollen and nectar availability; thereby it affects the honey bee population buildup (Stokstad 2007). In some parts of the world, 30% colony mortality was reported due to food scarcity, pollution and diseases (Neumann and Carreck 2010). Apart from the impact of climate adversities on pollination, insect pollinators have also the potential to help

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Table 12.4 List of insect pollinators in different crop ecosystems Order Hymenoptera

Family Apidae

Species Apis mellifera Apis dorsata Fab. Apis cerana indica Fab. Apis florea Tetragonula sp. Tetragonula iridipennis

Crops References Most of the fruits, vegetables and field crops like apple, citrus, grapes, papaya lady finger, brinjal, cotton, alfalfa, etc.

Tetragonula iridipennis Tetragonula laeviceps Tetragonula sp.

Sweet orange

Lophotrigona canifrons Melipona fasciculata Bombus sp. Centris sp. Ceratina tarsata Mor. Bombus impatiens Cresson B. haemorrhoidalis Smith Ceratina smaragdina Smith Xylocopa amethystina (Fabricius) Xylocopa violacea Xylocopa tenuiscapa Westwood

Citrus crop Cardamom

Mandarin orange Sweet orange Eggplant

Pradhan and Devy (2019) Singh (2016)

Safflower Apple

Khalil et al. (1986) Robertson et al. (2021)

Megachilidae

Andrenidae Halictidae

Nunes-Silva et al. (2013) Patricio et al. (2012)

Kaundil and Thakur (2020)

Pigeonpea Sweet orange

Scoliidae

Phartiyal et al. (2012) Chaudhary and Kumar (2000) Singh (2016)

Campsomeriella megachalis F. C. collaris F. Megachile velutina

Citrus crop

Megachile rotunda Oxaea sp. Solitary bees Homalictus dampieri Halictus sp.

Lucerne Eggplant Apple Coconut Apple

Pseudaugochloropsis graminea

Eggplant

Pigeonpea

Zheng-hong et al. (2012) Singh (2016), Vanlalhmangaiha et al. (2021) Phartiyal et al. (2012)

Zheng-hong et al. (2012) Anderson (2006) Patricio et al. (2012) Robertson et al. (2021) Ashburner et al. (2000) Kaundil and Thakur (2020) Patricio et al. (2012) (continued)

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Table 12.4 (continued) Order

Family

Vespidae

Ichneumonidae Formicidae

Diptera

Syrphidae

Species Nomia sp.

Crops Safflower Sweet orange

Seladonia sp. Lasioglossum sp. Polistes exclamans (Vier.) P. olivaceus (De Geer) P. macaensis (F.) P. maculipennis Vespa auraria Smith Xanthopimpla sp. Paratrechina longicornis (Latr.) Pheidole megacephala Eristalinus sp. E. taeniops (Wiedemann) Eristalis basifemorata (Brunetti) Episyrphus sp. Episyrphus viridaureus Eupeodes confrater (Wiedemann) Eristalis tenax (Linnaeus) Melanostoma sp. Episyrphus balteatus (De Geer) Sphaerophoria indiana Bigot Eupeodes sp. Metasyrphus confrater (Wiedemann) Ischiodon scutellaris (Fabricius) Eristalis tenax (Linnaeus) Episyrphus balteatus (De Geer) Sphaerophoria scripta L. Syrphus corollae Fab. Melanostoma sp.

Mandarin Orange

References Khalil et al. (1986) Singh (2016), Vanlalhmangaiha et al. (2021) Pradhan and Devy (2019)

Coconut

Sholdt (1966)

Apple

Kaundil and Thakur (2020)

Coconut

Sholdt (1966)

Eggplant Mandarin orange

Patricio et al. (2012) Pradhan and Devy (2019)

Apple

Kaundil and Thakur (2020)

Citrus crop

Phartiyal et al. (2012)

(continued)

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Table 12.4 (continued) Order

Coleoptera

Family

Species Syrphus sp.

Sepsidae Calliphoridae

Rhiniidae Sarcophagidae Muscidae

Sepsid fly Chrysomya megacephala Chrysomya sp. Rhinia sp. Wohlfahrtia sp. Musca sp.

Nitidulidae

Nitidulid beetles

Coccinellidae

Coccinella septempunctata Coccinella sp. Oenopia kirbyi (Mulsant) Hypomeces squamosus

Curculionidae Scarabaeidae

Lepidoptera

Chrysomelidae Pieridae

Papilionidae Noctuidae

Hemiptera

Lygaeidae

Largidae Thysanoptera

Dermaptera

Thripidae

Chelisochidae

Anomala sp. Clinteria sp. Chrysonopa sp. Pieris candida Pieris brassicae (Linnaeus) Vanessa cardui Vanessa caschmirensis Colias erate Papilio demoleus Mythimna unipuncta Haworth Peridroma saucia Hübner Spilostethus pandurus (Scopoli) Graptostethus incertus (Walker) Physopelta gutta gutta (Burmeister) Thrips sp.

Crops Sweet orange Apple

References Singh (2016)

Mandarin orange

Pradhan and Devy (2019)

Apple

Kaundil and Thakur (2020) George et al. (1989, 1991) Kaundil and Thakur (2020)

Annona sp. Apple

Kaundil and Thakur (2020)

Mandarin orange Sweet orange Mandarin orange

Pradhan and Devy (2019) Singh (2016)

Apple

Kaundil and Thakur (2020)

Sweet orange

Singh (2016) Robertson et al. (2021)

Mandarin orange

Pradhan and Devy (2019)

Apple

Kaundil and Thakur (2020) Shaw (1914) Anand (1926), Billes (1941) Sholdt (1966)

Thrips

Sugar beet Cacao

Chelisoches morio

Coconut

Pradhan and Devy (2019)

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Apis dorsata F. on bittergourd

Apis dorsata F. on cowpea

Apis dorsata F. on pigeonpea

Apis florea F. on bittergourd

Apis florea F. on cowpea

Apis florea F. on pigeonpea

Apis florea F. on muskmelon

Apis mellifera L. on chickpea

Fig. 12.1 Some of the common insect pollinators on different crops

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Ceratina binghami Cockerell on muskmelon

Megachile bicolor F. on cowpea

Syrphid on cowpea

Xylocopa fenestrata F. on cowpea Fig. 12.1 (continued)

277

Ceratina hieroglypiaca Smith on muskmelon

Megachile

lanata

F.

on

pigeonpea

Smith on bittergourd

Apis florea F. on coriander

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Xylocopa fenestrata F. on pigeonpea

Xylocopa latipes (Drury) on pigeonpea

Apis cerana indica F. on coriander

Xylocopa latipes (Drury) on cowpea

Apis mellifera L. on coriander

Tetragonula iridipennis Smith on coriander

Fig. 12.1 (continued)

compensate the loss caused by heat-stressed plants (Bishop et al. 2016). As the probability of heat waves increases, pollination by insects finds more importance in the changing climate scenario.

12.4

Conclusion and Area Addressable

It is evinced that insect pollinators are indispensable for life and deserve paramount importance for their conservation. Based on flower-insect suitability, a crop harbours a particular set of insect fauna and factors determining these need to be explored

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further. Their role in enhancing yield and quality of the produce as well as in maintaining a well-balanced ecosystem is incomparable. In context of hybrid seed production of major food crops, pollinators have greater preference towards restorer lines as compared to male sterile ones and are efficient in transferring pollens and accomplishing the seed setting thereby. Deployment of bee-boxes is done to improve yield and quality of the produce, but their required number per unit area still needs to be standardized for a range of crops in order to make balance between the foraging capacity (distance) and cost involvement of beehives. Standardization of ratio of arrangement of male sterile lines to fertile lines is required in many crops for efficient pollination and seed setting (Saxena et al. 2015). Indiscriminate use of synthetic chemical insecticides, habitat loss and impact of climate change are threats and should be considered as major causes of concern. These can be addressed through the approach of ecological engineering and judicious selection and use of insecticides to save and conserve these wonderful creatures.

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