Celebrating Professor Rajeev K. Varshney's transformative research odyssey from genomics to the field on his induction as Fellow of the Royal Society.

Professor Rajeev K. Varshney's transformative impact on crop genomics, genetics, and agriculture is the result of his passion, dedication, and unyielding commitment to harnessing the potential of genomics to address the most pressing challenges faced by the global agricultural community. Starting from a small town in India and reaching the global stage, Professor Varshney's academic and professional trajectory has inspired many scientists active in research today. His ground-breaking work, especially his effort to list orphan tropical crops to genomic resource-rich entities, has been transformative. Beyond his scientific achievements, Professor Varshney is recognized by his colleagues as an exemplary mentor, fostering the growth of future researchers, building institutional capacity, and strengthening scientific capability. His focus on translational genomics and strengthening seed system in developing countries for the improvement of agriculture has made a tangible impact on farmers' lives. His skills have been best utilized in roles at leading research centres where he has applied his expertise to deliver a new vision for crop improvement. These efforts have now been recognized by the Royal Society with the award of the Fellowship (FRS). As we mark this significant milestone in his career, we not only celebrate Professor Varshney's accomplishments but also his wider contributions that continue to transform the agricultural landscape.

Whole genome resequencing and phenotyping of MAGIC population for high resolution mapping of drought tolerance in chickpea.

Terminal drought is one of the major constraints to crop production in chickpea (Cicer arietinum L.). In order to map drought tolerance related traits at high resolution, we sequenced multi-parent advanced generation intercross (MAGIC) population using whole genome resequencing approach and phenotyped it under drought stress environments for two consecutive years (2013-14 and 2014-15). A total of 52.02 billion clean reads containing 4.67 TB clean data were generated on the 1136 MAGIC lines and eight parental lines. Alignment of clean data on to the reference genome enabled identification of a total, 932,172 of SNPs, 35,973 insertions, and 35,726 deletions among the parental lines. A high-density genetic map was constructed using 57,180 SNPs spanning a map distance of 1606.69 cM. Using compressed mixed linear model, genome-wide association study (GWAS) enabled us to identify 737 markers significantly associated with days to 50% flowering, days to maturity, plant height, 100 seed weight, biomass, and harvest index. In addition to the GWAS approach, an identity-by-descent (IBD)-based mixed model approach was used to map quantitative trait loci (QTLs). The IBD-based mixed model approach detected major QTLs that were comparable to those from the GWAS analysis as well as some exclusive QTLs with smaller effects. The candidate genes like FRIGIDA and CaTIFY4b can be used for enhancing drought tolerance in chickpea. The genomic resources, genetic map, marker-trait associations, and QTLs identified in the study are valuable resources for the chickpea community for developing climate resilient chickpeas.

High confidence QTLs and key genes identified using Meta-QTL analysis for enhancing heat tolerance in chickpea (Cicer arietinum L.).

The rising global temperatures seriously threaten sustainable crop production, particularly the productivity and production of heat-sensitive crops like chickpeas. Multiple QTLs have been identified to enhance the heat stress tolerance in chickpeas, but their successful use in breeding programs remains limited. Towards this direction, we constructed a high-density genetic map spanning 2233.5 cM with 1069 markers. Using 138 QTLs reported earlier, we identified six Meta-QTL regions for heat tolerance whose confidence interval was reduced by 2.7-folds compared to the reported QTLs. Meta-QTLs identified on CaLG01 and CaLG06 harbor QTLs for important traits, including days to 50% flowering, days to maturity, days to flower initiation, days to pod initiation, number of filled pods, visual score, seed yield per plant, biological yield per plant, chlorophyll content, and harvest index. In addition, key genes identified in Meta-QTL regions like Pollen receptor-like kinase 3 (CaPRK3), Flowering-promoting factor 1 (CaFPF1), Flowering Locus C (CaFLC), Heat stress transcription factor A-5 (CaHsfsA5), and Pollen-specific leucine-rich repeat extensins (CaLRXs) play an important role in regulating the flowering time, pollen germination, and growth. The consensus genomic regions, and the key genes reported in this study can be used in genomics-assisted breeding for enhancing heat tolerance and developing heat-resilient chickpea cultivars.

Major viral diseases in grain legumes: designing disease resistant legumes from plant breeding and OMICS integration.

Grain legumes play a crucial role in human nutrition and as a staple crop for low-income farmers in developing and underdeveloped nations, contributing to overall food security and agroecosystem services. Viral diseases are major biotic stresses that severely challenge global grain legume production. In this review, we discuss how exploring naturally resistant grain legume genotypes within germplasm, landraces, and crop wild relatives could be used as promising, economically viable, and eco-environmentally friendly solution to reduce yield losses. Studies based on Mendelian and classical genetics have enhanced our understanding of key genetic determinants that govern resistance to various viral diseases in grain legumes. Recent advances in molecular marker technology and genomic resources have enabled us to identify genomic regions controlling viral disease resistance in various grain legumes using techniques such as QTL mapping, genome-wide association studies, whole-genome resequencing, pangenome and 'omics' approaches. These comprehensive genomic resources have expedited the adoption of genomics-assisted breeding for developing virus-resistant grain legumes. Concurrently, progress in functional genomics, especially transcriptomics, has helped unravel underlying candidate gene(s) and their roles in viral disease resistance in legumes. This review also examines the progress in genetic engineering-based strategies, including RNA interference, and the potential of synthetic biology techniques, such as synthetic promoters and synthetic transcription factors, for creating viral-resistant grain legumes. It also elaborates on the prospects and limitations of cutting-edge breeding technologies and emerging biotechnological tools (e.g., genomic selection, rapid generation advances, and CRISPR/Cas9-based genome editing tool) in developing virus-disease-resistant grain legumes to ensure global food security.

Multi-locus genome-wide association study of chickpea reference set identifies genetic determinants of Pratylenchus thornei resistance.

Pratylenchus thornei is an economically important species of root-lesion nematode adversely affecting chickpea (Cicer arietinum) yields globally. Integration of resistant crops in farming systems is recognised as the most effective and sustainable management strategy for plant-parasitic nematodes. However, breeding for P. thornei resistance in chickpea is limited by the lack of genetic diversity. We deployed a genome-wide association approach to identify genomic regions and candidate genes associated with P. thornei resistance in 285 genetically diverse chickpea accessions. Chickpea accessions were phenotyped for P. thornei resistance in replicated glasshouse experiments performed for two years (2018 and 2020). Whole genome sequencing data comprising 492,849 SNPs were used to implement six multi-locus GWAS models. Fourteen chickpea genotypes were found to be resistant to P. thornei. Of the six multi-locus GWAS methods deployed, FASTmrMLM was found to be the best performing model. In all, 24 significant quantitative trait nucleotides (QTNs) were identified, of which 13 QTNs were associated with lower nematode population density and 11 QTNs with higher nematode population density. These QTNs were distributed across all of the chickpea chromosomes, except chromosome 8. We identified, receptor-linked kinases (RLKs) on chromosomes 1, 4 and 6, GDSL-like Lipase/Acylhydrolase on chromosome 3, Aspartic proteinase-like and Thaumatin-like protein on chromosome 4, AT-hook DNA-binding and HSPRO2 on chromosome 6 as candidate genes for P. thornei resistance in the chickpea reference set. New sources of P. thornei resistant genotypes were identified that can be harnessed into breeding programs and putative candidate P. thornei resistant genes were identified that can be explored further to develop molecular markers and accelerate the incorporation of improved P. thornei resistance into elite chickpea cultivars.

Delineating Marker-Trait Associations for Fusarium Wilt in Chickpea Using the Axiom® CicerSNP Array.

Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. ciceri is a devastating disease of chickpea (Cicer arietinum). To identify promising resistant genotypes and genomic loci for FW resistance, a core set of 179 genotypes of chickpea was tested for FW reactions at the seedling and reproductive stages under field conditions and controlled conditions in the greenhouse. Our results revealed that at the seedling stage, most of the genotypes were resistant, whereas at the reproductive stage, most of the genotypes were susceptible. Genotyping using a 50K Axiom® CicerSNP Array and trait data of FW together led to the identification of 26 significant (P ≤ E-05) marker-trait associations (MTAs) for FW resistance. Among the 26 MTAs, 12 were identified using trait data recorded in the field (three at the seedling and nine at the reproductive stage), and 14 were identified using trait data recorded under controlled conditions in the greenhouse (six at the seedling and eight at the reproductive stage). The phenotypic variation explained by these MTAs varied from 11.75 to 15.86%, with an average of 13.77%. Five MTAs were classified as major, explaining more than 15% of the phenotypic variation for FW, and two were declared stable, being identified in two environments. One of the promising stable and major MTAs (Affx_123280060) detected in field conditions at the reproductive stage was also detected in greenhouse conditions at the seedling and reproductive stages. The stable and major (>15% PVE) MTAs can be used in chickpea breeding programs.

Shoot transcriptome revealed widespread differential expression and potential molecular mechanisms of chickpea (Cicer arietinum L.) against Fusarium wilt.

Introduction: The yield of chickpea is severely hampered by infection wilt caused by several races of Fusarium oxysporum f. sp. ciceris (Foc). Methods: To understand the underlying molecular mechanisms of resistance against Foc4 Fusarium wilt, RNA sequencing-based shoot transcriptome data of two contrasting chickpea genotypes, namely KWR 108 (resistant) and GL 13001 (susceptible), were generated and analyzed. Results and Discussion: The shoot transcriptome data showed 1,103 and 1,221 significant DEGs in chickpea genotypes KWR 108 and GL 13001, respectively. Among these, 495 and 608 genes were significantly down and up-regulated in genotypes KWR 108, and 427 and 794 genes were significantly down and up-regulated in genotype GL 13001. The gene ontology (GO) analysis of significant DEGs was performed and the GO of the top 50 DEGs in two contrasting chickpea genotypes showed the highest cellular components as membrane and nucleus, and molecular functions including nucleotide binding, metal ion binding, transferase, kinase, and oxidoreductase activity involved in biological processes such as phosphorylation, oxidation-reduction, cell redox homeostasis process, and DNA repair. Compared to the susceptible genotype which showed significant up-regulation of genes involved in processes like DNA repair, the significantly up-regulated DEGs of the resistant genotypes were involved in processes like energy metabolism and environmental adaptation, particularly host-pathogen interaction. This indicates an efficient utilization of environmental adaptation pathways, energy homeostasis, and stable DNA molecules as the strategy to cope with Fusarium wilt infection in chickpea. The findings of the study will be useful in targeting the genes in designing gene-based markers for association mapping with the traits of interest in chickpea under Fusarium wilt which could be efficiently utilized in marker-assisted breeding of chickpea, particularly against Foc4 Fusarium wilt.

Legume Pangenome: Status and Scope for Crop Improvement.

In the last decade, legume genomics research has seen a paradigm shift due to advances in genome sequencing technologies, assembly algorithms, and computational genomics that enabled the construction of high-quality reference genome assemblies of major legume crops. These advances have certainly facilitated the identification of novel genetic variants underlying the traits of agronomic importance in many legume crops. Furthermore, these robust sequencing technologies have allowed us to study structural variations across the whole genome in multiple individuals and at the species level using 'pangenome analysis.' This review updates the progress of constructing pangenome assemblies for various legume crops and discusses the prospects for these pangenomes and how to harness the information to improve various traits of economic importance through molecular breeding to increase genetic gain in legumes and tackle the increasing global food crisis.

Genome-wide association analysis to delineate high-quality SNPs for seed micronutrient density in chickpea (Cicer arietinum L.).

Chickpea is the most important nutrient-rich grain legume crop in the world. A diverse core set of 147 chickpea genotypes was genotyped with a Axiom(®)50K CicerSNP array and trait phenotyped in two different environments for four seed micronutrients (Zn, Cu, Fe and Mn). The trait data and high-throughput 50K SNP genotypic data were used for the genome-wide association study (GWAS). The study led to the discovery of genes/QTLs for seed Zn, Cu, Fe and Mn, concentrations in chickpea. The analysis of seed micronutrient data revealed significant differences for all four micronutrient concentrations (P ≤ 0.05). The mean concentrations of seed Zn, Cu, Fe and Mn pooled over the 2 years were 45.9 ppm, 63.8 ppm 146.1 ppm, and 27.0 ppm, respectively. The analysis of results led to the identification of 35 SNPs significantly associated with seed Zn, Cu, Fe and Mn concentrations. Among these 35 marker-trait associations (MTAs), 5 were stable (consistently identified in different environments), 6 were major (explaining more than 15% of the phenotypic variation for an individual trait) and 3 were both major and stable MTAs. A set of 6 MTAs, MTAs (3 for Mn, 2 for Fe, and 1 for Cu) reported by us during the present study have been also reported in the same/almost same genomic regions in earlier studies and therefore declared as validated MTAs. The stable, major and validated MTAs identified during the present study will prove useful in future chickpea molecular breeding programs aimed at enhancing the seed nutrient density of chickpea.

Integrated breeding approaches to enhance the nutritional quality of food legumes.

Global food security, both in terms of quantity and quality remains as a challenge with the increasing population. In parallel, micronutrient deficiency in the human diet leads to malnutrition and several health-related problems collectively known as "hidden hunger" more prominent in developing countries around the globe. Biofortification is a potential tool to fortify grain legumes with micronutrients to mitigate the food and nutritional security of the ever-increasing population. Anti-nutritional factors like phytates, raffinose (RFO's), oxalates, tannin, etc. have adverse effects on human health upon consumption. Reduction of the anti-nutritional factors or preventing their accumulation offers opportunity for enhancing the intake of legumes in diet besides increasing the bioavailability of micronutrients. Integrated breeding methods are routinely being used to exploit the available genetic variability for micronutrients through modern "omic" technologies such as genomics, transcriptomics, ionomics, and metabolomics for developing biofortified grain legumes. Molecular mechanism of Fe/Zn uptake, phytate, and raffinose family oligosaccharides (RFOs) biosynthesis pathways have been elucidated. Transgenic, microRNAs and genome editing tools hold great promise for designing nutrient-dense and anti-nutrient-free grain legumes. In this review, we present the recent efforts toward manipulation of genes/QTLs regulating biofortification and Anti-nutrient accumulation in legumes using genetics-, genomics-, microRNA-, and genome editing-based approaches. We also discuss the success stories in legumes enrichment and recent advances in development of low Anti-nutrient lines. We hope that these emerging tools and techniques will expedite the efforts to develop micronutrient dense legume crop varieties devoid of Anti-nutritional factors that will serve to address the challenges like malnutrition and hidden hunger.

SSR markers in revealing extent of genetic diversity and phylogenetic relationships among chickpea core collection accessions for Western Himalayas.

BACKGROUND: The exploration of genetic diversity is the key source of germplasm conservation and potential to broaden its genetic base. The globally growing demand for chickpea suggests superior/climate-resilient varieties, which in turn necessitates the germplasm characterization to unravel underlying genetic variation. METHODOLOGY AND RESULTS: A chickpea core collection comprising of diverse 192 accessions which include cultivated Cicer arietinum, and wild C. reticulatum, C. echinospermum, and C. microphyllum species were investigated to analyze their genetic diversity and relationship, by assaying 33 unlinked simple sequence repeat (SSR) markers. The results amplified a total of 323 alleles (Na), ranging from 2 to 8 with an average of 4.25 alleles per locus. Expected heterozygosity (He) differed from 0.46 to 0.86 with an average of 0.68. Polymorphic information content (PIC) ranged from 0.73 to 0.98 with an average of 0.89. Analysis of molecular variance (AMOVA) showed that most of the variation was among individuals (87%). Cluster analysis resulted in the formation of four distinct clusters. Cluster I represented all cultivated and clusters II, III, and IV comprised a heterogeneous group of cultivated and wild chickpea accessions. CONCLUSION: We report considerable diversity and greater resolving power of SSR markers for assessing variability and interrelationship among the chickpea accessions. The chickpea core is expected to be an efficient resource for breeders for broadening the chickpea genetic base and could be useful for selective breeding of desirable traits and in the identification of target genes for genomics-assisted breeding.

High-Density Genetic Variation Map Reveals Key Candidate Loci and Genes Associated With Important Agronomic Traits in Peanut.

Peanut is one of the most important cash crops with high quality oil, high protein content, and many other nutritional elements, and grown globally. Cultivated peanut (Arachis hypogaea L.) is allotetraploid with a narrow genetic base, and its genetics and molecular mechanisms controlling the agronomic traits are poorly understood. Here, we report a comprehensive genome variation map based on the genotyping of a panel of 178 peanut cultivars using Axiom_Arachis2 SNP array, including 163 representative varieties of different provinces in China, and 15 cultivars from 9 other countries. According to principal component analysis (PCA) and phylogenetic analysis, the peanut varieties were divided into 7 groups, notable genetic divergences between the different areas were shaped by environment and domestication. Using genome-wide association study (GWAS) analysis, we identified several marker-trait associations (MTAs) and candidate genes potentially involved in regulating several agronomic traits of peanut, including one MTA related with hundred seed weight, one MTA related with total number of branches, and 14 MTAs related with pod shape. This study outlines the genetic basis of these peanut cultivars and provides 13,125 polymorphic SNP markers for further distinguishing and utility of these elite cultivars. In addition, the candidate loci and genes provide valuable information for further fine mapping of QTLs and improving the quality and yield of peanut using a genomic-assisted breeding method.

Raffinose Family Oligosaccharides: Friend or Foe for Human and Plant Health?

Raffinose family oligosaccharides (RFOs) are widespread across the plant kingdom, and their concentrations are related to the environment, genotype, and harvest time. RFOs are known to carry out many functions in plants and humans. In this paper, we provide a comprehensive review of RFOs, including their beneficial and anti-nutritional properties. RFOs are considered anti-nutritional factors since they cause flatulence in humans and animals. Flatulence is the single most important factor that deters consumption and utilization of legumes in human and animal diets. In plants, RFOs have been reported to impart tolerance to heat, drought, cold, salinity, and disease resistance besides regulating seed germination, vigor, and longevity. In humans, RFOs have beneficial effects in the large intestine and have shown prebiotic potential by promoting the growth of beneficial bacteria reducing pathogens and putrefactive bacteria present in the colon. In addition to their prebiotic potential, RFOs have many other biological functions in humans and animals, such as anti-allergic, anti-obesity, anti-diabetic, prevention of non-alcoholic fatty liver disease, and cryoprotection. The wide-ranging applications of RFOs make them useful in food, feed, cosmetics, health, pharmaceuticals, and plant stress tolerance; therefore, we review the composition and diversity of RFOs, describe the metabolism and genetics of RFOs, evaluate their role in plant and human health, with a primary focus in grain legumes.

BSA­seq and genetic mapping reveals AhRt2 as a candidate gene responsible for red testa of peanut.

KEY MESSAGE: The candidate recessive gene AhRt2 responsible for red testa of peanut was identified through combined BSA-seq and linkage mapping approaches. The testa color of peanuts (Arachis hypogaea L.) is an important trait, and those with red testa are particularly popular owing to the high-anthocyanin content. However, the identification of genes underlying the regulation of the red testa trait in peanut are rarely reported. In order to fine map red testa gene, two F2:4 populations were constructed through the cross of YZ9102 (pink testa) with ZH12 (red testa) and ZH2 (red testa). Genetic analysis indicated that red testa was controlled by a single recessive gene named as AhRt2 (Red testa gene 2). Using BSA-seq approach, AhRt2 was preliminary identified on chromosome 12, which was further mapped to a 530-kb interval using 220 recombinant lines through linkage mapping. Furthermore, functional annotation, expression profiling, and the analyses of sequence variation confirmed that the anthocyanin reductase namely (Arahy.IK60LM) was the most likely candidate gene for AhRt2. It was found that a SNP in the third exon of AhRt2 altered the encoding amino acids, and was associated with red testa in peanut. In addition, a closely linked molecular marker linked with red testa trait in peanut was also developed for future studies. Our results provide valuable insight into the molecular mechanism underlying peanut testa color and present significant diagnostic marker resources for marker-assisted selected breeding in peanut.

Two decades of association mapping: Insights on disease resistance in major crops.

Climate change across the globe has an impact on the occurrence, prevalence, and severity of plant diseases. About 30% of yield losses in major crops are due to plant diseases; emerging diseases are likely to worsen the sustainable production in the coming years. Plant diseases have led to increased hunger and mass migration of human populations in the past, thus a serious threat to global food security. Equipping the modern varieties/hybrids with enhanced genetic resistance is the most economic, sustainable and environmentally friendly solution. Plant geneticists have done tremendous work in identifying stable resistance in primary genepools and many times other than primary genepools to breed resistant varieties in different major crops. Over the last two decades, the availability of crop and pathogen genomes due to advances in next generation sequencing technologies improved our understanding of trait genetics using different approaches. Genome-wide association studies have been effectively used to identify candidate genes and map loci associated with different diseases in crop plants. In this review, we highlight successful examples for the discovery of resistance genes to many important diseases. In addition, major developments in association studies, statistical models and bioinformatic tools that improve the power, resolution and the efficiency of identifying marker-trait associations. Overall this review provides comprehensive insights into the two decades of advances in GWAS studies and discusses the challenges and opportunities this research area provides for breeding resistant varieties.

Translational Chickpea Genomics Consortium to Accelerate Genetic Gains in Chickpea (Cicer arietinum L.).

The Translational Chickpea Genomics Consortium (TCGC) was set up to increase the production and productivity of chickpea (Cicer arietinum L.). It represents research institutes from six major chickpea growing states (Madhya Pradesh, Maharashtra, Andhra Pradesh, Telangana, Karnataka and Uttar Pradesh) of India. The TCGC team has been engaged in deploying modern genomics approaches in breeding and popularizing improved varieties in farmers' fields across the states. Using marker-assisted backcrossing, introgression lines with enhanced drought tolerance and fusarium wilt resistance have been developed in the genetic background of 10 elite varieties of chickpea. Multi-location evaluation of 100 improved lines (70 desi and 30 kabuli) during 2016-2017 and 2018-2019 enabled the identification of top performing desi and kabuli lines. In total, 909 Farmer Participatory Varietal Selection trials were conducted in 158 villages in 16 districts of the five states, during 2017-2018, 2018-2019, and 2019-2020, involving 16 improved varieties. New molecular breeding lines developed in different genetic backgrounds are potential candidates for national trials under the ICAR-All India Coordinated Research Project on Chickpea. The comprehensive efforts of TCGC resulted in the development and adoption of high-yielding varieties that will increase chickpea productivity and the profitability of chickpea growing farmers.

A chickpea genetic variation map based on the sequencing of 3,366 genomes.

Zero hunger and good health could be realized by 2030 through effective conservation, characterization and utilization of germplasm resources1. So far, few chickpea (Cicer arietinum) germplasm accessions have been characterized at the genome sequence level2. Here we present a detailed map of variation in 3,171 cultivated and 195 wild accessions to provide publicly available resources for chickpea genomics research and breeding. We constructed a chickpea pan-genome to describe genomic diversity across cultivated chickpea and its wild progenitor accessions. A divergence tree using genes present in around 80% of individuals in one species allowed us to estimate the divergence of Cicer over the last 21 million years. Our analysis found chromosomal segments and genes that show signatures of selection during domestication, migration and improvement. The chromosomal locations of deleterious mutations responsible for limited genetic diversity and decreased fitness were identified in elite germplasm. We identified superior haplotypes for improvement-related traits in landraces that can be introgressed into elite breeding lines through haplotype-based breeding, and found targets for purging deleterious alleles through genomics-assisted breeding and/or gene editing. Finally, we propose three crop breeding strategies based on genomic prediction to enhance crop productivity for 16 traits while avoiding the erosion of genetic diversity through optimal contribution selection (OCS)-based pre-breeding. The predicted performance for 100-seed weight, an important yield-related trait, increased by up to 23% and 12% with OCS- and haplotype-based genomic approaches, respectively.