Multiomics of a rice population identifies genes and genomic regions that bestow low glycemic index and high protein content.

To counter the rising incidence of diabetes and to meet the daily protein needs, we created low glycemic index (GI) rice varieties with protein content (PC) surpassing 14%. In the development of recombinant inbred lines using Samba Mahsuri and IR36 amylose extender (IR36ae) as parental lines, we identified quantitative trait loci and genes associated with low GI, high amylose content (AC), and high PC. By integrating genetic techniques with classification models, this comprehensive approach identified candidate genes on chromosome 2 (qGI2.1/qAC2.1 spanning the region from 18.62 Mb to 19.95 Mb), exerting influence on low GI and high amylose. Notably, the phenotypic variant with high value was associated with the recessive allele of the starch branching enzyme 2b (sbeIIb). The genome-edited sbeIIb line confirmed low GI phenotype in milled rice grains. Further, combinations of alleles created by the highly significant SNPs from the targeted associations and epistatically interacting genes showed ultralow GI phenotypes with high amylose and high protein. Metabolomics analysis of rice with varying AC, PC, and GI revealed that the superior lines of high AC and PC, and low GI were preferentially enriched in glycolytic and amino acid metabolisms, whereas the inferior lines of low AC and PC and high GI were enriched with fatty acid metabolism. The high amylose high protein recombinant inbred line (HAHP_101) was enriched in essential amino acids like lysine. Such lines may be highly relevant for food product development to address diabetes and malnutrition.

Systems seed biology to understand and manipulate rice grain quality and nutrition.

Rice is one of the most essential crops since it meets the calorific needs of 3 billion people around the world. Rice seed development initiates upon fertilization, leading to the establishment of two distinct filial tissues, the endosperm and embryo, which accumulate distinct seed storage products, such as starch, storage proteins, and lipids. A range of systems biology tools deployed in dissecting the spatiotemporal dynamics of transcriptome data, methylation, and small RNA based regulation operative during seed development, influencing the accumulation of storage products was reviewed. Studies of other model systems are also considered due to the limited information on the rice transcriptome. This review highlights key genes identified through a holistic view of systems biology targeted to modify biochemical composition and influence rice grain quality and nutritional value with the target of improving rice as a functional food.

The genetics underlying metabolic signatures in a brown rice diversity panel and their vital role in human nutrition.

Brown rice (Oryza sativa) possesses various nutritionally dense bioactive phytochemicals exhibiting a wide range of antioxidant, anti-cancer, and anti-diabetic properties known to promote various human health benefits. However, despite the wide claims made about the importance of brown rice for human nutrition the underlying metabolic diversity has not been systematically explored. Non-targeted metabolite profiling of developing and mature seeds of a diverse genetic panel of 320 rice cultivars allowed quantification of 117 metabolites. The metabolite genome-wide association study (mGWAS) detected genetic variants influencing diverse metabolic targets in developing and mature seeds. We further interlinked genetic variants on chromosome 7 (6.06-6.43 Mb region) with complex epistatic genetic interactions impacting multi-dimensional nutritional targets, including complex carbohydrate starch quality, the glycemic index, antioxidant catechin, and rice grain color. Through this nutrigenomics approach rare gene bank accessions possessing genetic variants in bHLH and IPT5 genes were identified through haplotype enrichment. These variants were associated with a low glycemic index, higher catechin levels, elevated total flavonoid contents, and heightened antioxidant activity in the whole grain with elevated anti-cancer properties being confirmed in cancer cell lines. This multi-disciplinary nutrigenomics approach thus allowed us to discover the genetic basis of human health-conferring diversity in the metabolome of brown rice.

Pyramiding of genes for grain protein content, grain quality, and rust resistance in eleven Indian bread wheat cultivars: a multi-institutional effort.

Improvement of grain protein content (GPC), loaf volume, and resistance to rusts was achieved in 11 Indian wheat cultivars that are widely grown in four different agro-climatic zones of India. This involved use of marker-assisted backcross breeding (MABB) for introgression and pyramiding of the following genes: (i) the high GPC gene Gpc-B1; (ii) HMW glutenin subunits 5 + 10 at Glu-D1 loci, and (iii) rust resistance genes, Yr36, Yr15, Lr24, and Sr24. GPC increased by 0.8 to 3.3%, although high GPC was generally associated with yield penalty. Further selection among high GPC lines allowed identification of progenies with higher GPC associated with improvement in 1000-grain weight and grain yield in the backgrounds of the following four cultivars: NI5439, UP2338, UP2382, and HUW468. The high GPC progenies (derived from NI5439) were also improved for grain quality using HMW glutenin subunits 5 + 10 at Glu-D1 loci. Similarly, progenies combining high GPC and rust resistance were obtained in the backgrounds of following five cultivars: Lok1, HD2967, PBW550, PBW621, and DBW1. The improved pre-bred lines developed following multi-institutional effort should prove a valuable source for the development of cultivars with improved nutritional quality and rust resistance in the ongoing wheat breeding programmes. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01277-w.

OsTPR boosts the superior grains through increase in upper secondary rachis branches without incurring a grain quality penalty.

To address the future food security in Asia, we need to improve the genetic gain of grain yield while ensuring the consumer acceptance. This study aimed to identify novel genes influencing the number of upper secondary rachis branches (USRB) to elevate superior grains without compromising grain quality by studying the genetic variance of 310 diverse O. sativa var. indica panel using single- and multi-locus genome-wide association studies (GWAS), gene set analyses and gene regulatory network analysis. GWAS of USRB identified 230 significant (q-value < 0.05) SNPs from chromosomes 1 and 2. GWAS targets narrowed down using gene set analyses identified large effect association on an important locus LOC_Os02g50790/LOC_Os02g50799 encoding a nuclear-pore anchor protein (OsTPR). The superior haplotype derived from non-synonymous SNPs identified in OsTPR was specifically associated with increase in USRB with superior grains being low chalk. Through haplotype mining, we further demonstrated the synergy of offering added yield advantage due to superior allele of OsTPR in elite materials with low glycaemic index (GI) property. We further validated the importance of OsTPR using recombinant inbred lines (RILs) population by introgressing a superior allele of OsTPR into elite materials resulted in raise in productivity in high amylose background. This confirmed a critical role for OsTPR in influencing yield while maintaining grain and nutritional quality.

Genome-wide association coupled gene to gene interaction studies unveil novel epistatic targets among major effect loci impacting rice grain chalkiness.

Rice varieties whose quality is graded as excellent have a lower percent grain chalkiness (PGC) of two per cent and below with higher whole grain yields upon milling, leading to higher economic returns for farmers. We have conducted a genome-wide association study (GWAS) using a combined population panel of indica and japonica rice varieties, and identified a total of 746 single nucleotide polymorphisms (SNPs) that were strongly associated with the chalk phenotype, covered 78 Quantitative Trait Loci (QTL) regions. Among them, 21 were high-value QTLs, as they explained at least 10 % of the phenotypic variance for PGC. A combined epistasis and GWAS was applied to dissect the genetics of the complex chalkiness trait, and its regulatory cascades were validated using gene regulatory networks. Promising novel epistatic interactions were found between the loci of chromosomes 6 (PGC6.1) and 7 (PGC7.8) that contributed to lower PGC. Based on haplotype mining only a few modern rice varieties confounded with a lower chalkiness, and they possess several PGC QTLs. The importance of PGC6.1 was validated through multi-parent advanced generation intercrosses and several low-chalk lines possessing superior haplotypes were identified. The results of this investigation have deciphered the underlying genetic networks that can reduce PGC to 2%, and will thus support future breeding programs to improve the grain quality of elite genetic material with high-yielding potentials.

Integrating a genome-wide association study with a large-scale transcriptome analysis to predict genetic regions influencing the glycaemic index and texture in rice.

Reliably generating rice varieties with low glycaemic index (GI) is an important nutritional intervention given the high rates of Type II diabetes incidences in Asia where rice is staple diet. We integrated a genome-wide association study (GWAS) with a transcriptome-wide association study (TWAS) to determine the genetic basis of the GI in rice. GWAS utilized 305 re-sequenced diverse indica panel comprising ~2.4 million single nucleotide polymorphisms (SNPs) enriched in genic regions. A novel association signal was detected at a synonymous SNP in exon 2 of LOC_Os05g03600 for intermediate-to-high GI phenotypic variation. Another major hotspot region was predicted for contributing intermediate-to-high GI variation, involves 26 genes on chromosome 6 (GI6.1). These set of genes included GBSSI, two hydrolase genes, genes involved in signalling and chromatin modification. The TWAS and methylome sequencing data revealed cis-acting functionally relevant genetic variants with differential methylation patterns in the hot spot GI6.1 region, narrowing the target to 13 genes. Conversely, the promoter region of GBSSI and its alternative splicing allele (G allele of Wxa ) explained the intermediate-to-high GI variation. A SNP (C˃T) at exon-10 was also highlighted in the preceding analyses to influence final viscosity (FV), which is independent of amylose content/GI. The low GI line with GC haplotype confirmed soft texture, while other two low GI lines with GT haplotype were characterized as hard and cohesive. The low GI lines were further confirmed through clinical in vivo studies. Gene regulatory network analysis highlighted the role of the non-starch polysaccharide pathway in lowering GI.

Dissecting the genome-wide genetic variants of milling and appearance quality traits in rice.

Higher head rice yield (HRY), which represents the proportion of intact grains that survive milling, and lower grain chalkiness (opacity) are key quality traits. We investigated the genetic basis of HRY and chalkiness in 320 diverse resequenced accessions of indica rice with integrated single- and multi-locus genome-wide association studies using 2.26 million single-nucleotide polymorphisms. We identified novel haplotypes that underly higher HRY on chromosomes 3, 6, 8, and 11, and that lower grain chalkiness in a fine-mapped region on chromosome 5. Whole-genome sequencing of 92 IRRI breeding lines was performed to identify the genetic variants of HRY and chalkiness. Rare and novel haplotypes were found for lowering chalkiness, but missing alleles hindered progress towards enhancing HRY in breeding material. The novel haplotypes that we identified have potential use in breeding programs aimed at improving these important traits in the rice crop.

A genome-scale integrated approach aids in genetic dissection of complex flowering time trait in chickpea.

A combinatorial approach of candidate gene-based association analysis and genome-wide association study (GWAS) integrated with QTL mapping, differential gene expression profiling and molecular haplotyping was deployed in the present study for quantitative dissection of complex flowering time trait in chickpea. Candidate gene-based association mapping in a flowering time association panel (92 diverse desi and kabuli accessions) was performed by employing the genotyping information of 5724 SNPs discovered from 82 known flowering chickpea gene orthologs of Arabidopsis and legumes as well as 832 gene-encoding transcripts that are differentially expressed during flower development in chickpea. GWAS using both genome-wide GBS- and candidate gene-based genotyping data of 30,129 SNPs in a structured population of 92 sequenced accessions (with 200-250 kb LD decay) detected eight maximum effect genomic SNP loci (genes) associated (34% combined PVE) with flowering time. Six flowering time-associated major genomic loci harbouring five robust QTLs mapped on a high-resolution intra-specific genetic linkage map were validated (11.6-27.3% PVE at 5.4-11.7 LOD) further by traditional QTL mapping. The flower-specific expression, including differential up- and down-regulation (>three folds) of eight flowering time-associated genes (including six genes validated by QTL mapping) especially in early flowering than late flowering contrasting chickpea accessions/mapping individuals during flower development was evident. The gene haplotype-based LD mapping discovered diverse novel natural allelic variants and haplotypes in eight genes with high trait association potential (41% combined PVE) for flowering time differentiation in cultivated and wild chickpea. Taken together, eight potential known/candidate flowering time-regulating genes [efl1 (early flowering 1), FLD (Flowering locus D), GI (GIGANTEA), Myb (Myeloblastosis), SFH3 (SEC14-like 3), bZIP (basic-leucine zipper), bHLH (basic helix-loop-helix) and SBP (SQUAMOSA promoter binding protein)], including novel markers, QTLs, alleles and haplotypes delineated by aforesaid genome-wide integrated approach have potential for marker-assisted genetic improvement and unravelling the domestication pattern of flowering time in chickpea.

Genome-wide conserved non-coding microsatellite (CNMS) marker-based integrative genetical genomics for quantitative dissection of seed weight in chickpea.

Phylogenetic footprinting identified 666 genome-wide paralogous and orthologous CNMS (conserved non-coding microsatellite) markers from 5'-untranslated and regulatory regions (URRs) of 603 protein-coding chickpea genes. The (CT)n and (GA)n CNMS carrying CTRMCAMV35S and GAGA8BKN3 regulatory elements, respectively, are abundant in the chickpea genome. The mapped genic CNMS markers with robust amplification efficiencies (94.7%) detected higher intraspecific polymorphic potential (37.6%) among genotypes, implying their immense utility in chickpea breeding and genetic analyses. Seventeen differentially expressed CNMS marker-associated genes showing strong preferential and seed tissue/developmental stage-specific expression in contrasting genotypes were selected to narrow down the gene targets underlying seed weight quantitative trait loci (QTLs)/eQTLs (expression QTLs) through integrative genetical genomics. The integration of transcript profiling with seed weight QTL/eQTL mapping, molecular haplotyping, and association analyses identified potential molecular tags (GAGA8BKN3 and RAV1AAT regulatory elements and alleles/haplotypes) in the LOB-domain-containing protein- and KANADI protein-encoding transcription factor genes controlling the cis-regulated expression for seed weight in the chickpea. This emphasizes the potential of CNMS marker-based integrative genetical genomics for the quantitative genetic dissection of complex seed weight in chickpea.

Deciphering the Genetic Architecture of Cooked Rice Texture.

The textural attributes of cooked rice determine palatability and consumer acceptance. Henceforth, understanding the underlying genetic basis is pivotal for the genetic improvement of preferred textural attributes in breeding programs. We characterized diverse set of 236 Indica accessions from 37 countries for textural attributes, which includes adhesiveness (ADH), hardness (HRD), springiness (SPR), and cohesiveness (COH) as well as amylose content (AC). A set of 147,692 high quality SNPs resulting from genotyping data of 700K high Density Rice Array (HDRA) derived from the Indica diversity panels of 218 lines were retained for marker-trait associations of textural attributes using single-locus (SL) genome wide association studies (GWAS) which resulted in identifying hotspot on chromosome 6 for AC and ADH attributes. Four independent multi-locus approaches (ML-GWAS) including FASTmrEMMA, pLARmEB, mrMLM, and ISIS_EM-BLASSO were implemented to dissect additional loci of major/minor effects influencing the rice texture and to overcome limitations of SL-based GWAS approach. In total 224 significant quantitative trait nucleotide (QTNs) were identified using ML-GWAS, of which 97 were validated with at least two out of the four multi-locus methods. The GWAS results were in accordance with the very significant negative correlation (r = -0.83) observed between AC and ADH, and the significant correlation exhibited by AC (r < 0.4) with HRD, SPR, and COH. The novel haplotypes and putative candidate genes influencing textural properties beyond AC will be a useful resource for deployment into the marker assisted program to capture consumer preferences influencing rice texture and palatability.

Whole genome sequencing-based association study to unravel genetic architecture of cooked grain width and length traits in rice.

In this study, we used 2.9 million single nucleotide polymorphisms (SNP) and 393,429 indels derived from whole genome sequences of 591 rice landraces to determine the genetic basis of cooked and raw grain length, width and shape using genome-wide association study (GWAS). We identified a unique fine-mapped genetic region GWi7.1 significantly associated with cooked and raw grain width. Additionally, GWi7.2 that harbors GL7/GW7 a cloned gene for grain dimension was found. Novel regions in chromosomes 10 and 11 were also found to be associated with cooked grain shape and raw grain width, respectively. The indel-based GWAS identified fine-mapped genetic regions GL3.1 and GWi5.1 that matched synteny breakpoints between indica and japonica. GL3.1 was positioned a few kilobases away from GS3, a cloned gene for cooked and raw grain lengths in indica. GWi5.1 found to be significantly associated with cooked and raw grain width. It anchors upstream of cloned gene GW5, which varied between indica and japonica accessions. GWi11.1 is present inside the 3'-UTR of a functional gene in indica that corresponds to a syntenic break in chromosome 11 of japonica. Our results identified novel allelic structural variants and haplotypes confirmed using single locus and multilocus SNP and indel-based GWAS.

bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice.

Apposite development of anther and its dehiscence are important for the reproductive success of the flowering plants. Recently, bHLH142, a bHLH transcription factor encoding gene of rice has been found to show anther-specific expression and mutant analyses suggest its functions in regulating tapetum differentiation and degeneration during anther development. However, our study on protein level expression and gain-of-function phenotype revealed novel aspects of its regulation and function during anther development. Temporally dissimilar pattern of bHLH142 transcript and polypeptide accumulation suggested regulation of its expression beyond transcriptional level. Overexpression of bHLH142 in transgenic rice resulted in indehiscent anthers and aborted pollen grains. Defects in septum and stomium rupture caused anther indehiscence while pollen abortion phenotype attributed to abnormal degeneration of the tapetum. Furthermore, RNA-Seq-based transcriptome analysis of tetrad and mature pollen stage anthers of wild type and bHLH142OEplants suggested that it might regulate carbohydrate and lipid metabolism, cell wall modification, reactive oxygen species (ROS) homeostasis and cell death-related genes during rice anther development. Thus, bHLH142 is an anther-specific gene whose expression is regulated at transcriptional and post-transcriptional/translational levels. It plays a role in pollen maturation and anther dehiscence by regulating expression of various metabolic pathways-related genes.

An Efficient Strategy Combining SSR Markers- and Advanced QTL-seq-driven QTL Mapping Unravels Candidate Genes Regulating Grain Weight in Rice.

Development and use of genome-wide informative simple sequence repeat (SSR) markers and novel integrated genomic strategies are vital to drive genomics-assisted breeding applications and for efficient dissection of quantitative trait loci (QTLs) underlying complex traits in rice. The present study developed 6244 genome-wide informative SSR markers exhibiting in silico fragment length polymorphism based on repeat-unit variations among genomic sequences of 11 indica, japonica, aus, and wild rice accessions. These markers were mapped on diverse coding and non-coding sequence components of known cloned/candidate genes annotated from 12 chromosomes and revealed a much higher amplification (97%) and polymorphic potential (88%) along with wider genetic/functional diversity level (16-74% with a mean 53%) especially among accessions belonging to indica cultivar group, suggesting their utility in large-scale genomics-assisted breeding applications in rice. A high-density 3791 SSR markers-anchored genetic linkage map (IR 64 × Sonasal) spanning 2060 cM total map-length with an average inter-marker distance of 0.54 cM was generated. This reference genetic map identified six major genomic regions harboring robust QTLs (31% combined phenotypic variation explained with a 5.7-8.7 LOD) governing grain weight on six rice chromosomes. One strong grain weight major QTL region (OsqGW5.1) was narrowed-down by integrating traditional QTL mapping with high-resolution QTL region-specific integrated SSR and single nucleotide polymorphism markers-based QTL-seq analysis and differential expression profiling. This led us to delineate two natural allelic variants in two known cis-regulatory elements (RAV1AAT and CARGCW8GAT) of glycosyl hydrolase and serine carboxypeptidase genes exhibiting pronounced seed-specific differential regulation in low (Sonasal) and high (IR 64) grain weight mapping parental accessions. Our genome-wide SSR marker resource (polymorphic within/between diverse cultivar groups) and integrated genomic strategy can efficiently scan functionally relevant potential molecular tags (markers, candidate genes and alleles) regulating complex agronomic traits (grain weight) and expedite marker-assisted genetic enhancement in rice.

Genome-wide generation and use of informative intron-spanning and intron-length polymorphism markers for high-throughput genetic analysis in rice.

We developed genome-wide 84634 ISM (intron-spanning marker) and 16510 InDel-fragment length polymorphism-based ILP (intron-length polymorphism) markers from genes physically mapped on 12 rice chromosomes. These genic markers revealed much higher amplification-efficiency (80%) and polymorphic-potential (66%) among rice accessions even by a cost-effective agarose gel-based assay. A wider level of functional molecular diversity (17-79%) and well-defined precise admixed genetic structure was assayed by 3052 genome-wide markers in a structured population of indica, japonica, aromatic and wild rice. Six major grain weight QTLs (11.9-21.6% phenotypic variation explained) were mapped on five rice chromosomes of a high-density (inter-marker distance: 0.98 cM) genetic linkage map (IR 64 x Sonasal) anchored with 2785 known/candidate gene-derived ISM and ILP markers. The designing of multiple ISM and ILP markers (2 to 4 markers/gene) in an individual gene will broaden the user-preference to select suitable primer combination for efficient assaying of functional allelic variation/diversity and realistic estimation of differential gene expression profiles among rice accessions. The genomic information generated in our study is made publicly accessible through a user-friendly web-resource, "Oryza ISM-ILP marker" database. The known/candidate gene-derived ISM and ILP markers can be enormously deployed to identify functionally relevant trait-associated molecular tags by optimal-resource expenses, leading towards genomics-assisted crop improvement in rice.

Genome-wide high-throughput SNP discovery and genotyping for understanding natural (functional) allelic diversity and domestication patterns in wild chickpea.

We identified 82489 high-quality genome-wide SNPs from 93 wild and cultivated Cicer accessions through integrated reference genome- and de novo-based GBS assays. High intra- and inter-specific polymorphic potential (66-85%) and broader natural allelic diversity (6-64%) detected by genome-wide SNPs among accessions signify their efficacy for monitoring introgression and transferring target trait-regulating genomic (gene) regions/allelic variants from wild to cultivated Cicer gene pools for genetic improvement. The population-specific assignment of wild Cicer accessions pertaining to the primary gene pool are more influenced by geographical origin/phenotypic characteristics than species/gene-pools of origination. The functional significance of allelic variants (non-synonymous and regulatory SNPs) scanned from transcription factors and stress-responsive genes in differentiating wild accessions (with potential known sources of yield-contributing and stress tolerance traits) from cultivated desi and kabuli accessions, fine-mapping/map-based cloning of QTLs and determination of LD patterns across wild and cultivated gene-pools are suitably elucidated. The correlation between phenotypic (agromorphological traits) and molecular diversity-based admixed domestication patterns within six structured populations of wild and cultivated accessions via genome-wide SNPs was apparent. This suggests utility of whole genome SNPs as a potential resource for identifying naturally selected trait-regulating genomic targets/functional allelic variants adaptive to diverse agroclimatic regions for genetic enhancement of cultivated gene-pools.

Ultra-high density intra-specific genetic linkage maps accelerate identification of functionally relevant molecular tags governing important agronomic traits in chickpea.

We discovered 26785 and 16573 high-quality SNPs differentiating two parental genotypes of a RIL mapping population using reference desi and kabuli genome-based GBS assay. Of these, 3625 and 2177 SNPs have been integrated into eight desi and kabuli chromosomes, respectively in order to construct ultra-high density (0.20-0.37 cM) intra-specific chickpea genetic linkage maps. One of these constructed high-resolution genetic map has potential to identify 33 major genomic regions harbouring 35 robust QTLs (PVE: 17.9-39.7%) associated with three agronomic traits, which were mapped within <1 cM mean marker intervals on desi chromosomes. The extended LD (linkage disequilibrium) decay (~15 cM) in chromosomes of genetic maps have encouraged us to use a rapid integrated approach (comparative QTL mapping, QTL-region specific haplotype/LD-based trait association analysis, expression profiling and gene haplotype-based association mapping) rather than a traditional QTL map-based cloning method to narrow-down one major seed weight (SW) robust QTL region. It delineated favourable natural allelic variants and superior haplotype-containing one seed-specific candidate embryo defective gene regulating SW in chickpea. The ultra-high-resolution genetic maps, QTLs/genes and alleles/haplotypes-related genomic information generated and integrated strategy for rapid QTL/gene identification developed have potential to expedite genomics-assisted breeding applications in crop plants, including chickpea for their genetic enhancement.

A Genome-wide Combinatorial Strategy Dissects Complex Genetic Architecture of Seed Coat Color in Chickpea.

The study identified 9045 high-quality SNPs employing both genome-wide GBS- and candidate gene-based SNP genotyping assays in 172, including 93 cultivated (desi and kabuli) and 79 wild chickpea accessions. The GWAS in a structured population of 93 sequenced accessions detected 15 major genomic loci exhibiting significant association with seed coat color. Five seed color-associated major genomic loci underlying robust QTLs mapped on a high-density intra-specific genetic linkage map were validated by QTL mapping. The integration of association and QTL mapping with gene haplotype-specific LD mapping and transcript profiling identified novel allelic variants (non-synonymous SNPs) and haplotypes in a MATE secondary transporter gene regulating light/yellow brown and beige seed coat color differentiation in chickpea. The down-regulation and decreased transcript expression of beige seed coat color-associated MATE gene haplotype was correlated with reduced proanthocyanidins accumulation in the mature seed coats of beige than light/yellow brown seed colored desi and kabuli accessions for their coloration/pigmentation. This seed color-regulating MATE gene revealed strong purifying selection pressure primarily in LB/YB seed colored desi and wild Cicer reticulatum accessions compared with the BE seed colored kabuli accessions. The functionally relevant molecular tags identified have potential to decipher the complex transcriptional regulatory gene function of seed coat coloration and for understanding the selective sweep-based seed color trait evolutionary pattern in cultivated and wild accessions during chickpea domestication. The genome-wide integrated approach employed will expedite marker-assisted genetic enhancement for developing cultivars with desirable seed coat color types in chickpea.

A combinatorial approach of comprehensive QTL-based comparative genome mapping and transcript profiling identified a seed weight-regulating candidate gene in chickpea.

High experimental validation/genotyping success rate (94-96%) and intra-specific polymorphic potential (82-96%) of 1536 SNP and 472 SSR markers showing in silico polymorphism between desi ICC 4958 and kabuli ICC 12968 chickpea was obtained in a 190 mapping population (ICC 4958 × ICC 12968) and 92 diverse desi and kabuli genotypes. A high-density 2001 marker-based intra-specific genetic linkage map comprising of eight LGs constructed is comparatively much saturated (mean map-density: 0.94 cM) in contrast to existing intra-specific genetic maps in chickpea. Fifteen robust QTLs (PVE: 8.8-25.8% with LOD: 7.0-13.8) associated with pod and seed number/plant (PN and SN) and 100 seed weight (SW) were identified and mapped on 10 major genomic regions of eight LGs. One of 126.8 kb major genomic region harbouring a strong SW-associated robust QTL (Caq'SW1.1: 169.1-171.3 cM) has been delineated by integrating high-resolution QTL mapping with comprehensive marker-based comparative genome mapping and differential expression profiling. This identified one potential regulatory SNP (G/A) in the cis-acting element of candidate ERF (ethylene responsive factor) TF (transcription factor) gene governing seed weight in chickpea. The functionally relevant molecular tags identified have potential to be utilized for marker-assisted genetic improvement of chickpea.

Deploying QTL-seq for rapid delineation of a potential candidate gene underlying major trait-associated QTL in chickpea.

A rapid high-resolution genome-wide strategy for molecular mapping of major QTL(s)/gene(s) regulating important agronomic traits is vital for in-depth dissection of complex quantitative traits and genetic enhancement in chickpea. The present study for the first time employed a NGS-based whole-genome QTL-seq strategy to identify one major genomic region harbouring a robust 100-seed weight QTL using an intra-specific 221 chickpea mapping population (desi cv. ICC 7184 × desi cv. ICC 15061). The QTL-seq-derived major SW QTL (CaqSW1.1) was further validated by single-nucleotide polymorphism (SNP) and simple sequence repeat (SSR) marker-based traditional QTL mapping (47.6% R(2) at higher LOD >19). This reflects the reliability and efficacy of QTL-seq as a strategy for rapid genome-wide scanning and fine mapping of major trait regulatory QTLs in chickpea. The use of QTL-seq and classical QTL mapping in combination narrowed down the 1.37 Mb (comprising 177 genes) major SW QTL (CaqSW1.1) region into a 35 kb genomic interval on desi chickpea chromosome 1 containing six genes. One coding SNP (G/A)-carrying constitutive photomorphogenic9 (COP9) signalosome complex subunit 8 (CSN8) gene of these exhibited seed-specific expression, including pronounced differential up-/down-regulation in low and high seed weight mapping parents and homozygous individuals during seed development. The coding SNP mined in this potential seed weight-governing candidate CSN8 gene was found to be present exclusively in all cultivated species/genotypes, but not in any wild species/genotypes of primary, secondary and tertiary gene pools. This indicates the effect of strong artificial and/or natural selection pressure on target SW locus during chickpea domestication. The proposed QTL-seq-driven integrated genome-wide strategy has potential to delineate major candidate gene(s) harbouring a robust trait regulatory QTL rapidly with optimal use of resources. This will further assist us to extrapolate the molecular mechanism underlying complex quantitative traits at a genome-wide scale leading to fast-paced marker-assisted genetic improvement in diverse crop plants, including chickpea.