Sube Singh

Functionally Relevant Microsatellite Markers From Chickpea Transcription Factor Genes for Efficient Genotyping Applications and Trait Association Mapping

We developed 1108 transcription factor gene-derived microsatellite (TFGMS) and 161 transcription factor functional domain-associated microsatellite (TFFDMS) markers from 707 TFs of chickpea. The robust amplification efficiency (96.5%) and high intra-specific polymorphic potential (34%) detected by markers suggest their immense utilities in efficient large-scale genotyping applications, including construction of both physical and functional transcript maps and understanding population structure. Candidate gene-based association analysis revealed strong genetic association of TFFDMS markers with three major seed and pod traits. Further, TFGMS markers in the 5′ untranslated regions of TF genes showing differential expression during seed development had higher trait association potential. The significance of TFFDMS markers was demonstrated by correlating their allelic variation with amino acid sequence expansion/contraction in the functional domain and alteration of secondary protein structure encoded by genes. The seed weight-associated markers were validated through traditional bi-parental genetic mapping. The determination of gene-specific linkage disequilibrium (LD) patterns in desi and kabuli based on single nucleotide polymorphism-microsatellite marker haplotypes revealed extended LD decay, enhanced LD resolution and trait association potential of genes. The evolutionary history of a strong seed-size/weight-associated TF based on natural variation and haplotype sharing among desi, kabuli and wild unravelled useful information having implication for seed-size trait evolution during chickpea domestication.

Draft genome of the peanut A-genome progenitor (Arachis duranensis) provides insights into geocarpy, oil biosynthesis, and allergens

Significance We present a draft genome of the peanut A-genome progenitor, Arachis duranensis, providing details on total genes present in the genome. Genome analysis suggests that the peanut lineage was affected by at least three polyploidizations since the origin of eudicots. Resequencing of synthetic Arachis tetraploids reveals extensive gene conversion since their formation by human hands. The A. duranensis genome provides a major source of candidate genes for fructification, oil biosynthesis, and allergens, expanding knowledge of understudied areas of plant biology and human health impacts of plants. This study also provides millions of structural variations that can be used as genetic markers for the development of improved peanut varieties through genomics-assisted breeding. Peanut or groundnut (Arachis hypogaea L.), a legume of South American origin, has high seed oil content (45–56%) and is a staple crop in semiarid tropical and subtropical regions, partially because of drought tolerance conferred by its geocarpic reproductive strategy. We present a draft genome of the peanut A-genome progenitor, Arachis duranensis, and 50,324 protein-coding gene models. Patterns of gene duplication suggest the peanut lineage has been affected by at least three polyploidizations since the origin of eudicots. Resequencing of synthetic Arachis tetraploids reveals extensive gene conversion in only three seed-to-seed generations since their formation by human hands, indicating that this process begins virtually immediately following polyploid formation. Expansion of some specific gene families suggests roles in the unusual subterranean fructification of Arachis. For example, the S1Fa-like transcription factor family has 126 Arachis members, in contrast to no more than five members in other examined plant species, and is more highly expressed in roots and etiolated seedlings than green leaves. The A. duranensis genome provides a major source of candidate genes for fructification, oil biosynthesis, and allergens, expanding knowledge of understudied areas of plant biology and human health impacts of plants, informing peanut genetic improvement and aiding deeper sequencing of Arachis diversity.

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% R2 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.

Development of a composite collection for mining germplasm possessing allelic variation for beneficial traits in chickpea

Chickpea is one of the most important grain legume crops in the world. Large collections of genetic resources are maintained in the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) and International Center for Agricultural Research in the Dry Areas (ICARDA) genebanks. Association mapping using neutral markers has been suggested as a means to identify useful alleles in the vast reservoirs of genetic diversity existing in the germplasm collections that could be associated with the phenotypes among the population individuals. ICRISAT in collaboration with ICARDA developed a global composite collection of 3000 accessions that will be profiled using 50 polymorphic simple sequence repeat (SSR) markers. The data generated through this collaborative effort will be used to define the genetic structure of the global composite collection and to select a reference sample of 300 accessions representing the maximum diversity for the isolation of allelic variants of candidate gene associated with beneficial traits. It is then expected that molecular biologists and plant breeders will have opportunities to use diverse lines in functional and comparative genomics, in mapping and cloning gene(s), and in applied plant breeding to diversify the genetic base of the breeding populations which should lead to the development of broad-based elite breeding lines/cultivars with superior yield and enhanced adaptation to diverse environments.

Development of core collection in pigeonpea [Cajanus cajan (L.) Millspaugh] using geographic and qualitative morphological descriptors

Pigeonpea is an important pulse crop grown by smallholder farmers in the semi-arid tropics. Most of the pigeonpea cultivars grown to date are selections from the landraces, with a narrow genetic base. With the expansion of the crop to newer areas, problems of local importance are to be addressed. Hence, an economically feasible and faster germplasm evaluation mechanism, such as a core collection, is required. This article describes the development of core collection from 12,153 pigeonpea accessions collected from 56 countries and maintained at ICRISAT, Patancheru, India. The germplasm accessions from 56 countries were placed under 14 clusters based primarily on geographic origin. Data on 14 qualitative morphological traits were used for cluster formation by Ward’s method. From each cluster ≈10% accessions were randomly selected to constitute a core collection comprising 1290 accessions. Mean comparisons using Newman–Keuls test, variances’ comparisons by Levene’s test, and comparison of frequency distribution by χ2-test indicated that the core collection was similar to that of the entire collection for various traits and the genetic variability available in the entire collection is preserved in the core collection. The Shannon–Weaver diversity index for different traits was also similar for both entire and core collection. All the important phenotypic associations between different traits available in the entire collection were preserved in the core collection. The core collection constituted in the present study facilitates identification of useful traits economically and expeditiously for use in pigeonpea improvement.

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.

Employing genome-wide SNP discovery and genotyping strategy to extrapolate the natural allelic diversity and domestication patterns in chickpea

The genome-wide discovery and high-throughput genotyping of SNPs in chickpea natural germplasm lines is indispensable to extrapolate their natural allelic diversity, domestication, and linkage disequilibrium (LD) patterns leading to the genetic enhancement of this vital legume crop. We discovered 44,844 high-quality SNPs by sequencing of 93 diverse cultivated desi, kabuli, and wild chickpea accessions using reference genome- and de novo-based GBS (genotyping-by-sequencing) assays that were physically mapped across eight chromosomes of desi and kabuli. Of these, 22,542 SNPs were structurally annotated in different coding and non-coding sequence components of genes. Genes with 3296 non-synonymous and 269 regulatory SNPs could functionally differentiate accessions based on their contrasting agronomic traits. A high experimental validation success rate (92%) and reproducibility (100%) along with strong sensitivity (93–96%) and specificity (99%) of GBS-based SNPs was observed. This infers the robustness of GBS as a high-throughput assay for rapid large-scale mining and genotyping of genome-wide SNPs in chickpea with sub-optimal use of resources. With 23,798 genome-wide SNPs, a relatively high intra-specific polymorphic potential (49.5%) and broader molecular diversity (13–89%)/functional allelic diversity (18–77%) was apparent among 93 chickpea accessions, suggesting their tremendous applicability in rapid selection of desirable diverse accessions/inter-specific hybrids in chickpea crossbred varietal improvement program. The genome-wide SNPs revealed complex admixed domestication pattern, extensive LD estimates (0.54–0.68) and extended LD decay (400–500 kb) in a structured population inclusive of 93 accessions. These findings reflect the utility of our identified SNPs for subsequent genome-wide association study (GWAS) and selective sweep-based domestication trait dissection analysis to identify potential genomic loci (gene-associated targets) specifically regulating important complex quantitative agronomic traits in chickpea. The numerous informative genome-wide SNPs, natural allelic diversity-led domestication pattern, and LD-based information generated in our study have got multidimensional applicability with respect to chickpea genomics-assisted breeding.

Two major genes for seed size in chickpea ( Cicer arietinum L.)

SummarySeed size as determined by seed weight, is an important trait for trade and component of yield and adaptation in chickpea (Cicer arietinum L.). Inheritance of seed size in chickpea was studied in a cross between ICC11255, a normal seed size parent (average 120 mg seed−1) and ICC 5002, a small seed size parent (average 50 mg seed−1). Seed weight observations on individual plants of parents, F1, F2, and backcross generations, along with reciprocal cross generations revealed that the normal seed size was dominant over small seed size. No maternal effect was detected for seed size. The numbers of individuals with normal, small and medium (average 150 mg seed−1) seed sizes in F2 population were 1237, 323 and 111 fitting well to the expected ratio of 12:3:1 (χ2 = 0.923, P = 0.630). The segregation data of backcross generations also indicated that seed size in chickpea was controlled by two genes with dominance epistasis. We designate the genotype of ICC 11255 as Sd1Sd1sd2sd2, and ICC 5002 as sd1sd1Sd2Sd2 wherein Sd1 is epistatic to Sd2 and sd2 alleles.

Establishing a core collection of foxtail millet to enhance the utilization of germplasm of an underutilized crop

Foxtail millet (Setaria italica (L.) Beauv.) is one of the ten small millets and is cultivated in 23 countries. The foxtail millet is valued as a crop of short duration, which is good as food, feed and fodder. In general, grain yield levels of foxtail millet are low in comparison with other staple cereals. The greater use of diverse germplasm in breeding is suggested as a means to improve the productivity of this crop. The International Crops Research Institute for the Semi-Arid Tropics genebank is presently holding 1474 cultivated germplasm accessions from 23 countries. To utilize this diversity in research, a core collection (10% of the entire collection) was established using the taxonomic and qualitative traits data. The germplasm accessions were stratified into three taxonomic races (Indica, Maxima and Moharia). Principal coordinate analysis was performed on 12 qualitative traits for each of the biological races, separately that resulted in the formation of 29 clusters. From each cluster, 10% of the accessions were selected to constitute a core collection of 155 accessions. The composition and diversity of the core collection was validated by the x 2 -tests of the frequencies of origin, races, subraces and data on qualitative traits. The analysis of the quantitative traits for mean, range, variance, Shannon‐Weaver diversity index and phenotypic associations indicated that the diversity from the entire collection was optimally represented in the core collection. The core subset will be evaluated in replicated trials to make a more precise assessment of diversity and further efforts to identify the sources of agronomic and grain nutritional traits for utilization in breeding programmes.

Phenotypic diversity for morphological and agronomic characteristics in chickpea core collection

The chickpea (Cicer arietinum L.) core collection consists of 1956accessions, of which 1465 are desi, 433 kabuli, and 58 intermediate types. This core collection was evaluated for 7 morphological descriptors and 15agronomic characteristics to estimate phenotypic diversity. All the three groups differed significantly for flower colour, plant colour, dots on seed testa, seed testa texture, plant width, days to maturity, pods per plant,100-seed weight and plot yield. The kabuli and intermediate types were not significantly different for growth habit and seed colour, they differed,however, significantly from desi types for both traits. Desi, kabuli, and intermediate types were significantly different for plant width, days to maturity, pods per plant, 100-seed weight, and plot yield. Kabuli plants have broad plant width, matured late, have lowest average number of pods,highest 100-seed weight, and lowest plot yield. There were significant phenotypic correlations among the various characteristics. Two of these,between days to 50% flowering and flowering duration and between pod number and plant yield, explained 50% variation in the other trait in all three groups. Principal component analysis showed that days to 50%flowering, plant width, apical secondary branches, tertiary branches, dots on seed testa, 100-seed weight, flowering duration, basal secondary branches, seed colour, and seed testa texture were important traits in explaining multivariate polymorphism. Growth habit and basal primary branches did not significantly account for variation in the first five principal components of desi, kabuli, and intermediate types as well as for the entire core collection, indicating their low importance as chickpea descriptors. The average phenotypic diversity index was highest in the intermediate types(0.2653) and lowest in the kabuli types (0.1490). The Shannon-Weaver diversity index varied among traits between the three groups, and the diversity within a group depended upon the traits recorded.