Publications of NIPGR Scientists
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Item Identifying transcription factor genes associated with yield traits in chickpea(Springer, 2017) Shimray, Philanim Wungmarong; Bajaj, Deepak; Srivastava, Rishi; Daware, Anurag; Upadhyaya, Hari D.; Kumar, Rajendra; Bharadwaj, Chellapilla; Tyagi, Akhilesh K.; Parida, Swarup K.Identification of potential transcription factor (TF) gene-derived natural SNP allelic variants regulating pod and seed yield component traits by large-scale mining and genotyping of SNPs in natural germplasm accessions coupled with high-resolution association mapping is vital for understanding the complex genetic architecture of quantitative yield traits in chickpea. In these perspectives, the current study employed a genome-wide GBS (genotyping-by-sequencing) and targeted gene amplicon resequencing-based simultaneous SNP discovery and genotyping assays, which discovered 1611 novel SNPs from 736 TF genes physically mapped on eight chromosomes and unanchored scaffolds of kabuli chickpea genome. These SNPs were structurally and functionally annotated in diverse synonymous and non-synonymous coding as well as non-coding regulatory and intronic sequence components of chickpea TF genes. A high-resolution genetic association analysis was performed by correlating the genotyping information of 1611 TF gene-based SNPs with multi-location/years field phenotyping data of six major pod and seed yield traits evaluated in a constituted association panel (326 desi and kabuli germplasm accessions) of chickpea. This essentially identified 27 TF gene-derived SNPs exhibiting significant association with six major yield traits, namely days to 50% flowering (DF), plant height (PH), branch number (BN), pod number (PN), seed number (SN) and seed weight (SW) in chickpea. These trait-associated SNPs individually and in combination explained 10–23% and 32% phenotypic variation respectively for the studied yield component traits. Interestingly, novel non-synonymous coding SNP allelic variants in five potential candidate TF genes encoding SBP (squamosal promoter binding protein), SNF2 (sucrose non-fermenting 2), GRAS [Gibberellic acid insensitive (GAI)-Repressor of GAI (RGA)-SCARECROW (SCR)], bZIP (basic leucine zipper) and LOB (lateral organ boundaries)-domain proteins associated strongly with DF, PH, BN, PN, SN and SW traits respectively were found most promising in chickpea. The functionally relevant molecular signatures (TFs and natural SNP alleles) delineated by us have potential to accelerate marker-assisted genetic enhancement by developing high pod and seed yielding cultivars of chickpea.Item Genetic dissection of plant growth habit in chickpea(Springer, 2017) Upadhyaya, Hari D.; Bajaj, Deepak; Srivastava, Rishi; Daware, Anurag; Basu, Udita; Tripathi, Shailesh; Bharadwaj, Chellapilla; Tyagi, Akhilesh K.; Parida, Swarup K.A combinatorial genomics-assisted breeding strategy encompassing association analysis, genetic mapping and expression profiling is found most promising for quantitative dissection of complex traits in crop plants. The present study employed GWAS (genome-wide association study) using 24,405 SNPs (single nucleotide polymorphisms) obtained with genotyping-by-sequencing (GBS) of 92 sequenced desi and kabuli accessions of chickpea. This identified eight significant genomic loci associated with erect (E)/semi-erect (SE) vs. spreading (S)/semi-spreading (SS)/prostrate (P) plant growth habit (PGH) trait differentiation regardless of diverse desi and kabuli genetic backgrounds of chickpea. These associated SNPs in combination explained 23.8% phenotypic variation for PGH in chickpea. Five PGH-associated genes were validated successfully in E/SE and SS/S/P PGH-bearing parental accessions and homozygous individuals of three intra- and interspecific RIL (recombinant inbred line) mapping populations as well as 12 contrasting desi and kabuli chickpea germplasm accessions by selective genotyping through Sequenom MassARRAY. The shoot apical, inflorescence and floral meristems-specific expression, including upregulation (seven-fold) of five PGH-associated genes especially in germplasm accessions and homozygous RIL mapping individuals contrasting with E/SE PGH traits was apparent. Collectively, this integrated genomic strategy delineated diverse non-synonymous SNPs from five candidate genes with strong allelic effects on PGH trait variation in chickpea. Of these, two vernalization-responsive non-synonymous SNP alleles carrying SNF2 protein-coding gene and B3 transcription factor associated with PGH traits were found to be the most promising in chickpea. The SNP allelic variants associated with E/SE/SS/S PGH trait differentiation were exclusively present in all cultivated desi and kabuli chickpea accessions while wild species/accessions belonging to primary, secondary and tertiary gene pools mostly contained prostrate PGH-associated SNP alleles. This indicates strong adaptive natural/artificial selection pressure (Tajima's D 3.15 to 4.57) on PGH-associated target genomic loci during chickpea domestication. These vital leads thus have potential to decipher complex transcriptional regulatory gene function of PGH trait differentiation and for understanding the selective sweep-based PGH trait evolution and domestication pattern in cultivated and wild chickpea accessions adapted to diverse agroclimatic conditions. Collectively, the essential inputs generated will be of profound use in marker-assisted genetic enhancement to develop cultivars with desirable plant architecture of erect growth habit types in chickpea.Item A genome-wide mQTL-seq scan identifies potential molecular signatures regulating plant height in chickpea(Springer, 2017) Parida, Swarup K.; Srivastava, Rishi; Bajaj, DeepakThe current study employed a high-throughput genome-wide next-generation sequencing-led multiple QTL-seq (mQTL-seq) strategy in two inter- and intra-specific recombinant inbred line (RIL) mapping populations to identify the major genomic regions underlying robust quantitative trait loci (QTLs) regulating plant height in chickpea. The whole genome resequencing discovered 446,475 and 150,434 high-quality homozygous single nucleotide polymorphisms (SNPs) exhibiting polymorphism between tall and dwarf/semi-dwarf mapping parents and bulk/homozygous individuals selected from each of two chickpea RIL populations. These SNP-led mQTL-seq assays in RIL mapping populations scaled-down two longer major genomic regions (1.26–1.34 Mb) underlying robust plant height QTLs into the shorter high-resolution QTL intervals (653.2–756.3 kb) on chickpea chromosomes 3 and 8. This essentially delineated regulatory novel natural SNP allelic variants from brassinosteroid insensitive 1-receptor kinase 1 (BAK1) and gibberellin (GA) 20-oxidase genes governing plant height in chickpea. A strong impact of evolutionary bottlenecks including strong artificial/natural selection on two plant height gene loci during chickpea domestication was observed. The shoot apical meristem-specific expression aside from down-regulation of two plant height genes especially in dwarf/semi-dwarf as compared to tall parents and homozygous mapping individuals of two aforementioned RIL populations was apparent. The integrated genomics-assisted breeding strategy combining mQTL-seq with differential gene expression profiling and functional allelic diversity-based trait domestication study collectively identified potential natural allelic variants of candidate genes underlying major plant height QTLs in chickpea. These functionally relevant molecular signatures can be of immense use for marker-aided genetic enhancement to develop high seed- and pod-yielding non-lodging cultivars restructured with desirable plant height in chickpea.Item Genome-wide development and deployment of informative intron-spanning and intron-length polymorphism markers for genomics-assisted breeding applications in chickpea(Elsevier B.V., 2016) Srivastava, Rishi; Bajaj, Deepak; Sayal, Yogesh K.; Meher, Prabina K.; Upadhyaya, Hari D.; Kumar, Rajendra; Tripathi, Shailesh; Bharadwaj, Chellapilla; Rao, Atmakuri R.; Parida, Swarup K.The discovery and large-scale genotyping of informative gene-based markers is essential for rapid delineation of genes/QTLs governing stress tolerance and yield component traits in order to drive genetic enhancement in chickpea. A genome-wide 119169 and 110491 ISM (intron-spanning markers) from 23129 desi and 20386 kabuli protein-coding genes and 7454 in silico InDel (insertion-deletion) (1–45-bp)-based ILP (intron-length polymorphism) markers from 3283 genes were developed that were structurally and functionally annotated on eight chromosomes and unanchored scaffolds of chickpea. A much higher amplification efficiency (83%) and intra-specific polymorphic potential (86%) detected by these markers than that of other sequence-based genetic markers among desi and kabuli chickpea accessions was apparent even by a cost-effective agarose gel-based assay. The genome-wide physically mapped 1718 ILP markers assayed a wider level of functional genetic diversity (19–81%) and well-defined phylogenetics among domesticated chickpea accessions. The gene-derived 1424 ILP markers were anchored on a high-density (inter-marker distance: 0.65 cM) desi intra-specific genetic linkage map/functional transcript map (ICC 4958 × ICC 2263) of chickpea. This reference genetic map identified six major genomic regions harbouring six robust QTLs mapped on five chromosomes, which explained 11–23% seed weight trait variation (7.6–10.5 LOD) in chickpea. The integration of high-resolution QTL mapping with differential expression profiling detected six including one potential serine carboxypeptidase gene with ILP markers (linked tightly to the major seed weight QTLs) exhibiting seed-specific expression as well as pronounced up-regulation especially in seeds of high (ICC 4958) as compared to low (ICC 2263) seed weight mapping parental accessions. The marker information generated in the present study was made publicly accessible through a user-friendly web-resource, “Chickpea ISM-ILP Marker Database”. The designing of multiple ISM and ILP markers (2–5 markers/gene) from an individual gene (transcription factor) with numerous aforementioned desirable genetic attributes can widen the user-preference to select suitable primer combination for simultaneous large-scale assaying of functional allelic variation, natural allelic diversity, molecular mapping and expression profiling of genes among chickpea accessions. This will essentially accelerate the identification of functionally relevant molecular tags regulating vital agronomic traits for genomics-assisted crop improvement by optimal resource expenses in chickpea.Item Genome-wide insertion-deletion (InDel) marker discovery and genotyping for genomics- assisted breeding applications in chickpea(Oxford University Press, 2015) Das, Shouvik; Upadhyaya, Hari D.; Srivastava, Rishi; Bajaj, Deepak; Gowda, C.L.L.; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K.; Parida, Swarup K.We developed 21,499 genome-wide insertion–deletion (InDel) markers (2- to 54-bp in silico fragment length polymorphism) by comparing the genomic sequences of four (desi, kabuli and wild C. reticulatum) chickpea [Cicer arietinum (L.)] accessions. InDel markers showing 2- to 6-bp fragment length polymorphism among accessions were abundant (76.8%) in the chickpea genome. The physically mapped 7,643 and 13,856 markers on eight chromosomes and unanchored scaffolds, respectively, were structurally and functionally annotated. The 4,506 coding (23% large-effect frameshift mutations) and regulatory InDel markers were identified from 3,228 genes (representing 11.7% of total 27,571 desi genes), suggesting their functional relevance for trait association/genetic mapping. High amplification (97%) and intra-specific polymorphic (60–83%) potential and wider genetic diversity (15–89%) were detected by genome-wide 6,254 InDel markers among desi, kabuli and wild accessions using even a simpler cost-effective agarose gel-based assay. This signifies added advantages of this user-friendly genetic marker system for manifold large-scale genotyping applications in laboratories with limited infrastructure and resources. Utilizing 6,254 InDel markers-based high-density (inter-marker distance: 0.212 cM) inter-specific genetic linkage map (ICC 4958 × ICC 17160) of chickpea as a reference, three major genomic regions harboring six flowering and maturity time robust QTLs (16.4–27.5% phenotypic variation explained, 8.1–11.5 logarithm of odds) were identified. Integration of genetic and physical maps at these target QTL intervals mapped on three chromosomes delineated five InDel markers-containing candidate genes tightly linked to the QTLs governing flowering and maturity time in chickpea. Taken together, our study demonstrated the practical utility of developing and high-throughput genotyping of such beneficial InDel markers at a genome-wide scale to expedite genomics-assisted breeding applications in chickpea.