Publications of NIPGR Scientists
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Item Dissecting the genetic basis of seed-iron content in Chickpea using a combinatorial approach of QTL-Seq and molecular haplotyping(Springer Nature Publishing AG, 2026) Singh, Gourav; Chakraborty, Anirban; Singh, Sangeeta; Bhardwaj, Shubham; Parida, Swarup K.; Bhatia, SabhyataIn order to map the QTL(s) and genes regulating the complex seed-iron content (SFC) trait in chickpea, the quantitative trait locus (QTL)-seq approach was used. Whole genome re-sequencing of DNA bulks derived from a mapping population (ICC8261 × 1CC4958) contrasting for SFC led to the identification of three QTLs, [CaqFe4.1 (0.10 Mb), CaqFe4.2 (0.54 Mb) and CaqFe7.1 (0.83 Mb)] in chickpea. In-silico expression analysis of genes underlying the QTLs revealed their varied levels during stages of seed development. Moreover, estimation of Gʹ values of the SNPs identified in the QTL region revealed a SNP that generated synonymous variant of the MAIN-like-2 gene. Haplotype analysis of MAIN-like-2 in a diverse panel of chickpea germplasm varying for SFC further exemplified its haplotypes that displayed strong association to this trait. Homology-based protein interaction analysis coupled with quantitative-real time PCR based-expression analysis revealed several co-expressing co-chaperone and heat shock proteins including P23-1, HSP 90.5 and HSP90.6, having well established roles in seed development as protein components of MAIN-like-2 proteins in chickpea. The functional loci as well as the molecular signatures defined in this study have potential to expedite marker assisted breeding of iron-rich chickpea varieties.Item Global identification of metal ion transporters in chickpea and delineating the role of CaYSL4 in orchestrating iron content(Elsevier B.V., 2025) Singh, Gourav; Manivannan, Abinaya; Pandey, Vimal; Bhatia, SabhyataMetal ion transporters (MITs) are vital to maintain proper metal homeostasis during growth and development of plants thereby necessitating their identification and characterization. Considering the economic importance of chickpea in human nutrition, the molecular behaviour and biological functions of the metal ion transporters (MIT) encoding gene families remains highly relevant in recent times. Global identification of MITs revealed a total of 12 CAXs, 6 CTRs, 11 MGTs, 15 MTPs, 9 NRAMPs, 16 OPTs, and 14 ZIPs responsible for metal ion transport. Assessment of phylogenetic relationships, chromosomal distribution, gene structure and motif analysis of MITs suggested their diverse functions. The yellow stripe-like (YSL) family of transporters is an important family whose members have been suggested to have a role in metal ion translocation and assimilation. Expression analysis of key YSLs including CaYSL1, CaYSL4, CaYSL6 and CaYSL16 indicated their significant involvement in conferring tolerance to Fe starvation. Notable was the expression of CaYSL4 that showed specific expression in flower, leaf, shoot, seed at 30 DAA and 40DAA after 7 and 10 day of Fe-deficiency treatment. It was found to be localized in the plasma membrane. RNAi-mediated silencing of CaYSL4 demonstrated its critical role in orchestrating Fe, Zn, Cu and Mn translocation in chickpea seeds. Collectively, the comprehensive analysis of MITs coupled with the functional role of CaYSL4 provides critical insight into the complex regulation of Fe ion transport and distribution that will enable breeding of nutritionally enhanced chickpea varieties.Item Evolutionary and expression dynamics of LRR-RLKs and functional establishment of KLAVIER homolog in shoot mediated regulation of AON in chickpea symbiosis(Elsevier B.V., 2021) Tiwari, Manish; Pandey, Vimal; Singh, Baljinder; Yadav, Manisha; Bhatia, SabhyataChickpea shoot exogenously treated with cytokinin showed stunted phenotype of root, shoot and significantly reduced nodule numbers. Genome-wide identification of LRR-RLKs in chickpea and Medicago resulted in 200 and 371 genes respectively. Gene duplication analysis revealed that LRR-RLKs family expanded through segmental duplications in chickpea and tandem duplications in Medicago. Expression profiling of LRR-RLKs revealed their involvement in cytokinin signaling and plant organ development. Overexpression of KLAVIER ortholog of chickpea, Ca_LRR-RLK147, in roots revealed its localization in the membrane but showed no effect on root nodulation despite increased cle peptide levels. Two findings (i) drastic effect on nodule number by exogenous cytokinin treatment to only shoot and restoration to normal nodulation by treatment to both root and shoot tissue and (ii) no effect on nodule number by overexpression of Ca_LRR-RLK147 establishes the fact that despite presence of cle peptides in root, the function of Ca_LRR-RLK147 was shoot mediated during AON.Item Evolutionary and functional analysis of Two-Component System in chickpea reveals CaRR13, a TypeB RR, as positive regulator of symbiosis(John Wiley & Sons, 2021) Tiwari, Manish; Yadav, Manisha; Singh, Baljinder; Pandey, Vimal; Nawaz, Kashif; Bhatia, SabhyataThe critical role of cytokinin in early nodulation in legumes is well known. In our study, exogenous cytokinin application to roots of the important crop legume, chickpea (Cicer arietinum L.) led to the formation of pseudo nodules even in the absence of rhizobia. Hence a genome-wide analysis of the cytokinin signaling, Two-Component System (TCS) genes was conducted in chickpea, Medicago and Cajanus cajan. The integrated phylogenetic, evolutionary and expression analysis of the TCS genes was carried out which revealed that Histidine Kinases (HKs) were highly conserved, whereas, there was diversification leading to neofunctionalization at the level of Response Regulators (RRs) especially the TypeB RRs. Further, the functional role of the CaHKs in nodulation was established by complementation of the sln1Δ mutant of yeast and cre1 mutants of (Medicago) which led to restoration of the nodule deficient phenotype. Additionally, the highest expressing TypeB RR of chickpea, CaRR13 was functionally characterized. Its localization in the nucleus and its Y1H assay-based interaction with the promoter of the early nodulation gene CaNSP2 indicated its role as a transcription factor regulating early nodulation. Overexpression, RNAi lines and complementation of cre1 mutants with CaRR13 revealed its critical involvement as an important signaling molecule regulating early events of nodule organogenesis in chickpea.Item High throughput identification of miRNAs reveal novel interacting targets regulating chickpea-rhizobia symbiosis(Elsevier B.V., 2021) Tiwari, Manish; Singh, Baljinder; Yadav, Manisha; Pandey, Vimal; Bhatia, SabhyataLegumes developed symbiotic associations to meet its nitrogen requirement. The nitrogen fixation takes place in root nodules which involves bacterial colonization, organogenesis and nitrogen fixation. In order to unravel the miRNA mediated regulation of chickpea symbiosis, one microRNA and four parallel analysis of RNA ends (PARE) libraries were sequenced. Analysis of microRNA library identified a set of 91 miRNAs comprising of 84 conserved and 7 novel miRNAs. Additionally, PARE library analysis revealed 564 genes being targeted by 85 miRNAs. Phylogenetic analysis of the precursor sequences of the 91 miRNAs was carried out which revealed their ancestral relationships. Further, the mechanism of miRNAs biogenesis was predicted using the miRNAs information from other legumes. Reads from the nodule library were mapped to bacterial genomes to predict bacterial-encoded small RNAs. Real time expression analysis was used to validate the antagonistic expression pattern of important miRNA-mRNA target pairs. Four candidate miRNAs were selected for in planta study based on the antagonistic expression profiling as well as the novelty of their respective targets. miR171f, miR172c, miR394 and miR1509 targeted nodulation receptor kinase, Apetala2, histidine phosphotransferase, adenylate kinase respectively and were ectopically expressed in chickpea roots. The overexpression lines showed significant change in nodule numbers, the miR172c, miR394 and miR1509 resulted in an increase in nodule number whereas, miR171f overexpression led to a decrease in nodule number. Our analysis lays the foundation for functional characterization of novel miRNAs and their respective target pairs which control nodulation in chickpea and other leguminous crops.Item A high-density SNP-based linkage map using genotyping-by-sequencing and its utilization for improved genome assembly of chickpea (Cicer arietinum L.)(Springer Nature Publishing AG, 2020) Gaur, Rashmi; Verma, Subodh; Pradhan, Seema; Ambreen, Heena; Bhatia, SabhyataGenotyping-by-sequencing (GBS) allows rapid identification of markers for use in development of linkage maps, which expedite efficient breeding programs. In the present study, we have utilized GBS approach to identify and genotype single-nucleotide polymorphism (SNP) markers in an inter-specific RIL population of Cicer arietinum L. X C. reticulatum. A total of 141,639 raw SNPs were identified using the TASSEL-GBS pipeline. After stringent filtering, 8208 candidate SNPs were identified of which ~ 37% were localized in the intragenic regions followed by genic regions (~ 30%) and intergenic regions (~ 27%). We then utilized 6920 stringent selected SNPs from present study and 6714 SNPs and microsatellite markers available from previous studies for construction of linkage map. The resulting high-density linkage map comprising of eight linkage groups contained 13,590 markers which spanned 1299.14 cM of map length with an average marker density of 0.095 cM. Further, the derived linkage map was used to improve the available assembly of desi chickpea genome by anchoring 443 previously unplaced scaffolds onto eight linkage groups. The present efforts have refined anchoring of the desi chickpea genome assembly to 55.57% of the ~ 520 Mb of assembled desi genome. To the best of our knowledge, the linkage map generated in the present study represents one of the most dense linkage map developed for the crop till date. It will serve as a valuable resource for fine mapping and positional cloning of important quantitative trait loci (QTLs) associated with agronomical traits and also for anchoring and ordering of future genome sequence assemblies.Item A comprehensive analysis of the B3 superfamily identifies tissue-specific and stress-responsive genes in chickpea (Cicer arietinum L.)(Springer Nature Publishing AG, 2019) Verma, Subodh; Bhatia, SabhyataThe aim of this study was to provide a comprehensive analysis of the plant-specifc B3 domain-containing transcription factors (TFs) in chickpea. Scanning of the chickpea genome resulted in the identifcation of 51 B3 domain-containing TFs that were located on seven out of eight chickpea chromosomes. Based on the presence of additional domains other than the B3 domain, the candidates were classifed into four subfamilies, i.e., ARF (24), REM (19), LAV (6) and RAV (2). Phylogenetic analysis classifed them into four groups in which members of the same group had similar intron–exon organization and motif composition. Genome duplication analysis of the candidate B3 genes revealed an event of segmental duplication that was instrumental in the expansion of the B3 gene family. Ka/Ks analysis showed that the B3 gene family was under purifying selection. Further, chickpea B3 genes showed maximum orthology with Medicago followed by soybean and Arabidopsis. Promoter analyses of the B3 genes led to the identifcation of several tissue-specifc and stress-responsive cis-regulatory elements. Expression profling of the candidate B3 genes using publicly available RNA-seq data of several chickpea tissues indicated their putative role in plant development and abiotic stress response. These fndings were further validated by realtime expression analysis. Overall, this study provides a comprehensive analysis of the B3 domain-containing proteins in chickpea that would aid in devising strategies for crop manipulation in chickpea.Item Analysis of genes encoding seed storage proteins (SSPs) in chickpea (Cicer arietinum L.) reveals co-expressing transcription factors and a seed-specific promoter(Springer Nature, 2019) Verma, Subodh; Bhatia, SabhyataImprovement of the quality and quantity of chickpea seed protein can be greatly facilitated by an understanding of the genic organization and the genetic architecture of the genes encoding seed storage proteins (SSPs). The aim of this study was to provide a comprehensive analysis of the chickpea SSP genes, putative co-expressing transcription factors (TFs), and to identify a seedspecific SSP gene promoter. A genome-wide identification of SSP genes in chickpea led to the identification of 21 non-redundant SSP encoding genes located on 6 chromosomes. Phylogenetic analysis grouped SSP genes into 3 subgroups where members within the same clade demonstrated similar motif composition and intron-exon organization. Tandem duplications were identified to be the major contributors to the expansion of the SSP gene family in chickpea. Co-expression analysis revealed 14 TFs having expression profiles similar to the SSP genes that included members of important TF families that are known to regulate seed development. Expression analysis of SSP genes and TFs revealed significantly higher expression in late stages of seed development as well as in high seed protein content (HPC) genotypes. In silico analysis of the promoter regions of the SSP encoding genes revealed several seed-specific cis-regulatory elements such as RY repeats, ACGT motifs, CAANTG, and GCN4. A candidate promoter was analyzed for seed specificity by generating stable transgenics in Arabidopsis. Overall, this study provides a useful resource to explore the regulatory networks involved in SSP synthesis and/or accumulation for utilization in developing nutritionally improved chickpea genotypes.Item Dissecting the root nodule transcriptome of chickpea (Cicer arietinum L.)(PLOS, 2016) Kant, Chandra; Pradhan, Seema; Bhatia, SabhyataA hallmark trait of chickpea (Cicer arietinum L.), like other legumes, is the capability to convert atmospheric nitrogen (N2) into ammonia (NH3) in symbiotic association with Mesorhizobium ciceri. However, the complexity of molecular networks associated with the dynamics of nodule development in chickpea need to be analyzed in depth. Hence, in order to gain insights into the chickpea nodule development, the transcriptomes of nodules at early, middle and late stages of development were sequenced using the Roche 454 platform. This generated 490.84 Mb sequence data comprising 1,360,251 reads which were assembled into 83,405 unigenes. Transcripts were annotated using Gene Ontology (GO), Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways analysis. Differential expression analysis revealed that a total of 3760 transcripts were differentially expressed in at least one of three stages, whereas 935, 117 and 2707 transcripts were found to be differentially expressed in the early, middle and late stages of nodule development respectively. MapMan analysis revealed enrichment of metabolic pathways such as transport, protein synthesis, signaling and carbohydrate metabolism during root nodulation. Transcription factors were predicted and analyzed for their differential expression during nodule development. Putative nodule specific transcripts were identified and enriched for GO categories using BiNGO which revealed many categories to be enriched during nodule development, including transcription regulators and transporters. Further, the assembled transcriptome was also used to mine for genic SSR markers. In conclusion, this study will help in enriching the transcriptomic resources implicated in understanding of root nodulation events in chickpea.Item High-density linkage map construction and mapping of seed trait QTLs in chickpea (Cicer arietinum L.) using genotyping-by-sequencing (GBS)(Nature Publishing Group, 2015) Verma, Subodh; Gupta, Shefali; Bandhiwal, Nitesh; Kumar, Tapan; Bharadwaj, Chellapilla; Bhatia, SabhyataThis study reports the use of Genotyping-by-Sequencing (GBS) for large-scale SNP discovery and simultaneous genotyping of recombinant inbred lines (RILs) of an intra-specific mapping population of chickpea contrasting for seed traits. A total of 119,672 raw SNPs were discovered, which after stringent filtering revealed 3,977 high quality SNPs of which 39.5% were present in genic regions. Comparative analysis using physically mapped marker loci revealed a higher degree of synteny with Medicago in comparison to soybean. The SNP genotyping data was utilized to construct one of the most saturated intra-specific genetic linkage maps of chickpea having 3,363 mapped positions including 3,228 SNPs on 8 linkage groups spanning 1006.98 cM at an average inter marker distance of 0.33 cM. The map was utilized to identify 20 quantitative trait loci (QTLs) associated with seed traits accounting for phenotypic variations ranging from 9.97% to 29.71%. Analysis of the genomic sequence corresponding to five robust QTLs led to the identification of 684 putative candidate genes whose expression profiling revealed that 101 genes exhibited seed specific expression. The integrated approach utilizing the identified QTLs along with the available genome and transcriptome could serve as a platform for candidate gene identification for molecular breeding of chickpea.