Publications of NIPGR Scientists
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Item MicroRNA164e suppresses NAC100 transcription factor-mediated synthesis of seed storage proteins in chickpea(John Wiley & Sons, 2024) Chakraborty, Anirban; Singh, Baljinder; Pandey, Vimal; Parida, Swarup K.; Bhatia, SabhyataDevelopment of protein-enriched chickpea varieties necessitates an understanding of specific genes and key regulatory circuits that govern the synthesis of seed storage proteins (SSPs). Here, we demonstrated the novel involvement of Ca-miR164e-CaNAC100 in regulating SSP synthesis in chickpea. Ca-miRNA164e was significantly decreased during seed maturation, especially in high-protein accessions. The miRNA was found to directly target the transactivation conferring C-terminal region of a nuclear-localized transcription factor, CaNAC100 as revealed using RNA ligase-mediated-rapid amplification of cDNA ends and target mimic assays. The functional role of CaNAC100 was demonstrated through seed-specific overexpression (NACOE) resulting in significantly augmented seed protein content (SPC) consequential to increased SSP transcription. Further, NACOE lines displayed conspicuously enhanced seed weight but reduced numbers and yield. Conversely, a downregulation of CaNAC100 and SSP transcripts was evident in seed-specific overexpression lines of Ca-miR164e that culminated in significantly lowered SPC. CaNAC100 was additionally demonstrated to transactivate the SSP-encoding genes by directly binding to their promoters as demonstrated using electrophoretic mobility shift and dual-luciferase reporter assays. Taken together, our study for the first time established a distinct role of CaNAC100 in positively influencing SSP synthesis and its critical regulation by CamiR164e, thereby serving as an understanding that can be utilized for developing SPC-rich chickpea varieties.Item Comparative transcriptomic and metabolite profiling reveals genotype-specific responses to Fe starvation in chickpea(John Wiley & Sons, 2023) Singh, Gourav; Ambreen, Heena; Jain, Priyanka; Chakraborty, Anirban; Singh, Baljinder; Manivannan, Abinaya; Bhatia, SabhyataIron deficiency is a major nutritional stress that severely impacts crop productivity worldwide. However, molecular intricacies and subsequent physiological and metabolic changes in response to Fe starvation, especially in leguminous crops like chickpea, remain elusive. In the present study, we investigated physiological, transcriptional, and metabolic reprogramming in two chickpea genotypes (H6013 and L4958) with contrasting seed iron concentrations upon Fe deficiency. Our findings revealed that iron starvation affected growth and physiological parameters of both chickpea genotypes. Comparative transcriptome analysis led to the identification of differentially expressed genes (DEGs) between the genotypes related to strategy I uptake, metal ions transporters, reactive oxygen species (ROS) associated genes, transcription factors, and protein kinases that could mitigate Fe deficiency. Our gene correlation network discovered several putative candidate genes like CIPK25, CKX3, WRKY50, NAC29, MYB4 and PAP18, which could facilitate the investigation of the molecular rationale underlying Fe tolerance in chickpea. Furthermore, the metabolite analysis also illustrated the differential accumulation of organic acids, amino acids and other metabolites associated with Fe mobilization in chickpea genotypes. Overall, our study demonstrated the comparative transcriptional dynamics upon Fe starvation. The outcomes of the current endeavour will enable the development of Fe deficiency tolerant chickpea cultivars.Item Integrated genomic approaches delineate the novel role of ROP1 ENHANCER1 in regulating seed protein content of chickpea(Oxford University Press, 2023) Chakraborty, Anirban; Junaid, Alim; Parida, Swarup K.; Bhatia, SabhyataUtilizing a combinatorial approach of QTL-Seq and candidate gene-based association mapping, the QTLs and genes responsible for seed protein content (SPC), a major quality trait in chickpea were identified. Whole Genome Re-sequencing based QTL-Seq analysis of bulked RILs from a mapping population contrasting for SPC led to identification of two QTLs (0.94 Mb on Linkage Group (LG)5 and 1.16 Mb on LG6) encompassing three SNPs displaying the highest ΔSNP-index. These highly significant SNPs and their associated genes were validated in 211 chickpea mini-core accessions varying in SPC that revealed a tightly associated marker affecting CaREN1 (ROP1 ENHANCER1) with phenotypic variation explained of 23%. This SNP was subsequently converted into a cost effective allele specific PCR based marker that could be utilized for rapid screening of SPC during marker assisted breeding. Further, in planta functional validation via knockdown of CaREN1 led to significant reduction in SPC of chickpea. This decrease in seed protein is likely due to disruption in the formation of CaREN1 protein complexes comprising of chaperones, phosphopeptide-binding proteins and GTPases that mediate folding, transport and accumulation of seed storage proteins as indicated through AP-MS. Taken together, the information generated would expedite tailoring of chickpea cultivars with augmented SPC.Item A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.)(John Wiley & Sons, 2013) Jain, Mukesh; Misra, Gopal; Patel, Ravi K.; Priya, Pushp; Jhanwar, Shalu; Khan, Aamir W.; Shah, Niraj; Singh, Vikas K.; Garg, Rohini; Jeena, Ganga; Yadav, Manju; Kant, Chandra; Sharma, Priyanka; Yadav, Gitanjali; Bhatia, Sabhyata; Tyagi, Akhilesh K.; Chattopadhyay, DebasisCicer arietinum L. (chickpea) is the third most important food legume crop. We have generated the draft sequence of a desi-type chickpea genome using next-generation sequencing platforms, bacterial artificial chromosome end sequences and a genetic map. The 520-Mb assembly covers 70% of the predicted 740-Mb genome length, and more than 80% of the gene space. Genome analysis predicts the presence of 27,571 genes and 210 Mb as repeat elements. The gene expression analysis performed using 274 million RNA-Seq reads identified several tissue-specific and stress-responsive genes. Although segmental duplicated blocks are observed, the chickpea genome does not exhibit any indication of recent whole-genome duplication. Nucleotide diversity analysis provides an assessment of a narrow genetic base within the chickpea cultivars. We have developed a resource for genetic markers by comparing the genome sequences of one wild and three cultivated chickpea genotypes. The draft genome sequence is expected to facilitate genetic enhancement and breeding to develop improved chickpea varieties.Item EST-derived genic molecular markers: development and utilization for generating an advanced transcript map of chickpea(Springer Science, 2012) Choudhary, Shalu; Gaur, Rashmi; Gupta, Shefali; Bhatia, SabhyataWell-saturated linkage maps especially those based on expressed sequence tag (EST)-derived genic molecular markers (GMMs) are a pre-requisite for molecular breeding. This is especially true in important legumes such as chickpea where few simple sequence repeats (SSR) and even fewer GMM-based maps have been developed. Therefore, in this study, 2,496 ESTs were generated from chickpea seeds and utilized for the development of 487 novel EST-derived functional markers which included 125 EST-SSRs, 151 intron targeted primers (ITPs), 109 expressed sequence tag polymorphisms (ESTPs), and 102 single nucleotide polymorphisms (SNPs). Whereas ESTSSRs, ITPs, and ESTPs were developed by in silico analysis of the developed EST sequences, SNPs were identified by allele resequencing and their genotyping was performedusing the Illumina GoldenGate Assay. Parental polymorphism was analyzed between C. arietinum ICC4958 and C. reticulatum PI489777, parents of the reference chickpea mapping population, using a total of 872 markers: 487 new gene-based markers developed in this study along with 385 previously published markers, of which 318 (36.5%) were found to be polymorphic and were used for genotyping. The genotypic data were integrated with the previously published data of 108 markers and an advanced linkage map was generated that contained 406 loci distributed on eight linkage groups that spanned 1,497.7 cM. The average marker density was 3.68 cM and the average number of markers per LG was 50.8. Among the mapped markers, 303 new genomic locations were defined that included 177 gene-based and 126 gSSRs (genomic SSRs) thereby producing the most advanced gene-rich map of chickpea solely based on co-dominant markers.Item Development and use of genic molecular markers (GMMs) for construction of a transcript map of chickpea (Cicer arietinum L.)(Springer Science, 2011) Gujaria, Neha; Kumar, Ashish; Dauthal, Preeti; Dubey, Anuja; Hiremath, Pavana; Bhanu Prakash, A.; Farmer, Andrew; Bhide, Mangla; Shah, Trushar; Gaur, Pooran M.; Upadhyaya, Hari D.; Bhatia, Sabhyata; Cook, Douglas R.; May, Greg D.; Varshney, Rajeev K.A transcript map has been constructed by the development and integration of genic molecular markers (GMMs) including single nucleotide polymorphism (SNP), genic microsatellite or simple sequence repeat (SSR) and intron spanning region (ISR)-based markers, on an inter-specific mapping population of chickpea, the third food legume crop of the world and the first food legume crop of India. For SNP discovery through allele re-sequencing, primer pairs were designed for 688 genes/expressed sequence tags (ESTs) of chickpea and 657 genes/ESTs of closely related species of chickpea. High-quality sequence data obtained for 220 candidate genic regions on 2-20 genotypes representing 9 Cicer species provided 1,893 SNPs with an average frequency of 1/35.83 bp and 0.34 PIC (polymorphism information content) value. On an average 2.9 haplotypes were present in 220 candidate genic regions with an average haplotype diversity of 0.6326. SNP2CAPS analysis of 220 sequence alignments, as mentioned above, provided a total of 192 CAPS candidates. Experimental analysis of these 192 CAPS candidates together with 87 CAPS candidates identified earlier through in silico mining of ESTs provided scorable amplification in 173 (62.01%) cases of which predicted assays were validated in 143 (82.66%) cases (CGMM). Alignments of chickpea unigenes with Medicago truncatula genome were used to develop 121 intron spanning region (CISR) markers of which 87 yielded scorable products. In addition, optimization of 77 EST-derived SSR (ICCeM) markers provided 51 scorable markers. Screening of easily assayable 281 markers including 143 CGMMs, 87 CISRs and 51 ICCeMs on 5 parental genotypes of three mapping populations identified 104 polymorphic markers including 90 markers on the inter-specific mapping population. Sixty-two of these GMMs together with 218 earlier published markers (including 64 GMM loci) and 20 other unpublished markers could be integrated into this genetic map. A genetic map developed here, therefore, has a total of 300 loci including 126 GMM loci and spans 766.56 cM, with an average inter-marker distance of 2.55 cM. In summary, this is the first report on the development of large-scale genic markers including development of easily assayable markers and a transcript map of chickpea. These resources should be useful not only for genome analysis and genetics and breeding applications of chickpea, but also for comparative legume genomics.Item Advancing the STMS genomic resources for defining new locations on the intraspecific genetic linkage map of chickpea (Cicer arietinum L.)(BioMed Central, 2011) Gaur, Rashmi; Sethy, Niroj K; Choudhary, Shalu; Shokeen, Bhumika; Gupta, Varsha; Bhatia, SabhyataChickpea (Cicer arietinum L.) is an economically important cool season grain legume crop that is valued for its nutritive seeds having high protein content. However, several biotic and abiotic stresses and the low genetic variability in the chickpea genome have continuously hindered the chickpea molecular breeding programs. STMS (Sequence Tagged Microsatellite Sites) markers which are preferred for the construction of saturated linkage maps in several crop species, have also emerged as the most efficient and reliable source for detecting allelic diversity in chickpea. However, the number of STMS markers reported in chickpea is still limited and moreover exhibit low rates of both inter and intraspecific polymorphism, thereby limiting the positions of the SSR markers especially on the intraspecific linkage maps of chickpea. Hence, this study was undertaken with the aim of developing additional STMS markers and utilizing them for advancing the genetic linkage map of chickpea which would have applications in QTL identification, MAS and for de novo assembly of high throughput whole genome sequence data.Item Development of chickpea EST-SSR markers and analysis of allelic variation across related species(Springer, 2009) Choudhary, Shalu; Sethy, Niroj Kumar; Shokeen, Bhumika; Bhatia, SabhyataDespite chickpea being the third important grain legume, there is a limited availability of genomic resources, especially of the expressed sequence tag (EST)-based markers. In this study, we generated 822 chickpea ESTs from immature seeds as well as exploited 1,309 ESTs from the chickpea database, thus utilizing a total of 2,131 EST sequences for development of functional EST-SSR markers. Two hundred and forty-six simple sequence repeat (SSR) motifs were identified from which 183 primer pairs were designed and 60 validated as functional markers. Genetic diversity analysis across 30 chickpea accessions revealed ten markers to be polymorphic producing a total of 29 alleles and an observed heterozygosity average of 0.16 thereby exhibiting low levels of intra-specific polymorphism. However, the markers exhibited high cross-species transferability ranging from 68.3 to 96.6% across the six annual Cicer species and from 29.4 to 61.7% across the seven legume genera. Sequence analysis of size variant amplicons from various species revealed that size polymorphism was due to multiple events such as copy number variation, point mutations and insertions/deletions in the microsatellite repeat as well as in the flanking regions. Interestingly, a wide prevalence of crossability-group-specific sequence variations were observed among Cicer species that were phylogenetically informative. The neighbor joining dendrogram clearly separated the chickpea cultivars from the wild Cicer and validated the proximity of C. judaicum with C. pinnatifidum. Hence, this study for the first time provides an insight into the distribution of SSRs in the chickpea transcribed regions and also demonstrates the development and utilization of genic-SSRs. In addition to proving their suitability for genetic diversity analysis, their high rates of transferability also proved their potential for comparative genomic studies and for following gene introgressions and evolution in wild species, which constitute the valuable secondary genepool in chickpea.Item Development of sequence-tagged microsatellite site markers for chickpea (Cicer arietinum L.)(Wiley-Blackwell, 2006) Choudhary, Shalu; Sethy, Niroj K.; Shokeen, Bhumika; Bhatia, SabhyataMicrosatellite loci were identified from chickpea (Cicer arietinum L.), the third most important grain legume crop in the world. A total of 13 sequence-tagged microsatellite markers were developed using two different approaches: (i) amplification using degenerate primers and (ii) cloning of intersimple sequence repeat (ISSR)-amplified fragments. Thirty-five chickpea accessions were analysed, which resulted in a total of 30 alleles at the 13 loci. The observed heterozygosity ranged from 0.1143 to 0.4571 with an average of 0.2284. The cross-species transferability of the sequence-tagged microsatellite site (STMS) markers was checked in Cicer reticulatum, the wild annual progenitor of chickpea. These microsatellite markers will be useful for assessing the genetic diversity patterns within chickpea as well as aid in construction of intra- and interspecific genetic linkage maps.