Browsing by Author "Bhatia, Sabhyata"

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An advanced draft genome assembly of a desi type chickpea (Cicer arietinum L.)
(Nature Publishing Group, 2015) Parween, Sabiha; Nawaz, Kashif; Roy, Riti; Pole, Anil K.; Venkata Suresh, B.; Misra, Gopal; Jain, Mukesh; Yadav, Gitanjali; Parida, Swarup K.; Tyagi, Akhilesh K.; Bhatia, Sabhyata; Chattopadhyay, Debasis
Chickpea (Cicer arietinum L.) is an important pulse legume crop. We previously reported a draft genome assembly of the desi chickpea cultivar ICC 4958. Here we report an advanced version of the ICC 4958 genome assembly (version 2.0) generated using additional sequence data and an improved genetic map. This resulted in 2.7-fold increase in the length of the pseudomolecules and substantial reduction of sequence gaps. The genome assembly covered more than 94% of the estimated gene space and predicted the presence of 30,257 protein-coding genes including 2230 and 133 genes encoding potential transcription factors (TF) and resistance gene homologs, respectively. Gene expression analysis identified several TF and chickpea-specific genes with tissue-specific expression and displayed functional diversification of the paralogous genes. Pairwise comparison of pseudomolecules in the desi (ICC 4958) and the earlier reported kabuli (CDC Frontier) chickpea assemblies showed an extensive local collinearity with incongruity in the placement of large sequence blocks along the linkage groups, apparently due to use of different genetic maps. Single nucleotide polymorphism (SNP)-based mining of intra-specific polymorphism identified more than four thousand SNPs differentiating a desi group and a kabuli group of chickpea genotypes.
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, Sabhyata
Chickpea (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.
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, Sabhyata
Improvement 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.
Analysis of genomic-transcriptomic dynamics delineates key molecular signatures modulating seed size and weight in lentil
(John Wiley & Sons, 2026) Padhy, Asish Kumar; Singh, Sangeeta; Tripathi, Kuldeep; Parida, Swarup K.; Bhatia, Sabhyata
Delineating key genetic determinants associated with seed size/weight is crucial for increasing productivity. In this study, the advantages of an integrated approach combining QTL mapping, GWAS and transcriptomics to identify robust candidates governing seed size and weight were demonstrated in lentil, an important grain legume. QTL mapping identified three stable QTLs harbouring 5113 genes. GWAS identified 42 MTAs (5 consistent) containing 192 underlying genes. Comparative transcriptome analysis identified 1202 differentially expressed transcripts. Integrated analysis of the results obtained from QTL mapping and GWAS revealed nine SNPs located in the three robust QTLs harbouring 32 candidate genes. Upon integration with transcriptome data, only one (LcWDL1) was identified as the most promising candidate. LcWDL1 (a member of TPX2 family involved in microtubule organisation and cell expansion) and its predicted interacting partners that is, LcGLIPs are known to function as regulators of seed size. Candidate gene-based association analysis identified a SNP on second exon of LcWDL1 to be significantly associated with seed size and weight of lentil. The genomic loci/candidate gene identified in the study will serve to expedite the molecular breeding and gene editing programs for enhancing seed size and seed weight in lentils.
Characterization and genetic linkage mapping of the horticulturally important mutation leafless inflorescence (lli) in periwinkle Catharanthus roseus
(Elsevier, 2011) Chaudhary, Swati; Sharma, Vishakha; Prasad, Manoj; Bhatia, Sabhyata; Tripathi, Bhumi Nath; Yadav, Gitanjali; Kumar, Sushil
Catharanthus roseus is a seasonal to perennial garden plant and the exclusive source of the anticancer drugs vincristine and vinblastine. Its horticultural importance is due to the compound racemose inflorescence architecture of branches in which pairs of prominent flowers are subtended by one of the two leaves per node. Here is reported the construction of a molecular framework genetic linkage map and mapping on it of the LEAF-LESS INFLORESCENCE (LLI) locus. It is quantitatively shown that the adult lli mutant plants produce altered inflorescence of improved horticultural value, wherein axes are excessively branched, two flowers are formed per node that are bare of subtending leaves, and several times more open flowers are displayed each day, as compared to LLI plants.
The chickpea genomic web resource: visualization and analysis of the desi-type Cicer arietinum nuclear genome for comparative exploration of legumes
(BioMed Central Ltd, 2014) Misra, Gopal; Priya, Piyush; Bandhiwal, Nitesh; Bareja, Neha; Jain, Mukesh; Bhatia, Sabhyata; Chattopadhyay, Debasis; Tyagi, Akhilesh K.; Yadav, Gitanjali
Background: Availability of the draft nuclear genome sequences of small-seeded desi-type legume crop Cicer arietinum has provided an opportunity for investigating unique chickpea genomic features and evaluation of their biological significance. The increasing number of legume genome sequences also presents a challenge for developing reliable and information-driven bioinformatics applications suitable for comparative exploration of this important class of crop plants. Results: The Chickpea Genomic Web Resource (CGWR) is an implementation of a suite of web-based applications dedicated to chickpea genome visualization and comparative analysis, based on next generation sequencing and assembly of Cicer arietinum desi-type genotype ICC4958. CGWR has been designed and configured for mapping, scanning and browsing the significant chickpea genomic features in view of the important existing and potential roles played by the various legume genome projects in mutant mapping and cloning. It also enables comparative informatics of ICC4958 DNA sequence analysis with other wild and cultivated genotypes of chickpea, various other leguminous species as well as several non-leguminous model plants, to enable investigations into evolutionary processes that shape legume genomes. Conclusions: CGWR is an online database offering a comprehensive visual and functional genomic analysis of the chickpea genome, along with customized maps and gene-clustering options. It is also the only plant based web resource supporting display and analysis of nucleosome positioning patterns in the genome. The usefulness of CGWR has been demonstrated with discoveries of biological significance made using this server. The CGWR is compatible with all available operating systems and browsers, and is available freely under the open source license at http://www.nipgr.res.in/CGWR/home.php.
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, Sabhyata
Iron 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.
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, Sabhyata
The 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.
Construction of a genetic linkage map and identification of QTLs for seed weight and seed size traits in lentil (Lens culinaris Medik.)
(PLOS, 2015) Verma, Priyanka; Goyal, Richa; Chahota, R.K.; Sharma, Tilak R.; Abdin, M. Z.; Bhatia, Sabhyata
Seed weight and seed size both are quantitative traits and have been considered as important components of grain yield, thus identification of quantitative trait loci (QTL) for seed traits in lentil (Lens culinaris) would be beneficial for the improvement of grain yield. Hence the main objective of this study was to identify QTLs for seed traits using an intraspecific mapping population derived from a cross between L. culinaris cv. Precoz (seed weight-5.1g, seed size-5.7mm) and L. culinaris cv. L830 (seed weight-2.2g, seed size-4mm) comprising 126 F8-RILs. For this, two microsatellite genomic libraries enriched for (GA/CT) and (GAA/CTT) motif were constructed which resulted in the development of 501 new genomic SSR markers. Six hundred forty seven SSR markers (including 146 previously published) were screened for parental polymorphism and 219 (33.8%) were found to be polymorphic among the parents. Of these 216 were mapped on seven linkage groups at LOD4.0 spanning 1183.7cM with an average marker density of 5.48cM. Phenotypic data from the RILs was used to identify QTLs for the seed weight and seed size traits by single marker analysis (SMA) followed by composite interval mapping (CIM) which resulted in one QTL each for the 2 traits (qSW and qSS) that were co-localized on LG4 and explained 48.4% and 27.5% of phenotypic variance respectively. The current study would serve as a strong foundation for further validation and fine mapping for utilization in lentil breeding programs.
Construction of intersubspecific molecular genetic map of lentil based on ISSR, RAPD and SSR markers
(Indian Academy of Sciences, 2012) Gupta, Mamta; Verma, Bhawna; Kumar, Naresh; Chahota, Rakesh K.; Rathour, Rajeev; Sharma, Shyam K.; Bhatia, Sabhyata; Sharma, Tilak R.
Lentil (Lens culinaris ssp. culinaris), is a self-pollinating diploid (2n = 2x = 14), cool-season legume crop and is consumed worldwide as a rich source of protein (~24.0%), largely in vegetarian diets. Here we report development of a genetic linkage map of Lens using 114 F(2) plants derived from the intersubspecific cross between L 830 and ILWL 77. RAPD (random amplified polymorphic DNA) primers revealed more polymorphism than ISSR (intersimple sequence repeat) and SSR (simple sequence repeat) markers. The highest proportion (30.72%) of segregation distortion was observed in RAPD markers. Of the 235 markers (34 SSR, 9 ISSR and 192 RAPD) used in the mapping study, 199 (28 SSRs, 9 ISSRs and 162 RAPDs) were mapped into 11 linkage groups (LGs), varying between 17.3 and 433.8 cM and covering 3843.4 cM, with an average marker spacing of 19.3 cM. Linkage analysis revealed nine major groups with 15 or more markers each and two small LGs with two markers each, and 36 unlinked markers. The study reported assigning of 11 new SSRs on the linkage map. Of the 66 markers with aberrant segregation, 14 were unlinked and the remaining 52 were mapped. ISSR and RAPD markers were found to be useful in map construction and saturation. The current map represents maximum coverage of lentil genome and could be used for identification of QTL regions linked to agronomic traits, and for marker-assisted selection in lentil.
Delineating microRNA169-Nuclear Factor Y-Subunit a module for its potential implications in crop improvement
(John Wiley & Sons, 2026) Chakraborty, Anirban; Sharma, Shambhavi; Pandey, Girdhar K.; Bhatia, Sabhyata; Prasad, Manoj
Climate change considerably impacts plant growth and productivity by inducing stress responses. This, along with the problems of feeding the ever-increasing global population, could be mitigated by generating climate-resilient crop varieties with enhanced productivity. However, an exhaustive account of the key regulatory processes that underlie developmental and stress-responsive pathways is a prerequisite for generating improved crop varieties. Towards this, our study, for the first time, provides an exhaustive compilation of the potential regulatory pathways impacted by the miR169-NFYA network in plants. The NFYA transcription factors belong to a class of nuclear factor-encoding genes directly influencing the transcription of many genes involved in developmental and stress responses. Meanwhile, miR169 provides a layer to NFYA-mediated gene regulation by post-transcriptionally suppressing the expression of these transcription factors. Evidence from several studies shed light on key molecular signatures related to hormone synthesis and signaling, calcium signaling, epigenetic regulation, nutrient starvation and miRNA biogenesis that could serve as downstream components of the miR169-NFYA cascade in plants. This ability of miR169-NFYA nexus to impact a wide range of biological processes makes it a suitable toolbox for developing tailor-made crop varieties through appropriate genetic manipulation strategies.
Delineation of novel genomic loci and putative candidate genes associated with seed iron and zinc content in lentil (Lens culinaris Medik.)
(Elsevier B.V., 2023) Singh, Baljinder; Singh, Sangeeta; Mahato, Ajay Kumar; Dikshit, Harsh Kumar; Tripathi, Kuldeep; Bhatia, Sabhyata
The use of molecular breeding approaches for development of lentil genotypes biofortified with essential micro-nutrients such as iron and zinc, could serve as a promising solution to address the problem of global malnutrition. Thus, genome-wide association study (GWAS) strategy was adopted in this study to identify the genomic regions associated with seed iron and zinc content in lentil. A panel of 95 diverse lentil genotypes, grown across three different geographical locations and evaluated for seed iron and zinc content, exhibited a wide range of variation. Genotyping-by-sequencing (GBS) analysis of the panel identified 33,745 significant single nucleotide polymorphisms (SNPs) that were distributed across all the 7 lentil chromosomes. Association mapping revealed 23 SNPs associated with seed iron content that were distributed across all the chromosomes except chromosome 3. Similarly, 14 SNPs associated with seed zinc content were also identified that were distributed across chromosomes 1, 2, 4, 5 and 6. Further, 80 genes were identified in the proximity of iron associated markers and 36 genes were identified in the proximity of zinc associated markers. Functional annotation of these genes revealed their putative involvement in iron and zinc metabolism. For seed iron content, two highly significant SNPs were found to be located within two putative candidate genes namely iron-sulfur cluster assembly (ISCA) and flavin binding monooxygenase (FMO) respectively. For zinc content, a highly significant SNP was detected in a gene encoding UPF0678 fatty acid-binding protein. Expression analysis of these genes and their putative interacting partners suggests their involvement in iron and zinc metabolism in lentil. Overall, in this study we have identified markers, putative candidate genes and predicted putative interacting protein partners significantly associated with iron and zinc metabolism that could be utilized in future breeding studies of lentil for nutrient biofortification.
Development and characterization of recombinant inbreed lines for seed related traits in chickpea
(CABI, 2015) Kumar, Tapan; Bharadwaj, C.; Alam, Afroz; Singh, Puja; Singhal, Tripti; Chauhan, S.K.; Bhatia, Sabhyata; Sarker, Ashutosh
Seed traits are very important factors in improving yield and productivity of chickpea. However being quantitative in nature, gains through direct selection for these traits particularly for bolder seed size with higher yield were never successful and thus the way forward is to identify QTLs linked to these traits. Such a study requires development of mapping populations segregating for traits of interest. Mapping population of the cross SBD 377 (Desi Bold Type with 100 seed weight 48 g and seed number per plant of 34 per plant) and BGD 112 (Desi small seeded with 100 seed weight of 12-15 g and seed number per plant of 90) was developed at Division of Genetics, IARI. The recombinant inbred line (RILs) has been extensively phenotyped. Contrasting variability exists among the RILs for quantitative characters studied. Plant height ranged from 20 to 70 cm with 1-4 primary branches, 0-23 secondary branches and 7-54 total branches per plant. Number of seeds ranged 5 to 250 seeds per plant and pod number 5-180 per plant. The range of 100 seed weight was 13-50 g. Plant yield varied from 2-69 g. Availability of such a RIL population shall serve as a good base for the QTL analysis.
Development and integration of genome-wide polymorphic microsatellite markers onto a reference linkage map for constructing a high-density genetic map of chickpea
(PLOS, 2015) Khajuria, Yash Paul; Saxena, Maneesha S.; Gaur, Rashmi; Chattopadhyay, Debasis; Jain, Mukesh; Parida, Swarup K.; Bhatia, Sabhyata
The identification of informative in silico polymorphic genomic and genic microsatellite markers by comparing the genome and transcriptome sequences of crop genotypes is a rapid, cost-effective and non-laborious approach for large-scale marker validation and genotyping applications, including construction of high-density genetic maps. We designed 1494 markers, including 1016 genomic and 478 transcript-derived microsatellite markers showing in-silico fragment length polymorphism between two parental genotypes (Cicer arietinum ICC4958 and C. reticulatum PI489777) of an inter-specific reference mapping population. High amplification efficiency (87%), experimental validation success rate (81%) and polymorphic potential (55%) of these microsatellite markers suggest their effective use in various applications of chickpea genetics and breeding. Intra-specific polymorphic potential (48%) detected by microsatellite markers in 22 desi and kabuli chickpea genotypes was lower than inter-specific polymorphic potential (59%). An advanced, high-density, integrated and inter-specific chickpea genetic map (ICC4958 x PI489777) having 1697 map positions spanning 1061.16 cM with an average inter-marker distance of 0.625 cM was constructed by assigning 634 novel informative transcript-derived and genomic microsatellite markers on eight linkage groups (LGs) of our prior documented, 1063 marker-based genetic map. The constructed genome map identified 88, including four major (7-23 cM) longest high-resolution genomic regions on LGs 3, 5 and 8, where the maximum number of novel genomic and genic microsatellite markers were specifically clustered within 1 cM genetic distance. It was for the first time in chickpea that in silico FLP analysis at genome-wide level was carried out and such a large number of microsatellite markers were identified, experimentally validated and further used in genetic mapping. To best of our knowledge, in the presently constructed genetic map, we mapped highest number of new sequence-based robust microsatellite markers (634) which is an advancement over the previously documented (~300 markers) inter-specific genetic maps. This advanced high-density map will serve as a foundation for large-scale marker validation and genotyping applications, including identification and targeted mapping of trait-specific genes/QTLs (quantitative trait loci) with sub-optimal use of resources and labour in chickpea.
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.
Development of SSR and gene-targeted markers for construction of a framework linkage map of Catharanthus roseus
(Oxford University Press, 2011) Shokeen, Bhumika; Choudhary, Shalu; Sethy, Niroj Kumar; Bhatia, Sabhyata
BACKGROUND AND AIMS: Catharanthus roseus is a plant of great medicinal importance, yet inadequate knowledge of its genome structure and the unavailability of genomic resources have been major impediments in the development of improved varieties. The aims of this study were to develop co-dominant sequence-tagged microsatellite sites (STMS) and gene-targeted markers (GTMs) and utilize them for the construction of a framework intraspecific linkage map of C. roseus.
Development of an expressed gene catalogue and molecular markers from the de novo assembly of short sequence reads of the lentil (Lens culinaris Medik.) transcriptome
(John Wiley & Sons Ltd, 2013) Verma, Priyanka; Shah, Niraj; Bhatia, Sabhyata
Genomic resources such as ESTs, molecular markers and linkage maps are essential for crop improvement. However, these resources are still limited in important legumes such as lentil (Lens culinaris Medik.), which is valued world wide as a rich source of dietary protein. In this study, the de novo transcriptome assembly of 119,855,798 short reads, generated by Illumina paired-end sequencing, was performed using various assembly programs. This resulted in 42,196 nonredundant high-quality transcripts of average length 810 bases, N50 value of 1,432 and an average expression per transcript of 26.21 rpkm reads per kilobase per million(RPKM). Similarity search with the unigenes and protein sequences of other plants resulted in maximum similarity with soybean. A total of 20,009 nonredundant transcripts showed similarity with the UniProtKB database and of these, 18,064 transcripts were grouped into three main GO categories, that is, biological process (15,126), molecular function (15,505) and cellular component (9,434). Annotated transcripts were mapped to 289 predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 8,893 transcripts were classified into 24 functional categories based on Cluster of Orthologous Groups (COG) of proteins. Mining the data set for the presence of SSRs resulted in 8,722 SSRs with a frequency occurrence of one SSR per 3.92 kb. From these, 5,673 SSR primer pairs were designed, and a subset of these were utilized for diversity analysis. This study, which provides a large data set of annotated transcripts and gene-based SSR markers, would serve as a foundation for various applications in lentil breeding and genetics.
Development of chickpea EST-SSR markers and analysis of allelic variation across related species
(Springer, 2009) Choudhary, Shalu; Sethy, Niroj Kumar; Shokeen, Bhumika; Bhatia, Sabhyata
Despite 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.
Development of gene-based markers for use in construction of the chickpea (Cicer arietinum L.) genetic linkage map and identification of QTLs associated with seed weight and plant height
(Springer, 2015) Gupta, Shefali; Kumar, Tapan; Verma, Subodh; Bharadwaj, Chellapilla; Bhatia, Sabhyata
Seed weight and plant height are important agronomic traits and contribute to seed yield. The objective of this study was to identify QTLs underlying these traits using an intra-specific mapping population of chickpea. A F11 population of 177 recombinant inbred lines derived from a cross between SBD377 (100-seed weight- 48 g and plant height- 53 cm) and BGD112 (100-seed weight- 15 g and plant height- 65 cm) was used. A total of 367 novel EST-derived functional markers were developed which included 187 EST-SSRs, 130 potential intron polymorphisms (PIPs) and 50 expressed sequence tag polymorphisms (ESTPs). Along with these, 590 previously published markers including 385 EST-based markers and 205 genomic SSRs were utilized. Of the 957 markers tested for analysis of parental polymorphism between the two parents of the mapping population, 135 (14.64 %) were found to be polymorphic. Of these, 131 polymorphic markers could be mapped to the 8 linkage groups. The linkage map had a total length of 1140.54 cM with an average marker density of 8.7 cM. The map was further used for QTL identification using composite interval mapping method (CIM). Two QTLs each for seed weight, qSW-1 and qSW-2 (explaining 11.54 and 19.24 % of phenotypic variance, respectively) and plant height, qPH-1 and qPH-2 (explaining 13.98 and 12.17 % of phenotypic variance, respectively) were detected. The novel set of genic markers, the intra-specific linkage map and the QTLs identified in the present study will serve as valuable genomic resources in improving the chickpea seed yield using marker-assisted selection (MAS) strategies.
Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)
(Springer, 2006) Sethy, Niroj Kumar; Shokeen, Bhumika; Edwards, Keith J.; Bhatia, Sabhyata
Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT)n and (CT/GA)n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers devel- oped in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.