MicroRNA164e suppresses NAC100 transcription factor-mediated synthesis of seed storage proteins in chickpea.

Development 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.

Evolutionary insights into 3D genome organization and epigenetic landscape of Vigna mungo.

Eukaryotic genomes show an intricate three-dimensional (3D) organization within the nucleus that regulates multiple biological processes including gene expression. Contrary to animals, understanding of 3D genome organization in plants remains at a nascent stage. Here, we investigate the evolution of 3D chromatin architecture in legumes. By using cutting-edge PacBio, Illumina, and Hi-C contact reads, we report a gap-free, chromosome-scale reference genome assembly of Vigna mungo, an important minor legume cultivated in Southeast Asia. We spatially resolved V. mungo chromosomes into euchromatic, transcriptionally active A compartment and heterochromatic, transcriptionally-dormant B compartment. We report the presence of TAD-like-regions throughout the diagonal of the HiC matrix that resembled transcriptional quiescent centers based on their genomic and epigenomic features. We observed high syntenic breakpoints but also high coverage of syntenic sequences and conserved blocks in boundary regions than in the TAD-like region domains. Our findings present unprecedented evolutionary insights into spatial 3D genome organization and epigenetic patterns and their interaction within the V. mungo genome. This will aid future genomics and epigenomics research and breeding programs of V. mungo.

Delineation of novel genomic loci and putative candidate genes associated with seed iron and zinc content in lentil (Lens culinaris Medik.).

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.

Comparative transcriptomic and metabolite profiling reveals genotype-specific responses to Fe starvation in chickpea.

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 between the genotypes related to strategy I uptake, metal ions transporters, reactive oxygen species-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 endeavor will enable the development of Fe deficiency tolerant chickpea cultivars.

Integrated genomic approaches delineate a novel role of ROP1 ENHANCER1 in controlling seed protein content of chickpea.

Utilizing a combinatorial approach of quantitative trait locus (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 recombinant inbred lines from a mapping population contrasting for SPC led to the 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, revealing a tightly associated marker affecting CaREN1 (ROP1 ENHANCER1) and explaining a phenotypic variation 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. Furthermore, in planta functional validation via knockdown of CaREN1 transcripts 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 chaperones, phosphopeptide-binding proteins, and GTPases that mediate folding, transport and accumulation of seed storage proteins, as indicated through affinity purification-mass spectrometry. Taken together, our data will expedite tailoring of chickpea cultivars with augmented SPC.

Evolutionary and expression dynamics of LRR-RLKs and functional establishment of KLAVIER homolog in shoot mediated regulation of AON in chickpea symbiosis.

Chickpea 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.

Evolutionary and functional analysis of two-component system in chickpea reveals CaRR13, a TypeB RR, as positive regulator of symbiosis.

The 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 signalling, 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 signalling molecule regulating early events of nodule organogenesis in chickpea.

Identification and molecular characterization of miRNAs and their target genes associated with seed development through small RNA sequencing in chickpea.

Multiple studies have attempted to dissect the molecular mechanism underlying seed development in chickpea (Cicer arietinum L.). These studies highlight the need to focus on the role of miRNAs in regulating storage protein accumulation in seeds. Therefore, a total of 8,856,691 short-read sequences were generated from a small RNA library of developing chickpea seeds and were analyzed using miRDeep-P to identify 74 known and 26 novel miRNA sequences. Known miRNAs were classified into 22 miRNA families with miRNA156 family being most abundant. Of the 26 putative novel miRNAs identified, only 22 could be experimentally validated using stem loop end point PCR. Differential expression analyses led to the identification of known as well as novel miRNAs that could regulate various stages of chickpea seed development. In silico target prediction revealed several important target genes and transcription factors like SPL, mediator of RNA Polymerase II transcription subunit 12, aspartic proteinase and NACs, which were further validated by real-time PCR analysis. A comparative expression analysis in chickpea genotypes with contrasting seed protein content revealed one known (Car-miR156h) and two novel miRNA (Car-novmiR7 and Car-novmiR23) candidates to be highly expressed in the LPC (low protein content) chickpea genotypes, targets of which are known to regulate seed storage protein accumulation. Therefore, this study provides a useful resource in the form of miRNA and their targets which can be further utilized to understand and manipulate various regulatory mechanisms involved in seed development with the overall aim of improving yield and nutrition attributes in chickpea.

Dynamics of miRNA mediated regulation of legume symbiosis.

Symbiotic nitrogen fixation in legume nodules is important in soils with low nitrogen availability. The initiation and sustainability of symbiosis require cellular reprogramming that involves the miRNA-mediated inhibition or activation of specific nodulation genes. The high-throughput sequencing of small RNA libraries has identified miRNAs and their targets, which are the major players in the post-transcriptional gene regulation (PTGS) of the different stages of legume-rhizobia symbiosis ranging from bacterial colonization and organogenesis to symbiotic nitrogen fixation. Here, we present an overview of information obtained from the miRNA libraries from nodulating tissues that have been sequenced to date. The functional analysis of miRNAs has revealed roles in phytohormone homeostasis and spatio-temporal regulation, as well as the mobility of miRNAs and their functions in shoot to root signalling that affects diverse functions, including bacterial entry, meristem division and differentiation, nitrogen fixation and senescence. Furthermore, small RNA fragments of rhizobial origin repress complementary plant mRNAs. We also consider the roles of miRNAs in determinate or indeterminate nodules. Taken together, this overview confirms that miRNAs are master regulators of the legume-rhizobia symbiosis.

A high-density SNP-based linkage map using genotyping-by-sequencing and its utilization for improved genome assembly of chickpea (Cicer arietinum L.).

Genotyping-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.

A comprehensive analysis of the B3 superfamily identifies tissue-specific and stress-responsive genes in chickpea (Cicer arietinum L.).

The aim of this study was to provide a comprehensive analysis of the plant-specific B3 domain-containing transcription factors (TFs) in chickpea. Scanning of the chickpea genome resulted in the identification 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 classified into four subfamilies, i.e., ARF (24), REM (19), LAV (6) and RAV (2). Phylogenetic analysis classified 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 identification of several tissue-specific and stress-responsive cis-regulatory elements. Expression profiling 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 findings were further validated by real-time 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.

Analysis of genes encoding seed storage proteins (SSPs) in chickpea (Cicer arietinum L.) reveals co-expressing transcription factors and a seed-specific promoter.

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 seed-specific 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.

Pathways of nitric oxide metabolism and operation of phytoglobins in legume nodules: missing links and future directions.

The interaction between legumes and rhizobia leads to the establishment of a beneficial symbiotic relationship. Recent advances in legume - rhizobium symbiosis revealed that various reactive oxygen and nitrogen species including nitric oxide (NO) play important roles during this process. Nodule development occurs with a transition from a normoxic environment during the establishment of symbiosis to a microoxic environment in functional nodules. Such oxygen dynamics are required for activation and repression of various NO production and scavenging pathways. Both the plant and bacterial partners participate in the synthesis and degradation of NO. However, the pathways of NO production and degradation as well as their cross-talk and involvement in the metabolism are still a matter of debate. The plant-originated reductive pathways are known to contribute to the NO production in nodules under hypoxic conditions. Non-symbiotic hemoglobin (phytoglobin) (Pgb) possesses high NO oxygenation capacity, buffers and scavenges NO. Its operation, through a respiratory cycle called Pgb-NO cycle, leads to the maintenance of redox and energy balance in nodules. The role of Pgb/NO cycle under fluctuating NO production from soil needs further investigation for complete understanding of NO regulatory mechanism governing nodule development to attain optimal food security under changing environment.

Genome-wide analysis of the CCCH zinc finger family identifies tissue specific and stress responsive candidates in chickpea (Cicer arietinum L.).

The CCCH zinc finger is a group of proteins characterised by a typical motif consisting of three cysteine residues and one histidine residue. These proteins have been reported to play important roles in regulation of plant growth, developmental processes and environmental responses. In the present study, genome wide analysis of the CCCH zinc finger gene family was carried out in the available chickpea genome. Various bioinformatics tools were employed to predict 58 CCCH zinc finger genes in chickpea (designated CarC3H1-58), which were analysed for their physio-chemical properties. Phylogenetic analysis classified the proteins into 12 groups in which members of a particular group had similar structural organization. Further, the numbers as well as the types of CCCH motifs present in the CarC3H proteins were compared with those from Arabidopsis and Medicago truncatula. Synteny analysis revealed valuable information regarding the evolution of this gene family. Tandem and segmental duplication events were identified and their Ka/Ks values revealed that the CarC3H gene family in chickpea had undergone purifying selection. Digital, as well as real time qRT-PCR expression analysis was performed which helped in identification of several CarC3H members that expressed preferentially in specific chickpea tissues as well as during abiotic stresses (desiccation, cold, salinity). Moreover, molecular characterization of an important member CarC3H45 was carried out. This study provides comprehensive genomic information about the important CCCH zinc finger gene family in chickpea. The identified tissue specific and abiotic stress specific CCCH genes could be potential candidates for further characterization to delineate their functional roles in development and stress.

A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don.

Simple Sequence Repeats (SSRs) of polypurine-polypyrimidine type motifs occur very frequently in the 5' flanks of genes in plants and have recently been implicated to have a role in regulation of gene expression. In this study, 2 accessions of Catharanthus roseus having (CT)8 and (CT)21 varying motifs in the 5'UTR of Tryptophan decarboxylase (Tdc) gene, were investigated for its role in regulation of gene expression. Extensive Tdc gene expression analysis in the 2 accessions was carried out both at the level of transcription and translation. Transcript abundance was estimated using Northern analysis and qRT-PCR, whereas the rate of Tdc gene transcription was assessed using in-situ nuclear run-on transcription assay. Translation status of Tdc gene was monitored by quantification of polysome associated Tdc mRNA using qRT-PCR. These observations were validated through transient expression analysis using the fusion construct [CaM35S:(CT)8-21:GUS]. Our study demonstrated that not only does the length of (CT)n -SSRs influences the promoter activity, but the presence of SSRs per se in the 5'-UTR significantly enhances the level of gene expression. We termed this phenomenon as "microsatellite mediated enhancement" (MME) of gene expression. Results presented here will provide leads for engineering plants with enhanced amounts of medicinally important alkaloids.

Dissecting the Root Nodule Transcriptome of Chickpea (Cicer arietinum L.).

A 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.

Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea.

Drought and salinity are the major factors that limit chickpea production worldwide. We performed whole transcriptome analyses of chickpea genotypes to investigate the molecular basis of drought and salinity stress response/adaptation. Phenotypic analyses confirmed the contrasting responses of the chickpea genotypes to drought or salinity stress. RNA-seq of the roots of drought and salinity related genotypes was carried out under control and stress conditions at vegetative and/or reproductive stages. Comparative analysis of the transcriptomes revealed divergent gene expression in the chickpea genotypes at different developmental stages. We identified a total of 4954 and 5545 genes exclusively regulated in drought-tolerant and salinity-tolerant genotypes, respectively. A significant fraction (~47%) of the transcription factor encoding genes showed differential expression under stress. The key enzymes involved in metabolic pathways, such as carbohydrate metabolism, photosynthesis, lipid metabolism, generation of precursor metabolites/energy, protein modification, redox homeostasis and cell wall component biogenesis, were affected by drought and/or salinity stresses. Interestingly, transcript isoforms showed expression specificity across the chickpea genotypes and/or developmental stages as illustrated by the AP2-EREBP family members. Our findings provide insights into the transcriptome dynamics and components of regulatory network associated with drought and salinity stress responses in chickpea.

High-density linkage map construction and mapping of seed trait QTLs in chickpea (Cicer arietinum L.) using Genotyping-by-Sequencing (GBS).

This 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.

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.

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.

Construction of a Genetic Linkage Map and Identification of QTLs for Seed Weight and Seed Size Traits in Lentil (Lens culinaris Medik.).

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.