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.

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.

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.

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.

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.

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.

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.

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 survey and expression analysis of F-box genes in chickpea.

BACKGROUND: The F-box genes constitute one of the largest gene families in plants involved in degradation of cellular proteins. F-box proteins can recognize a wide array of substrates and regulate many important biological processes such as embryogenesis, floral development, plant growth and development, biotic and abiotic stress, hormonal responses and senescence, among others. However, little is known about the F-box genes in the important legume crop, chickpea. The available draft genome sequence of chickpea allowed us to conduct a genome-wide survey of the F-box gene family in chickpea. RESULTS: A total of 285 F-box genes were identified in chickpea which were classified based on their C-terminal domain structures into 10 subfamilies. Thirteen putative novel motifs were also identified in F-box proteins with no known functional domain at their C-termini. The F-box genes were physically mapped on the 8 chickpea chromosomes and duplication events were investigated which revealed that the F-box gene family expanded largely due to tandem duplications. Phylogenetic analysis classified the chickpea F-box genes into 9 clusters. Also, maximum syntenic relationship was observed with soybean followed by Medicago truncatula, Lotus japonicus and Arabidopsis. Digital expression analysis of F-box genes in various chickpea tissues as well as under abiotic stress conditions utilizing the available chickpea transcriptome data revealed differential expression patterns with several F-box genes specifically expressing in each tissue, few of which were validated by using quantitative real-time PCR. CONCLUSIONS: The genome-wide analysis of chickpea F-box genes provides new opportunities for characterization of candidate F-box genes and elucidation of their function in growth, development and stress responses for utilization in chickpea improvement.

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.

A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.).

Cicer 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.

Integrated physical, genetic and genome map of chickpea (Cicer arietinum L.).

Physical map of chickpea was developed for the reference chickpea genotype (ICC 4958) using bacterial artificial chromosome (BAC) libraries targeting 71,094 clones (~12× coverage). High information content fingerprinting (HICF) of these clones gave high-quality fingerprinting data for 67,483 clones, and 1,174 contigs comprising 46,112 clones and 3,256 singletons were defined. In brief, 574 Mb genome size was assembled in 1,174 contigs with an average of 0.49 Mb per contig and 3,256 singletons represent 407 Mb genome. The physical map was linked with two genetic maps with the help of 245 BAC-end sequence (BES)-derived simple sequence repeat (SSR) markers. This allowed locating some of the BACs in the vicinity of some important quantitative trait loci (QTLs) for drought tolerance and reistance to Fusarium wilt and Ascochyta blight. In addition, fingerprinted contig (FPC) assembly was also integrated with the draft genome sequence of chickpea. As a result, ~965 BACs including 163 minimum tilling path (MTP) clones could be mapped on eight pseudo-molecules of chickpea forming 491 hypothetical contigs representing 54,013,992 bp (~54 Mb) of the draft genome. Comprehensive analysis of markers in abiotic and biotic stress tolerance QTL regions led to identification of 654, 306 and 23 genes in drought tolerance "QTL-hotspot" region, Ascochyta blight resistance QTL region and Fusarium wilt resistance QTL region, respectively. Integrated physical, genetic and genome map should provide a foundation for cloning and isolation of QTLs/genes for molecular dissection of traits as well as markers for molecular breeding for chickpea improvement.

The chickpea genomic web resource: visualization and analysis of the desi-type Cicer arietinum nuclear genome for comparative exploration of legumes.

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.

Expanding the repertoire of microsatellite markers for polymorphism studies in Indian accessions of mung bean (Vigna radiata L. Wilczek).

Limited availability of validated, polymorphic microsatellite markers in mung bean (Vigna radiata), an important food legume of India, has been a major hurdle towards its improvement and higher yield. The present study was undertaken in order to develop a new set of microsatellite markers and utilize them for the analysis of genetic diversity within mung bean accessions from India. A GA/CT enriched library was constructed from V. radiata which resulted in 1,250 putative recombinant clones of which 850 were sequenced. SSR motifs were identified and their flanking sequences were utilized to design 328 SSR primer pairs. Of these, 48 SSR markers were employed for assessing genetic diversity among 76 mung bean accessions from various geographical locations in India. Two hundred and thirty four alleles with an average of 4.85 alleles per locus were detected at 48 loci. The polymorphic information content (PIC) per locus varied from 0.1 to 0.88 (average: 0.49 per locus). The observed and expected heterozygosities ranged from 0.40 to 0.95 and 0.40 to 0.81 respectively. Based on Jaccard's similarity matrix, a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA) analysis which revealed that one accession from Bundi, Rajasthan was clustered out separately while remaining accessions were grouped into two major clusters. The markers generated in this study will help in expanding the repertoire of the available SSR markers thereby facilitating analysis of genetic diversity, molecular mapping and ultimately broadening the scope for genetic improvement of this legume.

Exploring genetic variability within lentil (Lens culinaris Medik.) and across related legumes using a newly developed set of microsatellite markers.

Lentil (Lens culinaris Medik.) is an economically important grain legume, yet the genetic and genomic resources remain largely uncharacterized and unexploited in this crop. Microsatellites have become markers of choice for crop improvement applications. Hence, simple sequence repeat (SSR) markers were developed for lentil through the construction of genomic library enriched for GA/CT motifs. As a result 122 functional SSR primer pairs were developed from 151 microsatellite loci and validated in L. culinaris cv. Precoz. Thirty three SSR markers were utilized for the analysis of genetic relationships between cultivated and wild species of Lens and related legumes. A total of 123 alleles were amplified at 33 loci ranging from 2-5 alleles with an average of 3.73 alleles per locus. Polymorphic information content (PIC) for all the loci ranged from 0.13 to 0.99 with an average of 0.66 per locus. Varied levels of cross genera transferability were obtained ranging from 69.70 % across Pisum sativum to 12.12 % across Vigna radiata. The UPGMA based dendrogram was able to establish the uniqueness of each genotype and grouped them into two major clusters clearly resolving the genetic relationships within lentil and related species. The new set of SSR markers reported here were efficient and highly polymorphic and would add to the existing repertoire of lentil SSR markers to be utilized in molecular breeding. Moreover, the improved knowledge about intra- and inter-specific genetic relationships would facilitate germplasm utilization for lentil improvement.

Large scale in-silico identification and characterization of simple sequence repeats (SSRs) from de novo assembled transcriptome of Catharanthus roseus (L.) G. Don.

Transcriptomic data of C. roseus offering ample sequence resources for providing better insights into gene diversity: large resource of genic SSR markers to accelerate genomic studies and breeding in Catharanthus . Next-generation sequencing is an efficient system for generating high-throughput complete transcripts/genes and developing molecular markers. We present here the transcriptome sequencing of a 26-day-old Catharanthus roseus seedling tissue using Illumina GAIIX platform that resulted in a total of 3.37 Gb of nucleotide sequence data comprising 29,964,104 reads which were de novo assembled into 26,581 unigenes. Based on similarity searches 58 % of the unigenes were annotated of which 13,580 unique transcripts were assigned 5016 gene ontology terms. Further, 7,687 of the unigenes were found to have Cluster of Orthologous Group classifications, and 4,006 were assigned to 289 Kyoto Encyclopedia of Genes and Genome pathways. Also, 5,221 (19.64 %) of transcripts were distributed to 81 known transcription factor (TF) families. In-silico analysis of the transcriptome resulted in identification of 11,004 SSRs in 26.62 % transcripts from which 2,520 SSR markers were designed which exhibited a non-random pattern of distribution. The most abundant was the trinucleotide repeats (AAG/CTT) followed by the dinucleotide repeats (AG/CT). Location specific analysis of SSRs revealed that SSRs were preferentially associated with the 5'-UTRs with a predicted role in regulation of gene expression. A PCR validation of a set of 48 primers revealed 97.9 % successful amplification, and 76.6 % of them showed polymorphism across different Catharanthus species as well as accessions of C. roseus. In summary, this study will provide an insight into understanding the seedling development and resources for novel gene discovery and SSR development for utilization in marker-assisted selective breeding in C. roseus.

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.

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.

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.