SpeedFlower: a comprehensive speed breeding protocol for indica and japonica rice.

To increase rice yields and feed billions of people, it is essential to enhance genetic gains. However, the development of new varieties is hindered by longer generation times and seasonal constraints. To address these limitations, a speed breeding facility has been established and a robust speed breeding protocol, SpeedFlower is developed that allows growing 4-5 generations of indica and/or japonica rice in a year. Our findings reveal that a high red-to-blue (2R > 1B) spectrum ratio, followed by green, yellow and far-red (FR) light, along with a 24-h long day (LD) photoperiod for the initial 15 days of the vegetative phase, facilitated early flowering. This is further enhanced by 10-h short day (SD) photoperiod in the later stage and day and night temperatures of 32/30 °C, along with 65% humidity facilitated early flowering ranging from 52 to 60 days at high light intensity (800 µmol m-2 s-1). Additionally, the use of prematurely harvested seeds and gibberellic acid treatment reduced the maturity duration by 50%. Further, SpeedFlower was validated on a diverse subset of 198 rice accessions from 3K RGP panel encompassing all 12 distinct groups of Oryza sativa L. classes. Our results confirmed that using SpeedFlower one generation can be achieved within 58-71 days resulting in 5.1-6.3 generations per year across the 12 sub-groups. This breakthrough enables us to enhance genetic gain, which could feed half of the world's population dependent on rice.

A first-generation haplotype map (HapMap-1) of tea (Camellia sinensis L. O. Kuntz).

MOTIVATION: Tea is a cross-pollinated woody perennial plant, which is why, application of conventional breeding is limited for its genetic improvement. However, lack of the genome-wide high-density SNP markers and genome-wide haplotype information has greatly hampered the utilization of tea genetic resources toward fast-track tea breeding programs. To address this challenge, we have generated a first-generation haplotype map of tea (Tea HapMap-1). Out-crossing and highly heterozygous nature of tea plants, make them more complicated for DNA-level variant discovery. RESULTS: In this study, whole genome re-sequencing data of 369 tea genotypes were used to generate 2,334,564 biallelic SNPs and 1,447,985 InDels. Around 2928.04 million paired-end reads were used with an average mapping depth of ∼0.31× per accession. Identified polymorphic sites in this study will be useful in mapping the genomic regions responsible for important traits of tea. These resources lay the foundation for future research to understand the genetic diversity within tea germplasm and utilize genes that determine tea quality. This will further facilitate the understanding of tea genome evolution and tea metabolite pathways thus, offers an effective germplasm utilization for breeding the tea varieties. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pan-genomic, transcriptomic, and miRNA analyses to decipher genetic diversity and anthocyanin pathway genes among the traditional rice landraces.

Black rice is famous for containing high anthocyanin while Joha rice is aromatic with low anthocyanin containing rice from the North-Eastern Region (NER) of India. However, there are limited reports on the anthocyanin biosynthesis in Manipur Black rice. Therefore, the present study was aimed to understand the origin, domestication and anthocyanin biosynthesis pathways in Black rice using the next generation sequencing approaches. With the sequencing data, various analyses were carried out for differential expression and construction of a pan-genome. Protein coding RNA and small RNA sequencing analysis aided in determining 7415 and 131 differentially expressed transcripts and miRNAs, respectively in NER rice. This is the first extensive study on identification and expression analysis of miRNAs and their target genes in regulating anthocyanin biosynthesis in NER rice. This study will aid in better understanding for decoding the theory of high or low anthocyanin content in different rice genotypes.

Unexplored nutritive potential of tomato to combat global malnutrition.

Tomato, a widely consumed vegetable crop, offers a real potential to combat human nutritional deficiencies. Tomatoes are rich in micronutrients and other bioactive compounds (including vitamins, carotenoids, and minerals) that are known to be essential or beneficial for human health. This review highlights the current state of the art in the molecular understanding of the nutritional aspects, conventional and molecular breeding efforts, and biofortification studies undertaken to improve the nutritional content and quality of tomato. Transcriptomics and metabolomics studies, which offer a deeper understanding of the molecular regulation of the tomato's nutrients, are discussed. The potential uses of the wastes from the tomato processing industry (i.e., the peels and seed extracts) that are particularly rich in oils and proteins are also discussed. Recent advancements with CRISPR/Cas mediated gene-editing technology provide enormous opportunities to enhance the nutritional content of agricultural produces, including tomatoes. In this regard, genome editing efforts with respect to biofortification in the tomato plant are also discussed. The recent technological advancements and knowledge gaps described herein aim to help explore the unexplored nutritional potential of the tomato.

Contrasting ß-ODAP content correlates with stress gene expression in Lathyrus cultivars.

Lathyrus sativus, commonly known as grass pea, is a nutrient-rich pulse crop with remarkable climate-resilient attributes. However, wide use of this nutritious crop is not adopted owing to the presence of a non-protein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), which is neurotoxic if consumed in large quantities. We conducted a de novo transcriptomic profiling of two ODAP contrasting cultivars, Pusa-24 and its somaclonal variant Ratan, to understand the genetic changes leading to and associated with ß-ODAP levels. Differential gene expression analysis showed that a variety of genes are downregulated in low ß-ODAP cultivar Ratan and include genes involved in biotic/abiotic stress tolerance, redox metabolism, hormonal metabolism, and sucrose, and starch metabolism. Several genes related to chromatin remodeling are differentially expressed in cultivar Ratan. ß-ODAP biosynthetic genes in these cultivars showed differential upregulation upon stress. ODAP content of these cultivars varied differentially upon stress and development. Physiological experiments indicate reduced relative water content and perturbed abscisic acid levels in the low ODAP cultivar. Altogether, our results suggest that the low ODAP cultivar may have a reduced stress tolerance. The dataset provides insight into the biological role of ODAP and will be helpful for hypothesis-driven experiments to understand ODAP biosynthesis and regulation.

Decoding the genome of superior chapatti quality Indian wheat variety 'C 306' unravelled novel genomic variants for chapatti and nutrition quality related genes.

An Indian wheat variety, 'C 306' has good chapatti quality, which is controlled by multiple genes that have not been explored. We report the high quality de novo assembled genome of 'C 306' by combining short and long read sequencing data. The hybrid assembly covered 93% of gene space and identified about 142 K coding genes, 34% repetitive DNA and ~ 501 K SSR motifs. The phylogenetic analysis of about 83 K orthologous protein groups suggested the closest relationship with T. turgidum, T. aestivum and Ae. tauschii. Genome wide analysis annotated 69,217,536 genomic variants. Out of them, 1423 missense and 117 deleterious variants identified in processing, nutrition, and chapatti quality related genes such as alpha- and beta-gliadin, SSI, SSIII, SUT1, SBEI, CHS, YSL, DMAS, and NAS encoded proteins. These variants may affect quality genes. The genomic data will be potential genomic resources in wheat breeding programs for quality improvement.

Functional characterization of two type-1 diacylglycerol acyltransferase (DGAT1) genes from rice (Oryza sativa) embryo restoring the triacylglycerol accumulation in yeast.

KEY MESSAGE: Two OsDGAT1 genes showed the ability to restore TAG and LB synthesis in yeast H1246. Alterations in the N-terminal region of OsDGAT1-1 gene revealed its regulatory role in gene function. Accumulation of triacylglycerol (TAG) or oil in vegetative tissues has emerged as a promising approach to meet the global needs of food, feed, and fuel. Rice (Oryza sativa) has been recognized as an important cereal crop containing nutritional rice bran oil with high economic value for renewable energy production. To identify the key component involved in storage lipid biosynthesis, two type-1 diacylglycerol acyltransferases (DGAT1) from rice were characterized for its in vivo function in the H1246 (dga1, lro1, are1 and are2) yeast quadruple mutant. The ectopic expression of rice DGAT1 (designated as OsDGAT1-1 and OsDGAT1-2) genes restored the capability of TAG synthesis and lipid body (LB) formation in H1246. OsDGAT1-1 showed nearly equal substrate preferences to C16:0-CoA and 18:1-CoA whereas OsDGAT1-2 displayed substrate selectivity for C16:0-CoA over 18:1-CoA, indicating that these enzymes have contrasting substrate specificities. In parallel, we have identified the intrinsically disordered region (IDR) at the N-terminal domains of OsDGAT1 proteins. The regulatory role of the N-terminal domain was dissected. Single point mutations at the phosphorylation sites and truncations of the N-terminal region highlighted reduced lipid accumulation capabilities among different OsDGAT1-1 variants.

Dissecting the nutrient partitioning mechanism in rice grain using spatially resolved gene expression profiling.

Rice, a staple food worldwide, contains varying amounts of nutrients in different grain tissues. The underlying molecular mechanism of such distinct nutrient partitioning remains poorly investigated. Here, an optimized rapid laser capture microdissection (LCM) approach was used to individually collect pericarp, aleurone, embryo and endosperm from grains 10 days after fertilization. Subsequent RNA-Seq analysis in these tissues identified 7760 differentially expressed genes. Analysis of promoter sequences of tissue-specific genes identified many known and novel cis-elements important for grain filling and seed development. Using the identified differentially expressed genes, comprehensive spatial gene expression pathways were built for accumulation of starch, proteins, lipids, and iron. The extensive transcriptomic analysis provided novel insights about nutrient partitioning mechanisms; for example, it revealed a gradient in seed storage protein accumulation across the four tissue types analysed. The analysis also revealed that the partitioning of various minerals, such as iron, is most likely regulated through transcriptional control of their transporters. We present the extensive analysis from this study as an interactive online tool that provides a much-needed resource for future functional genomics studies aimed to improve grain quality and seed development.

Alleviating aluminum toxicity in plants: Implications of reactive oxygen species signaling and crosstalk with other signaling pathways.

Aluminum (Al) toxicity is a major limiting factor for plant growth and productivity in acidic soil. At pH lower than 5.0 (pH < 5.0), the soluble and toxic form of Al (Al3+ ions) enters root cells and inhibits root growth and uptake of water and nutrients. The organic acids malate, citrate, and oxalate are secreted by the roots and chelate Al3+ to form a non-toxic Al-OA complex, which decreases the entry of Al3+ into the root cells. When Al3+ enters, it leads to the production of reactive oxygen species (ROS) in cells, which are toxic and cause damage to biomolecules like lipids, carbohydrates, proteins, and nucleic acids. When ROS levels rise beyond the threshold, plants activate an antioxidant defense system that comprises of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione S-transferase (GST), ascorbic acid (ASA), phenolics and alkaloids etc., which protect plant cells from oxidative damage by scavenging and neutralizing ROS. Besides, ROS also play an important role in signal transduction and influence many molecular and cellular process like hormone signaling, gene expression, cell wall modification, cell cycle, programed cell death (PCD), and development. In the present review, the mechanisms of Al-induced ROS generation, ROS signaling, and crosstalk with other signaling pathways helping to combat Al toxicity have been summarized, which will help researchers to understand the intricacies of Al-induced plant response at cellular level and plan research for developing Al-toxicity tolerant crops for sustainable agriculture in acid soil-affected regions of the world.

Role of transporters in plant disease resistance.

Plants being sessile have evolved numerous mechanisms to meet the changing environmental and growth conditions. Plant pathogens are responsible for devastating disease epidemics in many species. Transporter proteins are an integral part of plant growth and development, and several studies have documented their role in pathogen disease resistance. In this review, we analyze the studies on genome-wide identifications of plant transporters like sugars will eventually be exported transporters (SWEET), multidrug and toxic compound extrusion (MATE) transporters, ATP-binding cassette (ABC) transporters, natural resistance-associated macrophage proteins (NRAMP), and sugar transport proteins (STPs), all having a significant role in plant disease resistance. The mechanism of action of these transporters, their solute specificity, and the potential application of recent molecular biology approaches deploying these transporters for the development of disease-resistant plants are also discussed. The applications of genome editing tools, such as CRIPSR/Cas9, are also presented. Altogether the information included in this article gives a better understanding of the role of transporter proteins during plant-pathogen interaction.

Significance of solute specificity, expression, and gating mechanism of tonoplast intrinsic protein during development and stress response in plants.

Tonoplast intrinsic proteins (TIPs), belonging to the aquaporin family, are transmembrane channels located mostly at the tonoplast of plant cells. The TIPs are known to transport water and many other small solutes such as ammonia, urea, hydrogen peroxide, and glycerol. In the present review, phylogenetic distribution, structure, transport dynamics, gating mechanism, sub-cellular localization, tissue-specific expression, and co-expression of TIPs are discussed to define their versatile role in plants. Based on the phylogenetic distribution, TIPs are classified into five distinct groups with aromatic-arginine (Ar/R) selectivity filters, typical pore-morphology, and tissue-specific gene expression patterns. The tissue-specific expression of TIPs is conserved among diverse plant species, more particularly for TIP3s, which are expressed exclusively in seeds. Studying TIP3 evolution will help to understand seed development and germination. The solute specificity of TIPs plays an imperative role in physiological processes like stomatal movement and vacuolar sequestration as well as in alleviating environmental stress. TIPs also play an important role in growth and developmental processes like radicle protrusion, anther dehiscence, seed germination, cell elongation, and expansion. The gating mechanism of TIPs regulates the solute flow in response to external signals, which helps to maintain the physiological functions of the cell. The information provided in this review is a base to explore TIP's potential in crop improvement programs.

Identification and functional prediction of long non-coding RNAs of rice (Oryza sativa L.) at reproductive stage under salinity stress.

Salinity adversely affects the yield and growth of rice (Oryza sativa L.) plants severely, particularly at reproductive stage. Long non-coding RNAs (lncRNAs) are key regulators of diverse molecular and cellular processes in plants. Till now, no systematic study has been reported for regulatory roles of lncRNAs in rice under salinity at reproductive stage. In this study, total 80 RNA-seq data of Horkuch (salt-tolerant) and IR-29 (salt-sensitive) genotypes of rice were used and found 1626 and 2208 transcripts as putative high confidence lncRNAs, among which 1529 and 2103 were found to be novel putative lncRNAs in root and leaf tissue respectively. In Horkuch and IR-29, 14 and 16 lncRNAs were differentially expressed in root tissue while 18 and 63 lncRNAs were differentially expressed in leaf tissue. Interaction analysis among the lncRNAs, miRNAs and corresponding mRNAs indicated that these modules are involved in different biochemical pathways e.g. phenyl propanoid pathway during salinity stress in rice. Interestingly, two differentially expressed lncRNAs such as TCONS_00008914 and TCONS_00008749 were found as putative target mimics of known rice miRNAs. This study indicates that lncRNAs are involved in salinity adaptation of rice at reproductive stage through certain biochemical pathways.

Differential proteins associated with plasma membrane in X- and/or Y-chromosome bearing spermatozoa in indicus cattle.

The differential proteins associated with plasma membrane of spermatozoa are less known, identification of which shall help overcome limitations of currently used methods of sperm sexing, considered as a high priority for livestock sector of many countries. This study has reported plasma membrane proteomics of unsorted spermatozoa and differential expression of plasma membrane-associated proteins between X- and Y-chromosome bearing spermatozoa of indicus cattle (Bos indicus). Isolation of plasma membrane fraction using percoll gradient, relatively a rapid method, from bovine spermatozoa has been reported to enrich isolation of plasma membrane proteins. Significant enrichment for plasma membrane-associated proteins was observed in plasma membrane fraction (p < .05) as compared to the total cell lysate using LC-MS/MS. Furthermore, these experiments were conducted in flow cytometry sorted, sexed-semen samples. Thirteen proteins were identified as differentially abundant between X- and Y-sorted spermatozoa. Among these, two proteins were downregulated in Y-sorted spermatozoa compared to the X-sorted spermatozoa (p < .05), while four and seven proteins could be noted in X- and Y-sorted spermatozoa, respectively. Proteins that are presumed to support sperm capacitation and sperm migration velocity were found to be abundant in Y-sorted spermatozoa while those associated with structural molecule activity were identified as abundant in X-sorted spermatozoa in the present study. Our study provides better insight into the plasma membrane proteomics of spermatozoa of indicus cattle and furnishes data that might aid in design and development of alternate and open technology for sex-sorting of semen.

Deciphering the role of microRNAs during Pi54 gene mediated Magnaporthe oryzae resistance response in rice.

The broad-spectrum resistance gene Pi54 confers resistance to multiple isolates of Magnaporthe oryzae in rice. In order to decipher the molecular mechanism underlying the Pi54 mediated resistance in rice line Taipei309 Pi54 (carrying Pi54), miRNAome study was performed at 24 h post-inoculation (hpi) with M. oryzae. A total of 222 known miRNAs representing 101 miRNA families were found in this study. Of these, 29 and 24 miRNAs were respectively up- and down-regulated in the resistant Taipei309 Pi54 . Defence response (DR) genes, like, NBSGO35, and OsWAK129b, and genes related to transcription factors were up-regulated in Taipei309 Pi54 line. The vast array of miRNA candidates identified here are miR159c, miR167c, miR2100, miR2118o, miR2118l, miR319a, miR393, miR395l, miR397a, miR397b, miR398, miR439g, miR531b, miR812f, and miR815c, and they manifest their role in balancing the interplay between various DR genes during Pi54 mediated resistance. We also validated miRNA/target gene pairs involved in hormone signalling, and cross-talk among hormone pathways regulating the rice immunity. This study suggests that the Pi54 gene mediated blast resistance is influenced by several microRNAs through PTI and ETI components in the rice line Taipei309 Pi54 , leading to incompatible host-pathogen interaction.

Identification and validation of reference genes for qRT-PCR based studies in horse gram (Macrotyloma uniflorum).

The quantitative real-time polymerase chain reaction (qRT-PCR) is the most sensitive and commonly used technique for gene expression studies in biological systems. However, the reliability of qRT-PCR results depends on the selection of reference gene(s) for data normalization. Horse gram (Macrotyloma uniflorum) is an important legume crop on which several molecular studies have been reported. However, the stability of reference genes has not been evaluated. In the present study, nine candidate reference genes were identified from horse gram RNA-seq data and evaluated in two horse gram genotypes, HPK4 and HPKM317 under six abiotic stresses viz. cold, drought, salinity, heat, abscisic acid and methyl viologen-induced oxidative stress. The results were evaluated using geNorm, Bestkeeper, Normfinder and delta-delta Ct methods and comprehensive ranking was assigned using RefFinder and RankAggreg software. The overall result showed that TCTP was one of the most stable genes in all samples and in genotype HPK4, while in HPKM317 profilin was most stably expressed. However, PSMA5 was identified as least stable in all the experimental conditions. Expression of target genes dehydrin and early response to dehydration 6 under drought stress was also validated using TCTP and profilin for data normalization, either alone or in combination, which confirmed their suitability for qRT-PCR data normalization. Thus, TCTP and profilin genes may be used for qRT-PCR data normalization for molecular and genomic studies in horse gram. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01104-0.

Identification and characterization of Dicer-like genes in leaf rust pathogen (Puccinia triticina) of wheat.

Puccinia triticina (P. triticina) is one of the most devastating fungal pathogens of wheat which causes significant annual yield loss to the crop. Understanding the gene regulatory mechanism of the biotrophic pathogen is one of the important aspects of host-pathogen interaction studies. Dicer-like genes are considered as important mediators of RNAi-based gene regulation. In this study, we report the presence of three Dicer-like genes (Pt-DCL1, Pt-DCL2, Pt-DCL3) in P. triticina genome identified through computational and biological analyses. Quantitative real-time PCR studies revealed an increase in the expression of these genes in germinating spore stages. Heterologous expression combined with mass spectrometry analysis of Pt-DCL2 confirmed the presence of a canonical Dicer-like gene in P. triticina. Phylogenetic analysis of the Pt-DCLs with the Dicer-like proteins from other organisms showed a distinct cluster of rust pathogens from the order Pucciniales. The results indicated a species-specific duplication of Dicer-like genes within the wheat rust pathogens. This study, for the first time, reports the presence of Dicer-dependent RNAi pathway in P. triticina that may play a role in gene regulatory mechanism of the pathogen during its development. Our study serves as a vital source of information for further RNAi-based molecular studies for better understanding and management of the wheat leaf rust disease.

Spatio-temporal distribution of micronutrients in rice grains and its regulation.

Rice has been a staple food for more than half of the global population. Different parts of rice grains contain different amounts of macro- and micro-nutrients. Polished white rice, which is the main form of rice consumption, mainly contains starch, however, the bran and germ, which are removed during polishing, contain large amounts of micronutrients and bioactive compounds. To engineer nutritionally superior rice varieties, it is imperative to understand the spatial and temporal distribution of different nutrients in different parts of the rice grain. Keeping this in mind, in this review, we have performed a comprehensive literature review to put together all the recent findings regarding the spatio-temporal distribution of all the important micronutrients in different cell-layers/tissues of developing seeds and mature seed grains. Furthermore, we have overviewed the underlying cell-layer specific possible regulatory mechanism responsible for the loading/partitioning for each of the micronutrients into specific tissue types. Most of the nutrient filling occurs between 7 and 18 days after fertilization (DAF) through the dorsal vascular bundle and the aleurone layer. During the last few years, spatio-temporal distribution of various minerals and the role of their transporters has been studied in great detail. However, with regard to vitamins and other bioactive compounds, such studies are still very limited. Distribution of minerals in the grain is mainly regulated by the distribution of their ligands and transporters, whereas the accumulation of various vitamins is mainly metabolic enzyme activity. Collective knowledge discussed here in this niche area would help to design new studies to improve the micronutrient content located in the inner part of the seed.

Applications and challenges for efficient exploration of omics interventions for the enhancement of nutritional quality in rice (Oryza sativa L.).

Rice nutritional quality is one of the major concerns along with productivity enhancement to feed the continuously growing population. To address wide-spread malnutrition influencing global health, novel high yielding rice cultivars with better nutritional quality need to be bred. No doubt, the conventional breeding approaches have helped to decrease the gap between demand and supply for yield and nutrition; however, to meet today's demands more advanced approaches need to be employed. This review discusses approaches for the improvement of nutritional quality of rice and gauges the availability of omics resources. Recent omics advances providing numerous tools and techniques for the efficient exploration of genetic resources as well as for the understanding of molecular mechanism involved in the trait development have been discussed. Understanding of genes or loci governing different traits has been found to be effective in accelerating the crop breeding programs. In this regard, approaches like QTL (quantitative trait loci) mapping, genome-wide association study and genomic selection are discussed in light of their utilization for rice nutritional quality improvements. Efficient integration of different omics approaches is recognized as a promising way to achieve the desired improvements in rice cultivars. Therefore, advances in omics branches like transcriptomics, proteomics, ionomics, and metabolomics being efficiently explored for rice improvement programs are also addressed. This article provides a catalog of genes, loci, mutants, online resources and computational approaches for rice improvement. The information provided here will be helpful for pursuing present progress and directing rice research program for better future.

Nitric oxide and hydrogen sulfide crosstalk during heavy metal stress in plants.

Gases such as ethylene, hydrogen peroxide (H2 O2 ), nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2 S) have been recognized as vital signaling molecules in plants and animals. Of these gasotransmitters, NO and H2 S have recently gained momentum mainly because of their involvement in numerous cellular processes. It is therefore important to study their various attributes including their biosynthetic and signaling pathways. The present review provides an insight into various routes for the biosynthesis of NO and H2 S as well as their signaling role in plant cells under different conditions, more particularly under heavy metal stress. Their beneficial roles in the plant's protection against abiotic and biotic stresses as well as their adverse effects have been addressed. This review describes how H2 S and NO, being very small-sized molecules, can quickly pass through the cell membranes and trigger a multitude of responses to various factors, notably to various stress conditions such as drought, heat, osmotic, heavy metal and multiple biotic stresses. The versatile interactions between H2 S and NO involved in the different molecular pathways have been discussed. In addition to the signaling role of H2 S and NO, their direct role in posttranslational modifications is also considered. The information provided here will be helpful to better understand the multifaceted roles of H2 S and NO in plants, particularly under stress conditions.

Development of transcriptome-wide SSR markers for genetic diversity and structure analysis in Macrotyloma uniflorum (Lam.) Verdc.

Horsegram is an important drought resistant pulse crop from Fabaceae and can be easily grown in dry lands with no irrigation facilities. However, it remained neglected since long and has been considered as orphan legume which requires immediate attention for its improvement and for the development of new promising varieties in future. In the present study, 7352 simple sequence repeat (SSR) markers were developed from the transcriptome data and 150 SSR were randomly synthesized for validation and diversity analysis in a panel of 58 horsegram genotypes. The synthesized primers included all types of repeats spanning direpeats to hexarepeats. Of the validated SSR markers, 33 markers were polymorphic and produced 40 loci which were used to analyze the genetic diversity and structure of horsegram. In total, 130 alleles were produced in a range of 2-9 alleles with maximum alleles produced by primer HTSSR 155. Expected heterozygosity (He) ranged from 0.03 to 1.00 and observed heterozygosity (Ho) ranged from 0.13 to 0.81. Polymorphism information content value ranged from 0.065 to 0.78. Dendrogram based on UPGMA and principal component analysis showed four groups of the 58 genotypes of horsegram. Structure analysis showed three genetic stocks for the analyzed germplasm. Thus, the developed SSRs can be useful in future population genetics analysis, molecular breeding studies and mapping works in horsegram germplasm as well as in related legume species.