gSSR Markers Research Articles

Molecular Characterization of Tinospora cordifolia (Willd.) Miers Using Novel g-SSR Markers and Their Comparison with EST-SSR and SCoT Markers for Genetic Diversity Study.

In the present study, novel genomic-SSR (g-SSR) markers generated in our laboratory were used to characterize Tinospora cordifolia and related species. The g-SSR marker was also compared with EST-SSR and SCoT markers used earlier in our laboratory to assess the genetic diversity of T. cordifolia. A total of 26 accessions of T. cordifolia and 1 accession each of Tinospora rumphii and Tinospora sinensis were characterized using 65 novel g-SSR markers. A total of 125 alleles were detected with 49 polymorphic g-SSR markers. The number of alleles per locus varied from 1-4 with a mean value of 2.55 alleles per locus. Mean PIC, gene diversity, and heterozygosity were estimated to be 0.33, 0.41, and 0.65, respectively. The two species, namely T. rumphii and T. sinensis, showed cross-species transferability of g-SSRs developed in T. cordifolia. The success rate of cross-species transferability in T. rumphii was 95.3% and 93.8% in T. sinensis, proving the usefulness of this marker in genetic diversity studies of related species. The Tinospora accessions were also used for molecular characterization using SCoT and EST-SSR markers and compared for genetic diversity and cross-species transferability. The PIC, gene diversity, heterozygosity, and principal coordinate analysis showed that g-SSR is the better maker for a genetic diversity study of T. cordifolia. Additionally, high cross-species transferability of g-SSRs was found (95.3% and 93.8%) compared to EST-SSRs (68.8% and 67.7%) in T. rumphii and T. sinensis, respectively.

Polymorphisms in the hypervariable control region of the mitochondrial DNA differentiate BPH populations.

The brown planthopper (BPH; Nilaparvata lugens) is one of India's most destructive pests of rice. BPH, a monophagous migratory insect, reported from all major rice-growing ecosystems of the country, is capable of traversing large distances and causing massive crop loss. A crucial step for developing viable management strategies is understanding its population dynamics. Very few reliable markers are currently available to screen BPH populations for their diversity. In the current investigation, we developed a combinatorial approach using the polymorphism present within the mitochondrial Control Region of BPH and in the nuclear genome (genomic simple sequence repeats; gSSRs) to unravel the diversity present in BPH populations collected from various rice-growing regions of India. Using two specific primer pairs, the complete Control Region (1112 to 2612 bp) was PCR amplified as two overlapping fragments, cloned and sequenced from BPH individuals representing nine different populations. Results revealed extensive polymorphism within this region due to a variable number of tandem repeats. The three selected gSSR markers also exhibited population-specific amplification patterns. Overall genetic diversity between the nine populations was high (>5%). Further, in silico double-digestion of the consensus sequences of the Control Region, with HpyCH4IV and Tsp45I restriction enzymes, revealed unique restriction fragment length polymorphisms (digital-RFLPs; dRFLPs) that differentiated all the nine BPH populations. To the best of our knowledge, this is the first report of markers developed from the Control Region of the BPH mitogenome that can differentiate populations. Eventually, such reliable and rapid marker-based identification of BPH populations will pave the way for an efficient pest management strategy.

Development and Characterization of 20 Genomic SSR Markers for Ornamental Cultivars of Weigela.

Weigela (Caprifoliaceae) is a genus of ornamental plants popular for its phenotypic variation and hardiness, that includes species hybridized to produce the commercially available cultivars. Despite its popularity, limited genetic resources exist for the genus. Twenty genomic simple sequence repeat (gSSR) markers distributed across the genome were developed using low coverage whole-genome sequencing data of Weigela Spilled Wine®. A cross-amplification evaluation with these 20 gSSR markers on a collection of 18 Weigela cultivars revealed a total of 111 unique alleles, including 36 private alleles. A diagrammatic key was constructed to identify cultivars using only six of the gSSR markers, demonstrating the newly developed gSSR markers are immediately useful for cultivar identification. Future uses could include breeding with marker-assisted selection, determining the history of hybridization of the current cultivated lines, aiding in the construction of genetic maps, and assessing the patterns of population genetic structure of Weigela spp.

Development of Genomic SSR for the Subtropical Hardwood Tree Dalbergia hupeana and Assessment of Their Transferability to Other Related Species

Dalbergia hupeana Hance (D. hupeana) is a precious hardwood tree of the genus Dalbergia. It is one of the few species widely distributed within subtropical areas and is important for timber production and forest restoration. At present, there is little published genetic information on D. hupeana. Therefore, we performed a genome survey using next generation sequencing (NGS) and developed a set of novel genomic SSR (gSSR) markers from the assembled data, and assessed the transferability of these markers to other Dalbergia species in Asia. The results of the genome survey show the genome size of D. hupeana to be about 664 Mb and highly heterozygous. The assembly of sequencing data produced 2,431,997 contigs, and the initial assembly of the NGS data alone resulted in contig N50 of 393 kb with a total of 720 Mb. A total of 127,742 perfect SSR markers were found in the assembled contigs. A total of 37 highly polymorphic and easily genotyped gSSR markers were developed in D. hupeana, while the majority of gSSR markers could be successfully transferred to nine other Dalbergia species in Asia. The transferability rate of gSSR markers was highest in D. balansae, which is more closely related to D. hupeana. Seven gSSR markers were able to be amplified in all tested species. In addition, a preliminary assessment of the genetic diversity of three tree species in the Dalbergia genus suggested a high level of genetic diversity within populations distributed in the subtropical area in China. However, the determination of the global status of their genetic variation still requires further and more comprehensive assessment. Our findings will enable further studies on the genetic diversity, phylogenetics, germplasm characterization, and taxonomy of various Dalbergia species.

Diversity and relationships of Miscanthus sinensis from 20 geographical distributions in China based on SSR markers

Miscanthus is a C4 herbaceous perennial genus, and it was chosen as a bioenergy crop due to high biomass yield. Miscanthus sinensis has many phenotypes which are adapted to various environments in China. In this study, 421 accessions of M. sinensis were collected from 22 provinces, and the genetic polymorphisms amongst these germplasm collections were identified using 20 primer pairs designed against 10 each from expressed sequence tag-simple sequence repeats [EST-SSR (eSSR)] and genomic SSR (gSSR) transferable markers from barley. A total of 95 SSR polymorphic bands were detected producing a 100% polymorphic rate among these M. sinensis accessions. The gSSR markers showed a richer genetic polymorphism than eSSR markers. Based on the unweighted pair group method with arithmetic mean (UPGMA) clustering, there was a distinct sub-population separation in M. sinensis, which indicates that geographical differences and natural selection are the driving forces for genetic variation and evolution in the species. The 20 pairs of barley markers matched to 26 polymorphic bands associated with date of heading, plant height, leaf weight, stem weight, leaf/stem ratio, and total biomass yield. Eleven marker polymorphic bands were associated with the date of heading, 4 with plant height, 10 with leaf weight, 7 with stem weight, 3 with leaf/stem ratio, and 10 with biomass yield.

Development of Novel Genomic Simple Sequence Repeat (g-SSR) Markers and Their Validation for Genetic Diversity Analyses in Kalmegh [Andrographis paniculata (Burm. F.) Nees

Kalmegh (Andrographis paniculata (Burm. F.) Nees) is one of the most important medicinal plants and has been widely explored as traditional medicine. To exploit its natural genetic diversity and initiations of molecular breeding to develop novel cultivars or varieties, developments of genomic resources are essential. Four microsatellite-enriched genomic libraries—(CT)14, (GT)12, (AG)15 and (AAC)8—were constructed using the genomic DNA of A. paniculata. Initially, 183 recombinant colonies were screened for the presence of CT, GT, AG, and AAC microsatellite repeats, out of which 47 clones found positive for the desired simple sequence repeats (SSRs). It was found that few colonies had more than one desirable SSR. Thus, a sum of 67 SSRs were designed and synthesized for their validation among 42 A. paniculata accessions. Out of the 67 SSRs used for genotyping, only 41 were found to be polymorphic. The developed set of g-SSR markers showed substantial genetic variability among the selected A. paniculata accessions, with an average polymorphic information content (PIC) value of 0.32. Neighbor-joining tree analysis, population structure analysis, analysis of molecular variance (AMOVA), and principal coordinate analysis (PCoA) illustrated the considerable genetic diversity among them. The novel g-SSR markers developed in the present study could be important genomic resources for future applications in A. paniculata.

The influence of breeding history, origin and growth type on population structure of barley as revealed by SSR markers

Natural and mass selection during domestication and cultivation favored particular traits of interest in barley. In the present study, population structure, and genetic relationships among 144 accessions of barley landraces and breeding materials from various countries were studied using a set of 77 and 72 EST-SSR and gSSR markers, respectively distributed on seven chromosomes of barley. In total, 262 and 429 alleles were amplified in 77 EST-SSRs and 72 gSSR loci, respectively. Out of which, 185 private/group-specific alleles were identified in the landraces compared with 14 in "cultivar and advanced breeding lines", indicating the possibility to introgress favorite alleles from landraces into breeding materials. Comparative analysis of genetic variation among breeding materials, Iranian landraces, and exotic landraces revealed higher genetic diversity in Iranian landraces compared with others. A total of 37, 15, and 14 private/group-specific alleles were identified in Iranian landraces, exotic landraces, and breeding materials, respectively. The most likely groups for 144 barley genotypes were three as inferred using model- and distance-based clustering as well as principal coordinate analysis which assigned the landraces and breeding materials into separate groups. The distribution of alleles was found to be correlated with population structure, domestication history and eco-geographical factors. The high allelic richness in the studied set of barley genotype provides insights into the available diversity and allows the construction of core groups based on maximizing allelic diversity for use in barley breeding programs.

Development of novel g-SSR markers in guava (Psidium guajava L.) cv. Allahabad Safeda and their application in genetic diversity, population structure and cross species transferability studies

Dearth of genomic resources particularly, microsatellite markers in nutritionally and commercially important fruit crop, guava necessitate the development of the novel genomic SSR markers through the library enrichment techniques. Three types of 3' -biotinylated oligonucleotide probes [(CT)14, (GT)12, and (AAC)8] were used to develop microsatellite enriched libraries. A total of 153 transformed colonies were screened of which 111 positive colonies were subjected for Sanger sequencing. The clones having more than five motif repeats were selected for primer designing and a total of 38 novel genomic simple sequence repeats could be identified. The g-SSRs had the motif groups ranging from monomer to pentamer out of which dimer group occurred the most (89.47%). Out of 38 g-SSRs markers developed, 26 were found polymorphic, which showed substantial genetic diversity among the guava genotypes including wild species. The average number of alleles per locus, major allele frequency, gene diversity, expected heterozygosity and polymorphic information content of 26 SSRs were 3.46, 0.56, 0.53, 0.29 and 0.46, respectively. The rate of cross-species transferability of the developed g-SSR loci varied from 38.46 to 80.77% among the studied wild Psidium species. Generation of N-J tree based on 26 SSRs grouped the 40 guava genotypes into six clades with two out-groups, the wild guava species showed genetic distinctness from cultivated genotypes. Furthermore, population structure analysis grouped the guava genotypes into three genetic groups, which were partly supported by PCoA and N-J tree. Further, AMOVA and PCoA deciphered high genetic diversity among the present set of guava genotypes including wild species. Thus, the developed novel g-SSRs were found efficient and informative for diversity and population structure analyses of the guava genotypes. These developed novel g-SSR loci would add to the new genomic resource in guava, which may be utilized in genomic-assisted guava breeding.

Assessment of population structure and genetic diversity study of landraces and elite rice varieties commonly grown in Nigeria

A set of 21 rice genotypes including both elite varieties and landraces were characterised based on their population structure using genomic simple sequence repeat (gSSR) markers. A model-based method was used to delineate clusters of individuals based on their genotypes at multiple loci using a Bayesian approach. The population structure analysis separated the 21 genotypes into 3 sub-populations and the grouping were consistent with the ancestral heritage of most of the genotypes whose pedigree is known. Whereas, further clustering analysis based on unweighted pair group method with arithmetic mean (UPGMA), separated the 21 genotypes into 5 clusters. The reason for the disparity in the number clusters may be because of the different grouping methods. The knowledge obtained from both population structure and genetic diversity of the selected rice genotypes is an important contribution to future development of rice varieties for deferent agro-ecological zones in Nigeria.

OPTIMASI SUHU ANNEALING UNTUK PRIMER g-SSR DAN EST-SSR PADA KACANG HIJAU (Vigna radiata L.)

Mungbean (Vigna radiata L.) is one of important legume in Indonesia. G-SSR and EST-SSR markers had been widely used in mungbean genetic diversity research. DNA isolation and DNA amplification are required to obtain genetic information about mungbean to obtain accurate data on the genetic diversity of mungbean. This study aims to determine the Temperature of annealing (Ta) of the g-SSR and EST-SSR primer pairs. The total DNA was isolated from young leaves mungbean origin Pelalawan and the eight primer pairs of the g-SSR and EST-SSR were optimized. The optimal Ta for G2436, G3598, G2516, G7472, G0483. G1671, G3302, and G3427 were 50,55ºC, 51,15ºC,51,25ºC, 51,2ºC, 51,6ºC, 49,0ºC, 49,8ºC, and 52,8ºC respectively. Meanwhile, the optimal Ta for E51985, E19823, E24080, E22860, E26637, E16266, E11659, and E10675 were 52,2ºC, 54,4ºC, 52,5ºC,51,25ºC, 53,25ºC, 54ºC, 54,35ºC, and 53,6ºC respectively.

Taraxacum kok-saghyz (rubber dandelion) genomic microsatellite loci reveal modest genetic diversity and cross-amplify broadly to related species

Taraxacum kok-saghyz (TKS) carries great potential as alternative natural rubber source. To better inform future breeding efforts with TKS and gain a deeper understanding of its genetic diversity, we utilized de novo sequencing to generate novel genomic simple sequence repeats markers (gSSRs). We utilized 25 gSSRs on a collection of genomic DNA (gDNA) samples from germplasm bank, and two gDNA samples from historical herbarium specimens. PCR coupled with capillary electrophoresis and an array of population genetics tools were employed to analyze the dataset of our study as well as a dataset of the recently published genic SSRs (eSSRs) generated on the same germplasm. Our results using both gSSRs and eSSRs revealed that TKS has low- to- moderate genetic diversity with most of it partitioned to the individuals and individuals within populations, whereas the species lacked population structure. Nineteen of the 25 gSSR markers cross-amplified to other Taraxacum spp. collected from Southeastern United States and identified as T. officinale by ITS sequencing. We used a subset of 14 gSSRs to estimate the genetic diversity of the T. officinale gDNA collection. In contrast to the obligatory outcrossing TKS, T. officinale presented evidence for population structure and clonal reproduction, which agreed with the species biology. We mapped the molecular markers sequences from this study and several others to the well-annotated sunflower genome. Our gSSRs present a functional tool for the biodiversity analyses in Taraxacum, but also in the related genera, as well as in the closely related tribes of the Asteraceae.

Genome-Wide Mining and Characterization of SSR Markers for Gene Mapping and Gene Diversity in Gossypium barbadense L. and Gossypium darwinii G. Watt Accessions

The present study aimed to characterize the simple sequence repeat markers in cotton using the cotton expressed sequence tags. A total of 111 EST-SSR polymorphic molecular markers with trinucleotide motifs were used to evaluate the 79 accessions of Gossypium L., (G. darwinii, 59 and G. barbadense, 20) collected from the Galapagos Islands. The allele number ranged from one to seven, with an average value of 2.85 alleles per locus, while polymorphism information content values varied from 0.008 to 0.995, with an average of 0.520. The discrimination power ranks high for the majority of the SSRs, with an average value of 0.98. Among 111 pairs of EST-SSRs and gSSRs, a total of 49 markers, comprising nine DPLs, one each of MonCGR, MUCS0064, and NAU1028, and 37 SWUs (D-genome), were found to be the best matched hits, similar to the 155 genes identified by BLASTx in the reference genome of G. barbadense, G. arboreum L., and G. raimondii Ulbr. Related genes GOBAR_DD21902, GOBAR_DD15579, GOBAR_DD27526, and GOBAR_AA04676 revealed highly significant expression 10, 15, 18, 21, and 28 days post-anthesis of fiber development. The identified EST-SSR and gSSR markers can be effectively used for mapping functional genes of segregating cotton populations, QTL identification, and marker-assisted selection in cotton breeding programs.

Chloroplast genome analyses and genomic resource development for epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae), using genome skimming data

BackgroundEpilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae) have an epilithic habitat (rocky slopes) and a parapatric distribution in East Asia, which makes them an ideal model for a more comprehensive understanding of the demographic and divergence history and the influence of climate changes in East Asia. However, the genetic background and resources for these two genera are scarce.ResultsThe complete chloroplast (cp) genomes of two Oresitrophe rupifraga and one Mukdenia rossii individuals were reconstructed and comparative analyses were conducted to examine the evolutionary pattern of chloroplast genomes in Saxifragaceae. The cp genomes ranged from 156,738 bp to 156,960 bp in length and had a typical quadripartite structure with a conserved genome arrangement. Comparative analysis revealed the intron of rpl2 has been lost in Heuchera parviflora, Tiarella polyphylla, M. rossii and O. rupifraga but presents in the reference genome of Penthorum chinense. Seven cp hotspot regions (trnH-psbA, trnR-atpA, atpI-rps2, rps2-rpoC2, petN-psbM, rps4-trnT and rpl33-rps18) were identified between Oresitrophe and Mukdenia, while four hotspots (trnQ-psbK, trnR-atpA, trnS-psbZ and rpl33-rps18) were identified within Oresitrophe. In addition, 24 polymorphic cpSSR loci were found between Oresitrophe and Mukdenia. Most importantly, we successfully developed 126 intergeneric polymorphic gSSR markers between Oresitrophe and Mukdenia, as well as 452 intrageneric ones within Oresitrophe. Twelve randomly selected intergeneric gSSRs have shown that these two genera exhibit a significant genetic structure.ConclusionsIn this study, we conducted genome skimming for Oresitrophe rupifraga and Mukdenia rossii. Using these data, we were able to not only assemble their complete chloroplast genomes, but also develop abundant genetic resources (cp hotspots, cpSSRs, polymorphic gSSRs). The genomic patterns and genetic resources presented here will contribute to further studies on population genetics, phylogeny and conservation biology in Saxifragaceae.

Construction of genetic linkage maps of ‘Fina Sodea’ clementine (Citrus clementina) and Byungkyul (C. platymamma), a Korean landrace, based on RAPD and SSR markers

In this study, genetic linkage maps were constructed for ‘Fina Sodea’ clementine (Citrus clementina) and Byungkyul (C. platymamma) using 152 F1 clones as a mapping population. Reference-guided whole-genome sequencing, using next-generation sequencing technology, along with 82 randomly amplified polymorphic DNA markers and seven genomic simple-sequence repeat (gSSR) loci, mined 41 polymorphic gSSR loci from the direct comparative analysis. The generated linkage map for ‘Fina Sodea’ clementine contained 22 markers distributed into 10 linkage groups, with a total length of 417.7 cM and an average interval of 19.0 cM. The map for Byungkyul contained 57 markers distributed into 10 linkage groups, with a total length of 684.9 cM and a smaller average interval of 12.0 cM. Comparison of shared gSSR markers between the two maps identified collinearities, such as linkage groups B3 and C7 and groups B9 and C7. The newly developed gSSR markers greatly enrich the limited amount of gSSR markers in Citrus, providing markers that can be useful for comparative mapping and other genetic studies.

Genetic diversity spectrum and marker trait association for agronomic traits in global accessions of Linum usitatissimum L.

A thorough understanding of genetic diversity in Linum usitatissimum L. will equip scientist to address ever expanding breeding challenges in the species. Present study was undertaken to assess the genetic diversity and marker trait association analysis using 130 Linum usitatissimum L. accessions of diverse geographic origin (75 exotic, 55 indigenous), by employing 26 morpho-metric (11 qualitative and 15 quantitative) traits along with 22 simple sequence repeat (SSR) markers including 07 expressed sequence tagged-SSR (EST-SSR) and 15 genic SSR (gSSR). In the study high estimates of variability were recorded for seed yield per plant (85.12%) followed by fibre weight per plant (69.24%). Fibre weight per plant was found significantly correlated with plant height and days to flowering while seed yield per plant had significant but negative association with plant height and fibre weight per plant. First four principal components explained a total of 98.98% of the morphological variations. Fifteen gSSR markers produced 47 polymorphic bands with mean polymeric information content (PIC) value 0.326 whereas, seven EST-SSR markers (13 bands) recorded lower mean PIC value (0.245). Quantitative traits based clustering grouped all accessions into six groups. Clustering based on individual markers was neither according to origin nor morphotypes. Finally, clustering using all markers together (Morphological+EST-SSR+gSSR) divided flax type and linseed type accessions into separate groups. Pooled markers based clustering well supplemented by Nei’s genetic distance. Multiple regression analysis using all markers together revealed 95 polymorphic marker alleles (gSSR: 48, EST-SSR: 10 and morphological markers: 37) associated with one or more quantitative traits. For fibre weight per plant 11 marker alleles were found associated and explained 62.4% of variations. However, finding of the present study can be further strengthened with inclusion of more number of markers putatively associated with economically important traits.

Rangewide population differentiation and population substructure in Quercus rubra L.

Genetic diversity and differentiation in Quercus rubra remains undescribed for most of the native range. Using 10 highly informative gSSR markers, we genotyped 23 populations (980 trees), from across the native range, at local and rangewide spatial scales. We used a hierarchical Bayesian method to generate population-specific F st estimates. The posterior probabilities of 3 spatial and 19 climate factors revealed that latitude and precipitation in the warmest quarter were the only plausible models for population-specific F st estimates. Population-specific F st estimates increased as a linear function of population latitude (R 2 = 0.745, p < 0.0001), the expected effect of postglacial range expansion. Measures of shared ancestry (pairwise local differentiation) among populations sampled at fine spatial scales within locations also increased with the latitude of the location (R 2 = 0.495, p = 0.014), suggesting an influence on differentiation that could not be attributed to postglacial range expansion. As precipitation in the warmest quarter drives radial growth rate in this species, we propose that growth rate influences generation time, which determines the time required to re-establish migration-drift equilibrium among local regenerating stands. Population substructure analysis detected four groups, only one of which was geographically coherent. Most other populations were moderately admixed. Sampling designs that include both fine and coarse spatial scales can begin to disentangle the effect of past range expansions from the effect of recent disturbances. Accounting for population substructure in this species will require additional studies involving functional genes, interspecific interactions, and species distribution modeling.

Molecular characterization and genetic diversity of Jatropha curcas L. in Costa Rica.

We estimated the genetic diversity of 50 Jatropha curcas samples from the Costa Rican germplasm bank using 18 EST-SSR, one G-SSR and nrDNA-ITS markers. We also evaluated the phylogenetic relationships among samples using nuclear ribosomal ITS markers. Non-toxicity was evaluated using G-SSRs and SCARs markers. A Neighbor-Joining (NJ) tree and a Maximum Likelihood (ML) tree were constructed using SSR markers and ITS sequences, respectively. Heterozygosity was moderate (He = 0.346), but considerable compared to worldwide values for J. curcas. The PIC (PIC = 0.274) and inbreeding coefficient (f = − 0.102) were both low. Clustering was not related to the geographical origin of accessions. International accessions clustered independently of collection sites, suggesting a lack of genetic structure, probably due to the wide distribution of this crop and ample gene flow. Molecular markers identified only one non-toxic accession (JCCR-24) from Mexico. This work is part of a countrywide effort to characterize the genetic diversity of the Jatropha curcas germplasm bank in Costa Rica.

SSR and morphological trait based population structure analysis of 130 diverse flax (Linum usitatissimum L.) accessions

A total of 130 flax accessions of diverse morphotypes and worldwide origin were assessed for genetic diversity and population structure using 11 morphological traits and microsatellite markers (15 gSSRs and 7 EST–SSRs). Analysis performed after classifying these accessions on the basis of plant height, branching pattern, seed size, Indian/foreign origin into six categories called sub-populations viz. fibre type exotic, fibre type indigenous, intermediate type exotic, intermediate type indigenous, linseed type exotic and linseed type indigenous. The study assessed different diversity indices, AMOVA, population structure and included a principal coordinate analysis based on different marker systems. The highest diversity was exhibited by gSSR markers (SI=0.46; He=0.31; P=85.11). AMOVA based on all markers explained significant difference among fibre type, intermediate type and linseed type populations of flax. In terms of variation explained by different markers, EST-SSR markers (12%) better differentiated flax populations compared to morphological (9%) and gSSR (6%) markers at P=0.01. The maximum Nei's unbiased genetic distance (D=0.11) was observed between fibre type and linseed type exotic sub-populations based on EST-SSR markers. The combined structure analysis by using all markers grouped Indian fibre type accessions (63.4%) in a separate cluster along with the Indian intermediate type (48.7%), whereas Indian accessions (82.16%) of linseed type constituted an independent cluster. These findings were supported by the results of the principal coordinate analysis. Morphological markers employed in the study found complementary with microsatellite based markers in deciphering genetic diversity and population structure of the flax germplasm.

Development of genomic simple sequence repeats (g-SSR) markers in Tinospora cordifolia and their application in diversity analyses

Tinospora cordifolia, a medicinal plant of economic importance, has very rich diversity and distributed across India growing in wild. However, for its judicious exploitation through targeted breeding program there is complete lack of genomic resources which is necessary for its desired utilization. In the present study, we have developed novel genomic-SSRs (g-SSR) markers, which due to their wider distribution across the genome find greater usefulness in assessing genetic diversity. For development of g-SSR markers, four microsatellite enriched libraries [(CT)14, (AC)10, (GT)12 and (AAC)8] were constructed from genomic DNA of T. cordifolia accession (IC-281965) by using a streptavidin-biotin based enrichment approach. The 536 random recombinant clones from the four SSR enriched libraries were further verified by colony PCR for the presence of insert. From such screening, 356 clones were found positive and were subjected to sequencing using Sanger sequencing method. The sequence analyses revealed that 114 clones (32%) were having SSR repeats. Further analysis, based on microsatellite library enrichment showed that 60% SSR repeats were found in clones from GT enriched libraries followed by 38%, 23% and 19% in CT, AAC and AC enriched libraries respectively. Altogether 90 SSR sequences were submitted to the National Center for Biotechnology Information (NCBI) with accession numbers from KT384079 to KT384168. For diversity study 42 primer pairs were designed and all primers reproducibly amplified across all accessions of T. cordifolia. However, when such markers were used in analysis for Tinospora rumphii and Tinospora sinensis, only 83.3% and 85.7% of the markers were transferred successfully. The alleles generated across 28 accessions of three Tinospora species were assessed for genetic relatedness and it grouped accessions into three major clusters suggesting sufficient diversity in Tinospora. The g-SSR markers generated in the present study are a valuable genomic resource for effective utilization in crop improvement of Tinospora germplasm.

Development of SSR markers and assessment of genetic diversity of adzuki bean in the Chinese germplasm collection

Adzuki bean is an important food legume crop in East Asia. China is ranked as the number one country for its production and consumption. A large number of adzuki bean accessions are maintained in the Chinese national seed genebank. Tapping its genetic diversity may have potential in assisting breeding programs for new cultivar development and improvement. However, its genetic diversity was not well evaluated due to lack of sufficient DNA markers. For marker development, we initiated de novo transcriptome sequencing of adzuki bean. From sequence assembly, 65,950 unigenes were identified, and some of them were used for EST-SSR (eSSR) marker development. A set of 53 SSR markers were developed from adzuki bean, and 110 SSR (including 57 already available) markers were used for genotyping of 261 selected accessions. These adzuki bean accessions were classified into 10 clusters. The accessions from North China were well separated from the accessions from South China. The average polymorphic information content of genomic SSR (gSSR) markers and eSSR markers was 0.5081 and 0.2382, respectively. Results indicate that gSSR markers were more polymorphic than eSSR markers. These polymorphic eSSR and gSSR markers will be useful for assisting adzuki-bean-breeding programs and future germplasm curation.