Sebastin Raveendar

Cross-Amplification of Vicia sativa subsp. sativa Microsatellites across 22 Other Vicia Species

The temperate and herbaceous genus Vicia L. is a member of the legume tribe Fabeae of the subfamily Papilionoideae. The genus Vicia comprises 166 annual or perennial species distributed mainly in Europe, Asia, and North America, but also extending to the temperate regions of South America and tropical Africa. The use of simple sequence repeat (SSR) markers for Vicia species has not been investigated as extensively as for other crop species. In this study, we assessed the potential for cross-species amplification of cDNA microsatellite markers developed from common vetch (Vicia sativa subsp. sativa). For cross-species amplification of the SSRs, amplification was carried out with genomic DNA isolated from two to eight accessions of 22 different Vicia species. For individual species or subspecies, the transferability rates ranged from 33% for V. ervilia to 82% for V. sativa subsp. nigra with an average rate of 52.0%. Because the rate of successful SSR marker amplification generally correlates with genetic distance, these SSR markers are potentially useful for analyzing genetic relationships between or within Vicia species.

The Complete Chloroplast Genome of Capsicum annuum var. glabriusculum Using Illumina Sequencing

Chloroplast (cp) genome sequences provide a valuable source for DNA barcoding. Molecular phylogenetic studies have concentrated on DNA sequencing of conserved gene loci. However, this approach is time consuming and more difficult to implement when gene organization differs among species. Here we report the complete re-sequencing of the cp genome of Capsicum pepper (Capsicum annuum var. glabriusculum) using the Illumina platform. The total length of the cp genome is 156,817 bp with a 37.7% overall GC content. A pair of inverted repeats (IRs) of 50,284 bp were separated by a small single copy (SSC; 18,948 bp) and a large single copy (LSC; 87,446 bp). The number of cp genes in C. annuum var. glabriusculum is the same as that in other Capsicum species. Variations in the lengths of LSC; SSC and IR regions were the main contributors to the size variation in the cp genome of this species. A total of 125 simple sequence repeat (SSR) and 48 insertions or deletions variants were found by sequence alignment of Capsicum cp genome. These findings provide a foundation for further investigation of cp genome evolution in Capsicum and other higher plants.

Transcriptome Analysis of Two Vicia sativa Subspecies: Mining Molecular Markers to Enhance Genomic Resources for Vetch Improvement

The vetch (Vicia sativa) is one of the most important annual forage legumes globally due to its multiple uses and high nutritional content. Despite these agronomical benefits, many drawbacks, including cyano-alanine toxin, has reduced the agronomic value of vetch varieties. Here, we used 454 technology to sequence the two V. sativa subspecies (ssp. sativa and ssp. nigra) to enrich functional information and genetic marker resources for the vetch research community. A total of 86,532 and 47,103 reads produced 35,202 and 18,808 unigenes with average lengths of 735 and 601 bp for V. sativa sativa and V. sativa nigra, respectively. Gene Ontology annotations and the cluster of orthologous gene classes were used to annotate the function of the Vicia transcriptomes. The Vicia transcriptome sequences were then mined for simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. About 13% and 3% of the Vicia unigenes contained the putative SSR and SNP sequences, respectively. Among those SSRs, 100 were chosen for the validation and the polymorphism test using the Vicia germplasm set. Thus, our approach takes advantage of the utility of transcriptomic data to expedite a vetch breeding program.

The complete chloroplast genome sequence of Korean landrace "Subicho" pepper (Capsicum annuum var. annuum).

Chloroplast DNA sequences are a versatile tool for species identification and phylogenetic reconstruction of land plants. Different chloroplast loci have been utilized for phylogenetic classification of plant species. However, there is no report for a short DNA sequence that can distinguish all plant species from each other. Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. Thus, the complete chloroplast genome sequence of Korean landrace “Subicho” pepper (Capsicum annuum var. annuum) has been determined here. The total length of the chloroplast genome is 156,878 bp, with 37.7% overall GC content. A pair of IRs (inverted repeats) of 25,801 bp was separated by a small single copy (SSC) region of 17,929 bp and a large single copy (LSC) region of 87,347 bp. The chloroplast genome harbors 132 known genes, including 87 protein-coding genes, 8 ribosomal RNA genes, and 37 tRNA genes. A total of seven of these genes are duplicated in the inverted repeat regions, nine genes and six tRNA genes contain one intron, while two genes and a ycf have two introns. Analysis revealed 144 simple sequence repeat (SSR) loci and 96 variants, mostly located in the intergenic regions. The types and abundances of repeat units in Capsicum species were relatively conserved and these loci will be useful for developing C. annuum cp genome vectors.

The complete chloroplast genome sequence of Glycyrrhiza lepidota (Nutt.) Pursh - An American wild licorice

The wild species are considered as primary and secondary genepools for the world’s most important food crops. Here, we sequenced the complete chloroplast (cp) genome sequence of an American wild licorice, Glycyrrhiza lepidota for the first time to investigate their phylogenetic relationship among inverted-repeat-lacking clade (IRLC) legumes. The total length of the chloroplast genome is 127,939 bp, with 34.2% overall GC content. The chloroplast genome harbors 110 known genes, including 76 protein-coding genes, four ribosomal RNA genes, and 30 tRNA genes. Similar to other closely related plastomes, rpl22 and rps16 are absent. A total of 464 cp microsatellites (cpSSRs) were analyzed in the G. lepidota. The majority of the SSRs in this cp genome are penta-nucleotides (61.6%). Locally collinear blocks (LCBs) identified between the Glycyrrhiza glabra and G. lepidota cp genomes were showed that they were well conserved with respect to gene organization and order. Moreover, the phylogenetic analysis indicates that G. lepidota is closely related to its confamilial counterparts than to the other taxa of the IRLC legumes.

The Complete Chloroplast Genome of Capsicum frutescens (Solanaceae)

Premise of the study: We report the complete sequence of the chloroplast genome of Capsicum frutescens (Solanaceae), a species of chili pepper. Methods and Results: Using an Illumina platform, we sequenced the chloroplast genome of C. frutescens. The total length of the genome is 156,817 bp, and the overall GC content is 37.7%. A pair of 25,792-bp inverted repeats is separated by small (17,853 bp) and large (87,380 bp) single-copy regions. The C. frutescens chloroplast genome encodes 132 unique genes, including 87 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. Of these, seven genes are duplicated in the inverted repeats and 12 genes contain one or two introns. Comparative analysis with the reference chloroplast genome revealed 125 simple sequence repeat motifs and 34 variants, mostly located in the noncoding regions. Conclusions: The complete chloroplast genome sequence of C. frutescens reported here is a valuable genetic resource for Capsicum species.

Potential use of ITS2 and matK as a Two-Locus DNA Barcode for Identification of Vicia Species

We investigated the species discriminatory efficiency of the proposed plant barcoding loci ITS2 and matK in Vicia species. In 2011, China Plant BOL Group proposed the addition of nuclear ITS2 to matK be accepted as a 2-locus DNA barcode to classify plant species. The matK region was chosen as a DNA barcode because of its effective species discriminating power, high quality sequence recovery, and easy experimental analysis. Integration of matK sequences into Vicia phylogeny could improve phylogenetic reconstruction of this species. To assess the ability of barcoding loci to resolve Vicia species, we sampled 36 of the taxonomically best known groups in the genus. Topologies of the phylogenetic trees based on ITS2 and matK analyses were similar but a few accessions were placed into distant phylogenetic groups. Neither ITS2 nor matK analyses alone could discriminate some closely related Vicia species. Thus, we have proposed a concatenated data approach to increase the resolving power of ITS2 and used matK as an additional tool for phylogenetic analysis in Vicia because characterization of the nucleotide sequences of the matK region was easier to recover and more cost-effective than those of the ITS region.

Identification of genus Vigna using ITS2 and matK as a two-locus DNA barcode.

DNA barcoding is the use of short DNA sequences of the genome for large scale species identification. The Consortium for the Barcode of Life (CBOL) plant-working group recommended a 2-locus combination as the standard plant barcode. The evolutions of the chloroplast regions combined with nuclear gens are sufficiently rapid to allow discrimination between closely related species. We evaluated the efficacy of the proposed plant barcoding loci, matK, along with ITS2 for barcoding the Vigna species. To assess the discriminatory ability of barcoding loci for identifying the Vigna species, we sampled 52 of the taxonomically best known groups in the genus. Topologies of the phylogenetic trees based on ITS2 and matK analyses were similar but a few accessions were placed into distant phylogenetic groups. Neither ITS2 nor matK analyses were able to discriminate some closely related Vigna species. Thus, we used concatenated data to increase the resolving power of ITS2 and used matK as an additional tool for phylogenetic analysis in Vigna because characterization of the nucleotide sequences of the matK region was easier and more cost-effective than that of the ITS region.

Development of cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers from the chloroplast genome of Glycyrrhiza species

Licorice (Glycyrrhiza glabra) is an important medicinal crop often used as health foods or medicine worldwide. The molecular genetics of licorice is under scarce owing to lack of molecular markers. Here, we have developed cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers based on single nucleotide polymorphisms (SNP) by comparing the chloroplast genomes of two Glycyrrhiza species (G. glabra and G. lepidota). The CAPS and HRM markers were tested for diversity analysis with 24 Glycyrrhiza accessions. The restriction profiles generated with CAPS markers classified the accessions (2–4 genotypes) and melting curves (2–3) were obtained from the HRM markers. The number of alleles and major allele frequency were 2−6 and 0.31–0.92, respectively. The genetic distance and polymorphism information content values were 0.16–0.76 and 0.15–0.72, respectively. The phylogenetic relationships among the 24 accessions were estimated using a dendrogram, which classified them into four clades. Except clade III, the remaining three clades included the same species, confirming interspecies genetic correlation. These 18 CAPS and HRM markers might be helpful for genetic diversity assessment and rapid identification of licorice species.

Comparative efficacy of four candidate DNA barcode regions for identification of Vicia species

The genus Vicia L., one of the earliest domesticated plant genera, is a member of the legume tribe Fabeae of the subfamily Papilionoideae ( Fabaceae ). The taxonomic history of this genus is extensive and controversial, which has hindered the development of taxonomic procedures and made it difficult to identify and share these economically important crop resources. Species identification through DNA barcoding is a valuable taxonomic classification tool. In this study, four DNA barcodes (ITS2, matK , rbcL and psbA-trnH ) were evaluated on 110 samples that represented 34 taxonomically best-known species in the Vicia genus. Topologies of the phylogenetic trees based on an individual locus were similar. Individual locus-based analyses could not discriminate closely related Vicia species. We proposed a concatenated data approach to increase the resolving power of ITS2. The DNA barcodes matK , psbA-trnH and rbcL were used as an additional tool for phylogenetic analysis. Among the four barcodes, three-barcode combinations that included psbA-trnH with any two of the other barcodes (ITS2, matK or rbcL ) provided the best discrimination among Vicia species. Species discrimination was assessed with bootstrap values and considered successful only when all the conspecific individuals formed a single clade. Through sequencing of these barcodes from additional Vicia accessions, 17 of the 34 known Vicia species could be identified with varying levels of confidence. From our analyses, the combined barcoding markers are useful in the early diagnosis of targeted Vicia species and can provide essential baseline data for conservation strategies, as well as guidance in assembling germplasm collections.