De novo Assembly, Characterization of Immature Seed Transcriptome and Development of Genic-SSR Markers in Black Gram [Vigna mungo (L.) Hepper]

Abstract

Black gram [V. mungo (L.) Hepper] is an important legume crop extensively grown in south and south-east Asia, where it is a major source of dietary protein for its predominantly vegetarian population. However, lack of genomic information and markers has become a limitation for genetic improvement of this crop. Here, we report the transcriptome sequencing of the immature seeds of black gram cv. TU94-2, by Illumina paired end sequencing technology to generate transcriptome sequences for gene discovery and genic-SSR marker development. A total of 17.2 million paired-end reads were generated and 48,291 transcript contigs (TCS) were assembled with an average length of 443 bp. Based on sequence similarity search, 33,766 TCS showed significant similarity to known proteins. Among these, only 29,564 TCS were annotated with gene ontology (GO) functional categories. A total number of 138 unique KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were identified, of which majority of TCS are grouped into purine metabolism (678) followed by pyrimidine metabolism (263). A total of 48,291 TCS were searched for SSRs and 1,840 SSRs were identified in 1,572 TCS with an average frequency of one SSR per 11.9 kb. The tri-nucleotide repeats were most abundant (35%) followed by di-nucleotide repeats (32%). PCR primer pairs were successfully designed for 933 SSR loci. Sequences analyses indicate that about 64.4% and 35.6% of the SSR motifs were present in the coding sequences (CDS) and untranslated regions (UTRs) respectively. Tri-nucleotide repeats (57.3%) were preferentially present in the CDS. The rate of successful amplification and polymorphism were investigated using selected primers among 18 black gram accessions. Genic-SSR markers developed from the Illumina paired end sequencing of black gram immature seed transcriptome will provide a valuable resource for genetic diversity, evolution, linkage mapping, comparative genomics and marker-assisted selection in black gram.

De novo sequencing and characterization of seed transcriptome of the tree legume Millettia pinnata for gene discovery and SSR marker development

“…In the present study, 5710 putative EST-SSRs were identified from the transcriptome dataset. The distribution density of one SSR per 7.39 kb was higher than those reported for alfalfa (1/12.06 kb) (Liu et al 2013) and black gram (1/11.90 kb) (Souframanien and Reddy 2015), but was lower than those in chickpea (1/ 5.80 kb) (Garg et al 2011) and common bean (1/ 4.70 kb) (Wu et al 2014). Though the transcriptomes of these legume species were all generated by Illumina sequencing, the differences in SSR abundance might still be caused by inconsistency in genome structure or composition, dataset size, search method and criteria.…”

“…Transcriptome sequencing and de novo assembly resulted in 53,586 Pongamia unigenes with a mean length of 787 bp. Compared with the results from other legume species generated by the Illumina sequencing platform, the mean length of unigenes for Pongamia was longer than those for chickpea (523 bp) (Garg et al 2011), peanut (619 bp) , common bean (777 bp) (Wu et al 2014) and black gram (443 bp) (Souframanien and Reddy 2015), but shorter than those for alfalfa (803 bp) (Liu et al 2013), adzuki bean (1213 bp) (Chen et al 2015a) and mung bean (874 bp) (Chen et al 2015b). Moreover, the average sequencing depth of all unigenes was up to 130.7-fold.…”

“…Similarly, the unigenes involved in lipid metabolism were abundant in the seed transcriptome of peanut (Yin et al 2013). Nevertheless, lipid metabolism was not well represented by unigenes in the seed transcriptome of black gram, another legume species not rich in seed oil (Souframanien and Reddy 2015). Based on KEGG assignments, we further singled out 364 unigenes within five pathways associated with fatty acid and glycerolipid metabolism.…”

“…In recent years, the NGS-based transcriptome analyses have already been applied in more than a dozen legume species (Garg et al 2011;Zhang et al 2012;Garg and Jain 2013;Liu et al 2013;Hiz et al 2014;Chen et al 2015a, b;Souframanien and Reddy 2015). In a previous study, we employed the Illumina sequencing platform to produce a large-scale collection of Pongamia transcripts from root and leaf tissues for studying their transcriptome changes under salt treatments (Huang et al 2012).…”

Cross Species Amplification of Cowpea Derived Unigene-SSR Markers and Diversity Analysis in Black Gram [Vigna mungo (L.) Hepper]

“…In black gram, there are no reports of SSR marker development except development of genic-SSR markers from transcriptome sequences of immature pods (Souframanien and Reddy 2015). Therefore, SSR markers from other species have been used for genetic analysis Gupta and Gopalakrishna 2009;Gupta et al 2013).…”