Abstract
BackgroundBlackgram [Vigna mungo (L.) Hepper], is an important legume crop of Asia with limited genomic resources. We report a comprehensive set of genic simple sequence repeat (SSR) and single nucleotide polymorphism (SNPs) markers using Illumina MiSeq sequencing of transcriptome and its application in genetic variation analysis and mapping.ResultsTranscriptome sequencing of immature seeds of wild blackgram, V. mungo var. silvestris by Illumina MiSeq technology generated 1.9 × 107 reads, which were assembled into 40,178 transcripts (TCS) with an average length of 446 bp covering 2.97 GB of the genome. A total of 38,753 CDS (Coding sequences) were predicted from 40,178 TCS and 28,984 CDS were annotated through BLASTX and mapped to GO and KEGG database resulting in 140 unique pathways. The tri-nucleotides were most abundant (39.9%) followed by di-nucleotide (30.2%). About 60.3 and 37.6% of SSR motifs were present in the coding sequences (CDS) and untranslated regions (UTRs) respectively. Among SNPs, the most abundant substitution type were transitions (Ts) (61%) followed by transversions (Tv) type (39%), with a Ts/Tv ratio of 1.58. A total of 2306 DEGs were identified by RNA Seq between wild and cultivar and validation was done by quantitative reverse transcription polymerase chain reaction. In this study, we genotyped SNPs with a validation rate of 78.87% by High Resolution Melting (HRM) Assay.ConclusionIn the present study, 1621genic-SSR and 1844 SNP markers were developed from immature seed transcriptome sequence of blackgram and 31 genic-SSR markers were used to study genetic variations among different blackgram accessions. Above developed markers contribute towards enriching available genomic resources for blackgram and aid in breeding programmes.
Highlights
Blackgram [Vigna mungo (L.) Hepper], is an important legume crop of Asia with limited genomic resources
Illumina paired end sequencing and de novo assembly of transcriptome The transcriptome sequencing of blackgram wild accession (TW) generated 19,690,124 high-quality reads which accounts for 2,970,339,385 bases (2.97 GB)
Among the 140 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified for Trombay Wild (TW), 524 coding sequences (CDS) were fall under purine metabolism which played role in ammonia assimilation and detoxification in specialized tissues [37], primary nitrogen metabolism in tropical legumes such as soybean [56], nucleotide biosynthesis and degradation in cotyledons and embryonic axes of blackgram [44]
Summary
Blackgram [Vigna mungo (L.) Hepper], is an important legume crop of Asia with limited genomic resources. India is the largest producer and consumer of blackgram and the world’s major contributor in blackgram production. It is grown in 5.44 million hectares of area with an annual production of about 3.56 million tons during 2017–2018 and an average productivity of 655 kg per hectare [3]. It is extensively grown in varied climatic conditions and soil types in India. There is a need to accelerate progress of crop improvement programmes with the use of molecular markers in conventional breeding more appropriately molecular breeding
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