Background
Blackgram [
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.
Results
Transcriptome sequencing of immature seeds of wild blackgram,
V. mungo
var
. silvestris
by Illumina MiSeq technology generated 1.9 × 10
7
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.
Conclusion
In 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.
Electronic supplementary material
The online version of this article (10.1186/s12870-019-1954-0) contains supplementary material, which is available to authorized users.
Blackgram [Vigna mungo (L.) Hepper] (2n = 2x = 22), an important Asiatic legume crop, is a major source of dietary protein for the predominantly vegetarian population. Here we construct a draft genome sequence of blackgram, for the first time, by employing hybrid genome assembly with Illumina reads and third generation Oxford Nanopore sequencing technology. The final de novo whole genome of blackgram is ~ 475 Mb (82% of the genome) and has maximum scaffold length of 6.3 Mb with scaffold N50 of 1.42 Mb. Genome analysis identified 42,115 genes with mean coding sequence length of 1131 bp. Around 80.6% of predicted genes were annotated. Nearly half of the assembled sequence is composed of repetitive elements with retrotransposons as major (47.3% of genome) transposable elements, whereas, DNA transposons made up only 2.29% of the genome. A total of 166,014 SSRs, including 65,180 compound SSRs, were identified and primer pairs for 34,816 SSRs were designed. Out of the 33,959 proteins, 1659 proteins showed presence of R-gene related domains. KIN class was found in majority of the proteins (905) followed by RLK (239) and RLP (188). The genome sequence of blackgram will facilitate identification of agronomically important genes and accelerate the genetic improvement of blackgram.
Blackgram [Vigna mungo (L.) Hepper] (2n = 2x = 22), an important Asiatic legume crop, is a major source of dietary protein for the predominantly vegetarian population. Here we construct a draft genome sequence of blackgram, for the first time, by employing hybrid genome assembly with Illumina reads and third generation Oxford Nanopore sequencing technology. The final de novo whole genome of blackgram is ~ 475 Mb (82 % of the genome) and has maximum scaffold length of 6.3 Mb with scaffold N50 of1.42 Mb. Genome analysis identified 18655 genes with mean coding sequence length of 970bp. Around 96.7 % of predicted genes were annotated. Nearly half of the assembled sequence is composed of repetitive elements with retrotransposons as major (47.3% of genome) transposable elements, whereas, DNA transposons made up only 2.29% of the genome. A total of 166014 SSRs, including 65180 compound SSRs, were identified and primer pairs for 34816 SSRs were designed. Out of the 18665 proteins, 678 proteins showed presence of R-gene related domains. KIN class was found in majority of the proteins (372) followed by RLK (79) and N (79). The genome sequence of blackgram will facilitate identification of agronomically important genes and accelerate the genetic improvement of blackgram.
Blackgram is a widely cultivated pulse crop in Asia. Bruchid pests and yellow mosaic disease (YMD) causes huge loss in legume production including blackgram. Blackgram wild accession (Vigna mungo var. silvestris), Trombay wild urd (INGR10133) conferred resistance to bruchids especially Callosobruchus maculatus, through antibiosis. However, the mechanisms still remains uncharacterized. We performed the comparative transcriptome analysis of the developing seeds of wild and cultivated blackgram with contrasting phenotypes for 3 traits, bruchids infestation, YMD and seed size. In this study,715differentially expressed genes(DEGs) were re-annotated with reference to NCBI nr database. RNA-Seq was validated by quantitative real-time PCR for 22 DEGs. In Trombay wild, defense related components such as acid phosphatase, vicilins, trypsin inhibitor, brassinosteroid signalling components were found up-regulated. While in cultivar, transcripts for LEA, cysteine protease, autophagy related proteins(ATG3, ATG5, ATG8C and ATG1t), DnaJ, tobamovirus multiplication protein, downy mildew resistance protein, LRR/F-box proteins were found up-regulated. In TW, three transcripts were found common for both bruchids pest and geminivirus resistance (LRR receptor kinase, transmembrane protein 87b and thaumatin like protein).Our study is the first report on transcriptomic differences between wild and cultivated blackgram with new insights into the molecular networks underlying seed development, resistance to pests and pathogens.
Understanding the mechanism and nature of resistance genes in crop plants is essential for its use in new breeding techniques. Previously, a dominant rust resistance gene was fine-mapped within a 1.2 cM interval in chromosome A03 of groundnut. Here, the rust resistance gene, VG9514-Rgene was isolated through map based cloning. Sequencing of the gene from resistant and susceptible plants revealed non-synonymous mutations in the TIR, NBS and LRR region of R-protein. Genetic mapping of these SNPs-based markers confirmed the position of VG9514-Rgene in between FRS 72 and SSR_GO340445 markers in chromosome A03. Homology searching identified four homologous R-genes in groundnut genome. Of them, Arahy.R8KUIR, Arahy.T6DCA5 and Arahy.ZZ0VZ9 are paralogues. These paralogous genes had several small InDels. Mapping of these InDels-based markers revealed tandem duplication of these paralogous R-genes at distal portion of chromosome A03. Ka/Ks calculation revealed that this unique VG9514-Rgene had undergone positive selection. Homology based structure modelling of this R-protein revealed a typical consensus three dimensional folding of TIR-NBS-LRR protein. Non-synonymous mutations in susceptible version of R-protein were mapped in this protein model and found E268Q mutation in hhGRExE motif, Y309F in RNBS-A motif and I579T in MHD motif of NB-ARC domain are probable candidates for loss of function.
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