Abstract
Mung bean (Vigna radiate (L.) Wilczek) is an important traditional food legume crop, with high economic and nutritional value. It is widely grown in China and other Asian countries. Despite its importance, genomic information is currently unavailable for this crop plant species or some of its close relatives in the Vigna genus. In this study, more than 103 million high quality cDNA sequence reads were obtained from mung bean using Illumina paired-end sequencing technology. The processed reads were assembled into 48,693 unigenes with an average length of 874 bp. Of these unigenes, 25,820 (53.0%) and 23,235 (47.7%) showed significant similarity to proteins in the NCBI non-redundant protein and nucleotide sequence databases, respectively. Furthermore, 19,242 (39.5%) could be classified into gene ontology categories, 18,316 (37.6%) into Swiss-Prot categories and 10,918 (22.4%) into KOG database categories (E-value < 1.0E-5). A total of 6,585 (8.3%) were mapped onto 244 pathways using the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database. Among the unigenes, 10,053 sequences contained a unique simple sequence repeat (SSR), and 2,303 sequences contained more than one SSR together in the same expressed sequence tag (EST). A total of 13,134 EST-SSRs were identified as potential molecular markers, with mono-nucleotide A/T repeats being the most abundant motif class and G/C repeats being rare. In this SSR analysis, we found five main repeat motifs: AG/CT (30.8%), GAA/TTC (12.6%), AAAT/ATTT (6.8%), AAAAT/ATTTT (6.2%) and AAAAAT/ATTTTT (1.9%). A total of 200 SSR loci were randomly selected for validation by PCR amplification as EST-SSR markers. Of these, 66 marker primer pairs produced reproducible amplicons that were polymorphic among 31 mung bean accessions selected from diverse geographical locations. The large number of SSR-containing sequences found in this study will be valuable for the construction of a high-resolution genetic linkage maps, association or comparative mapping and genetic analyses of various Vigna species.
Highlights
Mung bean belongs to the Vigna genus within the Phaseoleae tribe and is a diploid crop (2n = 2x = 22) with a genome size of approximately 560 Mb
Transcriptome sequencing and de novo assembly has proven to be an important tool for gene discovery in many organisms and an effective method for molecular marker development [30,31]
Our results proved that the short reads from Illumina paired-end sequencing of mung bean cDNAs can be assembled and used for transcriptome analysis, marker development and gene identification even without a reference genome for the crop
Summary
Map-based cloning and molecular breeding of mung bean have lagged behind other legume crops due to the lack of genomic information for this pulse crop species [2]. Previous efforts to develop molecular markers for mung bean have not generated sufficient markers for linkage saturated map construction because they were either monomorphic or not fully informative for bi-parental mapping populations. This has been the case for RFLP [3], RAPD [4], AFLP [5], CAPS [6] and SNP [7] markers. Microsatellites ( known as SSRs based on their Simple Sequence Repeat core) are a logical choice for broadening the scope of markers available to mung bean researchers
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