Transcriptome Sequencing of Mung Bean (Vigna radiate L.) Genes and the Identification of EST-SSR Markers

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.

[1]  S. Gupta,et al.  Development of EST-derived microsatellite markers in mungbean [Vigna radiata (L.) Wilczek] and their transferability to other Vigna species , 2012 .

[2]  Yong-Jin Park,et al.  Efficiency of PowerCore in core set development using amplified fragment length polymorphic markers in mungbean , 2012 .

[3]  S. Gupta,et al.  Development of unigene-derived SSR markers in cowpea (Vigna unguiculata) and their transferability to other Vigna species. , 2010, Genome.

[4]  I. Nookaew,et al.  A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene expression and cross-comparison with microarrays: a case study in Saccharomyces cerevisiae , 2012, Nucleic acids research.

[5]  Lijuan Cong,et al.  Characterization of soybean genomic features by analysis of its expressed sequence tags , 2004, Theoretical and Applied Genetics.

[6]  M. Gerstein,et al.  RNA-Seq: a revolutionary tool for transcriptomics , 2009, Nature Reviews Genetics.

[7]  S. Gupta,et al.  Development and characterization of genic SSR markers for mungbean (Vigna radiata (L.) Wilczek) , 2013, Euphytica.

[8]  P. Somta,et al.  Development, characterization and cross-species amplification of mungbean (Vigna radiata) genic microsatellite markers , 2009, Conservation Genetics.

[9]  Colin N. Dewey,et al.  De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis , 2013, Nature Protocols.

[10]  M. Englen,et al.  A rapid DNA isolation procedure for the identification of Campylobacter jejuni by the polymerase chain reaction , 2000, Letters in applied microbiology.

[11]  I. Métais,et al.  Characterization of AT-rich microsatellites in common bean (Phaseolus vulgaris L.) , 2008, Theoretical and Applied Genetics.

[12]  Mukesh Jain,et al.  Transcriptome sequencing of wild chickpea as a rich resource for marker development. , 2012, Plant biotechnology journal.

[13]  Yong-Jin Park,et al.  Sequence information on simple sequence repeats and single nucleotide polymorphisms through transcriptome analysis of mungbean. , 2011, Journal of integrative plant biology.

[14]  Hongwei Jiang,et al.  Identification and characterization of SSRs from soybean (Glycine max) ESTs , 2012, Molecular Biology Reports.

[15]  Akhilesh K. Tyagi,et al.  De Novo Assembly of Chickpea Transcriptome Using Short Reads for Gene Discovery and Marker Identification , 2011, DNA research : an international journal for rapid publication of reports on genes and genomes.

[16]  Jia-kuan Chen,et al.  Characterization and high cross‐species transferability of microsatellite markers from the floral transcriptome of Aspidistra saxicola (Asparagaceae) , 2014, Molecular ecology resources.

[17]  M. Blair,et al.  Genetic diversity of Chinese common bean (Phaseolus vulgaris L.) landraces assessed with simple sequence repeat markers , 2008, Theoretical and Applied Genetics.

[18]  R. Varshney,et al.  Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.) , 2003, Theoretical and Applied Genetics.

[19]  M. Ishimoto,et al.  Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean (Vigna radiata L. Wilczek) , 1998, Molecular and General Genetics MGG.

[20]  Huei-Mei Chen,et al.  Development of a molecular marker for a bruchid (Callosobruchus chinensis L.) resistance gene in mungbean , 2007, Euphytica.

[21]  M. Blair,et al.  Development of microsatellite markers for common bean (Phaseolus vulgaris L.) based on screening of non-enriched, small-insert genomic libraries. , 2009, Genome.

[22]  Chaozhu Yang,et al.  Development of microsatellite markers by transcriptome sequencing in two species of Amorphophallus (Araceae) , 2013, BMC Genomics.

[23]  N. Friedman,et al.  Comprehensive comparative analysis of strand-specific RNA sequencing methods , 2010, Nature Methods.

[24]  R. Nair,et al.  Biofortification of mungbean (Vigna radiata) as a whole food to enhance human health. , 2013, Journal of the science of food and agriculture.

[25]  Suk-Ha Lee,et al.  Genome-wide SNP discovery in mungbean by Illumina HiSeq , 2013, Theoretical and Applied Genetics.

[26]  Michael Krawczak,et al.  PopGen: Population-Based Recruitment of Patients and Controls for the Analysis of Complex Genotype-Phenotype Relationships , 2006, Public Health Genomics.

[27]  M. Gerstein,et al.  Comparison and calibration of transcriptome data from RNA-Seq and tiling arrays , 2010, BMC Genomics.

[28]  S. Isobe,et al.  Detection of Genome Donor Species of Neglected Tetraploid Crop Vigna reflexo-pilosa (Créole Bean), and Genetic Structure of Diploid Species Based on Newly Developed EST-SSR Markers from Azuki Bean (Vigna angularis) , 2014, PloS one.

[29]  K. Shinozaki,et al.  Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.) , 2011, BMC Plant Biology.

[30]  M. Blair,et al.  Development and diversity of Andean-derived, gene-based microsatellites for common bean (Phaseolus vulgaris L.) , 2009, BMC Plant Biology.

[31]  Ju-Kyung Yu,et al.  Nonrandom distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley , 2005, BMC Genomics.

[32]  R. Wing,et al.  Gene-based SSR markers for common bean (Phaseolus vulgaris L.) derived from root and leaf tissue ESTs: an integration of the BMc series , 2011, BMC Plant Biology.

[33]  Juan Miguel García-Gómez,et al.  BIOINFORMATICS APPLICATIONS NOTE Sequence analysis Manipulation of FASTQ data with Galaxy , 2005 .

[34]  Soon-Jae Kwon,et al.  Assessment of genetic diversity and population structure in mungbean , 2010, Genes & Genomics.

[35]  N. S. Talekar,et al.  RFLP mapping of a major bruchid resistance gene in mungbean (Vigna radiata, L. Wilczek) , 1992, Theoretical and Applied Genetics.

[36]  S. Tangphatsornruang,et al.  The Chloroplast Genome Sequence of Mungbean (Vigna radiata) Determined by High-throughput Pyrosequencing: Structural Organization and Phylogenetic Relationships , 2009, DNA research : an international journal for rapid publication of reports on genes and genomes.

[37]  T. Sharma,et al.  RESEARCH ARTICLE Open Access Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh] , 2022 .

[38]  N. Sethy,et al.  Development of chickpea EST-SSR markers and analysis of allelic variation across related species , 2009, Theoretical and Applied Genetics.