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Molecular Characterization of Mungbean

Md. Rezwan Molla
Scientific Officer
Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute(BARI), Joydebpur, Gazipur-1701, Bangladesh

M.A.N. Nazim-ud-Dowla
Scientific Officer
Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute(BARI), Joydebpur, Gazipur-1701, Bangladesh

Dr. Md. Khaled Sultan
Chief Scientific Officer
Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute(BARI), Joydebpur, Gazipur-1701, Bangladesh

Abstract/ Executive Summary:

Characterization of mungbean (Vigna radiata L.) accessions on the basis of DNA fingerprinting has become an efficient tool to link genotypic variation. This work is reporting the utilization of a small set of five previously developed mungbean microsatellite markers for the identification and discrimination of six HYVs and 36 local cultivars. All five microsatellite markers were found to be polymorphic. Using five primers across 42 accessions a total of 20 alleles with an average number of 4 alleles per locus were found of which GBssr-MB91 showed highest number of alleles (6) (size ranging from 135 bp to 152 bp. The primer GBssr-MB91 motif [(AG)34(GA)14] also yielded highest number of PIC value (0.803). Average genetic differentiation (Fst) values and gene flow (Nm) values were found 0.686 and 0.237 respectively. Our data indicated that a narrow genetic base among the mungbean accessions in this study. Over all Nei’s genetic distance value (D) from nil to 2.706 among 861 accessions pair resulting as a means of permutation combination of 42 mungbean accessions. The UPGMA dendogram based on Nei’s genetic distance separated the accessions. Out of 42 accessions, 36 accessions were identified with at least one and/or combination of 4 primers.

Keywords: Vigna radiata, DNA fingerprinting, Microsatellite marker, Genetic distance, Gene flow

Location: Molecular Biology Laboratory, Plant Genetic Resource Center, Bangladesh Agricultural Research Institute (BARI), Gazipur

Start Date: 01-08-2010

End Date: 01-03-2011

Program Area: Variety and Species

Commodity: Mungbean

Objectives:

1) To identify accessions with unique DNA banding pattern using SSRs marker 2) To estimate genetic diversity 3) To identify duplicate accessions among the collection 4) To establish genetic relationship

Methodology:

A total of 42 accessions including six commercial varieties, one most popular local cultivar and 35 local cultivars of mungbean representing a wide spectrum of variability were selected randomly for the present study. Bulked DNA was isolated from 2-5 fresh leaves of 10 days old seedlings using following the protocol described by Saghai-Maroof et al. (1984) and also used by Rahman et al. (2007) with some modifications. The amount of genomic DNA was quantified at 260 nm spectrophotometrically (Spectronic® GENESYS™ 10 Bio). Five primer pairs viz. LR7322 B, LR 7323 A, LR7323 B, GBssr-MB91 and GBssr-MB77 with clear and expected amplified product sizes were selected and used for microsatellite analysis in the present study. The Polymerase chain reactions was set up 10 μl volumes containing 100 ng template DNA, 10 x PCR Buffer, 0.25 mM each of the dNTPs, 2 μM of each of primer, 1 unit ampli Taq DNA polymerase (Genei, India) and a suitable amount of sterile deionized water. The reaction was performed in a oil free Techne, TC 312 thermal cycler. The success of the reaction product following 2% agarose gel electrophoresis. PCR-products were electrophoresed on a 6% denaturing polyacrylamide gel containing 19:1 acrylamide: bis-acrylamide and 8M urea. Electrophoresis was done using the SequiGen GT Sequencing Cell (BIO-RAD Laboratories, Hercules, CA, USA) electrophoresis system. After completion of electrophoresis, the DNA fragments were visualized following the Promega (Madison, WI) silver-staining protocol. The software DNA FRAG version 3.03 was used to estimate allelic length (Nash, 1991). Expected (He) and observed heterozygosity (Ho) were also calculated as per Nei (1972) formula. Estimation of Nei’s genetic distance values (D) and construction of UPGMA dendrogram was constructed using the software POPGENE (Version 1.31) (Yeh et al., 1999).

Source of Information: ANNUAL RESEARCH REPORT OF PLANT GENETIC RESOURCES CENTRE-2010-2011

Article Link (Online Journal):

Funding Source:

1. Government

Budget: Tk. 1,50,000.00

Total Budget (Tk.) : Tk. 1,50,000.00

Achievement/Findings:

The set of microsatellite markers used here provides a positive assessment to the ability of SSR marker to produce unique DNA profiles of mungbean accessions. The results of the present study could be applied as baseline information to maintain the appropriate identity and the construction of a database of all mungbean cultivars and their wild relatives grown in Bangladesh and in broad sense, to protect the plant varieties of Bangladesh. Inter-mating cultivars from the major distinct gene pools could provide new genetic recombination to exploit in cultivar development programme.

Knowledge Management: Report/Proceedings