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Random Amplified Polymorphic DNA (rapd) Analysis of Selected Mungbean [vigna Radiata (l.) Wilczek] Cultivars

M.A. Afzal
Pulse Research center, Bangladesh Agricultural Research Institute, Joydebpur, Bangladesh.

M. Moynul Haque
Department of Agronomy, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur, Bangladesh.

S. Shanmugasundaram
Asian Vegetable Research and Development Center, Shanhua, Tainan, Taiwan-741

Abstract/ Executive Summary:

A laboratory experiment was conducted at the Asian Vegetable Research and Development Center, Taiwan to assess the genetic diversity of Mungbean [Vigna radiata (L.) Wilczek] cultivars using DNA markers. In this study, 21 mungbean cultivars were subjected to RAPD analysis using 34 decamer primers. All the primers produced polymorphic amplification products with some extent of variation. A total of 204 bands were generated with an average of 6.0 per primer and exhibited 75.0% polymorphism. Jaccard’s similarity coefficient ranged from 0.54 to 0.85 and concentrated mostly between 0.61 to 0.74. This indicated a rather narrow genetic base of tested cultivars. Genetic similarity obtained in this study may be used for selecting parents for breeding purposes. Clustering of cultivars into four groups showed reasonable variability that might be exploited for yield improvement.

Keywords: Mungbean, Genetic diversity, RAPD marker

Location: Asian Vegetable Research and Development Center, Taiwan

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Mungbean

Objectives:

To evaluate the genetic diversityof selected mungbean cultivars using RAPD markers.

Methodology:

The experiment was carried out at the Asian Vegetable research and Development Center, Taiwan using 21 cultivars. The cultivars were chosen to compare the Bangladesh cultivars with some commonly grown Indian cultivars and newly improved lines from the Asian Vegetable Research and Development Center (AVRDC). Seeds were sown on 14 August 2002 in pots under nethouse conditions. Young, healthy leaves pooled from three 20-day-old plants were used for DNA extraction. The harvested leaves were treated with liquid nitrogen and stored at -85°C until total genomic DNA was extracted using the modified protocol CTAB method (6). The DNA concentration was tested by staining DNA with ethidium bromide after electrophoresis in 0.8% agarose gel at 100V for 45 min in 0.5XTBE buffer (0.089M Tris-borate, 0.002 M EDTA, pH 8.0) with known DNA concentration standards (λ DNA, uncut). These results were estimated by AlphaImager program (AlphaImager 2000: AlphaEase TM, Alpha Innotech Corporation). PCR reactions were carried out in a Thermolyne Thermal Cycler (Amptitron II). Each 25 μl reaction mixture contained 1X reaction buffer (10 mM Tris-HCl, pH 8.3 and 50 mM KCl), 3 mM MgCl₂, 1U Taq DNA polymerase; 100 μM of dNTPs (dATP, dTTP, dCTP and dGTP, all reagents from PROtech Technologies Inc.), 0.2 μM of primer and approximately 25 ng of template DNA. The initial step of PCR amplification consisted of cycles of 1 min at 94°C, 1 min at 40°C and 2 min at 72°C. This was followed by 40 cycles of denaturing at 94°C for 30 sec, primer annealing at 40°C for 30 sec, primer elongation at 72°C for 1 min, followed by an extended elongation step at 72°C for 10 min. Reaction products were mixed with 3.0 μl of 5X loading dye [70% glycerol, 0.02 MEDTA, 0.2% SDS, 0.2% bromophenol blue (w/v]). The amplification products were electrophoresed on 1.5% agarose gel at 100V for 2 h. Gels were stained with ethidium bromide and photographed under ultraviolet light using an AlphaImager Program. Photography of the gels was analyzed using NTSYS software. Presence or absence (1 or 0) of the band was considered as a single trait and the binary data were used to calculate pair-wise similarity coefficients following^.. This matrix of similarity coefficients was subjected to unweighted pair-group method analysis (UPGMA) to generate a dendrogram using linkage procedure.

Source of Information: Asian Journal of Plant Sciences Volume 3, Number 1, 20-24, 2004, ISSN 1682-3974

Article Link (Online Journal): DOI: 10.3923/ajps.2004.20.24, URL: http://scialert.net/abstract/?doi=ajps.2004.20.24

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The results revealed that, the genetic base among the tested Mungbean cultivars was rather narrow. Collection of diverse germplasm from centers of diversity and acquired from other sources could have broaden the genetic base. This might be done through the use of γ radiation, which is a drastic method of muta genesis resulting in a major reshuffle of the genome. The reasonable diversity observed in this study might be exploited for further yield improvement.

Knowledge Management: Journal