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Molecular Assessment of Genetic Diversity and Relationship in Selected Mungbean Germplasms

Saiful Islam Bhuyan
Department of Biotechnology, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

Md. Sanower Hossain
Department of Biotechnology, Faculty of Science, International Islamic University Malaysia, Kuantan-25200, Pahang, Malaysia.

Mirza Mofazzal Islam
Division of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

Shamsun Nahar Begum
Division of Plant Breeding, Bangladesh Institute of Nuclear Agriculture (BINA).

Zannat Urbi
Department of Biotechnology, Faculty of Science, International Islamic University Malaysia, Kuantan-25200, Pahang, Malaysia.

Md. Sumon Hossain
Department of Biotechnology, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

Abstract/ Executive Summary:

Mungbean is an important crop considering the nutritional supplementary of low fat, high fiber and protein but its production is very low compared to the daily requirements. Hence, the assessment of genetic diversity and relationships in the existing germ plasms is a major concern for the development of high yielding variety of mungbean. In this study, Random Amplified Polymorphic DNA (RAPD) marker was used to analyze 7 exotic and 3 advance germ plasms using 3 primers (OPA01, OPB06 and OPB07). On an average 6 amplified products/primer were formed with overall polymorphisms of 78.33%. The similarity coefficient was highest (0.93) between AVRDC-3 and AVRDC-4, indicating less divergence and was least (0.39) between AVRDC-5 and AVRDC-6, indicating more divergence. On the basis of UPGMA dandogram, genotypes AVRDC-5, AVRDC-6 and AVRDC-7 were found to be quite distinct and the simple matching coefficient varied from 0.1824 to 0.8109. These findings will be of significance in the development of intra species crossing variety of mungbean crop.

Keywords: Genetic distance, Mungbean, RAPD, Vigna radiata

Location: Laboratory’s field, Division of Biotechnology, BINA.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Mungbean

Objectives:

To assess the genetic variability and phylogenetic relationships among ten germ plasms of mungbean

Methodology:

The plant materials (seeds) of the present study were collected from AVRDC (7 germplasms) and Bangladesh Institute of Nuclear Agriculture (BINA) (3 germplasms). All the collected seeds were round shaped and these seeds were sown and grown at the Laboratory’s field, Division of Biotechnology, BINA. Young and fresh leaves were collected from 28 days old seedlings of each genotype, quickly washed with distilled water and dipped in ethanol for few second followed by dried on blotting paper and finally stored at -80°C using zip lock bags until total genomic DNA was extracted. The total genomic DNA was extracted the modified method. The frozen leaves (~0.7 g) were ground in liquid nitrogen to a fine powder using mortar and pestle. DNA extraction buffer containing 0.5 M Ethylene Diamine Tetra-acetic Acid (EDTA) (pH 8.0), 1 M tris-HCl at pH 8.0, 5 M NaCl) and 50 μL 20% SDS was added to the powder tissue (1 mL g^-1 fresh weight). The equal volume (100 μL) of 5 M NaCl and Cetyl Trimethyl Ammonium Bromide (CTAB) were added to the suspension, respectively after vortex and incubation each time. The homogenate was extracted with phenol-chloroform-iso-amyl alcohol at 12000 rpm for 15 min. The supernatant was extracted at 12000 rpm for 15 min after adding 600 μL ice cold isopropanol to the supernatant. Then the pellet was washed with 200 μL 70% ethanol. The air dried DNA sample was suspended in TE buffer (10 mM tris-HCl and 1 mM EDTA at pH 8.0) and stored at -20°C until further analysis. The isolated DNA (5 μL of preparations) from each genotype was run in 0.8% agarose gel for purity and also scanned in spectrophotometer’s (Spectronic Genesys, Thermo Scientific) at 260 nm to take the absorbance in order to take quantification of DNA. The pre-screening was done with 23 primers and three arbitrary decamer primers (Operon technologies, USA) namely, OPA01, OPB06 and OPB07, were selected based on their ability to detect distinct polymorphic amplified products of mungbean. The DNA amplification reactions were carried out in a DNA thermocycler (Biometra-4111255, Biotron). Each 10 μL reaction mixture contained about 50 ng of template DNA, 10X Ampli Taq polymerase buffer, 250 μM dNTP mix, 10 μM primer and 0.2 μL Taq DNA polymerase. The thermal cycling conditions were, 3 min preheat at 94°C, 40 cycles of 1 min at 94°C, 1 min annealing at 54°C and elongation or extension at 72°C for 2 min. Finally, all amplified fragments were allowed for 7 min at 72°C to complete extension and temperature was brought down to 4°C. The RAPD fragments were separated by performing 1.4% agarose gel electrophoresis and the gel was documented using UV transilluminator in a gel doc (Biometra, Japan). All the PCR reactions were repeated at least twice to check the reproducibility. Positions of clearly visible and scorable RAPD bands of all genotypes were thereby given identification numbers according to their position on gel and scored visually with 1 for presence and 0 for absence at particular position for each individual genotype against each primer. The 1000 bp DNA ladder was used as standards for estimating the molecular weights of the bands. All amplifications were repeated at least twice to get reproducible bands for further analyzes. A single data matrix was created using the scores obtained from the RAPD analysis and it was used to determine polymorphic loci, gene diversity, population differentiation (G_st), gene flow (N_m), genetic distance and to construct a Unweighted Pair Group Method of Arithmetic means (UPGMA) dendrogram using POPGENE computer program, version 1.31. The pair-wise homogeneity test was also performed using the same program throughout different loci. The assumption of a two-allele system was the basis for gene frequency estimation for RAPD loci. It was used to calculate the Nei’s genetic distance from the RAPD pattern. The Similarity Index (SI) of any two individuals on the same gel of the RAPD profiles was calculated manually.

Source of Information: Biotechnology, 13(3): 126-134, 2014, ISSN 1682-296X

Article Link (Online Journal): DOI: 10.3923/biotech.2014.126.134; URL: http://scialert.net/abstract/?doi=biotech.2014.126.134

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The present study would be effective for the identification of the parents genotypes based on their characteristics of high yielding in order to develop the high yielding new variety using hybridization, gene transfer technology or any other relevant molecular techniques. Therefore it can be concluded that for further research program, especially for hybridization, the aimed genotype could be selected from different clusters that might be useful for the evolution of desired genotypes.

Knowledge Management: Journal