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Molecular Characterization of Garlic Germplasms Using Rapd

M.S. Alam
Germplasm Centre, Bangladesh Agricultural University, Mymensingh

M.A. Rahim
Department of Horticulture, Bangladesh Agricultural University, Mymensingh

P.W. Simon
Department of Horticulture–Agricultural Research Service, Vegetable Crops Research Unit, 1575, Linden Drive, Madison, WI, 53706

Abstract/ Executive Summary:

Genetic variation and relationship among 25 garlic (Allium sativum L.) germplasm were analyzed using Random Amplified Polymorphic DNA (RAPD). Out of 25 primers screened, two were selected, which gave 10 clear bands, out of which 8 bands were considered polymorphic. The proportions of polymorphic loci were 79.16%. The UPGMA dendrogram based on genetic distance segregated the 25 garlic germplasm into three main clusters. Cluster II contained 17 germplasm, cluster II contained 5 germplasm and cluster III contained only 3 germplasm. The highest genetic distance was 1.60. The results of the present study indicated that the RAPD analysis could be utilized by breeders for further improvement of garlic varieties.

Keywords: Germplasm, Characterization, Garlic and RAPD

Location: Germplasm Centre, Department of Horticulture, Bangladesh Agricultural University, Mymensingh

Start Date: 00-00-2011

End Date: 00-00-2011

Program Area: Variety and Species

Commodity: Garlic

Objectives:

1. To evaluate genetic variation and relationships of some garlic germplasm by RAPD (Random Amplified polymorphic DNA) technique.

Methodology:

Twenty five garlic germplasm were used in this stud. In order to carry out RAPD analysis, young leaves from each of the 25 germplasm were collected randomly from different parts of Bangladesh and out side the country. Total genomic DNA was isolated from garlic leaves following, phenol: chloroform: isoamyl alcohol purification and ethanol precipitation method. Finally, the DNA samples were stored at -200C, DNA concentrations were determined at 260 nm spectrophotometrically and the quality verified by electrophoresis on a 1% agarose gel. DNA amplification was done using two arbitrary decamer primers (Operon Technologies, Inc., Alameda, California, USA OPC-12 and OPC-13). PCR reactions were performed on each DNA sample in a 10µl reaction mix containing 1µl of 10x Ampli Taq polymerase buffer, 0.25 µl of 10µl mM primer, 1µl of 250µl mM dNTPs, 1 unit of Ampli Taq DNA polymerase (Bangalore Genei, India) and 4µl of 100ng genomic DNA (25ng/µl) and rest amount of sterile deionized water. DNA amplification was performed in an oil free thermal cycler (Master Cycler Gradient, Eppendorf). The reaction mix was preheated at 95⁰C for 1 min. 48⁰C for 1 min, 72⁰C for 2 min, continuing with 40cycles at 94⁰C for 30s, 48⁰C for 40s, 72⁰C for 2 min, and an extension period of 72⁰C for 10 min. After completion of cycling programme, reactions were held at 4⁰C. The amplified products were separated electrophoretically on a 1.5% agarose gel. One molecular weight marker, 100 bp DNA ladder were electrophoresed alongside the RAPD reactions. Electrophoresis was carried out at 120 V for 1 hour and 20 min. DNA bands were observed under UV light on a Transilluminator and photographed. Since RAPD markers are dominant, we assumed that each band represented the phenotype at a single allelic locus (Williams et al. 1990). All distinct bands or fragments (RAPD markers) were thereby given identification numbers according to their position on gel and scored visually on the basis of their presence (1) or absence (0), separately for each individual and each primer. The scores obtained using all the primers in the RAPD analysis were then pooled to create a single data matrix and used to estimate polymorphic loci, gene diversity, genetic distance (D) and to construct a UPGMA (Unweighted Pair Group Method of Arithmetic Means) dendrogram among populations using a computer program, POPGENE (Version 1.31). Genetic similarity values defined as the fraction of shared bands between the RAPD profiles of any two individuals on the same gel were calculated manually from RAPD markers of the same molecular weight on the data matrix according to the formula: Similarity index (SI) = 2Nxy / Nx + Ny . Where, Nxy is the number of RAPD bands shared by individuals x and y respectively, and Nx and Ny are the number of bands in individual x and y, respectively.

Source of Information: J. Agrofor. Environ. 6 (1): 63-66, 2012; ISSN 1995-6983

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Out of 25 primers screened, two were selected, which gave 10 clear bands, out of which 8 bands were considered polymorphic. The proportions of polymorphic loci were 79.16%. The UPGMA dendrogram based on genetic distance segregated the 25 garlic germplasm into three main clusters. Cluster II contained 17 germplasm, cluster II contained 5 germplasm and cluster III contained only 3 germplasm. The highest genetic distance was 1.60. The results of the present study indicated that the RAPD analysis could be utilized by breeders for further improvement of garlic varieties.

Knowledge Management: Journal