Optimization and Inference of PCR Conditions for Genetic Variability Studies of Commercially Important Cluster Bean Varieties by RAPD Analysis

Abstract

Cyamopsis tetragonoloba (L.) Taub commonly known as guar belongs to leguminoseae family. Guar seed has an important place in industry because of its galactomannan rich endosperm. Good quality genomic DNA was isolated from 34 independent cluster bean varieties and conditions were optimized for further amplification by PCR using random ten decamer operon primers. An evaluation was made for the application of RAPD as a genetic marker system in commercially important cluster bean varieties and a dendrogram was prepared. Various factors influencing the RAPD amplification were optimized and the data revealed that 50ng of template DNA, 1.5mM Mg 2+ ion concentration, 0.5U of Taq DNA polymerase and denatur- ing temperature of 94 0 C was found to be essential for reproducible RAPD banding pattern. Cluster bean cultivars revealed significant polymorphism with reference to RAPD markers showing authentic genotypic diversity among its races. Out of a total of seventeen operon primers employed; ten OPT primers showed amplified products in the size range of 50, 1200bp, yielding 34 polymorphic and 58 monomorphic reproducible bands with 72.7% polymorphism. Highest polymorphism was obtained in elite cultivars HG-75 and PNB. All the genotypes could be grouped in two major and three minor sub-clusters, when binary matrix was subjected to PAST software analysis and clustered dendrogram was constructed. The results of the present study can be used for molecular breeding and improvement of cluster bean for various desired traits through hybridization in future.