Genomic DNA isolation and identification of chloroplast microsatellite markers in <i>Asparagus racemosus </i>Willd.<i> </i>through cross-amplification

Abstract

33-38Cross-species amplification of microsatellite loci is a time saving as well as a cost-effective approach for developing locus specific markers for new species. In an attempt to reveal the genetic variation in different accessions of Asparagus racemosus, chloroplast microsatellite primer pairs developed for Acorus calamus (Acoraceace) were examined for cross-species amplification and validation in A. racemosus. Out of the 18 microsatellite primer pairs screened, 5 i.e. 27.77% (AC-03; AC-05; AC-09; AC-13 and AC-17) yielded good cross-species amplification across 20 different individuals of A. racemosus. These cpSSR markers comprise 1-dinucleotide repeat type; 2-trinucleotide and 2-tetranucleotide repeat types. The product size of the amplified cpSSR primers ranged between 180 and 337 bp. All the 5 cross-amplified cpSSR markers were found polymorphic across the 20 individuals of A. racemosus. Besides this an easy and competent protocol for the extraction of high quality genomic DNA in A. racemosus for the PCR-based microsatellite marker analysis has been also reported. The DNA extraction protocol involved a modification of CTAB procedure given by Stange et al, which includes the use of high concentrations of polyvinyl pyrrolidone (PVP), addition of 8 M lithium chloride in extraction buffer; a repeated chloroform:isoamyl alcohol step and washing of DNA pellets with the wash buffer and with the 80% ethanol. The developed protocol yielded approximately ~77.30 μg DNA per 100 mg plant tissue with the purity ratio of ~1.85 at A260/A280 nm wavelength. Following the protocol and using the primers, genetic diversity analysis in A. racemosus was carried out