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Title: Genomic DNA isolation and identification of chloroplast microsatellite markers in <i>Asparagus racemosus </i>Willd.<i> </i>through cross-amplification
Authors: Ginwal, H S
Mittal, Neha
Maurya, Shalini Singh
Barthwal, Santan
Bhatt, Preeti
Keywords: CTAB
<i style="">Asparagus racemosus</i>
microsatellite markers
Issue Date: Jan-2011
Publisher: NISCAIR-CSIR, India
Abstract: Cross-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 <i>Asparagus racemosus</i>, chloroplast microsatellite primer pairs developed for <i>Acorus calamus </i>(Acoraceace) were examined for cross-species amplification and validation in <i>A. racemosus</i>. 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 <i>A. racemosus</i>. 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 <i>A. racemosus</i>. Besides this an easy and competent protocol for the extraction of high quality genomic DNA in <i>A. racemosus</i> for the PCR-based microsatellite marker analysis has been also reported. The DNA extraction protocol involved a modification of CTAB procedure given by Stange <i style="">et al</i>, 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 A<sub>260</sub>/A<sub>280 </sub>nm wavelength. Following the protocol and using the primers, genetic diversity analysis in <i>A. racemosus</i> was carried out.
Description: 33-38
ISSN: 0975-0967 (Online); 0972-5849 (Print)
Appears in Collections:IJBT Vol.10(1) [January 2011]

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