Molecular investigation between four Himalayan pines of India through random amplified polymorphic DNA markers

Studies were undertaken to identify genetic relationship in four different species of Pinus L. through randomly amplified polymorphic DNA (RAPD) markers. A total of 500 DNA fragments ranging from 234 to 1353 bp were amplified using 5 selected primers. The number of amplification products produced by a primer ranged from as low as 4 to a maximum of 13, with an average of 8 bands per primer. The cluster analysis revealed one major cluster and one outlier. In the major cluster, Pinus roxburghii from Malithi, Pinus wallichiana from Malithi, P. wallichiana from Taradevi H.P, Pinus kesiya from Taradevi H.P, Pinus gerardiana from Chamba and P. roxburghii from Chamba falls into subcluster 1 and P. kesiya from Jubbal (east) and P. kesiya from Jubbal (west) falls into subcluster 2. The similarity coefficient value varied from 0.54 to 0.88. The highest similarity coefficient (0.88) was detected between samples collected from P. wallichiana (Malithi) and P. roxburghii (Malithi) as well as between P. roxburghii (Malithi) & P. wallichiana (Taradevi, H.P) and the lowest (0.54) was detected between the P. gerardiana (Raspa) and P. kesiya (South Vietnam). The level of polymorphism in our study was not so much which showed that samples used for the analysis could have close relationship.Key words: Randomly amplified polymorphic DNA (RAPD), similarity coefficient, polymorphism, Pinus, primer

An efficient genomic DNA isolation protocol for RAPD and SSR analysis<i style=""> </i>in<i style=""> Acorus calamus</i> L.

213-216Molecular based genetic analysis requires good quality of DNA in sufficient quantity to generate robust DNA fingerprints. The leaves of sweet flag (Acorus calamus L.) contain high amounts of polyphenolic compounds and polysaccharides, which interfere in the amplification reactions. Four genomic DNA extraction methods were tested for yield, quality and suitability of genomic DNA for RAPD (random amplified polymorphic DNA) and SSR (simple sequence repeat) marker analysis in sweet flag. Fresh young leaves were subjected to the already available protocols and a procedure was devised after modification in the protocol of Stange et al for the isolation of total genomic DNA. The modified method employs high concentrations of polyvinyl pyrrolidone, addition of lithium chloride solution as well as additional washing step of DNA pellets. The yield was found approximately 200-500 µg DNA per 100 mg of plant material and the purity ratio was found 1.7-2.0. Usage of highly concentrated PVP extraction buffer and chloroform:isoamylalcohol repetition step was found useful to overcome problems from polyphenolic compounds and polysaccharides. The extracted DNA through this method was found suitable for RAPD and SSR analysis

Evaluation and optimization of DNA extraction method for <i style="">Dalbergia sissoo </i>leaf

69-73A modified protocol for Dalbergia sissoo genomic DNA isolation has been optimized based on a cetyl trimethyl ammonium bromide (CTAB) method, described for other forest species. Leaves obtained from macro-propagated clones and mature trees of D. sissoo were tested. The method involves mortar grinding of tissue, a modified CTAB extraction buffer incorporating high salt concentrations, polyvinyl pyrrolidone and successive isoamyl alcohol-chloroform extractions with modified temperature conditions. The modification involved the use of doubled concentration of polyvinyl pyrrolidone (4% instead of 2%), increased incubation time with extraction buffer (40 min instead of 30 min), use of freshly prepared CTAB buffer and increased timing of washing of DNA pellet with wash buffer (45 min instead of 30 min). The yield was approximately 100 to 400 µg DNA per 100 mg of leaf tissue. The genomic DNA obtained by this method was suitable to be used in RAPD and ISSR analysis. This extraction method would allow the molecular analysis of DNA from different clones of D. sissoo

Identification of allele specific primers in chir pine (<em>Pinus roxburghii</em> Sarg.) through data mining

538-540Pinus roxburghii Sarg. commonly known as Chir pine, is a commercially exploited species for resin in India. Resin is a phenotypic trait which is expressed only in mature trees. Due to this a large number of trees are cut every year which pose a serious ecological threat. Recent advancements in bioinformatics and molecular biology have enabled scientists to identify high resin yielding pine genotypes at nursery stage using molecular markers. In the present study, investigation, 337 terpene coding sequences from the database were analyzed using data mining and 45 microsatellites allele specific primers were designed for marker assisted selection. Out of these 45 primers, only 22.2% primer pairs showed polymorphism within the 53 genotypes of Chir pine examined. A wide range of fragment size was observed from 100 - 600 bp. PCR amplification using allele specific markers produced a total of 22 bands, out of which 14 were found to be polymorphic. The total number of bands amplified per marker varied from of 1 to 3 with an average of 1.4. Genetic divergence in terms of percent polymorphism ranged from 50 to 100% with a mean of 63.3% per marker

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

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