Extraction of total DNA from Pinus sylvestris L. for assessment of polymorphism - using SSR- and RAPD-markers
The article demonstrates the efficiency of genomic DNA extraction from the needles of Pinus sylvestris L. using the commercial diaGene kit as an alternative to the traditional method with CTAB buffer for polymorphism assessment using RAPD and SSR markers. The study included 8 samples of vegetative tissues of Pinus sylvestris L. from the southern border of the range in the Central Federal District (Kantemirovsky District, Voronezh Region). DNA obtained during isolation, both using the column method and using the CTAB buffer, was quantified and tested using a PCR reaction. A spectrophotometric study of the purity of the obtained samples showed that the absorption ratio at A260 /A280 for the column method was on average 1.68 versus 2.17, which indicates a higher DNA purity relative to the standard isolation method. It has been shown that the use of the CTAB-buffer protocol gives a higher yield of nucleic acid from the vegetative tissue of Pinus sylvestris L. compared to the commercial diaGene kit. Molecular genetic analysis of four EST-SSRs of nuclear microsatellite loci and two RAPD-markers in eight samples of Pinus sylvestris L. showed the presence of amplification products with EST-SSRs loci. All investigated DNA samples were characterized in accordance with the literature data and were reproducible for two methods of DNA extraction (the sizes of amplification fragments with EST-SSRs loci correspond to the data of the original source). It has been shown that this method of nucleic acid isolation can be used to assess the genetic variability of SSR-loci polymorphism in population and other molecular genetic studies of Pinus sylvestris L.