Shilkina Е. А., Oreshkova N. V., Ibe А. А., Deych K. O., Krutovsky K. V. Development of Cytoplasmatic SSR-markers for Population Genetic Studies of the Siberian Stone Pine (Pinus Sibirica Du Tour)

© Shilkina Е. А., Oreshkova N. V., Ibe А. А., Deych K. O., Krutovsky K. V., 2014

The objective of this study was identification of variable cytoplasmic microsatellite loci and the development of the SSR markers (Simple Sequence Repeats) on their basis for the Siberian stone pine (Pinus sibirica Du Tour). The study used all nucleotide sequences of chloroplast and mitochondrial genomes available in the Genbank (http://www.ncbi.nlm.nih.gov/genbank)to design primers, including the almost complete sequence of the chloroplast genome (gi|228016112|gb|FJ899558.1|). Microsatellite regions were identified using the SciRoKo computer program (Kofler et al., 2007). Four microsatellite loci were found in the chloroplast genome, and only one locus was revealed among all investigated mitochondrial sequences, in an intron of the nad1 gene (gi|12002773|gb|AF160260.1|). Using the Primer3 computer program 24 pairs of PCR primers were designed for the detected loci: five pairs of mitochondrial and nineteen pairs of chloroplast loci. These primers were tested using two samples of 30 trees aged 40-70 years from two populations of Siberian stone pine in Tomsk region of the Russian Federation, respectively. DNA was extracted from dried pine needles according to the standard protocol for plant tissue using cetyltrimethylammonium bromide (CTAB method). The PCR was carried out using the GenPak^TM PCR Core kit (Laboratory Isogen, LLC, Russia). The PCR products were separated by electrophoresis in the standard 6 % polyacrylamide vertical slab gels using the Tris-borate-EDTA electrode buffer. Gels were stained in a solution of ethidium bromide and visualized under the UV light. Plasmid DNA pBR322 digested with a restriction enzyme HpaII was used as a molecular weight marker. Based on the amplification and genotyping of Siberian stone pine four best primer pairs were selected for three chloroplast and one mitochondrial DNA markers among 24 pairs of PCR primers, respectively. According to the results of genotyping of 60 trees, two chloroplast loci were monomorphic and one polymorphic with two alleles. Thus, two haplotypes for the chloroplast loci and two alleles or haplotypes (mitotypes) for the mitochondrial marker were revealed.