A RAPD analysis on genetic differentiation of Stipa krylovii populations in central and eastern Inner Mongolia Steppe

Stipa krylovii in Inner Mongolia is a peculiar moderate-temperate steppe of the Central Asia Subzone. Affected by temperate continental climate, the obvious thermal difference due to latitude and the precipitation discrepancy due to monsoons provide the possibility of genetic diversity for S. krylovii in the region. Thus it is necessary to make a systematic study of population diversity at the individual, physiological, cellular or molecular levels. Seven plots, representative of the typical steppe, desert steppe and transition zone respectively, were selected in the belt with the range of 43.6～44.7°N and 113.5～117.8°E in Inner Mongolia. S. krylovii samples were collected and the habitats were investigated at the same time. The relationship between the differentiation of S. krylovii populations and the change in their habitats(mainly aridity) was studied in this paper to discuss the mechanism of population diversity and offer scientific data and academic basis for the genetic character protection of S. krylovii. The total genomic DNA was isolated from the mixed, dry, leaf material of S. krylovii by using the improved SDS protocol and was amplified by RAPD after purification. The reaction mixture contained about 30 ng template DNA in a 25μl reaction volume with 2. 5μl 10×reaction buffer, 2.0 mmol/L MgCl_2, 0.2 mmol/L each dNTPs, 1 U Taq DNA polymerase and 0.2 pmol/L primer. Amplification was performed in a Programmable Thermal Controller-100. 7μl reaction mixture of each sample was loaded and run on 1.5% agarose gels(containing ethidium bromide)in 1×TBE buffer at 4 V per centimeter, visualized on UV-light and photographed. The amplified DNA fragments with the size of 400～2000bp were scored by presence (1) or absence (0). All of the data were transferred into a computer and statistical analysis was performed. Jaccard's genetic similarity coefficient, simple matching coefficient (SM) and Dice genetic similarity coefficient were calculated respectively, with which un-weighted pair group method with arithmetic mean (UPGMA) cluster established three DNA molecular dendrograms. Principle component analyses(PCA)and principle axis factoring(PAF) were done to get an overview of the genetic variation in the materials studied. Twenty-one(21)oligonucleotides screened from 100 decamer primers were selected to analyze the different geographic populations of S. krylovii; they yielded profiles with intense and well-separated bands. A total of 229 RAPD markers were produced from the 21 selected primers. The number of total bands for each primer to all samples varied from 8 to 17 with an average of 10.9 bands per primer and the size of the amplified fragments ranged from 400 to 2000bp. Among the total 229 fragments, 171 appeared to be polymorphic(74.67%) with an average of 8.1 for each primer, which illuminated that S. krylovii populations had rich polymorphism and distinct genetic difference. Twenty-six(26)unique loci were obtained from RAPD results, which were 11.25% of the total amplified loci. The percentages of unique loci were related to their habitats to a certain extent. Correlative analysis demonstrated that the percentage of unique loci would increase with the increasing aridity(r=0.76, P0.05). The three dendrograms provided the same results. Seven S. krylovii populations were clustered into three groups according to their genetic similarity coefficients. Three populations located in the semi-arid typical steppe belonged to one group; two populations, which lied in the arid desert steppe, were mingled into another group. The remaining populations, whose habitats were between typical steppe and desert steppe, were clustered into the third group. In addition, the results of principle component analyses(PCA)and principle axis factoring(PAF), which are based on band patterns, sustained the results of the dendrograms.