青海云杉ISSR-PCR反应体系的建立与优化

1材料与方法1．1材料供试材料为祁连山区青海云杉天然分布区内的优良单株，基本能反映出祁连山青海云杉分布区的特点。1．2试剂所用TaqDNA聚合酶、dNTPs等均购于兰州佰澳生物公司；ISSR选择引物来自University of British Columbia Biotechnology Lab（UBCBL）primer set UBC9，由北京赛百盛公司合成。

Establishment and Optimization of ISSR-PCR Reaction System for Picea crassifolia kom.

[Objective] The experiment aimed to determine the optimum ISSR-PCR reaction system of Picea crassifolia kom. [Method] Picea crassifolia kom. was used as material to select and optimize influencing factors of ISSR-PCR such as Mg2+, dNTPs, Taq DNA polymerase, template DNA, primers, annealing temperature. [Result] The optimum ISSR-PCR reaction system in 20 μl reaction system was consisted of 1 μl 10×buffer, 1.5 mmol/L Mg2+, 0.2 mmol/L dNTPs, 1.0 U Taq DNA polymerase, 40 ng template DNA, 0.6 μmol/L primers. According to gradient test of annealing temperature in optimum ISSR-PCR reaction system of Picea crassifolia kom, it was found that the optimum annealing temperature of UBC 818 was 54.2 ℃ and the annealing temperature was different for different primers.[Conclusion]The construction of ISSR-PCR reaction system provided technical basis for classification of germplasm resources, construction of genetic map, gene mapping of Picea crassifolia kom. through using ISSR technology.