华东黄杉ISSR引物反应体系的优化与筛选

Optimization of Experiment Conditions and Primer Screening with ISSR Markers for Pseudotsuga gaussenii

在研究华东黄杉（Pseudotsuga gaussenii Flous）的遗传多样性过程中，为了获得清晰、重复性好的ISSR扩增结果，对影响ISSR—PCR的多个因素包括模板浓度、Mg^2＋浓度、dNTPs浓度、Taq酶的用量、退火温度和循环次数等指标进行了筛选和优化，确定了华东黄杉ISSR—PCR分析的最佳扩增条件为：20μL PCR反应体系中，2μL10×Taq酶配套缓冲液，1．5UTaq聚合酶（上海Promega公司），1．5—2．5mmol／L MgCl2，0．2μmol／L引物，0．2mmol／LdNTPs，10ng／L模板DNA。用来自江西三清山的4个个体，以100个ISSR引物进行PCR扩增，筛选出扩增效果较好的12个引物。

The factors which affect the PCR - ISSR analysis in the study of the genetic diversity of Pseudotsuga gaussenii Flous, such as concentrations of template DNA, units of DNA Taq polymerase, Mg^2＋ concentration and dNTPs concentration were optimized. The optimal ISSR -PCR conditions in the experiments were as the following in 20μL PCR reaction system： 2μL 10 ×DNA Taq polymerase buffer,1.5 U Taq DNA polymerase,1.5 -2.5 mmol/L MgCl2, 0.2 μmol/L primer, 0.2 mmol/L dNTPs,10 ng/L temper DNA. One hundred ISSR primers were used to screen the suitable primers with 4 samples from different populations in Sanqingshan mountain for assessing the genetic diversity of P. gaussenii, of which 12 ISSR primers with high resolution and multiple polymorphic bands were screened.