穗花杉ISSR引物反应条件的优化与筛选

Optimization of experiment conditions and primer screening with ISSR markers for Amentotaxus argotaenia

在研究穗花杉（Amentotaxus aragotaenia）的遗传多样性过程中，为了获得清晰、重复性好ISSR扩增结果，对影响ISSR-PCR的多个因素包括模板浓度、Taq酶的选择和用量、Mg^2＋和dNTPs浓度及退火温度等指标等进行了筛选和优化，确定了穗花杉ISSRPCR分析的最适扩增条件：20μL PCR反应体系中，2μL 10×Taq酶配套缓冲液，1．8UTaq聚合酶（上海生工公司），0．2μmol·L^-1引物，0．18mmol·L^-1 dNTP，1．5～2．5mmol·L^-1 MgCl2，10ng·μL^-1模板DNA。用来自不同居群7个个体，以100个ISSR引物进行PCR扩增，筛选出扩增效果较好的10个引物。得到了92个位点，其中45个多态性位点，多态性位点比例为49％。

The factors which affect the ISSR analysis in the study of the genetic deversity of Amentotaxus argotaenia, such as concentrations of template DNA, DNA Taq polymerase, Mg^2＋ and dNTPsn et al. , were examined. The optimal ISSR-PCR conditions in the experiments were as the following： in 20μL PCR reaction system,2μL 10 × DNA Taq polymerase buffer, 1.8 U Taq DNA polymerase, 0.2μmol × L^-1 primer, 0. 18 mmol · L^-1 dNTP, 1.5 -2.5 mmol · L^-1 MgCl2, 10 ng · μL^-1 temper DNA. One hundred ISSR primers were used to screen the suitable primers with 7 samples from different populations for assessing the genetic diversity of Amentotaxus argotaenia, of which 10 ISSR primers with high resolution and multiple polymorphic bands were screened. The total 92 ISSR bands were amplified with 10 primers, and produced 45 polymorphic bands, The percentage of polymorphic bands is 49%.