华中五味子ISSR-PCR反应体系优化及引物筛选

系统研究了华中五味子ISSR PCR反应体系中的主要影响因子，确立华中五味子ISSR-PCR最适反应体系，并筛选出12条有效引物。单因子实验结果显示，华中五味子ISSR-PCR反应体系中各主要成分的适宜浓度范围为，Mg²⁺ 1.50~3.50 mmol·L^-1，dNTPs 0.10~0.35 mmol·L^-1，引物0.25~0.60 μmol·L^-1，Taq酶0.50~1.50 U。4因子3水平正交实验确立了最适反应体系，即20 μL体系中包含2.50 mmol·L^-1 Mg²⁺、0.20 mmol·L^-1 dNTPs、0.25 μmol·L^-1引物、1.50 U Taq酶、60 ng DNA模板、2.50 μL 10×PCR Buffer。本研究为华中五味子种质资源的评估及遗传多样性分析奠定基础。

Optimization of ISSR-PCR Reaction System and Primers Screening in Schisandra sphenanthera

The main influential factors of ISSR-PCR reaction system in Schisandra sphenanthera Rehd. et Wils. were systematically studied, optimization of ISSR-PCR system was established, and 12 effective ISSR primers were selected. Single-factor experimental results showed that the suitable concentration of composition of ISSR-PCR systen was 1.50~3.50 mmol·L^-1 of Mg²⁺, 0.10~0.35 mmol·L^-1 of dNTPs, 0.25~0.60 μmol·L^-1 of primer and 0.50~1.50 U of Taq DNA polymerase, respectively. The optimal ISSR-PCR reaction system was defined by orthogonal experiment with four-factor of three-level, i.e. 20 μL reaction system contained 2.50 mmol·L^-1 of Mg²⁺, 0.20 mmol·L^-1 of dNTPs, 0.25 μmol·L^-1 of primer, 1.50 U of Taq DNA polymerase, 60 ng of DNA template, 2.50 μL of 10×PCR Buffer. The present study could be used in the research for evaluation of germplasm resources and analysis of genetic diversity in S.sphenanthera Rehd. et Wils..