关键词: 华中五味子, ISSR, 体系优化, 引物筛选

Abstract: 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..

Key words: Schisandra sphenanthera Rehd. et Wils., ISSR, optimization of system, primers screening