关键词: 木犀科, 苦丁茶, ISSR, 影响因子, 体系优化

Abstract: The influential factors of ISSR for Kudingcha species in Oleaceae of China were systematically studied, a set of stable ISSR-PCR reaction parameters was established. 10 effective ISSR primers were selected out, and with them 21 test germplasm materials from 8 Kudingcha species in Oleaceae of China were examined for the repeatability and polymorphism of the optimized reaction conditions. The optimum ISSR-PCR reaction system was that 2.5 μL 10×PCR buffer, 2.0~3.0 mmol·L^-1 MgCl₂, 150~300 μmol·L^-1 dNTPs, 1.0~1.5 Taq polymerase, 0.4~0.5 μmol·L^-1 primers, and 5~320 ng DNA template were contained in 25 μL reaction solution. The optimized amplification program was that pre-denaturing at 94℃ for 4 min, then denaturing at 94℃ for 40 s, primer annealing at 50℃~54℃ for 45 s, extension at 72℃ for 120 s, for 35 cycles, at last extension at 72℃ for 8 min. The productions were stored at 4℃. The optimized ISSR-PCR reaction system was suitable for the study of genetic diversity and relationship of Kudingcha germplasm resources in Oleaceae of China.

Key words: Kudingcha species, Oleaceae, ISSR, influential factors, Optimization of system