中国木犀科苦丁茶ISSR实验条件优化的研究

系统地研究了中国木犀科苦丁茶ISSR反应体系中的主要影响因子，建立了一套稳定的ISSR PCR反应参数。筛选出了10个有效引物，并以中国木犀科苦丁茶8个物种共21份种质材料为供试材料对优化后的反应条件的重复性、多态性进行了检测。优化后的反应体系为：10×buffer 2.5 μL，2.0~3.0 mmol·L^-1 MgCl₂，150~300 μmol·L^-1 dNTPs，Taq酶1.0~1.5 U，引物0.4~0.5 μmol·L^-1，DNA模板5~320 ng。PCR扩增程序为：94℃预变性4 min，然后按94℃变性40 s，50~54℃退火45 s，72℃延伸120 s，进行35个循环，最后72℃延伸8 min。该反应条件可应用于中国木犀科苦丁茶亲缘关系和遗传多样性分析。

Studies on Optimization of ISSR Amplified Conditions in Kudingcha Species in Oleaceae of China

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.