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作 者 ：郑道君;刘国民;*;梁远发;鄢东海;令狐昌弟;田永辉

期 刊 ：植物研究 2009年 29卷 2期 页码：234-241

关键词：木犀科；苦丁茶；ISSR；影响因子；体系优化；

Keywords:Kudingcha species, Oleaceae, ISSR, influential factors, Optimization of system,

摘 要 ：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。

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.

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