中国木犀科苦丁茶ISSR实验条件优化的研究

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

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×buffer2.5μL,2.0～3.0mmol·L-1 MgCl2,150～300μmol·L-1 dNTPs,Taq酶1.0～1.5U,引物0.4～0.5μmol·L-1,DNA模板5～320ng。PCR扩增程序为:94℃预变性4min,然后按94℃变性40s,50～54℃退火45s,72℃延伸120s,进行35个循环,最后72℃延伸8min。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。

The influential factors of ISSR for Kudingcha species in Oleaceae of China were systematically stud- ied, 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 poly- merase, 0.4 - 0.5 μmol · L- 1 primers, and 5 - 320 ng DNA template were contained in 25 μL reaction solu- tion. The optimized amplification program was that pre-denaturing at 94℃ for 4 rain, then denaturing at 94℃ for 40 s, primer annealing at 50℃-54℃ for45 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 Kndingcha germplasm resources in Oleaceae of China.