水藓科植物ISSR-PCR反应体系的优化

Optimization of ISSR - PCR Reaction System of Fontinaloideae

目的:对水藓科植物ISSR-PCR反应体系进行优化,为水藓科的遗传多样性研究奠定基础。方法:提取水藓的基因组DNA,利用正交设计法对水藓ISSR-PCR反应体系进行5因素4水平的优化,并通过计算极差分析各因素对PCR扩增的影响程度。结果:水藓科ISSR-PCR最佳的反应体系(20μL)为:2.0μL PCR Buffer溶液、1.52mmol.L-1Mg2+、1.28μmol.L-1引物、1.20mmol.L-1dNTPs、70.0 ng模板、0.50 UTaq酶。5个因素对该反应体系的影响顺序为:Mg2+﹥Taq酶﹥dNTPs﹥模板DNA=引物。在此反应体系下,可以得到重复性好、稳定且多态性高的条带。结论:该实验通过对水藓科基因组DNA的提取及体系优化,获得了水藓科植物最佳反应体系,为后续应用ISSR技术鉴定水藓科种质资源及其遗传多样性研究奠定了基础。

Objective：The ISSR - PCR reaction system was optimized to lay the foundation for genetic diversity of Fontinaloideae. Method： Genomic DNA was extracted from Fontinaloideae, five factors and four levels of ISSR - PCR reaction system were optimized with the or- thogonal experimental method, and the impact of the five factors for PCR amplification was observed based on calculating variance analysis. Result：The best ISSR - PCR reaction system of Fontinaloideae contained 2. 0μL PCR buffer, 1.52 mmol. L -1 Mg2＋ , 1.28 μmol ~ L-1 primer,1.20mmol. L-1 dNTPs,70.0 ng polymerase,0. 50 U Taq polymerase. The effect order of five factors was Mg2＋ 〉 Taq poly-merase 〉 dNTPs 〉 template DNA = primer, Conclusion：The best reaction system was obtained by isolated genomic DNA and optimized the system, which had laid the good foundation for ISSR analysis on studies of Fontinaloideae resources identification and genetic diversity.