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目的:优化岩白菜ISSR-PCR反应体系,为利用ISSR标记进行岩白菜遗传多样性研究服务。方法:采用5因子4水平正交设计法优化岩白菜ISSR-PCR反应体系。结果:五个因子从大到小的影响力排序结果为:dNTPs>Mg2+>模板DNA>引物=Taq酶。岩白菜ISSR-PCR最佳反应体系为:总体积25μL,内含10×PCR缓冲液2.5μL、2.5 mmol.L-1Mg2+、2.0 U Taq酶、0.2 mmol.L-1dNTPs、0.48μmol.L-1ISSR引物、125 ng模板DNA。结论:研究获得的最佳反应体系具有标记位点清晰、反应系统稳定、检测多态性能力强、重复性好等特点,为利用ISSR标记技术研究岩白菜遗传多样性奠定了基础。
OBJECTIVE: To optimize the ISSR-PCR reaction system of Brassica campestris to serve the genetic diversity of Brassica campestris using ISSR markers. Methods: The ISSR-PCR reaction system of Brassica campestris was optimized by orthogonal design of 5 factors and 4 levels. Results: The order of influence of five factors descending order: dNTPs> Mg2 +> template DNA> primer = Taq enzyme. The optimum reaction system of ISSR-PCR for Brassica campestris was as follows: total volume 25μL, containing 2.5μL of 10 × PCR buffer, 2.5mmol.L-1Mg2 +, 2.0U Taq enzyme, 0.2mmol.L-1dNTPs, 0.48μmol.L-1ISSR Primer, 125 ng template DNA. Conclusion: The best reaction system obtained has the characteristics of clear marker site, stable reaction system, strong ability of detecting polymorphism and good repeatability, which laid the foundation for the study of genetic diversity of Brassica campestris L. with ISSR marker technology.