岩白菜ISSR-PCR反应体系的优化

Optimization of ISSR-PCR Reaction System of Bergenia purpurascens

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：The ISSR-PCR reaction system of Bergenia purpurascens（Hook.f.et Thoms.） Engl.was optimized for identifying and revealing genetic diversity of B.purpurascens.Method：The orthogonal experimental design was used for optimization.Result：The effect sequence of 5 factors from big to small was dNTPs Mg2＋ template DNA primer = Taq DNA polymerase.The optimum ISSR-PCR reaction system contained 2.5μL 10×PCR buffer,2.5 mmol·L-1 Mg2＋,2.0 U Taq DNA polymerase,0.2 mmol·L-1 dNTPs,0.48 μmol·L-1 primer and 125 ng template DNA in total 25 μL reaction volume complemented with ddH2O.Conclusion：The optimum ISSR-PCR reaction system obtained in this research could provide clear,reliable and abundant polymorphic markers.This result provided a solid basis for revealing the genetic diversity of B.purpurascens.