杉木SSR-PCR体系优化

Optimization on SSR-PCR reaction system of Chinese fir

SSR—PCR反应体系优化是杉木SSR标记研究的基础。利用正交L16（4^5）设计实验对杉木SSR-PCR反应体系的Mg2＋、dNTPs、引物、Taq酶和DNA浓度5个因素的4个水平进行优化试验。并在正交直观分析和方差分析的基础上，分别对Mg2＋、dNTPs进行单因素确定。获得的最佳反应体系为：在20灿反应体系中，Mg2＋浓度为1．0mmol／L，引物浓度为0．25μmol／L，dNTPs浓度为0．15mmol／L，Taq酶为0．05U／μL，DNA为30ng，10×PCRBuffer2μL，不足部分用双蒸水补齐至20μL。在最佳反应体系的基础上，采用单因素实验确定了引物的最佳退火范围为63～53℃。利用此反应体系对10个F1代杉木材料及其亲本，进行4对SSR标记进行PCR扩增并电泳检测，其结果清晰、稳定。说明此体系适合进一步的杉木SSR研究工作。

It＇ s important to optimize the SSR-PCR（ Polymerase Chain Reaction）reaction system firstly when doing research with SSR markers in Chinese fir. The orthogonal design of L16 （4）5 was used to optimize the SSR-PCR system for Chinese fir with four levels and five factors, namely, Mg2＋, dNTPs, primer, DNA polymerase and DNA template. The results of PCR were evaluated by variance and intuitive analysis. Then, we respectively determined the concentration of Mg2＋ , dNTPs, primer and annealing temperature using single factor experiment design. The results showed that the optimum reac- tion system was 20 μL reaction solution, containing Mg2＋ 1.0mmol/L, dNTPs 0.15 retool/L, primer 0.25μmol/L, Tat/ polymerase 0.05 U/μL, DNA template 30 ng, 2 μL 10 × PCR buffer and adding ddH20 to 20 μL, and the annealing tem- perature was the 63 -53℃ To verify the reaction system, we used 4 pairs of EST-SSR markers to amplify l0 Fl Chinese fir plantlets and their parents, and we got clear and stable bands, which showed that the SSR-PCR system was suitable fur- ther SSR research in Chinese fir.