核桃SSR反应体系的优化

Optimization of SSR analysis system in walnut

以清香、香铃、爱米格为试材,利用CTAB法提取基因组DNA,将PCR体系的主要成分设定5个梯度,根据每个成分的变化引起的PCR-SSR的效果差异,探讨了核桃SSR技术中PCR体系的主要成分对扩增结果的影响,并对引物WGA321的适宜退火温度进行优化。最终确定了引物WGA321的最适退火温度为48～52℃,PCR反应体系的最佳条件为:15μL体系中,Mg2+1.0mmol/L,dNTPs浓度0.40mmol/L,TaqE用量为0.5U,DNA模板1.0ng/μL,引物浓度为0.4μmol/L。利用此反应体系对部分核桃品种进行PCR扩增并电泳检测,扩增结果清晰且有较高的多态性,表明该体系适合核桃的亲缘关系分析。

DNA of Qingxiang, Xiangling and Amigo walnut cultivars were distilled by CTAB. Every factor of PCR had five different concentrations and its variation changed the result of PCR-SSR. Then the factors affecting the SSR results of walnut were studied. The annealing temperature of primer WGA321 was optimized. The results showed that the optimal annealing temperature was 48 to 52 ℃, and the best reaction system of SSR-PCR was： 1.0 mmol/L Mg^2＋, 0.40 mmol/L dNTPs,0.5 U Taq pelymerase, 1.0 ng/μL template DNA,and 0.4 μmol/L primer in 15 μL reaction system. Using above PCR system, SSR fragments of 12 walnut cultivars were obtained. The clear and abundant pelymorphism indicated this system was suitable for analysis of phylogenetic relationship.