苹果砧木SRAP-PCR反应体系的优化及耐盐性标记的筛选

Optimization of the SRAP-PCR System and Screening of Markers Linked to Salt-tolerance Gene in Apple Rootstock

本研究以苹果砧木SH38为材料,利用正交试验设计对影响SRAP-PCR反应的4因素(Taq DNA聚合酶、模板DNA,d NTPs,引物)4水平进行优化,并构建苹果砧木SH38的SRAP-PCR的最佳反应体系。结果显示:15μL的总反应体系中,d NTPs浓度为0.3 mmol/L,Taq DNA聚合酶用量为0.75 U,上、下游引物为50 ng,DNA模板用量为60 ng。运用该反应体系并结合BSA法,从128对引物组合中筛选出4对引物,该4对引物扩增出的特异性片段与苹果砧木的耐盐基因存在连锁。研究结果可为苹果砧木利用SRAP分子标记进行耐盐性的分子标记辅助育种提供基础。

The SRAP- PCR reaction system of apple rootstock SH38 was optimized by means of an orthogonal experiment which was in four levels of four factors(Taq DNA polymerase, template DNA, d NTPs and Primer). The results showed that the optimized 15 μL reaction system of SRAP-PCR contained d NTPs 0.3 mmol/L, Taq DNA polymerase 0.75 U, each prime 50 ng, and DNA template 60 ng. Under this optimum reaction conditions, 4 primer combinations were screened from 128 ones by BSA method. The 4 primer combinations expressed polymorphism in parents and DNA pools and totally produced 4 polymorphic fragments which linked to salt-tolerance gene in apple rootstock.The optimized reaction system of SRAP-PCR and this 4 primer combinations would contribute to the basis for molecular assistant selection breeding in apples rootstock salt- tolerance.