山梨SSR反应体系的建立及其稳定性验证

Establishment for SSR Reaction System of Pyrus ussuriensis Maxim and Its Stability Assessment

以山梨为试材,提取基因组DNA,运用5因素5水平正交实验设计,对SSR反应体系中的DNA模板、Mg2+、Taq酶、dNTPs和引物用量进行优化试验,确立适宜的SSR反应体系。结果表明:20μL反应体系中,模板DNA 10ng、Mg2+(20mmol/L)2.5μL、Taq酶1.25U、dNTPs(10mmol/L)1.0μL、引物(10μmol/L)1.5μL、10×PCR buffer 2.0μL;应用该SSR体系,在引物筛选试验及山梨×"幸水"后代群体中进行扩增,扩增效果较好,证实了该体系的适用性和稳定性。

Genomic DNA of young leaves of Pyrus ussuriensis Maxim was tested as the template,optimization experiments on some important parameters affecting SSR-PCR amplification were conducted to establish an optimum system suitable for SSR-PCRPyrus ussuriensis Maxim.Orthogonal design was used to optimize SSR anplification system with five factors（including DNA template,Mg^2＋,Taq polymerase,dNTPs and primer）at five levels,respectively.The results showed that,a suitable SSR-PCR reaction system was established,its total volume was 20μL,containing 10 ng DNA template,2.5μL Mg^2＋（20mmol/L）,1.25 UTaq polymerase,1.0μL dNTPs（10mmol/L）,1.5μL primer（10μmol/L）and 2μL10×PCR buffer.The reaction system was successfully applied in primer screening experiment and the amplification of the lines fromPyrus ussuriensis MaximבKosui＇using primer CHg04 b,and indicated the suitability and stability of the system.