砂梨的ISSR-PCR反应体系的建立与优化

以砂梨基因组为材料，通过单因素试脸，对ISSR-PCR反应体系中各种影响因子如dNTPs浓度，DNA模板含量，Taq DNA聚合酶量，引物用量以及最适退火温度等进行了优化和筛选，建立了适合砂梨的ISSR-PCR反应体系和应用程序为为: 20 μl PCR体系中，1 x Taq 酶配套缓冲液，0.5 U Taq DNA 聚合酶，0.7μmol / L引物，100μmol / L dNTPs，2.25 mmol/LMgCl2，40ng模板DNA。最佳扩增程序为：预变性：94°C 5min； 94°C变性45sec，52°C退火45sec，72°C延伸1min；共40个循环后，最后72°C延伸10min，4°C保存。用引物836可以将这16份砂梨材料区分开。砂梨ISSR反应体系的建立为利用ISSR标记技术进行砂梨品种鉴别、分类、种质资源遗传多样性分析莫定了良好基础。

Establishment and Optimization of ISSR-PCR Reaction System in Pyrus py rifolia Nakai

The influenced factors of ISSR-RCR reaction were optimized and the effect of 5 factors such as annealing temperature,Taq DNA polymerase dosage,DNA templates concentration,primer concentration and dNTPs concentration using single factor experiment based on the genomic of Pyruspy rifolia Nakai,cultured in China southern.The total genome DNA had been obtained through the method of modified CTAB.Its reaction system and amplified procedure were established that was 20μL amplification reaction system containing lxTaq enzyme PCR buffer,0.5 U Taq DNA polymerase,100μmol /L dNTPs,0.7 μ mol/L primer,2.25 mmol/L MgCl 2,40ng template DNA.The optimized annealing temperature was 52℃.The optimal amplified procedure was as follows：after apre-denaturing of 5 min at 94℃,40 cycles were performed with 45 s for denaturing at 94℃,annealing of 45 s at 52℃,extension of 1 min at 72℃,10 minutes of extension at 72℃ in the final cycle and hold at 4℃.Using primers 863 was selected to separate 16 copies Pyrus pyrifolia material.The extablishment of the ISSR-PCR reaction system could settle favorable foundation for identification of Pyrus pyrifolia,classification and analysis of the genetic diversity of them using ISSR molecular marker techniques.