红三叶ISSR-PCR反应体系的建立与优化

采用正交试验设计,对影响红三叶ISSR-PCR反应较大的4个因素(Taq DNA聚合酶、Mg2+、dNTP及引物)在4个水平上进行筛选,建立并优化了适合红三叶ISSR分析的最佳反应体系。在25μL反应体系中各反应物的最适含量为40ng模板DNA,10×PCR-buffer 2.5μL,2.5 mmol/L Mg2+,2.5UTaq DNA聚合酶,引物1μmol/L,0.2mmol/L dNTP。反应程序为:94℃预变性5min;94℃变性30s,引物退火30s,72℃延伸1min,循环35次;72℃延伸7min,4℃保存。优化体系的建立为采用ISSR分子标记技术进行红三叶种质资源遗传多样性分析提供了技术参考。

Establishment and optimization of ISSR-PCR amplification system for red clover

With a orthogonal design,the paper studied four main factors(Taq DNA polymerase,Mg2 + ,prem-ier and dNTP)at four level that infulence ISSR-PCR amplification system for red clover and finally established an optimal reaction system.In the amplification system with 25 μL solution,the appropriate containing of react-ants were 40 ng template DNA,10 ×PCR-buffer 2.5 μL,2.5 mmol/L Mg2 + ,2.5 U Taq DNA polymerase,1μmol/L premier and 0.2 mmol/L dNTP.The optimized reaction program was 94 ℃ for 5min,followed by 35 cy-cles of 94 ℃ for 30 s,annealing 30 s,1 min at 72 ℃ and a final extension at 72 ℃ for 7 min,then preservation at 4 ℃.The establishment of the optimized amplification system provides a technical references for the analysis of genetic diversity of red clover with ISSR markers.