甜瓜SRAP-PCR程序和反应体系的优化

以甜瓜品种炮台红和皇后为试验材料,对影响甜瓜SRAP-PCR反应体系的各因子进行了梯度试验,并对退火温度进行了探索,得到扩增多态性高、重复性好、带型清晰的SRAP-PCR反应程序和体系。反应程序为:94℃预变性5 min;94℃变性1 min,35℃退火1 min,72℃延伸1 min,共5个循环;94℃变性1 min,51℃退火1 min,72℃延伸1 min,共35个循环;72℃延伸10 min;4℃保存。反应体系(共25μL):DNA 45 ng,Mg~2+2.0mmol/L,dNTPs0.15 mmoL/L,primer 0.24/μmol/L,Taq polymerase 1.5 U。结果表明,该程序和体系能够较好的满足甜瓜基因组SRAP扩增的要求。

The Optimization of SRAP-PCR Amplification Program and Reaction System for Cucumis melo L.

Cucumis melo L.varieties Paotaihong and Huanghou were taken as experimental materials.SRAP-PCR reaction program in Cucumis melo L.was investigated and the gradient experiment was conducted on each impact factors(Taq DNA polymerase,Mg~(2+),DNA template,dNTP,primer,and annealing temperature) in SRAP-PCR reaction system and reaction procedure to establish the optimum SRAP - PCR reaction program and system that could amplify high polymorphism,good repeatability and clear band pattern.The optimum SRAP - PCR reaction procedure was:pre - denaturation at 94℃for 5 min followed by 5 cycles of denaturation at 94℃for 1 min,anneal at 35℃for 1 min and extension at 72℃for 1 min;then 35cycles of 94℃for 1min,51℃for 1 min and 72℃for 1 min;and a final extension at 72℃for 10 min;kept at 4℃.The optimum SRAP- PCR system for a volume of 25μL was:DNA45ng,Mg~(2+) 2.0 mmol/L,dNTPs 0.15 mmol/L,primer 0.24μmol/L,polymerase 1.5U.The procedure and system could meet the demands for genome SRAP amplification in Cucumis melo L.