甜瓜SRAP-PCR程序和反应体系的优化

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

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。

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 rain 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 I min,51℃ for 1 min and 72℃ for 1 min;and a final extension at 72℃ for 10 rain;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 0.24 μmol/L, polymerase 1. 5U. The procedure and system could meet the demands for genome SRAP amplification in Cucumis melo L.