南瓜RAPD分析体系的优化*

 为确保南瓜RAPD反应结果的稳定性和重复性，对MgCl₂浓度、dNTPs浓度、Taq酶含量、引物浓度、模板DNA浓度、复性温度、PCR循环次数等影响南瓜RAPD结果的重要因素进行了初步研究。最终优化的南瓜 RAPD反应体系为25 μL反应液中：10×反应 buffer（100 mmol/L Tris-HCl,100 mmol/L KCl, 80 mmol/L (NH₄)₂SO₄ ,pH 9.0,NP-40）2.5 μL, 3 mmol/L MgCl₂，0.2 mmol/L dNTPs，Taq酶l.0 U,引物15 ng/μL ，DNA模板10 ng/μL 。本研究最终确立的PCR反应程序为: 94 ℃预变性5 min,然后按94 ℃变性30 s,37 ℃退火30 s,72 ℃延伸90 s，进行40个循环,最后72 ℃延伸5 min。在此条件下得到的RAPD图谱可为南瓜遗传多样性、分子标记及辅助育种等研究提供有效的手段。

Optimization of RAPD Analysis System in Squash

The optimum reaction system of RAPD in squash was studied in order to ensure stability and reproducibility of RAPD. After testing some important influencing factors of RAPD in squash such as MgCl₂, dNTPs, Taq DNA polymerase,primer, template DNA,the temperature for annealing and the number of cycles,the results showed that in 25 μL RAPD reaction system the optimum concentration were 3 mmol/L MgCl₂, 0.2 mmol/L dNTPs, Taq DNA polymerase 1.0 U, primer 15 ng/μL,template DNA 10 ng/μL. The PCR amplification procedure used in this study was as follow : pre-denature at 94 ℃ for 5 min, then 94 ℃ 30 s,37 ℃ 30 s 72 ℃ 90s,for 40 cycles, finally extended at 72 ℃ for 5 min. The results also indicated that the optimized conditions of RAPD could provide an effective means for the research of genetic diversity, molecular marker and auxiliary breeding in squash.