葡萄目标起始密码子多态性反应体系的优化

Optimization of Start Codon Targeted Polymorphism PCR(SCoT-PCR) system in Vitis vinifera

SCoT标记是一种新型目的基因分子标记方法,采用L16(45)正交试验和单因素试验2种方法优化了葡萄SCoT-PCR反应体系,结果表明正交试验中关键影响因子为dNTPs浓度,单因素试验中为dNTPs和Mg2+浓度。综合2种方法,优化的葡萄SCoT-PCR反应体系为:Mg2+2.0 mmol.L-1、dNTPs 0.3 mmol.L-1、Taq酶1.0 U、引物0.3μmol.L-1、DNA模板30 ng,总体积20μL。扩增程序为:94℃预变性5 min;94℃变性1 min,54℃退火1 min,72℃复性2 min,34个循环;最后72℃延伸8 min。该反应体系在20个葡萄品种的验证中表现出良好的稳定性和可重复性,该反应体系的建立为葡萄种质遗传多样性评价、基因组分析、指纹图谱构建、分子标记辅助选择育种等研究提供了新的技术手段。

The grape SCoT-PCR reaction system was optimized based on the L16（45） orthogonal experiment and single factor experiment design.The results showed that： the key factor was the dNTPs concentration in the orthogonal experiment,while in the single factor experiment,the key factors were the dNTPs and Mg2＋ concentration;Based on the results,it was suggested that the optimal SCoT-PCR system for grape was： total 20 μL reaction system containing 2.0 mmol·L^-1Mg2＋,0.3 mmol·L^-1dNTPs,1.0 U Taq polymerase,0.3 μmol·L^-1primer,and 30 ng DNA template.The PCR amplification profile was pre-denaturing at 94 ℃ for 5 min,,denaturing at 94 ℃ for 1 min,annealing at 54 ℃ for 1 min and extending at 72 ℃ for 2 min,34 cycles and extending at 72 ℃ for 8 min finally.The optimal reaction system was further verified in 20 grape varieties,which showed good stability and repeatability.The system provides a new technology for evaluation of genetic diversity,genomic analysis,construction of fingerprinting and molecular marker assisted breeding for the grape.