摘要
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.
出处
《果树学报》
CAS
CSCD
北大核心
2011年第2期209-214,共6页
Journal of Fruit Science
基金
国家自然科学基金(NSFC:30800742)
河南省教育厅自然科学研究计划(2009B210003)
国家葡萄产业技术体系(nycytx-30-zy-01)
关键词
葡萄
SCoT
正交设计
单因素试验
体系优化
Grape(Vitis vinifera)
SCoT
Orthogonal design
Single factor experiment
System optimization