摘要
采用正交试验设计,以山野豌豆叶片DNA为模板,从Mg2+、dNTP、引物和Taq DNA聚合酶4种因素3个水平,对山野豌豆SRAP-PCR反应体系进行优化,并比较了不同浓度模板DNA对扩增效果的影响,建立了山野豌豆的SRAP-PCR最佳反应体系。结果表明,山野豌豆SRAP-PCR最佳反应体系为:2μL 10×PCR buffer(不含Mg2+)、30ng的模板DNA、引物2.0μmol/L、Mg2+2.0mmol/L、Taq DNA聚合酶1.5U、dNTP 0.2mmol/L,总体积为25μL。各因素对扩增反应结果均有不同影响,其中以引物浓度影响最大,dNTP浓度的影响最小。运用该体系对3份山野豌豆种源进行验证,证明该体系稳定可靠,并从98对SRAP引物组合中筛选出扩增条带清晰、多态性丰富的20对引物组合。这一体系的建立及多态性引物组合的筛选为SRAP分子标记技术进行山野豌豆分子遗传学研究奠定了基础。
An orthogonal design was used to optimize a SRAP-PCR system with 4 factors（Mg2＋,dNTP,primer and Taq polymerase） at 3 levels plus the concentration of template DNA.The optimized SRAP-PCR system for Vicia amoena was： 2 μL 10×PCR buffer（Mg2＋ free）,30 ng template DNA,Mg2＋ 2.0 mmol/L,dNTP 0.2 mmol/L,primer 2.0 μmol/L,Taq DNA polymerase 1.5 U in a total of 25 μL reaction solution.Each factor had a different effect on the results of PCR.Primer concentration had the greatest effect and dNTP concentration had the least effect.The optimized SRAP-PCR system was tested on three V.amoena germplasm and was shown to be consistent and reliable.Twenty pairs of primer combinations with abundant polymorphisms were selected from 98 pairs of primer combinations.The optimized SRAP-PCR system and polymorphism primer combinations provide a basis for molecular genetic research on V.amoena.
出处
《草业学报》
CSCD
北大核心
2012年第5期325-330,共6页
Acta Prataculturae Sinica
基金
十二五国家科技支撑项目(2011BAD17B01-02)资助
关键词
山野豌豆
SRAP
PCR体系优化
正交设计
引物筛选
Vicia amoena
SRAP（sequence related amplified polymorphism）
optimization of system
orthogonal design
selection of primers
