山豆根ISSR-PCR反应体系优化及引物筛选

Optimization of ISSR-PCR Reaction System and Primer Selection of Sophora tonkinensis Gagnep

以山豆根为试验材料,采用正交试验考察DNA、引物、Mg^(2+)、dNTPs、Taq酶5个因素对ISSR-PCR反应体系的影响,建立并优化山豆根ISSR-PCR反应体系条件。结果表明:山豆根ISSR-PCR反应体系(20μL)最优条件为Mg^(2+)浓度3 mmol/L,dNTPs 0.2 mmol/L,Taq酶1 U,引物0.5μmol/L,模板DNA 100 ng,运用该体系从100条UBC引物中筛选到10条清晰、多态性好的引物。采用正交试验设计可有效建立山豆根ISSR-PCR反应的最优体系,为山豆根的亲缘关系,种质资源遗传多样性评价及品种鉴定和系统分类等研究提供科学依据。

Taking Sophora tonkinensis as materaial,orthogonal experiments were used to investigate the effects of DNA,primers,Mg^(2+),dNTPs,and Taq enzymes on the ISSR-PCR reaction system,and the conditions of ISSR-PCR reaction system of Sophora tonkinensis were established and optimized.The results showed that the optimal conditions for the ISSR-PCR reaction system(20μL)of Sophora tonkinensis including 3 mmol/L Mg^(2+),0.2 mmol/L dNTPs,1 U Taq enzyme,0.5μmol/L primer,100 ng template DNA.Ten primers with good polymorphism were selected from 100 UBC primers by this system.The optimal system of ISSR-PCR reaction can be effectively established by orthogonal experiment design,which provides a scientific basis for the research of genetic relationship,genetic diversity evaluation of germplasm resources,variety identification and systematic classification of Sophora tonkinensis.