金柑属ISSR反应体系的建立及优化

Establishment and Optimization of the ISSR Reaction System for Fortunella Swingle

以融安金弹(F.crassifolia Swingle)为试材,利用正交设计和单因素试验相结合的方法,对ISSR反应体系中各个主要影响因子进行了优化筛选,结果表明,20μL的ISSR反应体系中各组分的最适浓度分别为:Mg2+1.60 mmol/L,dNTPs 0.15 mmol/L,TaqDNA酶1.0 U,引物0.2μmol/L模板1.2 ng/μL.并利用该优化对金弹等21份金柑属材料进行了ISSR扩增,证实了该体系稳定和可靠性.

The orthogonal design experiment and the single-factor experiment were combined, in which Fortunella crassifolia Swingle genome DNA was used as the template, to screen for the optimum combinations of the main influencing factors of the ISSR reaction system of the fruit. The optimum concentrations of Mg^2＋, dNTPs, Taq DNA polymerase, primer, template DNA in 20 μL reaction system were 1.60mmol/L, 0. 15 mmol/ L, 1.0 U, 0.2 μmol/L, 1.2 ng/μL, respectively. Amplifications of 21 varieties of Fortunella were made with this system, the results of which confirmed its stabilization and reliability.