梨ISSR-PCR反应体系的正交优化研究

On Optimization of Reaction System in Pear-Using Orthogonal Design

以‘饼子梨’为试材,利用正交设计L16(45)研究了反应体系中引物、模板DNA浓度等5个因素4个水平对梨反应体系的影响,PCR结果应用极差分析和MINITAB软件对扩增结果进行方差分析.梨最佳ISSR-PCR反应体系:25μL反应体系中含2.5μL 10×Buffer,2.0mmol/L Mg2+,0.25mmol/L dNTPs,0.25μmol/L引物,60ng模板DNA,0.75UTaq DNA聚合酶.优化的ISSR-PCR反应体系稳定性高和重复性较好,为梨品种指纹图谱构建和遗传多样性分析奠定基础.

Orthogonal design has been applied to optimize ISSR‐PCR amplification system of in pear in five factors such as primer ,dNTPs ,template DNA ,Taq DNA polymerase and Mg2＋ at four levels .The result of PCR has been analyzed by range analysis and variance analysis .An optimal reaction system （25μL） has been established ,i .e .2 .5 μL 10 × Buffer ,2 .0 mmol/L M g2＋ ,0 .25 mmol/L dN T Ps ,0 .25 μmol/L prim‐er ,60 ng template DNA ,0 .75 U Taq DNA polymerase .The results indicate that the optimized reaction system was stable and repeatable .The objective of the study is to provide a scientific basis for the con‐structing DNA fingerprints and genetic diversity analysis of in pear .