草莓ISSR-PCR体系的建立和优化

Establishment and Orthogonal Optimization of ISSR-PCR Reaction System to Fragaria × ananassa

草莓ISSR-PCR反应体系中的5个影响因子:DNA模板(ng/(25μl)、Taq酶(U/(25μl)、引物(mmol/L)、dNTPs(mmol/L)、Mg2+(mmol/L),在4个水平上对其进行正交优化试验,并通过单因素试验验证各因素的最佳反应水平,建立起草莓的最佳反应体系:模板浓度为40 ng/(25μl),Taq酶浓度为0.5 U/(25μl),引物浓度为0.35 mmol/L,dNTPs浓度为0.20 mmol/L,Mg2+的浓度为2.0 mmol/L。研究为ISSR标记技术鉴定草莓的种质资源提供了科学依据。

In the present study,the five factors of the Inter Simple Sequence Repeats amplification system of Fragaria × anonassa,which are DNA template（ng/（25 μl）,Taq polymerase （U/（25 μl）,primers （mmol/L）,dNTPs（mmol/L）,Mg2 ＋ （mmol/L） separately,can influence the amplification polymorphism,experimental results and its reliability.The orthogonal design was used to optimize the experiment at four levels respectively,and the optimum reaction level of every factor was selected.The best ISSR amplification system of the strawberry was established as follows：the density of DNA Template was 40 ng/（25 pl）,and the density of Taq polymerase was 0.5 U/（25 μl）,and the density of primers was 0.35 mmol/L,and the density of dNTPs was 0.20 mtmol/L,and the density of Mg2 ＋ was 2.0 mmoL/L.The experiment could provide an experimental basis for later germplasm identification of strawberry.