Establishment and Optimization of an ISSR Reaction System for Simulium quinquestriatum

In this paper,the orthogonal design and single factor method of six critical factors (primer,DNA template,dNTP,Mg2+ ,BSA,Taq DNA polymerase) of ISSR-PCR reaction system were used to optimize the ISSRPCR amplification system on S. quinquestriatum. Finally,a satisfactory ISSR (Inter Simple Sequence Repeat) reaction system for S. quinquestriatum with desirable repeatability and polymorphic bands was established. The 20μl reaction system contained primer 1. 0μmol / L,DNA template 50. 0ng/μl,dNTP 0. 15mmol/L,Mg2+ 1. 50mmol/L, BSA 2. 00mg/ml and Taq DNA polymerase 0. 15 5U/μl. Eight stable and high polymorphic primers were screened from 24 reserve primers by temperature gradient method. This optimized ISSR reaction system would provide the basis for the analysis of genetic differentiation and genetic diversity among populations of S. quinquestriatum.