Optimization for ISSR-PCR reaction system on Schisandra henryi by orthogonal design

Objective To optimize the each factor affecting on ISSR-PCR reaction system of Schisandra henryi and establish the stable ISSR-PCR system.Methods Based on the analysis of orthogonal design test,an orthogonal design was used to optimize the ISSR-PCR amplification system on S.henryi by five factors(Taq polymerase,Mg2+,DNA template,dNTP,and primer) at four concentration levels,respectively.Results A suitable ISSR-PCR reaction system was constructed with the 20 μL reaction system containing 1.00 U Taq polymerase,Mg2+1.50 mmol/L,DNA template 40.00 ng,dNTP 0.25 mmol/L,and primer 0.50 μmol/L.Twelve effective ISSR primers were selected and the optional annealing temperature of every one primers was fixed.Conclusion ISSR-PCR is significantly influenced by the concentration of S.henryi.This ISSR-PCR system could provide clear bands,reliable reaction system,and abundant polymorphisms.It proves a reference for molecular research of S.henryi.