Establishment and Optimization of the ISSR-PCR System for Hypericum perforatum

Hypericum perforatum is an important medicinal plant,which was subjected to study ISSR-PCR system in order to investigate the genetic diversity within and among populations.The influencing factors of ISSR-PCR system were study and the experiment parameters were optimized.By adjusting template DNA concentration,Mg2+concentration,dNTP and Taq polymerase contents and annealing temperature,the PCR amplification conditions were optimized.The optimal experiment conditions were as follow: 20μL system contained 10 × PCR Buffer,200 μmol/LdNTP,0.5 μmol/L primmer,1.0 mmol/L MgCl2,2.0 U Taq polymerase and 50 ng DNA template.Moreover,25 ISSR primers were used to screen the suitable primers for assessing the genetic diversity of H.perforatum,and 13 ISSR primers with high resolution,multiple polymorphic and good repeatability bands were screened.It lays the foundation for the study of genetic diversity and germplasm resources of H.perforatum identification.