Establishment and optimization of ISSR-PCR reaction conditions for Rheum tanguticum

Inter-Simple Sequence Repeats(ISSR)is a good molecular marker for revealing genetic diversity. Reaction system differed in different species,so optimization of ISSR-PCR reaction is very important. Factors which affect the ISSR-PCR amplification,such as the concentration of Mg2+,Taq DNA polymerase,dNTP,primer and template DNA with different annealing temperatures,were optimized and selected by using the genomic DNA of Rheum tanguticum as material. Optimal PCR(20 μL)mix contained 1.5×PCR buffer(15 mmol/L Tris-HCl,75 mmol/L KCl),1.00 mmol/L MgCl2,0.6 U Taq DNA polymerase,0.125 mmol/L dNTP,0.5 μmol/L primer and 30ng template DNA. The suitable annealing temperature was 57.4 ℃ for primer UBC888. Establishment of the PCR reaction conditions could favor further studies on the genetic diversity of R.tanguticum by using ISSR molecular marker techniques.