Establishment and optimization of an ISSR reaction system for Populus tomentosa Carr

In order to use ISSR markers to study genetic variation,assistant breeding,cultivar identification and phylogenesis of Populus tomentosa,the annealing temperature,concentration of primer,dNTP,Mg²⁺ and Taq DNA polymerase dosage on ISSR-PCR amplications were tested to determine their optimal levels and establish the following optimal reaction system for ISSR analysis in P.tomentosa.The results showed that the optimal conditions for ISSR-PCR of P.tomentosa were as follows:PCR reaction volume of 20 μL,1×Taq buffer(10 mmol/L Tris-HCl,50 mmol/L KCl,0.1% Triton X-100,pH 9.0),1.0 U Taq DNA Polymerase,0.2 μmol/L primer,0.2 mmol/L dNTP,1.5 mmol/L MgCl-2 and 10 ng template DNA.The optimized annealing temperature was 61℃ for primer(GTG)-6.