Optimizing the inter simple sequence repeat(ISSR) reaction system with Gingko biloba

Key words: 
• forest tree breeding /
• Gingko biloba /
• ISSR-PCR /
• reaction system

Abstract: To optimize the inter simple sequence repeat(ISSR) reaction conditions with Ginkgo biloba,the concentrations of template DNA,Taq DNA polymerase,primers,deoxyribonucleotide triphosphate(dNTP),along with annealing temperature and their influence on polymerase chain reaction(PCR) amplification were studied. The sample leaves were collected in 97 old ginkgo trees from more than 20 provinces in April and May,2006. Results showed that the optimum reaction conditions for ISSR-PCR of ginkgo were: 0.3 μg Taq DNA,2 μL 10×Taq buffer(including 15 mmol·L-1 MgCl2),40 ng template DNA,0.2 mmol·L-1 dNTP,0.5 μmol·L-1 primer,and 1.5 mmol·L-1 Mg2+ with a total volume of 20 μL. Amplification consisted of one cycle with initial denaturation at 94 ℃ for 5 min,followed by 38 cycles of 30 s at 94 ℃,30 s annealing at 48-53 ℃(depending on the primer used), 1 min at 72 ℃,and a final 10 min extension at 72 ℃. Afterward,the samples were maintained at 4 ℃.