Optimization of SSR reaction system in Malus sieversii

DNA of four Malus sieversii accessions were distilled by CTAB.An optimal experimental design was employed to evaluate five factors (template DNA,Mg2+,dNTPs,Taq DNA polymerase and primer) at five different concentrations.The results indicated that the optimal SSR-PCR reaction system of terminal volume 25 μL consisted of 0.5 U Taq DNA polymerase,0.2 μmol/L primers,5 ng template DNA,0.2 mmol/ L dNTP and 1.0 mmol/L Mg2+.The suitable amplification temperature profiles were as following:2 min at 94℃for pre-denaturing, followed by 4 cycles of 30 s at 94℃for denaturing,1 min at 65℃for anncaling,1 min at 72℃for extending,then followed by 30 cycles of 30 s at 94℃for denaturing,1 min at 60℃for anncaling,1 min at 72℃for extending, and finally kept the reaction mixture at 4℃after a final extension step of 72℃for 5 min.Using the above optimal SSR-PCR reaction system,SSR fragments of 30 Malus sieversii accessions were obtained.The clear and abundant polymorphism indicated this reaction system was suitable for construction gene linkage map and QTL mapping.