Optimization of SSR-PCR Reaction System in Fraxinus and SSR Primer Selection

This study established and optimized SSR-PCR reaction system of Fraxinus, and used this systemselected 3 primer pairs from 50 primer pairs for the SSR analysis based on their reproducible and clearbanding patterns. These primer pairs could be used for further research. This experiment established throughL16(45) orthogonal design which selected from 4 levels of 5 factors(Taq DNA polymerase, DNA, dNTPsconcentration, primers concentration and Mg2 +concentration) in SSR- PCR system and optimized throughsingle factor experiment about the main factors of the PCR reactions. The optimal SSR-PCR reaction systemconsisted of Mg2 +(25 mmol/L) 0.8 μL, containing forward- and reversed- primer(10 μmol/L) 0.2 μLrespectively, dNTP(10 mmol/L) 0.3 μL, Taq DNA polymerase(5 U/μL) 0.05 μL、DNA(5-10 ng/μL) 2.00 μL,10×PCR Buffer 1.0 μL and ddH2O 5.45 μL. The reaction system was the most conformable one for Fraxinus’ SSR-PCR, and was established as the good foundation of SSR-PCR marker technique on Fraxinus.