Optimization of SRAP-PCR system on Poa pratensis using orthogonal design and selection of primers

An orthogonal design was used to optimize a SRAP-PCR system for Poa pratensis with 4 factors(Mg2+,dNTP,primer and Taq polymerase) at 3 levels plus the concentration of template DNA.The optimized SRAP-PCR system was: 2 μL 10 × PCR buffer,40 ng template DNA,Mg2+1.75 mmol·L-1,dNTP 220 μmol·L-1,primer 0.25 μmol·L-1,Taq DNA polymerase 1.0 U in a total of 20 μL reaction mixtures.With the optimized system,43 primer combinations were selected among 100 primer combinations,which produced abundant polymorphism bands.This study optimized SRAP-PCR system and selected the proper primers in P.pratensis,which would play an important role in genetic diversity analyses,map construction,germplasm identification in P.pratensis with SRAP markers.