Establishment and Optimization of SRAP Amplification System in Squash

In order to screen out the optimal concentrations of various components,the best annealing temperature and the best cycle times,we took an optimization experiment of 20μL SRAP-PCR amplification system with single factor design which focused on the concentrated of Mg 2+,dNTP,Taq DNA polymerase,primer,template DNA and the amplification procedures of annealing temperature and cycle times by using the Squash ‘Miben'as the material of filter system.Experimental results showed the optimal 20μL SRAP amplification of Squash contained 0.2mmol·L-1 dNTP,1.5U Taq DNA polymerase,80ng template DNA,0.16μmol·L-1 single primer,1.5 mmol·L-1 Mg2+,2μL 10×Buffer.And the best amplification procedure was pre-denaturation at 94℃for 5min. Next by 5cycles of denaturation at 94℃for 1min,anneal at 35℃for 1min and the extension at 72℃for 1min then 35cycles of 94℃for 1 min,52℃for 1 min and 72℃for 1 min and a final extension at 72℃ for 10min.The amplification system and the amplification procedures were tested by using 17variaties of Squash.The test results showed the amplification products which were clear and bright,abundant polymorphism,strong specificity and good repeatability.Accordingly,it was suitable for SRAP analysis of Squash.