In order to obtain the best SCoT-PCR reaction system of oil palm(Elaesis guineensis),and select sui-table SCoT primers,this study used the L16(45)orthogonal design and single fantor experiment to optimize the infl-uencing factors of the SCoT-PCR reaction system,and based on this,determine the optimal annealing temperature and cycle number.The results showed that the factors affecting SCoT-PCR amplification,in decreasing order of impact,were dNTPs concentration＞Mg2+concentration＞DNA template amount＞Taq DNA polymerase amount＞primer concentration;the optimized SCoT-PCR reaction system(25 μL)was composed of 120 ng DNA template,0.2 mmol/L dNTPs concentration,1.5 mmol/L Mg2+concentration,0.6 μmol/L primer concentration,2.0 U Taq DNA polymerase amount,and sterilized ddH2O supplemented to 25 μL.The optimized PCR amplification profile was pre-denaturation at 95 ℃ for 3 min,denaturing at 95 ℃ for 30 s,annealing at 55 ℃ for 30 s,and extension at 72 ℃ for 1 min.After 30 cycles,extengding at 72 ℃ for 5 min and stored at 4℃ finally.In addition,30 primers were selected which could amplify clear bands from oil palms based on the optimized reaction system.Using three primers which including SCoT-2 to amplify 30 oil palms materials,the PCR products exhibited good polymorphism,reliability and stability.These results provide a theoretical basis for germplasm resources identifi-cation,genetic diversity and variety fingerprint construction of oil palms.
维普
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