Abstract:

Abstract: Taking radish ‘Chun xue zong tai’ as materials, six key factors of AFLP analysis system such as the digestion time of DNA, the different levels of concentration of each enzyme , Mg2+, dNTPs, the dilution ratio of response template and the number of amplification cycles were studied. The optimization system of AFLP makers linked to bolting trait in radish was established. The result showed: 400ng radish DNA were digested with 0.2μL EcoR I and 0.2μL Mse I at 37℃ for 10min and then 65℃for 10 min. Then 5μL digestion products were made a ligation mixture with 0.5μL EcoR I adapter, 0.5μL Mse I adapter and 0.5μL T4 DNA Ligase at 22℃ for overnight in 20μL.The pre-amplification system in 15μL was set up with 5μL 5-times dilutions of Ligation products,10μmol/L pre-selective amplification primers 0.6μL respectively, Mg2+1μL, dNTPs1.5μL,Taq DNA Polymerase0.2μL and ddH2O 4.6μL by 35 cycles. After diluting the pre-amplification samples by 20 times, the selective amplification system was set up with 5μL the pre-amplification dilution products and selective amplification primers 0.6μLrespectively, the concentration of other components was the same as the pre-amplification system in 15μL. The applicability of the optimization system of AFLP makers was verified with 16 radish varieties, which belonged to five ecotypes. Its electrophoresis strips were clear and polymorphism was rich.