Abstract:

In order to establish suitable MSAP (Methylation Sensitive Amplification Polymorphism) reaction system for apple (Malus domestica Borkh.), ‘Gala’ was used as material to screen the key factors in the system. It found that 600 ng genomic DNA could be fully digested by EcoRⅠ, HapⅡ or MspⅠ, 10 U respectively, at 37℃ for 12 h. The 20 μL pre-amplification reaction mixture contained the follow factors, DNA template 1 μL, 2×PCR master mix 10 μL (Taq DNA polymerase 1 U, dNTPs 5 mM, Mg2+ 30 mM), ddH2O 2 μL, primers E/HM 3 μL, respectively. The amplification products were diluted 60 times for the selective amplification. The 20 μL selective pre-amplification reaction mixture contained DNA template 2 μL, 2×PCR master mix 10 μL (Taq DNA polymerase 1U, dNTPs 5 mM, Mg2+ 30 mM), ddH2O 2 μL, Primer EcoR-ACA/HpaII-MspI-TCAA 3 μL respectively. Two pairs of MSAP primer combinations were used in the methylation style analysis, clear and stable MSAP patterns could be obtained. Total 91 specific bands were detected, 12 specific bands for each MSAP paired marker, and the statistics showed that the methylation degree of callus (40%) was higher than leaf (25%).