苹果MSAP技术体系的优化及其应用

为建立适合苹果的MSAP(Methylation Sensitive Amplification Polymorphism)反应体系，以‘嘎啦’为材料对MSAP技术中的关键因素进行优化筛选，并用于苹果叶片和愈伤组织的甲基化模式分析。经过不同因素水平摸索，结果表明：600 ng基因组DNA用EcoRⅠ，HapⅡ或MspⅠ各10 U，37℃保温12 h，即可酶切完全。最佳预扩增体系为：酶连产物1 μL、上下游引物各3 μL、2×PCR mastermix 10 μL（Taq酶1 U、dNTPs 5 mM、Mg2+ 30 mM）和ddH2O 3 μL。20 μL选择性扩增体系中，含有60倍稀释的模板DNA 2 μL、上下游引物各3 μL、2×PCR mastermix 10 μL（Taq酶1 U、dNTPs 5 mM、Mg2+ 30 mM）和ddH2O 2 μL。在该体系下选用2对引物对苹果叶片和愈伤组织进行甲基化模式分析，获得了清晰、重复性好的银染指纹图谱。平均每对引物可获得特异性条带12条，共获得特异性条带91条，表明该体系可用于苹果不同发育阶段DNA甲基化模式分析。

Optimization and Establishment of MSAP Reaction System for Apple

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 was found that 600 ng genomic DNA could be fully digested by EcoR Ⅰ,Hap Ⅱ or Msp Ⅰ,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 mmol/L,Mg 2 ＋ 30 mmol/L）,ddH 2 O 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 1 U,dNTPs 5 mmol/L,Mg 2＋ 30 mmol/L）,ddH 2 O 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%）.