橄榄基因组AFLP扩增体系的优化

Optimization of AFLP reaction system in Canarium album

为建立适于橄榄研究用的AFLP技术体系,以10个橄榄品种为材料,对其基因组DNA从酶切、连接、预扩增和选择性扩增等方面进行条件优化。研究结果表明,DNA模板用量为500 ng,酶切体系中限制性内切酶EcoRⅠ和MseⅠ各加入3 U,37℃水浴4 h;连接体系中T4DNA连接酶加入1.5 U,16℃连接6 h;并利用优化后的预扩增体系和选择性扩增体系筛选出6对适合于橄榄AFLP分析的引物组合:E/AAC-M/CAT、E/AAC-M/CTT、E/ACA-M/CAA、E/AGG-M/CAA、E/ACC-M/CAA、E/ACT-M/CAT。采用该体系得到的电泳条带清晰可靠、多态性好,可用于对橄榄遗传多样性的研究。

To establish an appropriate AFLP（Amplifiied Fragment Length Polymorphism） analysis system for Canariurn album （Lour.） Raeusch, 10 cuhivars of C. album （Lour.） Raeusch were involved in optimization of enzyme digestion, ligation, pre-amplification and selective amplification. The results suggested that about 500 ng DNA should be digested with 3 units of EcoR I and Mse I enzymes respectively in a reaction volume of 20 μL, and incubated at 37 ℃ for 4 h. Double-stranded adaptors were legated to the restriction fragments with 1.5 units of T4DNA ligation enzyme at 16 ℃ for 6 h. With the optimized pre-amplifieation and selective amplification systems, 6 pairs of primers （E/AAC-M/CAT, E/AAC-M/CTT, E/ ACA-M/CAA, E/ACC-M/CAT, E/ACC-M/CAA, E/ACT-M/CAT） were selected for the AFLP reaction system of Canarium album. The clear and highly polymorphic AFLP bands could be obtained by this reaction system, which was useful for the further research on genetic diversity of Canarium album in Guangdong province.