华北落叶松AFLP反应体系的建立和优化

目的]建立和优化华北落叶松（Larixprincipis-rupprechtii Mayr.）的AFLP反应体系。方法]以华北落叶松为材料,对其AFLP分析过程中的5个主要影响因素（酶切时间、内切酶用量、DNA模板用量、连接产物和预扩增产物稀释倍数）进行了研究。结果]确立了适于华北落叶松AFLP分析的最佳反应体系,即在反应体积均为20.0μl的前提下,酶切时间为7 h（37℃3.5 h;65℃3.5 h）,EcoRⅠ和MseⅠ内切酶用量均为1 U,DNA模板用量为3.0μl（50 ng/μl）,连接产物稀释10倍取5.0μl用于预扩增,预扩增产物稀释70倍取4.0μl用于选择性扩增。结论]采用该体系得到的电泳条带清晰可辨、多态性好、重复性强,可用于对华北落叶松遗传多样性和分子进化特征的深入研究。

Establishment and Optimization of AFLP Reaction System in Prince Rupprecht's Larch

Objective] The aim was to establish and optimize the AFLP reaction system in prince rupprecht's larch.Method] Taking Larix principis-rupprechtii Mayr.as tested material,5 main influencing factors like the time of DNA digestion,the amount of endonucleases EcoR Ⅰand Mse Ⅰ,the amount of template DNA and the dilution times for ligated product and pre-amplification products were experimentally examined with 20.0 μl reaction system.Result] An efficient and stable AFLP reaction system was established as follows: 7 hours(37 ℃ 3.5 h;65 ℃ 3.5 h) of DNA digestion;1 U of EcoR Ⅰand Mse Ⅰenzymes,respectively;3.0 μl(50 ng/μl) of template DNA;5.0 μl of ligated product diluted by 10 times;and 4.0 μl of pre-amplification product diluted by 70 times.Conclusion] AFLP bands obtained through the reaction system were clear,highly polymorphic and reproducible,so the AFLP reaction system could be used in further studies on genetic diversity and molecular evolution of Larix principis-rupprechtii.