小麦锈菌AFLP分子标记技术体系的建立*

 以小麦条锈菌、杆锈菌、叶锈菌的夏孢子为材料，通过DNA提取、酶切、PCR扩增、凝胶电泳等系列程序摸索和优化，建立了锈菌的AFLP分子标记体系如下: 40μL酶切体系中采用了EcoRI,TrulI各5U,37℃3h,65℃3h双酶切4μL100ng/μL的DNA; 然后加入10μL连接混合液22℃连接3h,16℃10h; 连接产物5μL,10μmol/LEcoRI,10μmol/LTru1I预扩引物各1.5μL,PCR反应液25μL,ddH₂O17μL进行预扩;预扩产物稀释20倍后取 5μL,50ng/μL EcoRI,Tru1I选扩引物各1μL,PCR反应液10μL,ddH₂O3μL体系进行选择性扩增。为研究小麦锈病和其他真菌性病害的分子标记克隆及抗病育种的辅助选择提供了有力工具。

The Establish of AFLP Markers Technical System for Wheat Rust Fungus

Now amplified fragment length polymorphism (AFLP) is one of the usest molecular marker methods, In this paper,we used wheat rust fungus as materials.Through extracting DNA, the digestion DNA of wheat rust fungus, PCR amplification and so on, we established the AFLP markers technical system for rust fungus400 ng quantity of DNA was digested with 5U EcoRⅠand 5U TrulⅠat 37℃for 3h and then 65℃ for 3h,then added ligation mixture at 22℃ for 3h and 16℃ for 10h;pre-selective amplification were set up 5μL restrictiom-ligatiom samples DNA,10μmol/L pre-selective amplification primers each 1.5μL,PCR mixture 25μL,ddH₂O 17μL; selective amplification were set up DNA 5μL, selective amplification primers each 1μL, PCR mixture 10μL,ddH₂O 3μL.These results provided an useful tool to research the molecular cloning of wheat rust fungus and other fungus disase and breeding assisted selection.