小麦锈菌AFLP分子标记技术体系的建立  

The Establish of AFLP Markers Technical System for Wheat Rust Fungus

作　　者：唐静[1] 蒲志刚[2] 张敏[1] 阎文昭[2] 

机构地区：[1]四川农业大学农学院,四川雅安625014 [2]四川农业科学院生物技术核技术研究所,四川成都610066

出　　处：《云南农业大学学报》2005年第6期753-757,共5页Journal of Yunnan Agricultural University

基　　金：国家"863"计划课题资助(2002AA245041)

摘　　要：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EcoR I,Tru lI各5U,37℃3 h,65℃3h双酶切4μL 100 ng/μL的DNA;然后加入10μL连接混合液22℃连接3 h,16℃10 h;连接产物5μL,10μmol/LEcoR I,10μmol/LTru1 I预扩引物各1.5μL,PCR反应液25μL,ddH2O 17μL进行预扩;预扩产物稀释20倍后取5μL,50 ng/μL EcoR I,Tru1 I选扩引物各1μL,PCR反应液10μL,ddH2O 3μL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。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 fungus：400 ng quantity of DNA was digested with 5U EcoR I and 5U Trul I at 37 ℃for 3 h and then 65 ℃ for 3 h,then added ligation mixture at 22 ℃ for 3 h and 16 ℃ for 10 h;pre-selective amplification were set up 5 μLrestrictiom-ligatiom samples DNA, 10 μmol/L pre-selective amplification primers each 1.5 μL,PCR mixture 25 μL,ddH20 17 μL; selective amplification were set up DNA 5 IxL, selective amplification primers each 1 μL, PCR mixture 10 μL, ddH2O3 μL. These results provided an useful tool to research the molecular cloning of wheat rust fungus and other fungus disase and breeding assisted selection.

关 键 词：小麦锈菌 AFLP 分子标记 抗病育种 

分 类 号：S435.121.43[农业科学—农业昆虫与害虫防治]