小麦锈菌AFLP分子标记技术体系的建立  

The Establishment 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,雅安625014 [2]四川农业科学院生物技术核技术研究所,成都610066

出　　处：《中国农学通报》2005年第8期325-329,共5页Chinese Agricultural Science Bulletin

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,10μMEcoRI、10μMTru1I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/μlEcoRI、Tru1I选扩引物各1μl,PCR反应液10μl,ddH2O3μl体系进行选择性扩增,为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 f DNAof wheat rust fungus,PCR amplification and so on, we established the AFLP markers technical system for rust fungus： 400ng quantity of DNA was digested with 5U EcoRI and 5U TrulI at 37℃ for 3hours and then 65℃ for 3 hours,then added ligation mixture at 22℃ for 3hours and 16℃ for 10 hours; pre-selective amplification were set up 5μl restrictiom-ligatiom samples DNA,10uM 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 disease and breeding assisted selection.

关 键 词：小麦 条锈菌 杆锈菌 叶锈菌 AFLP分子标记技术体系 抗病育种 

分 类 号：S435.121.4[农业科学—农业昆虫与害虫防治]