小麦抗叶锈病基因Lr45的表达序列标签-酶切扩增多态性(EST-CAPS)分析  

Analysis of Expressed Sequence Tags-Cleaved Amplified Polymorphic Sequences(EST-CAPS) for Wheat Leaf Rust Resistance Gene Lr45

作　　者：郭楠[1] 刘春燕[2] 赵盼盼[1] 杨文香[1] 闫红飞[1] 刘大群[1] 

机构地区：[1]河北农业大学植物保护学院、河北省农作物病虫害生物防治工程技术研究中心、国家北方山区农业工程技术研究中心,保定071001 [2]河北北方学院动物科技学院,张家口075131

出　　处：《农业生物技术学报》2013年第6期668-676,共9页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展计划(No.2013CB127700);国家公益性行业(农业)科研专项(No.200903035);教育部博士点基金(No.2010130212005);河北省留学人员科技活动项目择优资助项目(No.20120340);河北省教育厅自然科学青年基金(NO.Q2012010)

摘　　要：为了获得与小麦(Triticum aestivum L.)抗叶锈基因Lr45更多的分子标记,建立更丰富的遗传连锁图谱,本研究利用表达序列标签-酶切扩增多态性(EST-CAPS)标记技术,选择小麦2A染色体短臂的26对EST引物与4种限制性内切酶共104对组合,对小麦抗叶锈近等基因系(near-isogenic line,NILs)TcLr45和感病对照Thatcher进行了多态性分析。EST引物BE426158与BE442876的PCR产物在亲本间存在差异,多态性检出率为7.7%;104个引物/酶切组合中,CD454036/MspⅠ、CD454036/HaeⅢ、BE406923/MspⅠ和BE425962/RsaⅠ共4组在Thatcher和TcLr45之间呈现多态性,多态性检出率为3.8%。将BE426158标记成功转化为一个更稳定的序列标签位点(sequence tagged site,STS)标记,命名为LR45-1,经在TcLr45×Thatcher F2群体、抗叶锈近等基因系及黑麦品种中验证,LR45-1与Lr45连锁,遗传距离为8.2 cM。研究结果提示,EST-CAPS技术可应用于小麦抗叶锈病基因的多态性分析及分子标记的开发。The objective of this study is to develop more molecular markers for construction of high density genetic map of the leaf rust resistance gene Lr45 in wheat （Triticum aestivum L.）. Twenty-six pairs ofexpressed sequence tag（EST） primers located on the short arm of wheat chromosome 2A in combination with 4 kinds of restriction enzyme, resulting in a total of 104 pairs of EST-CAPS（expressed sequence tags-cleavedamplified polymorphic sequences） markers were used to analyze the polymorphism between TcLr45 and Thatcher. The EST primers BE426158 and BE442876 expressed polymorphsim of the PCR products betweenTcLr45（resistant leaf rest parent） and Thatcher（susceptible leaf rust parent） with the polymorphism rate of 7.7%. Four of 104 primers/restriction enzyme combinations （3.8%）, CD454036/Msp Ⅱ, CD454036/Hae Ⅱ,BE406923/Msp Ⅰ and BE425962 / Rsa Ⅰ, were polymorphic between Thatcher and TcLr45. In addition, a pair of primers designed based on the sequence of band amplified with BE426158 in TcLr45 and convertedsuccessfully to a stable STS（sequence tagged site） marker, LR45-1. The linkage distance of the LR45-1 with Lr45 was 8.2 cM based on the data of a F2 population derived from TcLr45 × Thatcher. The specificity andstability were validated by testing the wheat leaf rust resistance near isogenic lines and rye（Secale cereale） varieties. The results described above indicate that the EST-CAPS markers can be used in analysising ofpolymorphisms and development of molecular markers in wheat leaf rust resistance genes.

关 键 词：小麦 抗叶锈基因 TcLr45 EST-CAPS 分子标记 

分 类 号：S565.4[农业科学—作物学]