【作者】 王海；

【导师】 陈维多；

【作者基本信息】 东北农业大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 小麦条锈病是由小麦条锈菌(Puccinia striiformis West.f.sp.tritici)侵染引起的世界性气传病害,在我国发生尤为普遍而严重。小麦条锈菌是一种专化寄生菌,至今尚未发现有性阶段,利用寄主抗病基因是鉴定小种致病基因的唯一途径。明确小麦条锈菌鉴别寄主的抗条锈病基因及其遗传特点,并标记、定位其抗条锈病基因,可将小种生理专化研究和品种抗病性分析提高到基因分析水平,将基因分析工作纳入国际轨道。Chinese166是重要的小麦条锈菌国际鉴别寄主,其含有的抗条锈病基因Yr1存在于我国部分小麦品种中。明确并标记、定位其抗条锈基因Yr1,有重要理论与实际意义。本研究采用常规遗传分析方法和微卫星标记技术,利用由Chinese166与感病品种Taichang29回交6代转育而成的抗条锈病近等基因系Taichang29*6/ Yr1,用近等基因系转育时的小麦条锈菌系2E16,对Chinese166抗条锈病基因进行标记定位。结果显示,Taichang29与Taichang29*6/ Yr1杂交的245个F2代分离群体中,有181株为抗病株,64个单株为感病株,符合3R:1S的理论比例,经由45株BC1和1560株F3代分析验证,表明近等基因系Taichang29*6/ Yr1(N1)对2E16的抗性由1对显性基因控制。以近等基因系Taichung29*6/Yr1为试材,用Yr1基因所在2A染色体上120对微卫星引物,对Chinese166、近等基因系及其轮回亲本Taichung29进行DNA多态性分析,结果显示,引物Xgwm311、Xgwm382和Xcfa2086在近等基因系Taichung29*6/Yr1(N1)与轮回亲本间能稳定扩增出特异的DNA片段,且近等基因系和抗病基因供体Chinese166间存在相同扩增片段。经F2代抗性遗传连锁性检验确认,这3个引物与近等基因系中的抗条锈病基因Yr1连锁。用3对特异性引物对近等基因系株系的245个F2代单株基因组DNA进行PCR扩增和聚丙烯酰胺凝胶电泳分析,并用Mapmaker3.0b软件处理,对各引物在200个F2代单株的扩增带型结合其抗感情况进行统计分析,表明引物Xgwm311、Xgwm382和Xcfa2086与抗条锈病基因Yr1的遗传距离分别为5.6cM、1.2cM和11.4cM。根据小麦2A染色体遗传图谱,Yr1基因在小麦2AL染色体上的具体位置为: Cent—Xcfa2086—Xgwm382—Yr1—Xgwm311。同时,试验一种新的分子标记技术—MFLP技术应用于小麦DNA多态性研究的可行性。结果显示,该方法在小麦上能产生较高多态性,可用于小麦分子标记和遗传多样性分析。更多还原

【Abstract】 Wheat stripe rust, caused by Puccinia striiformis West. f. sp. Tritici (PST), is one of the most destructive diseases on wheat in many wheat-grown regions in the world,and it is especially heavy and popular in China. Puccinia striiformis is a specific fungus on wheat and its perfect stage has not yet been found .Therefore it is the only method to use host resistance genes to identify the pathogens’virulent genes. After finding out the genetic characteristics, markers and chromosome localization of stripe rust resistance genes, we can improve research levels of pathogens’specificity and host resistance gene analysis. Chinese166 is a very important international wheat stripe rust differential host containing the stripe rust resistance gene Yr1 which exists in many Chinese cultivars. Molecular marker development and chromosome localization for Yr1 is of great theoretical and practice sense.Conventional genetic analysis and SSR were used to develop markers linked closely with Yr1 and to localize it on chromosome genetic map in present study. We analyzed the Taichang29*6/Yr1 near isogenic line (NIL) N1 through crossing the Yr1 gene donor Chinese166 with susceptible recurrent cultivar Taichang29 with pathotype 2E16 of P. striiformis. Through seedling resistance identification of F2 population, the results showed that 181 lines were resistant and 64 were susceptible, and the ratio of resistance lines to susceptible ones was 3R:1S. We also tested the 45-line BC1 population and 1560-line F3 population and further confirmed that the resistance to 2E16 in Taichung29*6/Yr1 N1 line was only controlled under only one dominant gene.Yr1 has been identified to be on wheat 2A Chromosome, 120 SSR primers on 2A were used to test the polymorphisms among Taichung29*6/Yr1 NILs, the recurrent parent Taichung29 and resistance gene donor Chinese166 in this research. The results showed that different DNA bands between NIL, Chinese166 and Taichang29 were produced by PCR amplification with three markers Xgwm311, Xgwm382 and Xcfa2086 and the those markers were further confirmed to be linked with Yr1 by a F2 population.We tested Xgwm311, Xgwm382 and Xcfa2086 on 245 lines of Taichung29*6/Yr1 F2 population through PCR amplification and PAGE electrophoresis. Based on the analysis results of DNA band types and corresponding resistance phenotypes of the 200 F2 lines with software Mapmaker3.0b, the genetic distance between molecular marker and Yr1 were 5.6cM, 1.2cM and 11.4cM respectively, and we proposed that the resistance gene Yr1 was at the position of Cent—Xcfa2086—10.2cM—Xgwm382—1.2cM—Yr1—5.6cM —Xgwm311 on wheat chromosome 2AL in wheat SSR genetic map.We also tested a new method MFLP on wheat. The result showed that it can produce more polymorphisms on wheat genome. So it can be used to develop molecular markers and to identify genetic polymorphism among wheat varieties.更多还原