【作者】 张娜；

【导师】 杨文香； 刘大群；

【作者基本信息】 河北农业大学 ， 植物病理学， 2005， 硕士

【摘要】 小麦叶锈病是国内外影响小麦生产的重要病害。因此,对小麦抗叶锈病基因进行标记、定位,可以为分子标记辅助选择提供便利,并对随后抗病育种效率的提高和基因克隆有重要作用。 小麦抗叶锈病基因Lr45发现于一个日本小麦与黑麦（Secale cereale）的杂交后代中,并定位于2A染色体上。国内外至今尚未发现对它表现有毒力的菌株,是一非常有效的小麦抗叶锈基因,目前还未见有Lr45分子标记的报道。TcLr45×Thatcher F₂苗期室内接菌鉴定表明,Lr45抗病性由显性单基因控制。 本研究选取Thatcher及23个以Thatcher为遗传背景的小麦抗叶锈病近等基因系（NILs）和F₂（TcLr45×Thatcher）分离世代的种子,采用本实验室杨文香等建立的小麦AFLP（amplified fragment length polymorphism,扩增片段长度多态性）技术体系和程序,并摸索适合小麦扩增的SSR（simple sequence repeat,简单重复序列）体系及不同引物的退火温度,开展抗叶锈基因Lr45的分子标记研究,寻找与Lr45紧密连锁的AFLP和SSR分子标记,为构建更加稠密的遗传连锁图和抗小麦叶锈病育种的分子辅助选择提供有力工具。 利用304对AFLP引物对23个近等基因系筛选出对Lr45有多态性的引物84对,得到5个与Lr45紧密连锁的AFLP分子标记,其中标记P-AGG/M-GAG_261bp和P-ACA/M-GGT_105bp与抗病基因Lr45的遗传距离为0.6cM和1.3cM,且位于Lr45的两侧。前者扩增的目的片段经克隆测序确定长度为261 bp,经BLAST同源性比较,与大麦属Vulgare HotrⅠ基因部分序列同源性达86%。引物P-ACA/M-GGT扩增的目的片段之一经克隆测序确定长度为105 bp,序列经BLAST同源性比较,与一粒小麦磷脂酰丝氨酸脱羧酶基因部分序列同源性高达96%。两个测序片段均包含开放阅读框（ORF）序列。 利用微卫星技术对小麦抗叶锈基因Lr45进行了标记,从31对定位于小麦2A染色体的SSR引物中筛选出9对在亲本及F₂抗感群体间揭示多态性的引物,最终得到4个与Lr45紧密连锁的SSR分子标记,且位于Lr45的同一侧,其中两个标记Xgwm95和Xgwm122与Lr45的遗传距离分别为2.8cM和3.5 cM。Xgwm95扩出的抗感差异性片段经测序后发现微卫星序列分别为（GT）₁₁和（GT）₇GA（GT）₈,侧翼序列相同,与小麦基因组无同源序列;Xgwm122扩出的特异性片段经序列比较与小麦A基因组重复DNA序列部分同源,但未发现微卫星序列。 本研究得到的5个AFLP标记和4个SSR标记经MAPMAKER 3.0b分析,定位于同一染色体上。据Somer的微卫星图谱,Xgwm95位于小麦2AS染色体的近着丝点附近,故Lr45基因有可能位于2AS着丝点区域。更多还原

【Abstract】 Wheat leaf rust is one of the most destructive diseases on wheat production throughout the world. Therefore the exploitation of marking, location of leaf rust resistance （Lr） genes would facilitate MAS （marker assisted selection） and benefit breeding the effective leaf rust resistant cultivars, and the gene cloning.Lr45 was a resistant gene against wheat leaf rust disease originated from Secale cereale and was localized onto the chromosome 2A. No virulent races to the gene have been found so far, and molecular markers have not been reported for the gene based on our knowledge at the present time. Segregation ratio of resistance vs. susceptibility from TcLr45 ×Thatcher F₂ progenies in greenhouse test showed that the resistance of Lr45 to the disease is controlled by a single dominant gene. The successful marking of this gene would pave a solid way to MAS and cloning of the resistance gene.AFLP （amplified fragment length polymorphism） analysis were carried out in Thatcher, near isogenic lines （NILs） carrying different genes conferring resistance against wheat leaf rust and TcLr45×Thatcher F₂ progenies to develop markers for Lr45. 304 AFLP primer combinations were screened and PCR-amplified products were isolated on urea denaturing 6.0% poly-acrylamide gel, and then were visualized by silver straining. Most of them provided clear amplification products, and 84 primer pairs displayed polymorphism in NILs. Five AFLP markers closely linked to gene Lr45 were acquired. P-AGG/M-GAG261 bp was found closely linked to the Lr45 locus at a distance of 0.6cM on one side, and P-ACA/M-GGT₁₀₅ bP at a distance of 1.3cM on the other. The specific bands were cloned and sequenced subsequently. The fragment of 261bp produced by P-AGG/M-GAG was 86 percent similarity with the sequence of Vulgare HortI gene. The 105bp produced by P-ACA/M-GGT was 96 percent with the phosphatidylserine decarboxylase gene of the Triticum monococcum. Both of them included an open reading frame （ORF）.SSR protocol was also used to marks Lr45. A total of 31 microsatellite primer combinations specific to the chromosome 2A of wheat genome were used to detect polymorphism between the four NILs located on chromosome 2A. Of them nine primer更多还原