【作者】 陈长卿；

【导师】 康振生；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2008， 博士

【摘要】 小麦条锈病是我国小麦安全生产最严重的威胁之一，引起该病害的小麦条锈病菌(Puccinia striiformis f．sp．tritici)是一种远程气流传播的病原真菌，从而致使病害具有大区流行的特点，因此明确认识条锈菌在我国小麦主产区群体遗传结构对病害的有效控制至关重要。五十多年来锈病工作者对病原菌群体的持续研究获得了宝贵的理论成果，对控制病害的流行起到了重要作用，然而利用传统的病原菌毒性鉴定及病害调查方法对我国条锈菌大区流行体系的推断需要分子群体遗传学研究的证实和完善，而且对有些重要地区间的菌源传播关系还需要深入的研究。本研究旨在构建小麦条锈菌微卫星分子标记体系，并利用该体系对我国病害主要发生区域的病原菌进行群体遗传分析，以期明确我国小麦条锈菌不同地理群体遗传多样性情况，确定遗传多样性的中心，同时了解大区范围内群体遗传结构及群体间分化程度，并结合群体间基因流强度及个体共享指纹的统计结果推断地区间病原菌的传播关系。本研究获得了以下结果：1．本研究首次对小麦条锈菌cDNA文库蕴含的EST-SSR进行了的分析，明确了小麦条锈菌EST-SSR的基本特征。在1307条无冗余EST序列中，发现了135序列包含着170个SSR，平均长度为16．48 bp，相对较短，平均分布频率是1／3．69 kb，小麦条锈菌EST-SSR以三核苷酸重复最多，重复基元类型较为丰富，出现最多的重复基序是GTT／CAA类型。小麦条锈菌EST中SSR信息的明确为进一步建立和应用EST-SSR标记奠定了基础。2．对本研究开发的小麦条锈菌EST-SSR和已报道的适合条锈菌研究的SSR进行了多态性筛选，并结合TP-M13-SSR技术建立了适合中国小麦条锈菌群体遗传研究的微卫星分子标记体系，具有多态性丰富、灵敏度高、重复性好、高效简捷等优点，为进行大规模高通量的群体遗传研究奠定了基础。3．利用微卫星分子标记体系对17个小麦条锈菌模式生理小种和致病类型的遗传多样性分析显示，微卫星分子标记对中国小麦条锈菌生理小种的分析揭示出较高的遗传多样性水平，可为群体遗传研究提供宝贵的标记资源，研究证明两种标记特征相关性不显著。4．本研究首次利用微卫星分子标记对中国9个省市的20个种群共980份小麦条锈菌个体进行了大规模、大范围的群体遗传分析，获得了以下结论：(1)从分子水平上明确了中国小麦条锈菌保持着较高的遗传多样性水平，各种群间存在着差异，其中天水种群是中国小麦条锈菌遗传多样性水平最高的种群，襄樊种群最低。(2)中国小麦条锈菌群体间和群体内都存在着一定的遗传分化(Gst=0．2335，Fst=0．2215)，遗传变异主要存在于群体内部，不同种群间分化水平有所差异，个别种群间分化水平相对较大。研究发现种群间遗传距离与地理距离极显著相关(P＜0．001)，符合隔离分化模型。20个种群间基因流(Nm)为0．8787，说明种群间的基因流相对较弱，基因流的阻断可能是导致遗传分化的主要原因。(3)基于微卫星分子标记进行的UPGMA聚类分析结果呈现一定的地理区域性，20个种群被分为5个类群，最大的一个中心类群聚集了12(60%)个种群，其中除昭通种群外，包括了我国最主要的小麦条锈菌越夏区一西北和川西北2个越夏区，以及与两个越夏区相邻的3个盆地一关中盆地、陕西南部和四川盆地的所有种群。其余8个种群较为分散，被分为4个类群，主要来源于四川盆地以南区域和东部地区。(4)根据聚类结果及种群所在区域的地理分布特点，将20个种群分为6个不同地理区域的群体，分别为西北-川西北越夏区群体、关中盆地群体、陕南盆地群体、四川盆地群体、四川盆地以南群体和东部地区群体。明确了西北—川西北越夏区是中国小麦条锈菌遗传多样性最为丰富的地区，即为病原菌遗传多样性的中心区域，东部地区最低。各区域群体内种群间遗传分化程度最大是东部地区群体，最小的为陕南盆地群体。(5)证明了西北越夏区和川西北越夏区存在着一定的基因交流，种群间的遗传关系与其所属的地理区划并不完全相关，同时明确了西北-川西北越夏区种群与3个相邻盆地的群体遗传关系，其中关中盆地与平凉和天水，四川盆地与阿坝、陕南盆地与陇南的小麦条锈菌群体遗传关系最近。(6)获得了中国小麦条锈菌部分种群间个体共享分子指纹的结果，提供了中国小麦条锈菌远距离传播的分子证据，并结合群体基因流强度参数和种群间遗传分化关系，在分子水平上为传统流行学对一些地区间存在菌源关系的推断提供了证据，如平凉与关中盆地间、汉中与安康间等；同时也发现了目前尚未报道过的一些地区间存在菌源交流的可能，如西北越夏区种群与四川盆地以南种群之间。更多还原

【Abstract】 Puccinia striiformis f.sp. tritici (PST) is an obligate biotrophic pathogen, causing wheat yellow rust (stripe rust), one of the most economical important diseases of wheat (Triticum aestivum). Spores of fungal plant pathogens may be spread by wind over long distances, thereby enhancing disease epidemics across large areas. It is very essential for the development of more effective disease control to understand the population genetic structure in the regions wheat stripe rust occurs frequently. A great deal of valuable theory was obtained through continual study on population biology of pathogens since 1950s in China, which play an important role to prevent and control diseases epidemic. However, the inferring results about the epidemiological study of pathogens over a large area were mainly based on traditional methods of virulence identification and observation for disease degree, and some of relation between the regions is still unknown. So those need to be approved and complemented using the molecular population genetic methods. Our goal of the present research is to establish a system of SSR molecular marker and then to analyze the genetic diversity and population genetic structure and to infer the dispersal relations of pathogens according to the information of gene flow and shared fingerprints among the regions. The main results are summarized as following:1. The frequency and characteristics of SSR derived from ESTs by sequencing complementary DNA (cDNA) library of PST were analyzed. Of the 1307 nonredundant EST sequences, 170 SSR loci distribute in 135 ESTs (10.33%). The average length of EST-SSR searched is 16.48 bp and the average distance of distribution is 1/3.69 kb. The trinucleotide repeat is the dominant types with repeat motif GTT/CAA being the most common. These results lay the foundation for the future development and application of EST-SSR markers in PST.2. The SSRs markers derived from PST cDNA library and three others Puccinia species reported were tested for molecular polymorphism by isolates sampled from different geographical regions. The fluorescent labeling system of TP-M13-SSR (simple sequence repeat with tailed primer M13) was established based on polymorphic markers screened and the fluorescent labeling technique of TP-M13. The results showed that the TP-M13-SSR approach is of high polymorphism, sensitivity and good steady. The new system molecular marker provides technological support for large-scale and high-throughput population genetic analysis.3. The 17 physiological races and pathotypes were tested for diversity using the TP-M13-SSR system. The results showed that there is much diversity revealed by SSR markers. However, there is no significant correspondence between two measures of labeling. These molecular markers would become the useful labeling resources for population genetic research of PST.4. The 980 isolates grouped into 20 different geographical populations sampled from nine provinces in China were analyzed for the population genetic structure of PST using the TP-M13-SSR system. The results as followed:(1) Results showed that the genetic diversity level of PST in China is relatively high and is different from each other among 20 populations. The highest one was found in Tianshui population which means the central population of diversity and the lowest in Xiangfan population.(2) The results of genetic differentiation among 20 natural populations showed that it could be found within and among populations (Gst=0.2335, Fst=0.2215). The majority of genetic variation occurred within populations. But the level of differentiation was different from each other between two populations and parts of it were higher. The mantle test showed significant correlation between genetic and geographic distances among natural populations(P <0.001). Gene flow among natural populations was relatively weak (Nm=0.8787), which might have caused the genetic differentiation among the populations.(3) The 20 natural populations were clustered into five groups by UPGMA method according to their genetic distances. The largest group included 12(60%) populations which were mainly sampled from the oversummer zone of PST in northwest China and northwest Sichuan province and three neighboring basins except for Zhongtong population collected from Yunnan province. Eight others populations were clusterd into four groups, which were mostly from south of Sichuan basin and eastern China.(4) The 20 natural populations were divided into six zones according to their geographic distribution. These were the oversummer zone of PST in northwest China and northwest Sichuan province, GuanZhong basin, Southern Shaanxi province basin, Sichuan basin, south of Sichuan basin and eastern China zones, respectively. On the one hand, the genetic diversity of oversummer zone is highest among six zones, which showed the central zone of genetic diversity in China, and the lowest in eastern China zone. On the other hand, the maximal genetic differentiation was found among populations within eastern China zone and the minimum one within Southern Shaanxi province basin zone.(5) The evidence of gene flow between the two oversummer zones in northwest China and northwest Sichuan province was obtained, which showed that genetic relationship among populations located in two regions was not correlative with their geographical attribution. The genetic relationship of PST among the populations within the oversummer zone of PST in northwest China and northwest Sichuan province and the three neighboring basins was also clarified. The closest genetic relationships were found between GuanZhong basin and Pingliang, Sichuan basin and Aba, Southern Shaanxi province basin and Longnan populations, respectively.(6) The direct molecular evidence for long-distance dispersal in PST was obtained according to the finding of some identical fingerprints between and among different geographical populations. Parts of results about migration direction in PST inferred using traditional methods of virulence identification were proved. For instance, the relations of pathogens dispersal occurred between Pingliang and GuanZhong basin, also between Hanzhong and Ankang regions. The strong possibility of pathogens dispersal was found for the first time over very long distances like between the oversummer zone in northwest China and south of Sichuan basin regions.更多还原