【作者】 陆宁海；

【导师】 康振生；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2009， 博士

【摘要】 小麦条锈病是由小麦条锈菌(Puccinia striiformis f.sp.tritici)引起的气流传播性病害,是我国小麦上最重要的病害。西北地区特别是甘肃陇南地区是我国小麦条锈菌最重要的越夏区之一,也是我国小麦条锈菌新小种的策源地,该区秋季的菌源可向东部广大冬麦区传播,越冬后春季菌源可向北部和西部麦区蔓延,对周边地区有重要影响。新毒性小种的出现和群体数量的增长,使病原菌群体结构的改变,一些品种的抗病性丧失,导致病害的发生和流行。认识越夏易变区小麦条锈菌自然群体的遗传结构对抗病育种、抗病基因的合理布局以及开发更有效的病害防治策略具有重要的参考价值。本研究采用SSR分子标记技术,对1447个菌系进行了DNA指纹分析,包括甘肃和青海2省20个县市,其中,青海菌系270个,临夏州菌系206个,平凉菌系50个,陇南菌系971个;对西北越夏区的小麦条锈菌群体结构进行全面系统的研究,了解甘肃省小麦条锈菌在地区间的遗传多样性和遗传分化水平,分析陇南地区小麦条锈菌群体结构在季节间和年度间的稳定性,推断西北越夏区小麦条锈菌的传播途径。本研究主要取得如下结果:(1)小麦条锈菌毒性变异与DNA多态性的相关性分析。为了了解毒性和DNA多态性之间的相关性,对90个代表性的菌系进行了毒性和DNA多态性分析。毒性分析发现了14种致病类型或生理小种, SSR分子标记分析发现了75个基因型, DNA指纹的多态性远比毒性的多态性丰富。不同的小种有相同或相近的DNA指纹,同一小种可能有不同的DNA指纹。因此,毒性特征和DNA多态性之间并不存在相关性。(2)菌系数量对条锈菌群体遗传结构的影响。为了确定一个群体或亚群体的菌系数量,即能反映条锈菌遗传结构的真实水平,也不要增加额外的工作量,分析了菌系数量对条锈菌遗传结构的影响。结果表明,当菌系的数量小于10时,反映的遗传结构偏离真实水平,菌系的数量大于20时,基本接近真实水平,当菌系的数量大于30时,反映的遗传结构无限接近真实水平。因此,分析小麦条锈菌群体遗传结构时,一个群体或亚群体的菌系数量应大于30个,不少于20个。(3)甘肃省小麦条锈菌群体空间遗传结构的SSR分析。为了了解甘肃小麦条锈菌群体遗传多样性水平,明确地区间存在广泛的菌源交流,测试陇南地区是甘肃小麦条锈菌的菌源中心这一假说,对甘肃省小麦条锈菌群体结构进行了分析。结果表明甘肃省小麦条锈菌遗传多样性比较丰富,在亚群体之间,遗传多样性有显著的差异,其中,天水种群(H =0.28,I =0.41)和陇南种群(H =0.26,I =0.42 )的遗传多样性最高,临夏种群(H =0.22,I =0.36)次之,平凉种群(H =0.18,I =0.28)最低,表明陇南和天水地区是甘肃省小麦条锈菌的中心。SSR标记系统揭示甘肃小麦条锈菌群体遗传分化程度较小(Gst=0.15),地区间的遗传变异仅占1.38%,群体间的遗传变异占总变异的15.57% (P<0.001),群体内遗传变异占83.5%,遗传变异主要存在于群体内部。甘肃小麦条锈菌群体的基因流(Nm)为1.38,表明小麦条锈菌在甘肃各地区间存在广泛的交换。因此,甘肃小麦条锈菌群体遗传多样性丰富,遗传分化较小,各地区内和地区间存在广泛的菌源交流。(4)陇南地区不同海拔区域内小麦条锈菌群体遗传多样性的分析。为了了解不同海拔高度区域内小麦条锈菌群体的遗传结构和分化程度,明确海拔高度对小麦群体遗传多样性的影响,测试小麦条锈菌在陇南地区可以完成周年循环的假说,对陇南地区不同海拔高度的地区内小麦条锈菌群体遗传多样性进行了研究。结果表明陇南地区小麦条锈菌遗传多样性比较丰富,各亚群体存在显著差异,高山种群和半山种群的多样性最丰富,川道种群的相对较低。不同生态区域内,小麦条锈菌群体遗传分化程度不同,高山区和半山区遗传分化程度大,川道区群体遗传分化程度比较小。不同海拔区域的条锈菌群体之间有基因流的存在,从分子水平证实了小麦条锈菌在高海拔山区与川地之间进行移动,可就地完成周年循环这一假说。(5)陇南地区小麦条锈菌群体结构的季节性变化。为了了解小麦条锈菌群体遗传多样性的季节性变化规律和分化程度水平,评价小麦条锈菌群体结构的稳定性,对陇南地区小麦条锈菌群体结构的季节性变化进行了研究,结果表明,不同季节间,小麦条锈菌群体间遗传多样性没有显著的差异,群体遗传分化程度不同,越夏种群遗传分化程度较大,越冬和春季流行群体遗传分化程度比较小。陇南地区小麦条锈菌群体结构的季节性变化分析,表明陇南地区小麦条锈菌群体结构虽有局部的变化,但整体保持稳定。(6)陇南地区小麦条锈菌群体结构的周年时空变化。为了了解不同年度间小麦条锈菌群体的遗传多样性变化和分化程度水平,评价小麦条锈菌群体结构的稳定性,对陇南地区小麦条锈菌群体结构的年度时空变化进行了研究。结果表明,不同年度间,小麦条锈菌群体间遗传多样性和遗传分化没有显著的差异,陇南地区小麦条锈菌群体结构的年度间时空变化分析,表明陇南地区小麦条锈菌群体结构虽有局部的变化,但整体保持稳定。(7)西北越夏区小麦条锈菌长距离传播的分子证据。为了探索西北越夏区小麦条锈菌的传播途径,对西北越夏区小麦条锈菌群体结构进行了分析。结果表明,西北越夏区小麦条锈菌群体遗传多样性比较丰富,地区之间有显著的差异,陇南地区小麦条锈菌遗传多样性明显比临夏和青海地区的高。西北越夏区小麦条锈菌群体的基因流(Nm)为1.37,表明小麦条锈菌在西北越夏区各地区间存在广泛的交换或长距离的移动。我们的分子数据证实了在西北越夏区各地区之间有基因流的存在或病原菌的传播,陇南到青海的传播路线,是陇南直接传播到青海为主,辅助临夏到青海的传播。(8)小麦条锈菌体细胞遗传重组的分子证据。11对SSR引物中4对能够检测到陇南地区小麦条锈菌群体存在体细胞遗传重组,而且重组体出现的频率不同,CPS15揭示的条锈菌重组体出现的频率为20.0%, CPS34揭示的条锈菌重组体出现的频率为18.5%,RJ20揭示的条锈菌重组体出现的频率为12.8%, RJ18揭示的条锈菌重组体出现的频率为15.0,陇南地区小麦条锈菌群体重组体出现频率平均为16.6%。本文揭示的遗传重组现象表明陇南地区条锈菌体细胞结合十分普遍,由此推测我国小麦条锈菌在自然条件下通过遗传重组而导致毒性变异的可能性。更多还原

【Abstract】 Stripe rust (or yellow rust), caused by Puccinia striiformis Westend. f. sp. tritici Eriks., is one of the most important diseases of wheat (Triticum aestivum L.) worldwide. In China, stripe rust has been considered the most important disease of wheat since the first major epidemic in 1950. Gansu province, located in northwestern China, is one of the largest and the most important over-summering areas in China. The region provides inoculum to the major wheat-growing regions to the east. Almost all Chinese races of P. striiformis f. sp. tritici were first detected in the Longnan regions of Gansu, which serve as sources of new races and inoculum for rust development in the other regions of China because of their unique geographic topography and ideal environmental and cropping conditions. The objectives of this study were to (1) characterize genetic diversity of P. striiformis f. sp. tritici , (2)determine the levels of migration among different regions , (3) evaluate stability of genetic structure of P. striiformis f. sp. tritici in different seasons and years, (4) infer the pathway of pathogen spread in northwest China, used SSR marker. To determine population structures of P. striiformis f. sp. tritici , a total of 1447 isolates were studied using 11 microsatellite markers, including 270 isolates from Qinghai, 206 isolates from Linxia, 50 isolates from Pingliang and 971 isolates from Longnan, in which two provinces and 20 countries. The main results are summarized as following:(1) To determine the relativity between virulence and DNA polymorphism, a total of 90 isolates of Puccinia striiformis f. sp. tritici were studied. 14 pathotypes and 75 genotypes were found, suggesting DNA polymorphism was much more diversity than virulence. Moreover, there were the same or near genotype in different races, and the same race may be had different genotype. Therefore, there was no relativity between virulence and DNA polymorphism.(2) To determine the number of isolates for a population or subpopulation, we analyzed the effect of number of isolates to population genetic structure of P. striiformis f. sp. tritici. The result showed that population genetic structure was untrue when isolates less than 10. Which isolates more than 20, population genetic structure were be close to true, when isolates more than 30, population genetic structure was actually ture. Therefore a population should have more than 30 isolates.(3) To determine population structures of P. striiformis f. sp. tritici in Gansu, a total of 850 isolates were studied using 11 microsatellite markers. The genetic diversities in the Tianshui and Longnan populations were much higher than those of the Linxia and Pingliang populations. The Nei’s gene diversity and Shannon information index of the Tianshui (H = 0.28; I = 0.41) and Longnan (H = 0.26; I = 0.42) populations were higher than those of the Linxia (H = 0.22; I = 0.36) and Pingliang (H = 0.18; I = 0.28) populations. For the Gansu population, the genetic differentiation was low (Gst = 0.15). Thus, the results suggest there are extensive gene exchanges or short-distance migrations of pathogen among regions in Gansu. The most (83.05%) of the total variation presented within subpopulations; 15.57% was among subpopulations; and only 1.38% was among the four regions. These data supported considerable gene flow and insignificant separation among the regional populations. Our molecular data support the hypothesis that the Longnan regions are the center of wheat stripe rust in Gansu .(4) To determine population structures of P. striiformis f. sp. tritici in this region, a total of 330 isolates from 11 collections were studied using SSR markers. The populations of P. striiformis f. sp. tritici possessed relatively high levels of genetic diversity in high mountain area but a low gene flow. Analysis of molecular variation showed that only 1.43% of the total variation presented among areas, 24.21% among collections within areas, and the remaining 74.36% within collections. There is extensive gene exchange within areas and short-distance migration of pathogen among areas in different agro-ecological area in southeast of Gansu, China. The most significant finding of this study is that recombination of P. striiformis f. sp. tritici was detected in the southeast of Gansu. Our molecular data confirmed the hypothesis that the stripe rust pathogen is able to over-winter in the lowland areas and over-summer in high mountain areas, which finish its life cycle in southeast of Gansu .(5) To determine the change of population genetic diversity and evaluate stability of genetic structure of P. striiformis f. sp. tritici in different seasons, a total of 685 isolates were studied using SSR markers. There was no significant difference of genetic diversity in different seasons. While genetic differentiation was significant difference in different seasons. Although there was a little change, the population genetic structure of P. striiformis f. sp. tritici was stability in different seasons in Longnan.(6) To determine the change of population genetic diversity and evaluate stability of genetic structure of P. striiformis f. sp. tritici in different years, a total of 728 isolates were studied using SSR markers. There was no significant difference of genetic diversity and genetic differentiation in different years. Although there was a little change, the population genetic structure of P. striiformis f. sp. tritici was stability in different seasons in Longnan.(7) Interregional long-distance spread of wheat stripe rust and the pathway in the Northwest China were inferred by disease surveys and molecular markers. Thirteen genotypes in Linxia and 12 genotypes in Qinghai were also found in Longnan, where 17 genotypes were identified. The genetic diversity in the Longnan population was much higher than those in the Linxia and Qinghai populations. The low genetic differentiation (Gst = 0.15) and the extensive gene flow (Nm=1.37) were found among these regions. The most important molecular finding was that collections from different regions were clustered together and isolates from different regions shared the same DNA fingerprinting. Therefore, the most important conclusion of this study is that the stripe rust inoculum in Qinghai can from both Longnan and Linxia, but mainly directly from Longan in the spring.(8) The somatic genetic recombination of P. striiformis f. sp. tritici in Longnan of Gansu Province was detected by 4 of 11 SSR marker. Recombinant frequency of P. striiformis f. sp. tritici was different. Recombinant frequency were 20.0%, 18.5%, 12.8% and 15.0% detected by RJ CPS15, CPS34, RJ20 and RJ18. The average recombinant frequency of P. striiformis f. sp. tritici was 16.6% in Longnan. This study revealed genetic recombination by molecular markers and thus strongly suggests that genetic recombination is another way of producing a new race of P. striiformis f. sp. tritici and lead to virulence variation.更多还原