【作者】 孙石；

【导师】 邢邯；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 由大豆疫霉（Phytophthora sojae）引起的大豆疫霉根腐病（Phytophthora root rot,PRR）是一种毁灭性的大豆病害,在大豆主要生产国美国、巴西、阿根廷、中国等均有发生,对大豆生产造成很大的危害,培育和种植抗病品种是防治该病害最经济、安全、有效的途径。大豆对疫霉根腐病存在完全抗性和部分抗性,完全抗性是小种专化性抗性,通过限制大豆疫霉的侵染来实现的,是由Rps基因控制的,迄今已鉴定并定名了14个抗病基因,它们分布在4条大豆连锁群的8个位点上。然而,大豆疫霉变异迅速、复杂,自从1948年首次发现此病害以来,已鉴定六十多个生理小种和许多未定名的分离物,所有已知抗病基因均被“击败”。而大多数品种抗源单一,常使抗病品种在推广8～15年后就会“丧失”原有的抗性。因此,迫切需要拓宽新的抗病基因。迄今为止,各国的抗性品种选育主要集中在完全抗性品种的选育,但完全抗性品种在大面积推广种植后会因新的致病小种的流行而丧失抗性,并且单基因抗性品种的大面积推广容易对大豆疫霉产生选择压力从而产生新的生理小种。部分抗性（partial resistance）是另一类具有非小种专化性的数量抗性,能够减轻和减缓新生理小种产生的压力。黄淮地区是我国的大豆主产区之一。本研究旨在筛选出黄淮地区的优异抗源,发掘新的抗病基因,明确大豆疫霉根腐病抗性的遗传机制,对新的抗病基因进行分子作图,从而为抗病育种提供理论指导和物质基础,推动大豆抗疫霉根腐病育种的发展。1、抗性鉴定。利用7个具有不同毒力公式的大豆疫霉菌株,对黄淮夏大豆主产区的96个大豆品种（系）和一套含已知13个Rps基因的大豆鉴别寄主进行了接种鉴定,96个大豆品种（系）对7个大豆疫霉菌株共产生38种反应型,有4种反应型分别与单个抗病基因的反应型一致,有10种反应型与2个已知基因组合的反应型相同,有5种反应型与3个已知基因组合的反应型相同,其它反应型为新的类型。根据“基因对基因”学说,抗病基因的推导结果有7个品种可能含有Rps3a,有4个品种可能含有Rps3b,有1个品种可能含有Rps3c,有5个品种可能含有Rps7,有一些品种可能含有国际上尚未命名的新的抗病基因,这些抗源品种是我国大豆抗疫霉根腐病育种的有效抗源。2、根部接种法及部分抗性的抗源筛选。用根部接种法接种已知抗性水平的3个大豆品种对大豆疫霉根腐病的部分抗性,结果表明本方法所获鉴定结果稳定、可靠、效率较高,建议作为我国鉴定大豆种质资源对大豆疫霉根腐病部分抗性的基本方法。另外,用大豆疫霉菌株PNJ1接种鉴定38份黄淮地区大豆栽培品种和地方品种,其中高抗品种10个,占鉴定品种的26.3%。3、遗传分析。利用分别具有完全抗性和部分抗性的的品种配置4个杂交组合,在分别采用下胚轴伤口接种法和根部接种法接种大豆疫霉菌株PNJ1条件下,研究两类抗性的遗传模式。结果表明,大豆对疫霉根腐病完全抗性和部分抗性分属不同遗传体系,完全抗性由1对主基因控制,抗病对感病表现为显性;部分抗性由1对加性主基因+加性-显性多基因控制,F₂代主基因和多基因遗传率分别为41.31%～74.84%和15.60%～50.34%,F_2:3代主基因和多基因遗传率分别为54.21%～77.05%和13.52%～38.24%。两类抗性都有育种价值,并且在早世代选择是有效的。选育聚合有完全抗性和部分抗性的品种是使大豆获得疫霉根腐病高水平抗性和持久抗性的最佳途径。4、基因定位。大豆品种豫豆25和郑92116具有良好的抗大豆疫霉根腐病特性,经基因推导发现它们可能含有多个抗性基因组合或者含有新的抗病基因。用豫豆25与感病材料NG6255、郑92116与NH₅分别杂交构建了两个F_2:3群体,用大豆疫霉菌株对这两个群体进行抗性分析,通过SSR结合BSA的方法进行了分子作图,再结合系谱分析,结果显示豫豆25和郑92116的抗性是由同一个显性单基因控制,该基因可能为新的抗病基因,暂定名为RpsYu25,根据SSR标记的位置,该基因被定位在大豆连锁群N上。5、RGA分析。根据水稻抗白叶枯病Xa21基因的富含亮氨酸重复区域（LRR）和番茄抗细菌性斑点病的Pto基因编码蛋白激酶的DNA序列,设计2对引物用于扩增48个品种中的抗病基因同源序列,经聚丙烯酰胺凝胶电泳和聚类分析,结果表明供试品种间具有较丰富的RGA多态性,从48个大豆品种的抗病基因同源序列中,共扩增出53条谱带,各品种之间谱带较清晰且呈现明显的多态性,以相似系数0.746聚类,48个大豆品种可以分成7类。尽管RGA聚类和抗性表型聚类的类与类之间没有一一对应关系,但抗谱广的品种,能较好地聚在一类,如丰收黄、科丰36、即墨油豆等。因此,综合利用抗性表型和RGA分析可以为大豆疫霉根腐病抗病基因鉴定、品种的培育和布局提供一定的理论依据。更多还原

【Abstract】 Phytophthora root rot（PRR）,caused by Phytophthora sojae M.J.Kaufmann & J.W. Gerdemann,is one of the most devastating diseases in soybean[Glycine max（L.） Merr.]in the main soybean production countries such as USA,Brazil,Argentina and China. Utilization of resistant varieties is the most economical and environmentally safe method for controlling this disease.Two types of resistance to Phytophthora root rot have been described in soybean. Specific resistance confers immunity or near immunity to the plant through a hypersensitive response and is mediated by Rps genes.Fourteen genes at 8 loci（Rps1 to Rps8） for resistance to PRR have been designated and mapped to four molecular linkage groups in soybean:F,G,J and N,respectively.Single allele resistance has provided adequate disease management;however,following deployment of each single Rps gene,races of P.sojae were subsequently identified that had a susceptible interaction with the Rps gene.Single allele deployed in a soybean cultivar is only effective for 8 to 15 years,depending on inoculum density and environmental conditions.Continuous and enhanced uses of stable Rps genes in soybean cultivars against P.sojae races has created selection pressures for the evolution of new pathogenic P.sojae races that can overcome resistance conferred by these genes.P.sojae evolves constantly,and the number of physiological races of this oomycete pathogen is rapidly increasing.Partial resistance limits the lesion growth rate of the pathogen in host tissue,and it is understood to be quantitatively inherited.Huanghuai valley is one of main soybean production areas in China.The main goals of this study were:（1） to investigate the distribution and diversity of Phytophthora-resistant soybean and identify sources that confer resistance to multiple races for implementation into breeding programs; （2） to discover genetic mechanism of resistance to P.sojae;（3） to map resistant gene.The results of this study will be helpful for the development of resistant cultivars in this area.1.Resistance identification.96 soybean cultivars or lines from Huanhuai valley were evaluated for their responses to 7 strains of P.sojae using the hypocotyls inoculation technique.96 cultivars or lines elicited 38 different reaction types with the 7 strains of the pathogen.Among them 4 reaction types accorded to that of single gene,ten reaction types were consistent with two gene combinations,five reaction types were consistent with three gene combinations and the others were new reaction types.The 7 of 96 cultivars probably carried gene Rps3a,The 4 cultivars probably carried gene Rps3b,The 1 cultivar probably carried gene Rps3c,while 5 cultivars probably carried gene Rps7.Some cultivars or lines possibly carry new Rps genes that are effective to control Phytophthora root rot of soybean in China.These accessions may provide sources of resistance for control of Phytophthora rot of soybean in the future.2.Method of partial resistance to Phytophthora root rot and screening of soybean cultivar resistance.Three soybean cultivars with known resistance were evaluated for partial resistance to P.sojae with root inoculation.The result was that the root inoculation was simple,rapid and reliable and could be used as basal identification method of partial resistance of soybean to Phytophthora root rot.38 accessions from Huanghuai basin were evaluated for partial resistance to P.sojae using root inoculation technique.The results showed that 10 cultivars（26.3%） were high resistant to PNJ1 of P.sojae.3.Inheritance analysis.In the present study,four crosses between materials with complete or partial resistance were used to reveal the inheritance of resistance.The results showed that the complete resistance and partial resistance were controlled by different genetic systems:the former by a single dominant gene（Rps）,while the latter by one additive major gene plus additive-dominant polygene.The heritability values of resistance in development were about 90%,those of major and polygene were 41.31%～74.84%and 15.60%～50.34%in F₂,and 54.21%～77.05%and 13.52%～38.24%in F_2:3,respectively. Both kinds of resistance can be used in soybean improvement,and it would be effective to conduct selection in early generations for high resistance in breeding,combining partial resistance with specific Rps genes to provide long term management of Phytophthora root rot as well as to avoid the boom-and-bust cycle of single gene deployment.4.Gene Mapping.Chinese soybean cultivar（line） Yudou25 and Zheng92116 had the same reaction to the 7 P.sojae isolates and might contain novel resistance loci or alleles.In order to further identify the allele（s） for resistance to P.sojae in Yudou25 and Zheng92116 and to map these allele（s） to a linkage group,two soybean populations,Yudou25×NG6255 and Zheng92116×NH5,were evaluated following inoculation with P.sojae and with SSR markers.Results indicated that the resistance in Yudou25 and Zheng92116 was conferred by a same single dominant gene,and it maybe a new resistance gene,temporarily designated RpsYu25,located on molecular linkage group N in two populations.5.RGA analysis.Most known plant disease-resistance（R） genes include nucleotide binding site（NBS） or leucine-rich repeats（LRRs） and serine/threonine protein kinase（STK） in their encoded products domains.Two primers,XLRRfor/XLRRrev and Pto-kin1/Pto-kin2,were designed based on these conserved domains.By polymerase chain reaction（PCR） and denatured polyacrylamide-gel electrophoresis techniques,disease resistance gene analogs（RGA） have been amplified on 48 soybean materials.The RGA analysis of 48 cultivars produced 53 amplification bands,39 of the 53（73.6%） bands showed polymorphic,and they could be divided into 7 groups at 0.746 similarity coefficient. Although there is no parallelism relationship between group and group in two different types of the clustering,the varieties,such as Fengshouhuang,Kefeng36,and Jimoyoudou with broad spectrum can be fallen into the same group.The result indicated RGAP （resistance gene analog polymorphism） technique,combining the resistant spectrum, provides a useful and efficient way to improve the efficiency of parent materials selection in soybean breeding and to accelerate the process of developing soybean cultivars with resistance.更多还原