【作者】 张吉清；

【导师】 朱振东；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2013， 博士

【摘要】 由大豆疫霉（Phytophthora sojae Kaufmann&Gerdermann）引起的大豆疫病（Phytophthora rootrot，PRR）是毁灭性大豆病害之一，严重影响大豆产量和品质。因此，发掘和作图大豆抗疫病新基因对防治该病害和抗病育种具有重要的意义。本研究通过接种不同毒力的大豆疫霉菌株对34个大豆品种（系）可能携带的抗病基因分析，利用与Rps1座位连锁的分子标记进行标记基因鉴定，并利用TRAP标记分析了这些品种（系）的多样性；作图和精细定位了2个广谱抗性大豆品种豫豆29和皖豆15抗疫病基因。主要结果如下：1.利用下胚轴创伤接种法鉴定了30个豫豆系列品种（系）及其4个原始亲本对26个不同毒力的大豆疫霉菌株的抗性，应用基因对基因理论对品种（系）进行了抗疫病基因推导。结果表明，34个大豆品种（系）分别抗3个或3个以上大豆疫霉菌株，并产生34种不同的反应型。通过与一套含有单个已知抗病基因大豆品种（系）反应型比较发现，品种周豆17的反应型与含有Rps5品系L85–3059的反应型一致，可能携带抗病基因Rps5；郑77249可能携带抗病基因Rps3a或Rps3a+5抗病基因组合。其它32个大豆品种（系）反应型与已知的抗病基因或抗病基因组合反应型不同。因此，推测这32个大豆品种（系）可能携带新的抗病基因或抗病基因组合。聚类分析表明，当相似系数为0.65时，34个大豆品种（系）、17个鉴别寄主和感病品种Williams共聚为10组。本研究结果表明，河南地区培育的豫豆系列品种（系）对大豆疫霉的抗性具有极其丰富的多样性。这些具有广谱抗性的大豆品种（系）为有效地控制大豆疫病提供了新的抗源。2.利用靶位区域扩增多态性（Target Region Amplified Polymorphism, TRAP）标记对34个品种（系）、17个鉴别寄主和感病品种Williams的遗传多样性进行了分析。32对TRAP标记扩增34个品种（系）共产生251个位点，平均每对引物产生7.84个位点，每个位点均具有多态性，多态性位点比例占100%。基于TRAP标记分析，这34个品种（系）间的遗传相似性位于0.20-0.94，平均相似性为0.57。UPGMA聚类分析，在相似系数为0.57时，52个大豆品种（系）分为9个组。通过与抗性鉴定聚类结果分析表明，大豆对疫病的抗性与TRAP标记聚类结果无关。3.豫豆29是一个对大豆疫霉具有广谱抗性的大豆品种。接种25个不同毒力的大豆疫霉菌株进行抗性分析表明，豫豆29产生的反应型不同于携带已知单个抗病基因的鉴别寄主的反应型。以豫豆29为父本，感病品种吉科豆2号为母本，杂交产生的214个F2:3家系作为作图群体对豫豆29进行抗大豆疫病基因分析。抗性遗传分析表明，F2:3群体中纯合抗病、杂合和纯合感病家系分离比符合1：2：1的分离比，表明豫豆29对大豆疫霉的抗性由一个显性单基因控制，暂时命名为RpsYD29。利用SSR标记，基因RpsYD29被定位在了大豆基因组第3号染色体上（Molecularlinkage group N, MLG N），位于引物SattWM82–50和Satt1k4b之间，与两个标记的遗传距离分别为0.5和0.2cM。对定位区域内的基因序列分析表明，具有NBS-LRR结构的基因Glyma03g04030.1和Glyma03g04080.1可能是RpsYD29基因的参照候选基因。经候选基因克隆测序和序列分析表明，豫豆29和吉科豆2号的两个候选基因在核苷酸和编码的预测氨基酸序列上均存在差异。这些差异可能是造成豫豆29和吉科豆2号抗、感差异的原因。根据抗性鉴定和分子鉴定结果，可知RpsYD29基因可能是Rps1座位上一个新的等位基因或与Rps1座位紧密连锁的基因。4.利用与Rps1座位紧密连锁的分子标记Satt530和Sat₁86，和与RpsYD29基因紧密连锁的分子标记SattWM82–50和Satt1k4b，对34份大豆品种（系）进行了分子标记基因型分析。发现这4对引物共产生31种标记基因型。这34份大豆品种（系）在这4个位点具有丰富多样性。分子标记基因型分析表明，豫豆23、豫豆24和周豆11可能携带RpsYD25基因；郑77249、郑84285、郑120、郑90007和郑92116可能携带RpsYD29基因；豫豆12、豫豆22和赤黄豆可能携带Rps1a/1d基因；豫豆15可能携带Rps1b基因；齐黄1号、郑7104、山东四角齐、郑135、豫豆13和郑85558可能携带Rps1c/1k基因。5.以大豆品种皖豆15为父本，感病品种Williams为母本杂交产生的102个F2:3家系作为作图群体对皖豆15的抗大豆疫病基因进行分析。抗性遗传分析表明，F2:3群体中纯合抗病、杂合和纯合感病家系分离比符合1：2：1的分离比，表明皖豆15对大豆疫霉菌株PsMC1的抗性由一个显性单基因控制。连锁分析表明，皖豆15携带的抗病基因被定位在了大豆第17号染色体上（MLGD2），位于引物Sattwd15–24/25和Sattwd15–47之间，与这两个引物之间的遗传距离分别为0.5和0.8cM。同时还获得与抗病基因共分离的两对引物Sattwd15–28和Sattwd15–32。这是首次在该染色体上发现和定位的大豆抗病基因，因此将该基因命名为Rps10。对定位区域内的基因序列分析表明，具有丝氨酸/苏氨酸（Ser/Thr）蛋白激酶结构的基因Gly17g28950.1和Gly17g28970.1可能是Rps10的参照候选基因。经侯选基因克隆测序分析，皖豆15和Williams的两个候选基因在核苷酸和编码的氨基酸序列上均存在差异。这些差异可能是造成皖豆15和Williams对大豆疫霉菌株抗、感差异的原因。更多还原

【Abstract】 Phytophthora root rot （PRR） caused by Phytophthora sojae Kaufmann&Gerdemann, is one of themost destructive diseases on soybean （Glycine max （L.） Merr.）, and could result in a total yield loss and thelower quality. So, it is meaningful for the disease control and the resistant breeding to further study on thePhytophthora resistance.The34soybean cultivars （lines） were employed to characterize the Phytophthora resistance andpostulate the Phytophthora resistance gene. Then the genetic relationship of the cultivar （lines） was analyzedusing the TRAP （Target Region Amplified Polymorphism） markers. Based on the result of the Phytophthoraresistance, the inheritance, mapping and the candidate gene（s） of the Rps gene（s） in soybean cultivarYudou29with broad spevtrum resistance was further analysis. In addition, the pyhtophthpra resistance ofsoybean cultivar Wandou15was also analyzed as well as the Yudou29. The main conclusions are as follows:1. The objective of this study was to characterize the Phytophthora resistance in34soybean cultivars（lines） by inoculating26P.sojae isolates with different virulence phenotypes. The34soybean cultivars （lines）showed resistance to between3and26isolates, and34reaction types were produced. The reaction typesproduced on the cultivars were compared with those produced by the same isolates on the differential lines topostulate which Rps gene were present. The gene Rps5and Rps3a or gene combination Rps3a+5werepostulated to be present in Zhoudou17and Zheng77249, respectively. And the other32cultivars/linescharacterized novel reaction types, of which were different from the known Rps genes or two-genecombinations. The cluster analysis of the reaction types revealed10groups among the34soybean cultivars（lines）,17differentials and the cultivar Williams at the similarity coefficient0.654. This study indicated thatPhytophthora resistance was extremely diverse in this region. The cultivars （lines） with broad spectrumresistance could provide effective sources of resistance for the control of PRR in the future.2. This study investigated the genetic diversity and relationships among the34soybean cultivars（lines）,17differential lines carrying a single Rps gene and the susceptible cultivar Williams using thetarget region amplification polymorphism （TRAP） marker technique. Thirty-two primer combinationsgenerated a total of251polymorphic loci, of which each had the polymorphisms. The average numberof polymorphic locus for each primer combination was7.84. The pairwise genetic similarity coefficientsbased on the TRAP markers ranged from0.20to0.94with a mean of0.57, indicating that there issubstantial genetic variability in these soybean cultivars/lines. Cluster analysis using the UPGMAmethod, grouped the52cultivars （lines） into9classes. There is no relationship among the resistanceanalysis and the TRAP markers analysis of the34soybean cultivars （lines）.3. The soybean cultivar Yudou29is resistant to many P.sojae isolates in China. In response to25P.sojae isolates, Yudou29displayed a new resistance reaction pattern distinct from those of differentialscarrying known Rps genes. The genetic basis of the resistance in Yudou29was elucidated through aninheritance study and molecular mapping. A population of214F2:3families from a cross between Jikedou2（PRR susceptible） and Yudou29was used for Rps gene mapping. The segregation fit a1:2:1ratio for resistance: segregation: susceptibility within this population, indicating that resistance in Yudou29iscontrolled by a single dominant gene. This gene was temporarily named RpsYD29and mapped on soybeanchromosome03（molecular linkage group N; MLG N） flanked by SSR markers SattWM82-50and Satt1k4bat a genetic distance of0.5and0.2cM, respectively. Two nucleotide binding site-leucine rich repeat（NBS-LRR） type genes Glyma03g04030.1and Glyma03g04080.1were detected in the204.8kb regionbetween SattWM82-50and Satt1k4b, which were might be the referenced candidate genes of RpsYD29. Andthe differences in genomic sequence and the putative amino acid sequence, respectively, were identifiedwithin each candidate gene between Yudou29and Jikedou2, which might cause the resistance/susceptibltityin both cultivars. Based on the phenotype reactions and the physical position on soybean chromosome03,RpsYD29might be a novel allele at, or a novel gene tightly-linked to, the Rps1locus.4. The molecular marker phenotype of the34soybean cultivars （lines） were analyzed using thetightly-linked molecualr markers Satt530and Sat₁86with Rps1locus, SattWM82–50and Satt1k4bwith RpsYD29gene. Thirty-one marker phenotypes were generated using these four primers, indicatingthat34soybean cultivars （lines） had the high diversity at these four loci. The results of the molecularmarker phenotype showed that Yudou23, Yudou24and Zhoudou11might carry RpsYD25gene;Zheng7729, Zheng84285, Zheng120, Zheng90007and Zheng92116might carry RpsYD29gene;Yudou12, Yudou22and Chihuangdou might carry Rps1a/1d gene; Yudou15might carry Rps1b gene.And Qihuang1, Zheng7104, Shandongsijiaoqi, Zheng135, Yudou13and Zheng85558might carryRps1c/1k gene.5. Resistance to Phytophthora sojae isolate PsMC1was evaluated in102F2:3families derived froma cross between the resistant soybean cultivar Wandou15and the susceptible cultivar Williams andgenotyped using simple sequence repeat （SSR） markers. The segregation ratio of resistant, segregating,and susceptible phenotypes in the population suggested that the resistance in Wandou15was dominantand monogenic. Twenty-six polymorphic SSR markers were identified on soybean chromosome17（MLG D2）, which were linked to the resistance gene based on bulked segregation analysis （BSA）.Markers Sattwd15–24/25and Sattwd15–47flanked the resistance gene at a distance of0.5and0.8cM,respectively. Two cosegregating markers, Sattwd15–28and Sattwd15–32, were also screened in thisregion. This is the first Rps resistance gene mapped on chromosome17, which is designated as Rps10.Eight putative genes were found in the mapped region between markers Sattwd15–24/25andSattwd15–47, of which two gene models Gly17g28950.1and Gly17g28970.1might be the referencedcandidate genes of Rps10. The candidate genes encoding serine/threonine （Ser/Thr） protein kinases inWandou15and Williams were identified and sequenced.And the differences in genomic sequence andthe putative amino acid sequence, respectively, were identified within each candidate gene betweenWandou15and Williams. This novel gene Rps10and the linked markers should be useful in developingsoybean cultivars with durable resistance to P.sojae.更多还原