The Evaluation Of The Phytophthora Resistance In Soybean Cultivars(Lines), Mining And Analysis Of The Rps Candidate Gene

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.