Studies On Resistance Evaluation, Gene Mapping And Resistance Related Genes To P. Sojae In Soybean

Phytophthora root rot caused by Phytophthora sojae Kaufmann &. Gerdemann （P. sojae）, is one of the most devastating diseases of soybean throughout soybean-growing regions all over the world. P. sojae can infect soybean plants throughout the growing season under saturated soil conditions. In China, P. sojae mainly exists in three soybean ecological regions including the Northeast region, the Huang-Huai-Hai region and the Southern region, and induces loss in yield and seed quality in main soybean production regions.Utilization of resistance varieties is the most economical and environmentally safe method for controlling this disease. The scientific basis of effectively selecting resistance varieties is to know the type of physiological race. The keys of breeding for resistance are rational using of resistance, screening and searching new sources of resistance. Broadening the genetic resources, reducing the pressure of new pathogenic P. sojae races is the guarantee of resistance breeding process. The genetics of resistance to P. sojae is complex. The major sources of resistance are a series of single dominant host resistance genes （Rps genes）. There were 14 Rps genes mapped to four molecular linkage groups at 8 different loci that provide race-specific resistance. Molecular markers linked to these of the known phytophthora resistance genes have been reported. However, 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. In addition to the Rps genes, soybean has partial resistance, which has been shown to limit the lesion growth rate of pathogen in host tissue. The resistance is a relatively high heritable, quantitative trait, and controlled by several genes. Therefore, we should pay attention to the screening of the partial resistance to P. sojae, except for the complete resistance.The main goals of this study were:（1） to isolate and identify P. sojae in Nanjing; （2） to investigate the distribution of Phytophthora resistant sources, identify new sources of complete resistance and partial resistant resources to P. sojae; （3） to discover genetic mechanism of reisistance to P. sojae and map reisistant gene; （4） to understand the resistant mechanism to P. sojae in soybean.1. Identification and race of Phytophthora sojae. Soybean root rot caused severely losses in Jiangpu Farm, Nanjing Agricultural University during the growing season of 2005 and 2006. P. sojae was detected by a rapid and specific PCR in diseased tissues. Four isolates （PNJ1, PNJ2, PNJ3 and PNJ4） were isolated from diseased tissues of soybean and soil samples, and identified as Phytophthora sojae Kanfman & Gerdemann. By using the method of hypocotyls inoculation on 14 differential hosts, PNJ1, PNJ2, PNJ3 and PNJ4 were identified as new races, and their virulence formula is 1d,2,3b,3c,4,6,7; 1d,2,3b,3c,4,6,7; 1a,1b,1c,1d,1k,2,3b,3c,5,7; and 1a,1b,1c,1d,1k,2,3b,3c,4,6, respectively. This research may provide useful information for breeding and using resistant varieties in Jiangsu area.2. Resistance identifity.611 soybean germplasm candidate resources from three ecological zones were evaluated for their responses to 3 P. sojae strains PNJ1, PNJ3 and PNJ4 using the hypocotyl inoculation technique.611 soybean germplasm resources elicited 8 different reaction types with 3 strains. Of all resources,106 were resistant and 253 were susceptible to the 3 strains. Among 611 soybean germplasms, the germplasm from Huang-Huai-Hai region were highly resistant to 3 P. sojae, while soybean germplasm from Southern region were lower and the Northeast region were the lowest. The percentage of germplasm with resistance to 3 P. sojae was the highest from the province of Jiangsu, and relativity high from Henan, Shandong and Anhui. The 123 soybean germplasm lines, which exhibited good agronomic characters and were identified as susceptible to three P. sojae isolates in the hypocotyl inoculation test, were evaluated for partial resistance to PNJ1 using the slant board assay. The results showed that 47 cultivars （38.2%） were high level of partial resistance to PNJ1 of P.sojae.3. Inheritance analysis and gene mapping of complete resistance. Chinese soybean cultivar （line） Ludou4 and Su88-M21 had the different reaction to the 9 P. sojae isolates and might contain novel resistance loci or alleles. Two soybean populations, the F2:3 population derived from Ludou4 （resistant）×Youchu4 （susceptible） and the F2:7:11 RIL derived from Su88-M21 （resistant）×Xinyixiaoheidou （susceptible）, were identified the genes for resistance to P. sojae and mapped these genes to linkage groups. The results showed that the complete resistances in Ludou4 to strain Pm28 and Su88-M21 to strain Pm14 were controlled by a single dominant gene （Rps）, and the two resistance genes were temporarily designated RpsLu4 and RpsSu. RpsLu4 was located on molecular linkage group N and closely linked with Satt631 and Sat₁86. RpsSu was a novel phytophthora resistance gene to be first found on molecular linkage group O, and flanked by Satt358 and Sat₂42 with genetic distances 3.5 cM and 7.4 cM.4. Inheritance analysis and gene mapping of partial resistance. The partial resistance of the RIL population NJRISX derived from Su88-M21×Xinyixiaoheidou was used to study the inheritance of partial resistance. Genetic analysis was performed under major gene plus polygene mixed inheritance model in the P1, P2, F2:7:11 of the cross "Su88-M21×Xinyixiaoheidou". QTL mapping for partial resistance in soybean was carried out by the methods of composite interval mapping （CIM） and multiple interval mapping （MIM） of software WinQTL Cartographer Version 2.5. The results showed that the partial resistance of Su88-M21 to P. sojae P6497 was controlled by two major genes plus polygenes in the RIL population with the major gene heritability being 74.13%, and polygene heritability being 23.79%. With CIM, two QTLs （qPR-15-1 and qPR-10-2） associated with partial resistance were identified on MLG E, MLG O, explaining 13.95%,8.25% of the phenotypic variation, respectively. With MIM, three QTLs （qPR-15-1, qPR-10-2 and qPR-6-3）, explaining 4.30%～5.90% of the total phenotypic variation were detected on three linkage groups, of which two QTLs （qPR-15-1 and qPR-10-2） were located on the same regions of linkage group E and O as that under CIM.5. Analysis of resistance related gene to P. sojae in soybean. The expression of genes in hypocotyls inoculated P. sojae of soybean was studied by using soybean genome chip （Genechip（?） SoyGenome Array, Affymetrix）. A totally of 688 probe sets, on behalf of 665 transcripts were identified with differential expressions at different times post inoculation, including pathogen related proteins, defense genes, transcription factors, signal transduction cell structure etc. The upregulated genes with similarity to PR5 protein, peroxidases, glutathione transferase fell into the functional category of defense. Downregulated genes include those with similarity to cell-wall-associated protein, such as proline-rich protein, and xyloglucan endotransglycosylase. To confirm gene chip hybridization result,9 resistant related genes of soybean and 1 gene of P. sojae were selected for real time RT-PCR analysis. The similar results were obtained between gene chip and real time RT-PCR.