Discovery And Specific Marker Development Of Phytophthora Sojae Resistance Gene In Soybean

Phytophthora root rot caused by Phytophthora sojae is a destructive disease of soybean.The most effective disease-control strategy is to deploy resistant cultivars carrying Rps genes.1.The soybean cultivar Huachun 18 has a broad and distinct resistance spectrum,and is postulated to contain new resistant gene.Huachun 18 and the susceptible cultivar Jikedou 2 were used to produce the mapping F_2:3 population.The phenotypic identification results showed that the resistance to P.sojae in Huachun 18 was controlled by a single dominant gene and was designated as RpsHC18.QTL-seq revealed one 767-kb genomic region on chromosome 3 was identified as the RpsHC18 candidate region.The candidate region was reduced to a 146-kb region by fine mapping.Nonsynonymous SNP and haplotype analyses were carried out in the 146-kb region among 10 soybean genotypes using WGRS.Four specific nsSNPs were identified in two NBS-LRR genes,RpsHC18-NBL1 and RpsHC18-NBL2,which were considered the candidate genes.Specific SNP marker were developed based on nsSNP in candidate genes,and the three markers were found to be an RpsHC18 co-segregation markers and can effectively distinguish RpsHC18 and other known Rps genes.2.We previously detected a novel Phytophthora resistance gene,RpsZS18,on chromosome 2 of the soybean cultivar Zaoshu18.The aim of the present study was to identify and finely map RpsZS18.We used 232 F_2:3 families generated from a cross between Zaoshu18（resistant）and Williams（susceptible）as the mapping population.SSR markers distributed on chromosome 2 were used to map RpsZS18,and RpsZS18 were identified between developed SSR markers ZCSSR33（0.9 cM）and ZCSSR46（0.5 cM）.Second,PCR-based InDel markers were developed between the two parents and used to further narrow down the mapping region of RpsZS18 to 71.3 kb.Third,haplotype analyses were carried out in the RpsZS18 region using 14 soybean genotypes with whole-genome resequencing.We detected six genes with unique haplotype sequences in Zaoshu18.Finally,quantitative real-time PCR assays of the six genes revealed an EF-hand calcium-binding domain containing protein encoding gene（Glyma.02g245700）,a pfkB carbohydrate kinase encoding gene（Glyma.02g245800）,and a gene with no functional annotation（Glyma.02g246300）,are putative candidate PRR resistance genes.3.Previous studies have located the Rps genes in Yudou 25 on the short arm of chromosome 3.Therefore,RpsYD25 is further finely mapped.Based on the differences in resistance to P.sojae between the Zaoshu18 and Yudou25,the selection of the best isolates of Phytophthora was used to identify the phenotypes of the Zaoshu18 and Yudou25-derived populations.F_2:3 populations containing 165 families constructed a genetic linkage map for RpsYD25,locating RpsYD25 between SSR markers Satt1k3（2.2cM）and BARCSOYSSR₀3₀253（4.5 cM）.1127 F_3:4 families derived from Zaoshu18 and Yudou25were screened for recombinants using the two SSR markers.Based on the whole genome re-sequencing results of the two parents,PCR-based SNPs,InDel and SSR markers were developed,and the recombination sites of 1127 families were identified and the RpsYD25 co-segregated regions were obtained.RpsYD25 was located in the 101.3 kb region of soybean chromosome 3.This region contains three gene models,one of which is the gene Glyma.03g034700 with a zinc binding protein and nucleic acid binding protein domain,and the other two are the NBS-LRR genes Glyma.03g034800 and Glyma.03g034900 with the typical disease resistance structure.These three genes were predicted as candidate genes of RpsYD25.SSR markers co-segregated with RpsYD25 were screened and found that SSR40 can be effectively used for marker-assisted selection breeding to detect RpsYD25.