| Phytophthora sojae causing soybean Phytophthora root rot is one of the most devastating pathogens of soybean,it is also quarantine important plant pathogen in China. Developing fast, effective ways for identifying P. sojae and developing effective molecular markers to study its population variation and evolution were very important.This study aimed at developing molecular markers specific to P. sojae, establishing PCR assays based on the specific markers for detecting infected soybean and soil. In order to offer effective markers for studying the genetic variation, molecular mapping and phylogenetic of P. sojae, SSR markers were developed by screening P. sojae complete genome sequence, investigated the transferability, and analyzed genetic diversity of P. sojae. Main research results were followed:Three specific primer pairs of P. sojae: Cox3-F/Cox3-R, PSEL1-F/PSEL1-R and PSEL2-F/PSEL2-R, which could produce 450 bp, 289 bp and 370 bp fragments in P. sojae respectively, were developed based on cytochrome oxidaseⅡgene sequence and elicitin ESTs of P. sojae. The sensitivities of the 3 specific primer pairs for detection of P. sojae genomic DNA were 20 pg/μL, 2 pg/μL and 2 pg/μL, respectively. They could be used to detect P. sojae in infected soybean plants.Based on EST-SSR marker Psc239 specific to P. sojae, another specific primer pair Psc239EF/ Psc239ER and nested PCR assays were developed. Against 36 different isolates of P. sojae, 25 control pathogen isolates and 2 soybean varieties, Psc239EF/ Psc239ER, Psc239 and nested PCR assay were specific to P. sojae and produced 519bp, 242bp and 242bp fragments in P. sojae respectively. The sensitivities of Psc239EF/ Psc239ER, Psc239 were 50 pg/μL, while the nested PCR assay was 50 fg/μL. The nested PCR assay was also used to detect P. sojae in infected soybean plants and soil samples, it could detect as few as 0.4 oospores per 1 gram soil. Using nested PCR assay, 16 positive soil samples were detected among 22 soil samples, whereas, 12 positive soil samples were detected using leaf disc baiting technique.260 SSR primer pairs were designed based on 260 SSR sequences selected by screening P. sojae complete genome sequence. Of the 260 primer pairs, 213 (81.9%) could amplify characteristic SSR fragments against 10 different isolates of P. sojae, 114 (53.5%) were polymorphism, 72 (33.8%) amplified different fragments in 10 isolates of P. sojae. Some SSR primers could amplify producers in 11 isolates of Phytophthora spp, but the fragments were different with fragments amplified in P. sojae. Cluster Phytophthora spp and P. sojae based on different fragments of 10 SSR markers, these markers could classify Phytophthora spp and P. sojae.Using 18 SSR markers, the genetic diversity of 108 P. sojae were assessed. On the 18 SSR loci, a total of 119 alleles were detected in 108 P. sojae. At each locus,4 to 9 alleles could be detected,with an average of 6.61. These markers were high polymorphism and culd be used to analysis genetic diversity of P. sojae.Analysis of genetic diversity and clustering of 108 different isolates of P. sojae from 6 different regions. The results indicated that, high genetic diversity existed in Chinese population. Genetic diversity levels in Heilongjiang, Anhui and United States were higher and genetic variation might widely present in these regions. The populations of Xinjiang and Heilongjiang are closer to each other than to others, P. sojae in Xinjiang might come from Heilongjiang. The populations of Anhui and Heilongjiang might have the same genetic background. Against relatively few isolates, the genetic diversity of United States P. sojae was higher and the genetic background of United States was different from Chinese population indicated by the results of clustering and analysis of genetic distance.
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