| As an important plant pathogenic fungus, Rhizoctonia solani is a large complex containing lots of genetically distinct groups and affecting many plant species worldwide. R. solani is the cause agent of sheath blight against rice and barley causing significant yield loss every year. The knowledge has been accumulated on R. solani isolated from rice, which is sorted into AG1-IA anastomosis group since the past decades. However, due to the lack of quality resistance resources, the prevention and control of rice sheath blight is still heavily dependent on chemicals. Intensive use of chemicals has resulted in the increase of resistance of some strains in some areas. The understanding of the population genetic characteristics in different regions of the AG1-IA strains is not only beneficial to understand the population genetics of R. solani structure and evolutionary characteristics, but also help to explore resistant resources and delay the development of drug tolerance.In order to understand the genetic diversity and population genetic characteristics of the R. solani strains from Zhejiang Province, in present study, the sequence analysis of5.8S rDNA-ITS and SSR were used to investigate the population characteristics of86R. solani strains from7different regions in Zhejiang Province. At the meantime, the whole genome search and experimental verification of SSR markers based on the AG3complete genome sequence was carried out, which was about to provide a basis for the further use of the SSR to the population genetics. A brief summary of the research progress and the significance of this study were summarized as following:1) Eight-two R. solani strains and13standard anastomosis group strains were used in the sequence analysis of5.8S rDNA-ITS (ITS) interval. The differences in sequences of ITS interval between anastomosis group strains were much larger than them between the AG1-IA strains. Phylogenetic analysis showed that the differences in sequences of ITS interval can effectively distinguish strains from different anastomosis groups. But when the stains belonging to the same anastomosis group were concerned, the usage of the differences in sequences of ITS interval was quite limited. Haplotype analysis showed that82rice strains can be divided into10haplotypes. Haplotype diversity (Hd) of these field isolates was0.2087, and the total nucleotide diversity was0.00033. HI was the dominant haplotype, accounting for87.8%of all strains, the other nine haplotypes were each only a strain. Neutral test results showed that Tajima’s D test value-2.26012(P<0.01), Fu and Li’s D test value-5.20827(P<0.02), implied rice sheath blight strains significantly deviating from ZTE hypothesis which suggested the R. solani population may under expansion in the history, and there were strong selection effect exist.2) Due to the limited distinguishability of the differences in sequences of ITS interval in the same anastomosis group and also in order to understand the characteristics and distribution of SSR in the R. solani genome. We carried out the whole genome survey of SSR loci, designed the related primers and verified some of these primers based on AG3genome sequences. The results showed that pentanucleotide repeat SSR was dominant type in R. solani AG3genome. A total of63,578pairs of SSR amplification primers was designed through SSR Locator software and checked by electronic PCR. To further validate the authenticity of these SSR primers and the existence of the SSR loci, we synthesized281pairs of primers, which were all used in PCR amplification of the13anastomosis group strains. The results showed that the amplification rate and the percentage of specific band amplification of AG3anastomosis group strains was of90.39%and81.49%, respectively. The sequencing results also verified the authenticity of the SSR loci, which laid the foundation for further screening of specific SSR markers for population genetics studies.3) In order to further understand the characteristics of population genetics of the strains from Zhejiang Province and because we did not get any qualified SSR markers from our previous study of the whole genome SSR search and validation based on AG3genome sequence, we used10SSR markers reported in former studies to investigate the characteristics of population genetics of85R. solani strains. The results showed that all markers can show certain polymorphisms, of which TC06exhibited highest PIC value0.7112. In these seven regions, strains from Shaoxing had the height clonal fraction of0.33, and strains from Hangzhou area had the most specific sites. Regional groups showed varying degrees of admixture, ranged from67%-97%. Studies have shown that the difference of diversity in R. solani strains from Zhejiang Province comes from the individual strains, accounting for58.52%of the total difference. Population Differentiation analysis revealed that the differentiation between Ningbo region and other regions does not reach a significant level strains, the population of Wenzhou area showed significant difference from the population of Hangzhou area, but no significant differentiation with population from other regions. Genetic differentiation of the different groups does not follow the geographic separation mode based on Mantel test, strongly suggest that the gene exchange was quite often.The genetic diversity of R. solani is an effective means to understand the population variation of R. solani strains, but also can provide some theoretical support for the screening of resistant varieties and avoid drug resistance. |
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