| Soybean root rot is one of soil-borne fungal diseases on soybean [Glycine max(L.)Merr.],which occurs throughout the growth and development of soybean.Fusarium spp.is a pathogen causing soybean root rot that were reported and studied worldwide.Fusarium majorly acts on vascular bundle of soybean,and inhibits the growth of soybean roots,making soybean leaves appear yellowing and brown necrotic spots,and even causing the plants to death durling the infection.Planting resistant varieties is the most efficient and sustainable means to control Fusarium spp.on soybean.Identification of resistance genes is the precondition to identify resistantance,breed resistant varieties and research resistance mechanism.This study majorly focused on the following aspects:(1)isolated and identified the pathogens in soybean wilt plants by morphological and molecular methods,assayed the pathogenicity of the obtained isolates to soybean seedlings by dipping-root method,meanwhile,and isolated the pathogenic substances in Fusarium equiseti by activated carbon abosrbtion and organic solvent extraction methods;(2)developed the PI437654 and zhonghuang13 mutant populations by mutagenesis of ethyl methanesulfonate(EMS),and statistically analyzed the morphological phenotypes in the field;(3)screened and identified the mutants with phenotypic alteration from the developed PI437654 mutant population by infection of Fusarium equiseti to soybean seedlings,and performed whole genome resequencing of the identified mutants with alteration of Fusarium equiseti-infection phenotype,and identified the SNPs and InDels associated with resistance of soybean PI437654 to Fusarium equiseti through analyses of sequencing data.In this study,11 susceptible plants of root rot disease were found in a field planted with a 2016PI437654 mutant population,and 17 Fusarium isolates and a Framete shirsute were isolated and identified.These 17 Fusarium isolates were classified into 4 species,of which 16 Fusarium isolates were virulent to both Zhonghuang 13 and PI437654,while the #18 isolate,a Fusarium equiseti,was avirulent to PI437654 but virulent to Zhonghuang 13.In addition,the pathogenic substances were initially separated from Fusarium equiseti,and the results indicated that the pathogenic substances belong to water-soluble compounds.Two new soybean mutant populations,PI437654 mutant population and Zhonghuang 13 mutant population,were developed by EMS mutagenesis.The morphological phenotypes of M1 and M2 mutant populations were observed in the field,and statistical analyses showed that the total mutant rate was18.72% in M2 plants.The main morphological phenotypes included yellowing,shrinkage,bushy dwarf,multi-leaf,seed coat color change and etc.Some mutants with high yield and late maturity were initially screened.The phenotyping of PI437654 mutants were conducted with the infection of #18 Fusarium equiseti.From 500 randomly-selected PI437654 mutants,6 mutants were screened to alter significantly the phenotype to the infection of #18 Fusarium equiseti.The mutant rate was 1.2%.Nine plants of the L63 and L78 mutant lines were used to perform whole genome resequencing,and 62 unique SNPs and InDels were identified to be associated with resistance of soybean PI437654 to Fusarium equiseti by bioinformatic analyses.Using EMS-mutagenesis to develop the ideal genetic germplasm mutant populations not only can provide good genetic materials for the identification of genes involved in resistance of soybean to Fusarium spp.and subsequent researches on gene functions but also can be used for identification of other disease-resistant genes in soybean and mutation breeding.The results obtained in this study will be the bases for further exploration of soybean resistance genes and identification of Fusarium virulent factors,and understanding the molecular mechanism of soybean resistance to Fusarium equiseti,high-throughput screening of soybean resistant varieties and making molecular marker-assisted breeding. |
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