| Downy mildew of foxtail millet is one of the most important diseases that endanger the yield and quality of millet.In order to discover the key genes of foxtail millet resistance to bacterial blight and explore the interaction mechanism of foxtail millet resistance to Sclerospora graminicola,this study first evaluated the resistance of 360 foxtail millet germplasm resources to S.graminicola.At the same time,in order to further screen resistant and high-quality germplasm,the agronomic,quality and sheath blight resistance traits of the evaluated resistant varieties were investigated.Subsequently,genome-wide association analysis(GWAS)was performed on the disease-grade phenotype of foxtail millet at different stages.At the same time,transcriptome analysis was performed on the screened extremely resistant and susceptible varieties Yugu 1and ’ xiaomi ’ after inoculation with S.graminicola.The key candidate genes for foxtail millet resistance to S.graminicola were comprehensively excavated,and the key resistance genes were preliminarily functionally verified.The main results are as follows:1.A total of 360 foxtail millet materials were identified for resistance to S.graminicola,for many years.Correlation analysis showed that there was a strong positive correlation between the incidence of different years in the same location,and there was a significant positive correlation between the incidence of different locations,indicating that the incidence of each material was relatively stable.Combined with the results of resistance identification in two environments,62 high resistance materials were screened.2.6 agronomic traits such as plant height,heading date,panicle length,panicle diameter,stem diameter and tillering,and 2 quality traits such as the grain episperm color and amylose content were investigated.Phenotypic variation,heritability and Shannon-Weaver index analysis showed that these 8 traits of 62 resistant materials had rich genetic variation and breeding value.After comprehensive scoring evaluation by principal component analysis,10 high-quality foxtail millet germplasms with high resistance to S.graminicola were obtained.Combined with the results of multi-resistance investigation of sheath blight,three high-quality multi-resistant materials B359(Cang 555),B346(Jixiang 1)and B303(An 04-5014)were finally obtained.3.The MLM model was used to perform GWAS on the disease grade of all varieties.A total of 106 SNP loci significantly related to the resistance to S.graminicola were detected.and 14 disease-resistant candidate genes were screened in the 50 kb regions above and below the significant SNP loci.Functional annotation showed that these genes are mainly related to plant disease resistance-related lipolytic enzyme proteins,disease resistance RPP13-like proteins,cell wall-associated kinases,plant immune receptor proteins and laccases.Combined with haplotype analysis and q RT-PCR expression verification,three important candidate genes for resistance to S.graminicola were predicted.4.Transcriptome technology was used to analyze the changes of gene expression profiles of high-susceptible variety ’Xiaomi’ and high-resistant variety Yugu1 at different times after infection by S.graminicola.The results showed that the differential genes were significantly enriched in GO items related to photosynthesis,suggesting that S.graminicola may affect the photosynthesis of foxtail millet and promote its own invasion.In the plant-pathogen interaction pathway,the signal recognition protein recognizes the effectors produced by pathogens,and activates the up-regulated expression of downstream disease resistance-related genes including RPP13-like resistance genes and RPM1 resistance genes through various pathways to resist pathogen invasion.In the MAPK signal transduction pathway,the serine/threonine protein kinase signal transduction system induces significant up-regulation of downstream BAK1-related genes,PR1-related genes,and FRK1-related genes in the PTI pathway to produce hypersensitivity reactions to resist pathogen infection.As immune signaling molecules,ABA,JA and ET can activate a variety of defense responses to resist pathogen infection.Combined with GWAS association analysis,a key candidate gene Seita.8G191900 was predicted for the next step of gene function verification.5.The function of Si RPP gene was verified by constructing Arabidopsis heterologous transformation overexpression lines and Arabidopsis homologous gene T-DNA insertion mutants.The results showed that the expression level of Si RPP in overexpression lines was significantly up-regulated,while the expression level of Arabidopsis homologous gene At3g46730 mutant was significantly down-regulated.Phenotypic observation showed that Si RPP overexpression lines enhanced the resistance of Arabidopsis plants to S.graminicola,while the mutants reduced the resistance of Arabidopsis plants to S.graminicola.It is speculated that Si RPP is a positive regulatory gene for foxtail millet downy mildew disease resistance.Bioinformatics analysis showed that the end of chromosome 8 may play an important role in the defense of foxtail millet against the infection of S.graminicola in the form of disease-resistant gene cluster.In summary,this study has selected reliable and abundant germplasm resources of disease-resistant and high-quality foxtail millet.Combined with GWAS association analysis and RNA-seq technology,the key candidate gene of foxtail millet resistance to powdery mildew was targeted.The gene has been preliminarily functionally verified by Arabidopsis heterologous overexpression and T-DNA insertion mutants.The results can provide theoretical and application basis for in-depth analysis of the regulatory mechanism of disease-resistant genes and the breeding of disease-resistant and high-quality foxtail millet varieties. |
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