| Soybean is an important food crop in China.Each year,China imports more than 100 million tons of soybean from abroad for feed processing.The domestic soybean is mainly used for food processing.Heilongjiang Province is the largest soybean planting base in China,with an annual planting area of about 60 million mu.But soybean bacterial disease is a major disease decreasing soybean production in Heilongjiang Province.Soybean bacterial leaf pustule is a bacterial disease commonly found in soybean,which can cause severe yield loss.In view of the serious harm of soybean bacterial leaf pustule,we started with the isolation and identification of soybean bacterial leaf pustule pathogen,and the pathogenicity identification of the isolated pathogens.Combined with genome sequencing technology,the genome sequencing and annotation of pathogens have been completed.The substituted line material containing wild soybean genome information was used to analyze the genes that respond to pathogenic bacterial type III effect factors in soybean.Based on the QTL mapping results of the soybean recombinant inbred line population(RIL)after inoculation with pathogens,and the RNA-seq analysis on the substituted line,the differentially expressed genes and hub genes in soybeans that respond to type III effectors were obtained.Further more,we used the sequenced germplasm resources and substituted line materials to carry out haplotype analysis of candidate genes,and finally determined the key candidate genes in response to pathogenic bacteria type III effector factors in soybean.The results laid a theoretical foundation and material foundation for in-depth and detailed research on the molecular mechanism of soybean resistance to bacterial leaf pustule in Heilongjiang Province and the genetic improvement of soybean.The specific research results are as follows:(1)In this study,the disease spots of soybean leaves were isolated and identified by selecting the onset period of bacterial diseases after rain in a field environment.Eight pathogens that can cause bacterial diseases in soybeans have been identified.Seven of them belong to the genus Pseudomonas and one belongs to the genus Xanthomonas.According to16 S r DNA amplification and sequencing comparison analysis,it is found that this strain is compatible with Xanthomonasvasicola has very high homology.Combined with the previous research foundation,it is generally believed that Xanthomonas is the pathogen causing soybean bacterial leaf pustule.Therefore,the Xanthomonas isolated from this research report will be the focus for follow-up research.Through inoculation and identification on the main cultivated varieties in different accumulative temperature zones in Heilongjiang Province,it was found that the isolated Xanthomonas spp.belong to highly pathogenic strains,which could cause obvious pathogenicity and disease as well as large areas of lesions in the main cultivated varieties in Heilongjiang Province.Due to the strain belongs to Xanthomonas,it was named Xv NEAU001WT(Xanthomonas vasicola Northeast Agrcultural University Wild Type).For the need of follow-up research,the resistance of Xv NEAU001 WT was analyzed.The results show that Xv NEAU001 WT is resistant to spectinomycin,but not to antibiotics such as rifampicin,kanamycin,chloramphenicol,gentamicin and tetracycline.This ensures that spectinomycin can be used for resistance protection to Xv NEAU001 WT to ensure that no other bacteria will be contaminated during the cultivation,storage and operation of the bacteria.(2)After completing the isolation and identification and antibiotics resistance analysis of Xv NEAU001 WT,this project further performed sequencing analysis on the genome information of Xv NEAU001 WT.Using next-generation sequencing technology,the genome sequencing and gene annotation analysis of Xv NEAU001 WT have been completed.Using the previously published genome of Xanthomonas as a reference,the sequence of the Xv NEAU001 WT genome was finally assembled into a large plasmid with a total of 5,221,943 bp,and the sequence information was uploaded to the NCBI database.The GC content of this genome is 65.7%.According to genome annotation analysis,it was found that this strain could encode 15 hrp genes related to type III effectors.Because the hrpG gene is a key gene that affects the pathogenicity of Xanthomonas,this study conducted in-depth research on the regulatory gene hrpG of the hrp gene.A deletion mutant of the hrpG gene was constructed by means of three-parental mating.Further pathogenicity analysis was carried out on the soybean germplasm resource Suinong 14,and it was found that the mutation of hrpG gene can significantly affect the pathogenicity of Xv NEAU001 WT.This result indicates that there are disease resistance-related genes in soybeans that respond to the type III effector regulated byhrpG.(3)In order to identify and analyze the key genes that respond to hrpG-regulated type III effectors in soybean,we use a combination of RNA-seq sequencing and QTL mapping.Using substituted line populations containing wild soybean genome information and high-generation recombinant inbred line populations as soybean material,wild-type Xv NEAU001 WT and hrpG mutant Xv NEAU001WTâ–³ hrpG as strain materials,the analysis of differential genes and QTL mapping were performed.Provide reliable information for finding key candidate genes for disease resistance.By inoculating the wild-type pathogen Xv NEAU001 WT in the substituted line,it was found that the substituted line materials F1680 and F1011 were significantly different from the substituted line recurrent parent Suinong 14 in the susceptible phenotype.Among them,comparing to Suinong 14,F1680 is more susceptible,and F1011 is more resistant.This result indicates that the substitution of wild soybean genome has led to changes in soybean disease resistance,and also that the introduced segments contain key genes that regulate soybean disease resistance.Therefore,the genetic background of F1011 and F1680 was analyzed first to determine the location of the imported segments.Then use Suinong 14,F1011 and F1680 to perform RNA-seq analysis.Through sampling of 81 samples at 3 time points,the soybeans were inoculated with pathogenic bacteria at 6 h,12 h,and 24 h,and 4,624 differentially expressed genes were obtained.It was found that the genes in four genome clusters C4,C5,C6 and C8 were significantly related to the resistance of F1011 and F1680.Further through gene co-expression network analysis,35 hub genes were obtained.These hub genes are distributed on chromosomes 01-17 and 19.By combining the results of QTL mapping,two key genes substituted by hrpG-encoded type III effector regulatory genes were identified on chromosomes 08 and 11,namely Glyma.08G009900.1 and Glyma.11G056200.1.(4)In order to determine the accuracy of these two candidate genes,the genetic diversity of the two genes in 200 populations of substituted lines was further analyzed.Through comparison and analysis with the genetic background of Suinong 14,it is found that there are significant haplotypes in Glyma.08G009900.1 and Glyma.11G056200.1,and there are significant differences in haplotypes between F1011 and F1680.The disease resistance of materials has a consistent compliance relationship.It further proves that these two genes are type III effector response genes in soybeans that respond to hrpG regulation.Based on the above research results,it shows that the two key genes Glyma.08G009900.1 and Glyma.11G056200.1 obtained in this study can be used as important disease resistance candidate genes for in-depth mechanism research.At the same time,the materials and concepts adopted in this research provide an important theoretical basis and material basis for in-depth and detailed research on soybean resistance to bacterial disease.Also,the discovered QTL and genetic diversity can be used as a theoretical basis for marker development.The research results have important theoretical significance and application value for ensuring the stable and high yield of soybeans in Heilongjiang Province. |
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