Identification Of Bacterial Pustule Pathogen And Mapping QTL Of Resistance To The Disease In Soybean

Soybean bacterial pustule spot disease is one of the major diseases of soybean and has been reported in the major soybean producing areas in the world.Soybean plants infected with the disease produces brown spots with yellow halos on the leaves.In severe cases,leaves will die impacting seriously on soybean yield.The use of disease-resistant varieties is an effective way for managing bacterial pustule spot diease.Discovering of disease-resistant germplasm and studying its resistance mechanism are the key to develop of disease-resistant varieties.At present,there is limited information on genetic breeding of soybean resistance to bacterial spot.This study collected soybean bacterial spot disease strains from China to evaluated their pathogenicity and their characteristics.Again,soybean recombinant inbred line populations with different resistance were evaluated for disease resistance of the identified strains.Also,disease resistance QTL mapping,and screening for disease resistance candidate genes,analyzing their structural characteristics,and using gene editing technology to create mutants,which serves as the foundation for the discovery and breeding of soybean resistance genes.The main results are as follows:1.Isolation and identification of the pathogen of bacterial pustule spot of soybean.11isolated strains were identified as the pathogens causing bacterial pustule spot of soybean by infecting soybeans plant.Morphological,physiological and biochemical identification,together with the analysis of bacterial 16Sr DNA and gyr B gene sequence and phylogenetic tree analysis were using to identify bacterial species.11 isolated strains were considered to be Xanthomonas axonopodis pv.glycines by comparing the bacterial 16Sr DNA and gyr B gene sequence and known sequences in NCBI database.Soybean varieties Tianlong No.1showed different susceptibility after inoculation with 11 isolated strains which implying pathogenic differentiation between strains.After inoculating 10 soybean materials with 11strains,it was found that Nannong 493-1 and Nannong 86-4 showed different resistance to susceptibility.The outcome of the investigation is to provide a new source of pathogens for the evaluation of soybean germplasm resistant to bacterial pustule spot of soybean,and a reference for screening excellent germplasm resistant to bacterial pustule spot of soybean and control bacterial pustule spot of soybean.2.QTL mapping and candidate gene discovery of soybean resistance to bacterial spot disease D2 strain.Soybean ZN and K3N recombinant inbred line populations showed a skewed distribution of disease resistance to inoculate bacterial spot disease D2 strain,with average disease resistance ratings of 2.89 and 3.3 respectively.The proportion of high resistant families in ZN population was higher than that in K3N population.Using Win QTLCart 2.5 and mixed linear model compound interval mapping method to perform QTL mapping analysis on the resistance level data of two populations,one major QTL was co-located on chromosome 17 in the two populations,and the contribution rate was 29.16%and 35.01%,and its position overlaps with the segment where the rxp gene for resistance to bacterial spot disease was reported by previous reports.It was further found that there were97 annotated genes in the 889 k bp physical interval co-localized by the QTLs of the two populations,and candidate genes such as Glyma.17g085000、Glyma.17g086200、Glyma.17g089700、Glyma.17g090200、Glyma.17g090400 were preliminarily screened based on the function of disease resistance genes.The results obtained laid the foundation for the discovery and functional research of soybean resistance to bacterial spotted disease.3.The structural characteristics of soybean anti-spot disease candidate genes Glyma.17g089700 and Glyma.17g090200 and the creation of gene editing materials.Glyma.17g089700 encodes the peroxidase synthase Pex14,has 11 exons,the CDS sequence is 1491 bp,and encodes 496 amino acids.Glyma.17g090200 encodes a ring finger protein with 10 exons.The CDS sequence is 1152bp and encodes 383 amino acids.The soybean CRISPR/Cas9 gene editing vectors of Glyma.17g089700 and Glyma.17g090200 were further constructed,and Williams 82 was used as the recipient material for genetic transformation,and 45 and 35 explant seedlings were obtained,respectively.The T₀ generation regenerated seedlings were herbicides and transgenic.Component PCR detection,16 and 8 T₀ transgenic positive seedlings were obtained.The results obtained will provide materials for the further study of the biological functions of Glyma.17g089700 and Glyma.17g090200 genes,and provide reference for the genetic breeding of soybean resistance to bacterial spot disease.