Identification And Fine Mapping Of Xa-48(t)Gene With High Resistance To Bacterial Blight In Rice

Rice bacterical blight,caused by Xanthomonas oryzae pv.Oryzae(Xoo),is one of the three major diseases of rice in China,which not only threatens rice yield but also seriously affects rice quality.The cultivation of disease-resistant varieties is the most economical,safe and effective means to prevent and control this disease,and the excavation of disease-resistant genes is the prerequisite for cultivation of disease-resistant varieties,while the identification of disease-resistant resources is the basis for excavation of disease-resistant genes.With the continuous mutation of B albicans under both natural and artificial selection pressure,the 46 reported rice B albicans resistance genes can no longer meet the needs of existing rice breeding applications,and the identification of new B albicans resistance genes and their application in breeding has become an urgent problem for scientists from various countries.Based on the South China rice region,we selected 315 Asian cultivated rice with high genetic diversity from 3K materials and conducted three replicate inoculation tests for each of two years to identify material 117,which showed high resistance to type Ⅳ of South China rice region and was found to be free of the resistance genes xa5,Xa21 and Xa23,which are well used in South China rice region.In order to investigate the genetic mechanism of disease resistance in Asian cultivated rice 117,we carried out research through experimental techniques such as phenotype identification,resistance spectrum analysis,genetic mapping population construction,BSA analysis,marker localization,and candidate gene prediction,and the main results obtained are as follows:1.The leaves of JG30 and 117 at the gestation stage were inoculated with the dominant type Ⅳ fungus of Guangdong.The experimental results showed that JG30 was highly susceptible with a spot length between 18.9-23.4 cm,117 was highly resistant with a spot length between 0.4-0.6 cm,and F1 showed a highly resistant phenotype with a spot length between 0.8-1.2 cm,and both parents and F1 showed highly significant differences,which also indicated that the trait was controlled by a non-fully dominant gene.2.Field inoculations of JG30 and 117 at the tiller stage using 12 strains were identified at the gestation stage,and the lengths of sword leaf spots were counted.The results showed that JG30 showed obvious susceptible type to pathogenic strains R2,R3,R4,R5,R8,R9,PXO99,type Ⅳ bacterium and type Ⅸ bacterium with spot length between 16.7-27.3cm,and showed some resistant type to R1,R6 and R7 with spot length between 0.5-1.1cm.While 117 showed significant resistance to pathogenic minor species R1,R2,R3,R4,R5,R6,R7,R9,PXO99,and type Ⅳ with lesion lengths between 0.3-2.1cm,moderate resistance to pathogenic minor species R8 with lesion lengths between 5.9-7.3cm,and susceptible to type Ⅸ with lesion lengths between 10.2-10.6cm.The resistance phenotypes of JG30 and 117 were not significantly different for three strains R1,R6 and R7,and were significantly different for nine strains R2,R3,R4,R5,R8,R9,PXO99,Ⅳ and Ⅸ.The frequency of resistance spectrum of JG30 against 12 strains was about 25%,while the frequency of resistance spectrum of 117 against these 12 strains was about 92%.The above results indicated that Asian cultivated rice 117 had a broad spectrum of resistance to white bacterial blight.3.The F1 material was obtained from the cross between parents JG30 and 117,F1 was selfed to obtain F2 generation,and when the sword leaves were drawn at the gestation period of rice,the F2 population was inoculated with Guangdong dominant type Ⅳ and the recorded data were analyzed,the results showed that the number of resistant plants was 746 and the number of susceptible plants was 240,the ratio of resistant to susceptible plants in the F2 population was 3:1 and P>0.5 indicating a significant differences,and the F2 population showed a continuous distribution of positive skewness for white bacterial blight resistance,indicating that the resistance of 117 to Guangdong Ⅳ bacterial blight was controlled by a pair of main effect QTL.4.The parental susceptible indica rice variety JG30 was crossed with leaf blight resistant indica rice variety 117 to produce 986 single plants of F2 generation as the mapping population.50 disease resistant and 50 disease susceptible single plants were selected for BSA sequencing,and the data quality control showed that the base error rate of 117 was relatively low,ranging from 0.000%to 0.009%,and AT and GC basically did not occur the curve was smooth and gentle.The base coverage on the reference genome and the base coverage depth distribution curve and coverage distribution curve of the samples were calculated by variant detection,and the results showed that the average coverage depth of the genome was about 44.00X and the genome coverage was about 93.95%.By testing the SNPs of the samples,the results showed that the six mutation types of SNPs of the samples had the most mutation types of C:G>T:A type,followed by T:A>C:G.The number of SNPs in the parental and mixed pools between genomes was also counted,and the parental pools contained 790883 SNPs and the mixed pools contained 138800 SNPs,and the base deletion types were more than the insertion types,and the insertion.The deletion sites were counted,and 170183 InDel sites were detected among the parents and 39020 InDel sites were detected among the mixed pools,and the results of the association regions corresponding to SNPs and InDel were taken to intersect,and finally 1 candidate region associated with the trait with a total length of 2.39 Mb was obtained,and this region contained 356 genes,among which 65 were shift mutant genes and non After analyzing the sequencing results,the location of the selected gene was initially targeted to the right side of rice chromosome 11,which was between 265900000bp-28980000bp,totaling 2.39M.5.Based on the sequence differences,54 pairs of polymorphic primers were designed on the initial physical position 26590386bp-28980000bp of rice chromosome 11,using DNA from JG30,117 and F1,a total of 19 pairs of markers with polymorphism were screened.From the F2 isolates of JG30/117,219 highly susceptible single leaves(disease spot length from 27.23-23.23 cm)were selected for target gene linkage analysis using 19 pairs of polymorphic molecular markers,and the target genes were targeted between markers JG30/117-25 and JG30/117-46.6.The NCBI search for the number of genes and related information in the above interval resulted in the screening of 23 candidate genes LOC_Os11g46250,LOC_Os11g46280,LOC_Os11g46300,LOC_Os11g46820,LOC_Os11g46890which are genes for five expressed proteins of unknown function,LOC_Os11g46260,LOC_Os11g46270,LOC_Os11g46290,LOC_Os11g46806,LOC_Os11g46810,LOC_Os11g46830,LOC_Os11g46840,LOC_Os11g46910,LOC_Os11g46920,LOC_Os11g46930,LOC_Os11g46940 are 11 transposonand antitransposon protein-related genes.LOC_Os11g46850,LOC_Os11g46860,LOC_Os11g46900,LOC_Os11g46950 are four cell wall-associated kinase genes.LOC_Os11g46870,LOC_Os11g46880 are two genes related to protein kinases.LOC_Os11g46960 is a class of signal peptide proteins.Four of these cell wall-associated class kinase genes were considered to be the best candidates.