| Black point disease is serious in wheat in recent years.Planting resistant wheat cultivars is an effective way to control this disease.It is important to create excellent germplasm and analyze its resistance mechanism for breeding resistant wheat cultivars.In this study,based on the result of investigation of agronomic characters and resistance identification of black point,a mutant wyb-18 with stable resistance to black point disease and similar agronomic characters to the wild type were obtained from ethyl methane sulfonate（EMS）-mutagenized library from highly susceptible wheat line Wanyuanbai1 under artificial inoculation of Bipolaris sorokiniana in three years.To preliminarily analyze the disease resistance mechanism of wyb-18 and select the candidate genes for black point resistance,the development of black point symptoms on kernels from wyb-18 and its wild type was observed,and the activity of main antioxidant enzymes were measured at different days after inoculation and transcriptome analysis of kernels samples from wyb-18 and its wild type was performed at 9 d after inoculation with isolate Ta-BP33 of Bipolaris sorokiniana（the dominant pathogen causing black point in Henan province）,using the patented technology of spraying spore suspension and bagging for moisture.The main results of this study are as follows:1.A total of 499 mutants with different traits were selected from 10008EMS-mutagenized lines of the highly susceptible line Wanyuanbai 1,including 48 mutants of plant height and oligo-tillers,80 mutants of spike trait,51 mutants of leaf,17 mutants of male sterility,17 mutants of late anthesis,271 mutants of kernel,and 15 mutants with different resistance to black point,under artificial inoculation with B.sorokiniana,the average black point incidence of mutant wyb-18 was 7.5% in two years,which was significantly lower（P < 0.01）than that of its wild type（53.6%）.Except for resistance,the other agronomic traits of wyb-18,including leaf type,anthesis,plant height and grain traits,was similar to the wild type.Sixty-three SSR markers（three markers per chromosome）were used for molecular detection,and the results showed that there was no polymorphic SSR locus between wyb-18 and the wild type,and their genetic background was consistent,indicating that wyb-18 was the EMS mutant of Wanyuanbai 1.2.The resistant mutant wyb-18 showed black point symptoms at 12 days after inoculation with B.sorokiniana,which was three days later than that of the wild-type（9 days）.When 15 days after inoculation,black point symptoms on kernels from wyb-18 was scattered black/brown spots,while the whole embryo of the kernels from wild type had turned black,reflecting the symptoms of wyb-18 appeared later and developed more slowly than those of the wild type.At different days after inoculation,the activities of antioxidant enzymes,including superoxide dismutase（SOD）,catalase（CAT）,and peroxidase（POD）in kernels from wyb-18 were generally higher than those of the wild type.For example,at 9 days after inoculation,SOD and CAT activities of wyb-18 were 59.0% and 33.7% higher than those of the wild type,respectively（P < 0.01）,indicating that antioxidant enzymes may play an important role in wyb-18 response to B.sorokiniana.3.The transcriptomic analysis of wyb-18 and its wild-type kernels at 9 days after inoculation（control was sprayed with sterilized distilled water at the same time）was completed and 37,170 new genes in different samples revealed,among which 15,376 had functional annotations.A total of 543 differentially expressed genes（DEGs）were detected between wyb-18 and its wild type,and 1470 and 1790 DEGs were detected between wyb-18 and wild type samples and their control samples,respectively.Further GO analysis showed that DEGs included in toxin activity and antioxidant activity accounted for a large proportion in the molecular function group;DEGs in extracellular regions and membrane-closed cavities accounted for a large proportion in the cell component group;DEGs in detoxification and stress response accounted for a large proportion in biological processes.KEGG enrichment analysis showed that plant-MAPK signaling pathway（ko04016）,plant hormone signaling pathway（ko04075）,ubiquitin-mediated proteolysis（ko04120）was enriched.4.Expression analysis of 12 differentially expressed genes was carried out by RT-q PCR method.The expression trends of fluorescent quantitative PCR results were basically consistent with the transcriptome sequencing results,which indicated the accuracy of transcriptome sequencing results.The analysis of differentially expressed genes related to antioxidant enzymes,pathogenesis-related proteins and transcription factors were carried out and 72 genes including 14 antioxidant enzyme genes（e.g.POD gene Traes CS2D02G107800,SOD genes Traes CS5D02G372700 and Traes CS5B02G365600,CAT gene Traes CS4B02G325800）,31 PR protein genes（e.g.endo-β-1,3-glucanase gene Traes CS3A02G144600,defensin gene Traes CS1B02G067100）,and 27 WRKY transcription factor genes,were selected to related to the defense process of wheat black point.These differentially expressed genes provided important information for the selection of candidate genes associated with black point resistance.5.The main metabolic pathway enriched in KEGG（plant-MAPK signaling pathway）was further analyzed.There are 10 genes were up-regulated expression in wyb-18,including PR gene Traes CS5A02G439700,chitinase genes Traes CS1A02G326800,Traes CS1D02G207000,Traes CS1D02G207100 and Traes CS3D02G391100,WRKY22 gene Traes CS2A02G433000,serine/threonine protein kinase genes Traes CS2A02G426200 and Traes CS2A02G566700,CAT gene Traes CS4B02G325800,and abscisic acid-inducible protein kinase gene Traes CS2A02G493800.Two genes,ethylene receptor gene Triticumaestivumnew Gene5891 and vegetative storage protein gene Traes CS7D02G053700,were down-regulated expression in wyb-18,including.These 12 genes may be candidate genes resistance to black point caused by B.sorokiniana in wyb-18.A disease-resistant mutant wyb-18 was identified from 10008 EMS mutagenic lines of Wanyuanbai 1,a highly susceptible wheat strain,which was similar to the wild type and had stable resistance,which provided germplasm resources for breeding wheat resistant to black point disease.The higher antioxidant enzyme activity of wyb-18 may be an important reason for its anti-B.sorokiniana black point disease.The up-regulated genes involved in the plant-MAPK signaling pathway,such as PR1 gene,chitinase gene and CAT gene,may be important candidate genes of wyb-18 against B.sorokiniana black point disease. |
