Karyotype Analysis And Expression Of Sexual Reproduction Related Genes In Setosphaeria Turcica

Setosphaeria turcica in heterothallism is an important pathogenic fungus in maize,which has the ability to reproduce asexually and sexually.However,the chromosome distribution and sexual reproduction mechanism of the fungus are still unclear.In this study,the number of nuclei of S.turcica in different development stages was observed by staining,and then the germ tube stage with more nuclei was selected.The well-dispersed chromosomes were obtained from the germ tube using the modified germ tube burst method（GTBM）.The chromosomes were stained with giemsa and the fluorescent dye DAPI,respectively.The number and morphology of chromosomes were observed under the microscope,and the chromosome was mapped by drawing software.The lengths of each chromosome were measured by Image J software,and the karyotype pattern was drawn.The whole genome was sequenced and assembled by using single molecule real-time（SMRT）DNA sequencing and Hi-C sequencing technology,and the assembled scaffold was anchored on different chromosomes.The mutant strain ΔStMAT1-1 and "a"mating type strain（Et52B）,the mutant strains ΔStMAT1-2 and "A" mating type strain（Et28A）,ΔSTMAT1-1 and ΔSTMAT1-2 were conducted to sexual induction respectively.The wild-type strains were used as the control group to detect the expression level of sexual reproduction genes in different combinations and different induction periods.The main research results are as follows:1.The nuclei of conidia,germ tube,appressorium,primary hypha and mature hypha were stained with 10 μg/mL DAPI.The changes in the number of nuclei were observed under fluorescence microscope.The results showed that the number of nuclei in a single conidia was about 38,and the total number of nuclei increased to 47 in the germ tube stage.The number of nuclei at the tip of the germ tube increased continuously with the elongation of the germ tube.When the conidia germinated to the formation of appressorium,there were no significant changes in the total number of nuclei with the germ tube period.The nuclei with different numbers migrated from the germ tube to the appressorium.There are more nuclei in the new hyphae,about 13～14 in each cell,and fewer nuclei in the mature hyphae,about 3～4 nuclei in each cell.2.The shape and number of chromosomes were observed by Giemsa and DAPI staining,and it was found that there were 18-19 chromosomes of S.turcica,and the karyotype pattern of S.turcica was successfully drawn.3.A genome sequence of S.turcica at chromosome level was obtained by Pac Bio Sequel,Hi-C,and Illumina sequencing technology.The assembled genome of S.turcica was 42.45 Mb in size and anchored on 19 chromosomes.The corresponding functional annotation was performed on the gene prediction set of S.turcica through eight different databases including TF,IPR,SWISS-PROT,COG,GO,KEGG,NR,and CWDE.A total of 13,366 protein-encoding genes of S.turcica were predicted,of which 12,957 were well annotated,accounting for 96.93%of the whole gene set.Among them,there were 1,237 genes on chromosome 16 with the largest number;There are only two on chromosome 19,with the least number of genes.4.The genes related to sexual reproduction were located by Tbtools,and it was confirmed that StMAT1-1 was located on chromosome 4,Settu1₁02477 and Settu1₁63080 were located on chromosome 11,Settu1₁57970 was located on chromosome 10,Settu1₁34561 was located on chromosome 9 and Settu1₂6706 was located on Chromosome 2.5.During sexual induction of different strain combinations,the expression of genes related to sexual reproduction was higher in the ascus formation stage and lower in the early stage of sexual induction.In the combination composed of the two mutants,the expression level of the sexual reproduction-related gene was significantly decreased compared with that in the wild-type combination,but the expression level of Settu1₁63080 gene in the two mutant induction combinations was increased compared with that in the wild-type combination.It indicated that the mating type gene was very important for the expression of sexually related genes,but these genes were not completely regulated by the mating type gene.