Fine Mapping And Candidate Gene Analysis Of TASSELSEED10 A Sex Determining Gene In Maize

Maize is the largest food crop in China and an important source of food,feed and industrial raw materials.Increasing maize production is a key factor in resolving the current tension between food supply and demand.Maize is a dioecious and dioecious plant,and its inflorescence development has an important impact on maize yield.The study of molecular biology of maize inflorescence development,especially the molecular mechanism of sex determination,is of great significance to improve the inflorescence structure of maize and increase maize yield.In this study,A stable sex-determined mutant tasselseed10（ts10）was obtained from maize B73 inbred line pollen treated with ethyl methyl sulfonate.In this study,the phenotype of ts10 was observed,and the trait inheritance mode was analyzed;Using the method of map cloning,ts10 was finely located,candidate genes were identified,and its expression patterns were analyzed,and the main research contents were as follows:1.Careful observation of mutants and wild types in F₂isolates in the field showed that there was no significant difference in the development of ts10 and wild type plants during the vegetative growth period.After male pumping,some spikelets at the top of the ts10 male spikelet can be normally loose powder,but the middle and lower parts and lateral branches of the main spikelet of the male spike are feminized,producing a large number of filaments,and the upper and lower flowers of the spikelets can bear fruit normally after pollination;Under scanning electron microscopy,it was found that when the length of the unmature male spike of ts10 florets was about 12 mm,the upper flowers produced a cardimentized structure with the characteristic pistils of the small flowers except for the three stamens primordiums.2.The F₂isolate population was constructed by hybridizing with the ts10 mutant and Mo17,and the phenotypes of 168 single plants in the F₂isolated population were counted,and the results showed that there were 38 ts10 mutant plants and 130 normal plants,and the fitness test yieldedχ²=0.51<χ²_（0.05,1）=3.84,the difference was not significant,indicating that the isolation of ts10 conformed to a 3:1 separation ratio,so ts10 is a recessive mutant controlled by a single gene.The constructed wild-type DNA pool and mutant DNA pool were amplified with 432 SSR markers covering the entire maize genome and polymorphism between B73 and Mo17,and the molecular marker umc1972 linked to ts10 was identified.Subsequently,610 ts10 mutant plants in the F₂isolated population were genotypically identified,and recombinant single plants were screened to locate the target gene within 600 kb of chromosome one.Through analysis,it can be seen that there are a total of seven annotated genes in the localization interval,including transcription factors ZmNAC43,ZmVQ5,ZmMYC7 and ZmPLATZ3,and each gene sequence is amplified with gene-specific primers,and compared with the gene sequences in the B73 inbred line,and no differences in other gene sequences except ZmMYC7 are found.The ZmMYC7 gene consists of only one exon,CDS is 2118 bp in length,and the total length of the coding protein is composed of 705 amino acids.In the ts10 mutant,the 1594th base guanine G in the CDS sequence of this gene is replaced with adenine A,resulting in the replacement of the 532nd amino acid glutamate that codes for the protein with lysine,a mutation site in the conserved domain of the ZmMYC7 transcription factor helix-loop-helix（HLH）.Through pan-genomic analysis,it was found that this site was G base in 29 other inbred lines such as B73,and it was speculated that the mutation of this site may lead to the generation of the ts10 mutation phenotype.3.ZmMYC7 gene-specific primers were designed to amplify the full length of CDS,and the overexpression vector ubiquitin::ZmMYC7-Myc with the unterminated end of the maize ubiquitin promoter was constructed,and the subcellular localization vector35S::ZmMYC7-GFP was constructed.Genetic transformation of young embryos by maize yielded 39 transgenic positive plants identified by PCR,and genetic complementarity experiments are currently being carried out.Subcellular localization of35S::ZmMYC7-GFP transfected maize leaf protoplasts showed that nuclear Marker overlapped with ZmMYC7-GFP fluorescence signals and ZmMYC7 gene localized in the nucleus.4.Transcriptome results showed that a total of 1073 differentially expressed genes were obtained between the two samples of ts10 and wild-type,including 785 down-regulated genes and 258 up-regulated genes.DEGs are significantly enriched in metabolic pathways closely related to sex determination,such as plant hormone signaling and sequence-specific DNA-binding domains.Sex determination related genes in DEGs were analyzed and it was found that genes encoding sex determination such as cytochrome P450 enzyme,pollen protein and MADS-BOX family were downregulated by 1.57～7.17 times.The genes encoding cytochrome P450 enzyme,pollen protein,MYB,MADS-BOX and MYC family related genes were upregulated by 0.27～3.05 times.