Character Identification And Genetic Analysis Of A Thick Tassel Mutant In Maize Tassel

Maize is a very important food and feed crop,and its yield has an important influence on the development of national economy.As one of the main reproductive organs of maize,tassel plays an important physiological function in the whole life cycle of maize.Tassel development is an extremely complex and critical reproductive growth process derived from the cell activity of male inflorescence meristem.Previous studies on male inflorescence development mutants of various types of maize have made a deep understanding of male inflorescence development of maize.we identified a mutant ts2016 with abnormal tassel development（large,compact and dense branches with female filaments）,and its defect phenotype was different from that of most male inflorescence mutants reported.In this study,the tassel mutant was used as the material to analyze the morphological variation of tassel development,and candidate gene location and prediction analysis were conducted by cloning method,which provided certain basis for in-depth analysis of tassel development mechanism.The main results are as follows:1.The preliminary phenotypic identification results showed that the tassel spindle and branch of the mutant ts2016 were significantly shortened and thickened compared with the wild type 18-599,and the branches were compact and increased.At the same time,female filaments are produced on the spindles and at the top of the branches.Subsequently,the mutant ts2016 and wild type 18-599 were dynamically observed at different stages of tassel development.In addition,at the later stage of male inflorescence,spikelet differentiation and development were abnormal,forming densely arranged spikelet.Above results indicated that the mutant ts2016 tassel showed abnormal development in spindle,branch,sex determination and spikelet differentiation,indicating that it was related to the differentiation and development of male inflorescence meristem.In addition,compared with the wild type18-599,the female spike of mutant ts2016 also showed the phenotype variation of shortening and thickening,and the mutant ts2016 showed irregular grain arrangement,spike row disorder and partial double-grain phenomenon.At the early stage of male and female inflorescences of maize,the form of bisexual flowers had already developed.After sex determination,the female carpel in the male spike degenerated and the stamen in the female spike degenerated,and then the male spike and female spike peak development were completed respectively.Therefore,ts2016 mutation affected the development and differentiation of inflorescences（male and female inflorescences）from the early stage.2.The mutant ts2016 was hybridized with wild type 18-599 to construct F₁,F₂ and BC₁populations for genetic analysis.The results showed that the tassels of F₁ plants showed normal wild-type type,while tassels of F₂ and BC₁ were separated by variation.The chi-square test showed that the normal/mutant phenotypic separation ratios of F₂ and BC₁populations were in line with 3:1 and 1:1,respectively,indicating that the tassel-mutant phenotypic traits were consistent with Mendelian inheritance rules and controlled by a recessive single gene.3.Based on the strategy of map-based cloning,the inbred line HL9047 with different genetic backgrounds was used to construct F₂ population with the mutant ts2016 to locate the target gene.The target gene was preliminarily located on chromosome 3 of maize by using BSA method,between SSR marker umc2263 and bnlg1505（48943589-153033963）.Based on the results of initial localization,new InDel markers were designed in the localization region to expand the localization population.Finally,1293 mutant individual strains in the F₂population were used to locate the target gene between the marker xyb10-8 on chromosome 3of maize and xyb7-7（145177620-145802504）,and the physical range of this region was 625KB.9 candidate genes were found.Among them,the two genes zma-mir172e and ENSRNA049474486（precursor sequence of mir172 family）were included,while TS4 was known to encode MIR172e,which resulted in abnormal tassel development,increased branches of female tassel and male tassel,and female filament in tassel.It is worth noting that ts2016 mutant phenotype is different from ts4 in many ways,indicating that ts2016 is not an allele mutation of ts4.