Identification And Characterization Of An F-Box Gene Regulating Tassel Branch Number In Maize

Tassel branch number（TBN）is one of the important agronomic traits of maize,which has a direct relationship with maize grain yield.Maize is a crop with high utilization of heterosis,and the female parents and hybrids are required for suitable number of TBN,while the male parents need more TBN to ensure sufficient pollen.Therefore,it is of great significance to identify the genes that can precisely regulate TBN in maize.In this study,Q^Dtbn1,a TBN related gene encoding an F-box/kelch repeat protein,was identified by a testcrossing association population.Genetic analysis with an F₂population and an EMS mutant as well as overexpression analysis further proved that Q^Dtbn1 could negatively regulate of TBN by a dominant model.In addition,the mechanism of Q^Dtbn1 controlling TBN in maize was studied by protein interaction assay and the other experiments.The main results are as follows,1.A QTN(chr4:227,485,251 B73＿Refgen V4,named Q^Dtbn1)was identified to be significantly associated with TBN in Wuhan and Hainan by a testcrossing association population constructed previouly by our research group.The QTN is located in the 5’UTR region of Zm00001d053358.Zm00001d053358 contains one exon,and encodes a F-box/kelch repaet protein with 406 amino acids.Partial promoter region of this gene was resequenced and ten Indels/SNPs were screened out（MAF≥0.1）.Association analysis was then carried out,and the Indel4 and SNP4（the same as the QTN）were significantly associated with TBN in Wuhan and Hainan,simultaneously.In addition,the Phylogenetic tree analysis of the homologous genes of Zm00001d053358 in rice and Arabidopsis showed that Zm00001d053358 has a close evolutionary relationship and highly homologous with a gene governing Larger Panicle in rice.Therefore,Zm00001d053358is considered as the candidate gene of the QTN,and named Q^Dtbn1.2.According to the sequence difference of the Indel4 and SNP4 in the promoter region of Q^Dtbn1,five haplotypes were identified in the association mapping population.Difference of TBN among the haplotypes was significant.TBN in the inbreds of the haplotype with the largest members（Hap7）and their testcrossing hybrids was compared,and it was found that TBN in the hybrids was significantly less than that in the inbreds in Hainan.3.An F₂ population derived from a cross of maize inbreds B73 and Huangzao 4（HZ4）was used to analyze the gene action of Q^Dtbn1.The two years’experiments showed that significant difference existed between the two parents for the four traits investigated,but only TBN was significantly associated with Q^Dtbn1.Multiple comparison analysis for TBN furthure indicated that there was no difference between the homozygous and heterozygous genotypes with the B73 allele of Q^Dtbn1,but it was significantly less than that in the homozygous genotype of the HZ4 allele.These results indicate that Q^Dtbn1 was associated with TBN,and Q^Dtbn1 regulated the TBN by a dominant model.4.The spatial and temporal expression pattern of Q^Dtbn1 in the two inbreds,B73（with less TBN）and HZ4（with more TBN）was compared.Q^Dtbn1 was constitutively expressed,and the highest expression was found in the tassels at the stage of V8.Moreover,expression in B73 tassels at the stages of V8,V10 and V12 was significantly higher than that in HZ4,suggesting that Q^Dtbn1 negatively regulated TBN in maize.Promoter activity of HZ4,B73 and two B73 truncated promoters was detected.It was found that the promoter activity of B73 was significantly higher than that of HZ4,which was consistent with the fact that expression of Q^Dtbn1 in B73 was significantly higher than that in HZ4.Compared with the full-length sequence of B73 promoter,the activity of B73（△Indel4）（truncating Indel4）increased significantly,suggesting that the length of poly（d A:d T）in Indel4 is important for the expression of Q^Dtbn1.5.Branch number in the main stems of the positive antisense expression Q^Dtbn1Arabidopsis plants was significantly more than that in their negative controls.Moreover,TBN in the positive overexpression Q^Dtbn1 maize plants was significantly less than that in their negative controls in different field trails.While TBN in the homozygous or heterozygous mutants of Q^Dtbn1 was signifcantly higher than that in the wild types.The results further confirmed that Q^Dtbn1 negatively regulated TBN in maize.6.The BiFC assay was conducted to verify the interactions of Q^Dtbn1 with the proteins previously identified by yeast two hybrid experiment（Y2H）,and a Skp1-like protein was confirmed to be interacted with Q^Dtbn1.Therefore,Q^Dtbn1might negatively regulate TBN through the ubiquitin degradation system by the SCF complex.Y2H assey also showed that Q^Dtbn1 could interact with a SnRK1 protein,and which could interact with a SnRK2 protein and the Skp1-like protein.Moreover,expression of some downstream genes of SnRK2 involved in ABA pathway was significantly decreased in the Q^Dtbn1overexpression lines.These results suggest that Q^Dtbn1 may regulate TBN development in maize by the ABA pathway through SnRKs.7.An ABA treatment experiment was conducted on the Q^Dtbn1overexpressed maize lines and their negative controls in field.The results showed that there was no significant difference between the treatment of ABA and the control for TBN in the overexpression lines.Hovever,TBN was significantly reduced by ABA treatment in the negative controls.This was also suggested that Q^Dtbn1 regulated TBN by ABA.In conclusion,this study firstly identified a dominant F-box gene that negatively regulated TBN in maize.The preliminary results showed that Q^Dtbn1 was involved in the development of maize TBN by the ABA signaling pathway through SnRKs.In addition,the dominant and negative action model of Q^Dtbn1 makes it is very useful in maize heterosis utilization to solve the contradiction of different requirements of the male,female parents and their hybrids on TBN.