Fine Mapping And Identification A Candidate Gene For The Major Gene Ub4 Which Related To Tassel Branch In Maize

As an important reproductive organ,the tassel plays an important role in the growth and development of maize.The size of maize tassel is closely related to the size of ear and the photosynthetic efficiency of canopy.Thus,the study of genes related to the development of tassel branches in maize can provide a theoretical basis for breeders to improve the structure of tassel inflorescence.In this study,we used the inbred line Lx1,without tassel branch,which was isolated in breeding and its sister line Lx2 and six multi-branched inbred lines with different backgrounds to construct of genetic segregation population,and preformed to genetic analysis and QTL mapping.The genetic segregation population from the Lx1/Lx2 was used to fine mapping the major gene and identified the candidate gene.Meanwhile,using transcriptome sequencing to analyze the reason of the difference of tassel branches was formed.Based on this,the following results were obtained in this study:1.Under the background of PH4CV,8112,E28,Chang7-2,Liaobai 371 and M54,the unbranched tassel phenotype of Lx1 was controlled by two nuclear genes,while under the background of Lx2,the unbranched tassel phenotype of Lx1 was controlled by one pair of genes.The genetic populations constructed by Lx1/PH4CV and Lx1/Lx2 were used for gene mapping.It was found that ub4 was located on chromosome 6 in both populations.In addition,a gene on chromosome 4,named ub5,was detected in Lx1/PH4CV populations.2.To fine mapping of ub4 and the loci was narrowed to an 88.2 kb interval on chromosome 6,which contained five genes.Quantitative real-time polymerase chain reaction(qRT-PCR)analysis showed that the unique Zm00001d038546 gene in this region exhibited significantly different expression between Lx1 and Lx2.CDS sequence analysis revealed that only Zm00001d038546 gene had sense mutations,including 12 SNPs and an 8 bp deletion.The Zm00001d038546 proteins from Lx1 to Lx2 were analyzed and the result showed that 257 amino acid residues were encoded in Lx2 and including two domains,while 105 amino acid residues were encoded in Lx1 and without the Myb_CC_LHEQLE domain.The differences of predicted protein structure and functions might stem from the 8-bp deletion in the third exon generated a premature stop codon(TGA),leading to the termination of transcription.The g DNA of Zm00001d038546 gene in Lx1,Lx2 and 15 inbred lines was amplified.The result shown that the Zm00001d038546 sequences in 15 inbred lines were all shared an 8-bp insertion with Lx2,while other difference in intron and exon showed no correlation with tassel branch phenotype.In addition,an In Del marker,In Del546,was designed based on the 8bp deletion,and the marker was co-segregated with the unbranched tassel phenotype.Therefore,Zm00001d038546 gene was identified as the most likely candidate gene for ub4.3.RNA-seq analysis was carried out on the tassel tissues of Lx1 and Lx2 during the three critical stages of tassel branching development,i.e.Stage I,Stage II and Stage III,and cluster analysis of DEGs was carried out.The results showed that the plant hormone signal transduction pathway might be involved in the process of tassel branching development.Among them,only Zm00001d038546 gene was differentially expressed.In addition,it was found that ub4 gene and AP2-like ethylene response transcription factor were enriched in the same GO Term,and there was interaction between ub4 and the transcription factor in Arabidopsis thaliana,indicating that AP2-like ethylene response transcription factors may co-regulate the tassel branching with ub4.