Identification And Gene Mapping Of The Corn Cob Fasciation Mutant

Maize is an important food,feed and industrial raw materials,which plays an important role in China’s national economy.Maize ear as a library organ,which directly determine the yield level.It will changes the fruit size,ear row number and grain number per row in the corn cob development mutation,and affects the maize yield.Therefore,exploring the key gene which determine rows per ear traits in maize ear development process,identifying and cloning mutant genes have a great significance in breeding new high yield maize.In this study,we use the Mu transposon insertion mutants which ordering from the Maizegdb stock center as materials(the genetic background is W22),through several generations self-crossing to select the genetic stable,phenotype obvious cob mutants which named ccf-1.UniformMu stock number is UFMu-07360.Through phenotype identifying,genetic analysing,Mu insertion site detecting,gene mapping,transcriptome sequencing,gene cloning technology to find and research the candidate gene.Expected to lay the foundation for the molecular mechanism of spike development.The main results are as follows:1.Morphological identification and SEM analysis showed that the mutant tissue ccf-1 ear compared with the control group of W22 showed obvious differences,such as mutant ear meristem with unnormal hyperplasia,there are a number of growth points,and the plurality of growth points are irregularly arranged to closed loops.It was significantly enlarged of the position close to the ear tip,and the number of rows per ear was disordered,so the number of spikes per panicle was increased.The phenotype was observed when the panicle length was 1-2cm.Through phenotypic identification and analysis,found that ear length,ear diameter of flattening parts,corn cob diameter of flattening parts,ear row number of flattening parts of the mutant ccf-1 were significantly increased compared with the control,indicating that the mutation gene has significant impact on the development of ear growth,the general trend is the bigger ear,ear upper fascinated and thickening,ear row number increased,hundred-kernel weight decreased.2.The Maize GDB database showed that there were 7 known insertion sites in the mutant ccf-1,The results of Mu insertion site detection showed that there were 5 Mu insertion sites in the mutant actually,Respectively they were mu1056077::Mu(exonl),mu1033815::Mu(5’UTR),mu1037462::Mu(5’UTR),mu10570 87::Mu(exon),mu1055340::Mu(5’UTR+intron),just mu1055340::Mu is homozygous insertion sites.The homozygous insertion sites was amplified by PCR detecting,but it is not associated with phenotypic variation demonstrated by the F2 segration population,then we Speculating that the mutant is not inserted by mu1055340::Mu transposon insertion,The other 4 insertion sites are located in the UTR region,which may result in a change in the amount of gene expression,but not a homozygous insertion.Phenotypic variation and insertion site association analysis also demonstrate this.3.The maize inbred line B73 and the panicle axon variant ccf-1 were used as parents to construct F2 segregating populations.The results of genetic analysis showed that the phenotypic traits of ccf-1 mutant was recessive traits controlled by single gene.The target region was positioned between the molecular marker insert101 and insertl21 on chromosome 4(4.01bin)by the F2 segregating populations.The genetic distance between two molecular markers is 0.4cM,the physical distance is about 1.5Mb.4.Ear tip RNA-sequencing data is believable,it is indicated that there had 5654 differentially expressed genes between ccf-1 mutants 2mm ear tip samples and control in the developmental process,GO analysis result indicated that GO:0008150(biological process),GO:1903293(phosphatase complex),GO:0008287(protein serine/threon ine phosphatase complex),G0:0001071(nucleic acid binding transcriptio n factor activity),GO:0003700(transcription factor activity,sequence-specific DNA binding),GO:0043565(sequence-specific DNA binding)were the most important GO Which significantly enriched.Pathways analysis results indicated that 9 Pathways’s P values were less than 0.05,These signal pathway contacted with plant pathogen interactions,galactose metabolism,plant hormone signal transduction,photosynthesis antenna proteins,secondary metabolites biosynthesis,terpenoid biosynthesis,skeleton N metabolism.5.Candidate gene analysis and cloning sequencing:We detected 4 SNP sites and 3 differentially expressed genes in the candidate region by RNA-sequencing data analysis,totally related 4candidate genes.All genes have already sequencing,respectively,there was no variation.The results indicated that the mutant was caused by other genes in the region.At the same time,it was found that there were a lot of repeated sequences and gap sequences in the mapping region,which made it difficult to select the candidate genes,to develop markers and to clone genes.