Genetic Analysis And Gene Mapping Of A Maize Grain Mutant Smk-313

As a kind of main feed,grain and industrial raw material,it is critical to the daily life of the people and to the strategic development of the country.The size of the maize grains is one of the determinants of its yield;the study of genes regulating the development of the maize grains can not only help us to understand the molecular mechanism regulating grain development,It can also provide germplasm resources for maize breeding with high yield and high quality.In this study,one of the small grain mutants smk313(small kernel-313)was selected from the mutant bank constructed by maize pollen and induced by EMS.Then,after the genetic analysis of the mutant,RNA-seq sequencing and the Map-based cloning method were used to localize the candidate gene that led to the small grain mutation phenotype.The results of the study are as follows:1.The grain size of small grain mutant smk-313 was significantly smaller than that of wild type,The differences of grain length,grain width,grain thickness and 100-grain weight were significant.Ask for the grain of 20 DAP,the observation of the stained paraffin section of its powdery endosperm showed that the mutant smk-313 was significantly smaller than wild-type RP125 cells.After observing the smk-313 and wild-type RP125 seeds of 20 DAP,The protein bodies were not observed in the powdery endosperm cells of smk-313,then in smk-313 and wild-type RP125 matured grain silt endosperm cells.Field genetic experiments showed that the small grain mutation phenotype of smk-313 was a single gene controlled recessive variation.2.F2 Generation of B73 and smk-313 Hybridization for Map-based cloning,Finally,the target gene was located in maize chromosome seventh Bin7.02 by BSA mixing pool method,and the physical distance was 640 Kb.There were 13 genes in this region,In combination with RP125 and smk-313 the sequencing results of RNA-seq in this fine location region found a SNP that led to the early termination of mRNA coding amino acids.It was proved that the SNP existed in the small grains separated from the F2 generation of maize inbred lines and mutants in different backgrounds.3.Biological information analysis results show the candidate gene of the small grain mutant was identified as Zm00001d018977.There were 56 transcripts of this gene,the largest one contained 15 exons,encoding 1589 amino acids,with a total length of 14577 bp.The conserved domain of BAH and the activity site of cytosine C-5 methyltransferase exist in the protein structure.Functional prediction showed that the protein encoded by the gene was a S-adenosine-L-methionine dependent cytosine C-5 methyltransferase.The phylogenetic analysis showed that the gene was highly conserved among different species and had higher homology in Chinese sorghum and Oryza sativa than in Arabidopsis thaliana.The expression analysis of candidate gene and its homologous gene ZmMET8 in different tissues revealed that they were highly expressed in young ear and tassel,and were expressed in leaf,root,stem and 9 DAP and 12 DAP kernels.