Fine Mapping And Candidate Gene Cloning Of The Main QTL QED3 Of Ear Diameter In Maize

The ear diameter directly affects the kernel rows number in maize,is an important yield trait and the main screen target in breeding practice.In previous a few of genes controling maize ear differentiation and development and yield-related traits were cloned,but most of them were based on kinds of mutants.In this study,Segment substitution lines?CSSLs?of Chang7-2 chromosome with genetic background of lx9801 as base material,a major effect QTL which is related to ear diameter by multi-year analysis as target QTL,fine mapping and clonning of the ear diameter major QTL were carried out by constructing separation group,design ing molecular marker and,selecting recombined plants and bioinformatics analysis.Finally a candidate gene was identified by sequence analyzing,expression analysis and function forecast.The main results were as follows:Compared with background material?lx9801?,the ear phenotype of lx9801^C7-2 is thick and flat,kernel row number is increased,disorderly in the middle of the ear,diameter of ear increased significantly and ear deformity is flat.The difference in grain length and grain width is not significant.The phenotypic difference between the two materials was found to begin at the time of spikelet meristem?SM?by using scanning electron microscopy to observe the different developmental stages of undifferentiated ears.The top of the ear gradually enlarged and the appearance of depression phenotype.By crossing with lx9801 and lx9801^C7-2 and then backcrossing to construct BC1 population,we used 300 plants derived from separation groups to preliminary mapping of the QTL.We found that the QTL is located between mmc0071 and umc1844,and the physical distance between two markers is 4.42 Mb.Based that we designed 569 pairs of SSR and indels markers and construct 17,000 BC1 segregating population to fine mapping,and finally the QTL was mapped between M84 and L944 with the 140 kb physical distance between the two markers.Two functional genes ZM00001d043128 and ZM00001d043729 were forecasted in the prediction interval.The target QTL was finally located in the 89.9 kb region and contained only one functional gene ZM00001d043728 by designing 46 pair SSR marker in 140 k region and screening the recombination plants.Sequencing results revealed that the candidate gene was 4415 bp in length,including 3 exons and 2 introns.The candidate gene has difference sites in two materials during DNA sequence analysis.We used 150 lines toassociation analysis the differences sites between the two parental materials,and found that the 6 bp deletion of the candidate gene in lx9801^C7-2 is a specific site.In the association group the locus is the same as the lx9801 and the other two different sites in the exon region are non-specific sites.The amino acid sequence of the candidate gene was missing 2 amino acids and 3 amino acid mutations due to the 6 bp deletion in DNA sequence in lx9801^C7-2.These mutations cause changes in the tertiary structure of their proteins and may affect normal biological activity.Using Real-time Quantitative PCR to analysis the expression of ear diameter candidate gene in lx9801 and lx9801^C7-2.The differences in expression levels of different ear maize differentiation stages showed that the expression level of lx9801^C7-2 was significantly higher than that of lx9801 in SM stage,and no significant difference in expression between IM,SPM and FM.