Fine-mapping Cloning Of Maize Seed Mutant 1764 And Primary Functional Characterization Of Mutant 1763

Maize is one of the most important food and feed crops in the world.The quality and yield of maize kernel directly determine the application value of maize.The research on maize kernels is helpful to provide theoretical basis for improving maize yield and quality.Maize kernel mutants provide good materials for maize kernel research.The 1764 mutant is a defective kernel(dek)mutant controlled by a single recessive nuclear gene with the shrunken,inadequately filled endosperm and ungerminable embryos.In the same developmental stages,paraffin section showed that the development of mutant embryos was seriously affected and almost undeveloped compared with the wild type.Transmission electron microscopy(TEM)analysis of immature kernels showed that the 1764 mutant endosperm cells were inadequately filled and the volume and quantity of protein body were reduced.Biochemical components analysis of mature kernels further showed that the contents of zein and non-zein proteins decreased significantly in mutant kernels,especially the 19 kDa and 27 kDa zeins.However,the total starch in mutant kernels increased slightly.In this study,the candidate gene was located between 4.705933 M and 5.212037 M of chromosome 8 by map-based cloning.Total of 14 genes were identified in this region.For the 12 genes,no sequence change was found that could cause changes in amino acid coding regions between 1764 mutant and the wild types For the other two genes,sequence differences will be further analyzed and confirmed.1763 mutant also belongs to dek mutant.The mature kernels of the mutant were shrunken and empty,but the seeds could germinate and continue to grow.In our previous study,the 1763 gene was isolated by map-based cloning combined with the fine mapping of other allelic mutations of 1763.Finally,the mutant gene was located between molecular markers 3B-1 and 3B-5,with an interval of 340 kb in physical distance from 216.132312 M to 216.472712 M on chromosome 3.In this study,dCAPS markers were used to identify the candidate genes in this region combined with transcriptome data analysis.Finally,the gene GRMZM2G429899 was considered as the candidate gene.A C/T transition at the 286 th base of the GRMZM2G429899 causes an amino acid change from Gln(CAG)to stop codon(TAG)in 1763 mutant and results in a premature stop codon in the mRNA.The candidate gene is known as Shrunken2(Sh2),which encodes the large subunit of Adenosine diphosphate glucose pyrophosphorylases(AGPase),a key rate-limiting enzyme in the pathway of starch synthesis and metabolism.Quantitative analysis showed that the content of total starch was decreased significantly in mature kernels of 1763 mutant,but the content of soluble sugar was increased in immature kernels of the mutant.