Functional Analysis Of Maize Kernel Mutant 5601Q And Fine Mapping Of Maize Kernel Mutant 1748 And Et2

Maize is the highest yield crop in the world,and the kernel is the main place for the synthesis and storage of maize nutrients.The yield and quality of maize directly determine the economic value of maize.It is of great theoretical significance to improve maize yield and improve maize quality by studying maize kernel mutants and deeply analyzing their regulatory mechanism and function.1.Functional analysis of maize kernel mutant 5601Q Maize kernel mutant 5601Q was obtained from the Maize Genetics Cooperation Stock Center.This material was a shrunken kernel mutant which was generated by a random transposon insertion.After several successive generations of self-crossing,the kernel shrinkage of the mutant could be inherited stably and separation ratio of WT and mutant kernel followed the Mendelian heredity law,indicating this phenotype was controlled by recessive single gene.The F₂ segregating population was constructed by crossing5601Q into B73 inbred line,the target gene was located in the 2.39 Mb region of 60.19 Mb ～ 62.58 Mb on chromosome 4 of maize by map-based cloning using 868 seeds of F₂ population.Through the analysis of the gene annotation information in the region,it was found that there was a Brittle endosperm2（Bt2）gene that has been reported to be involved in kernel development,which encodes the small subunit of adenosine diphosphate glucose pyrophosphorylase（AGPase）,a rate-limiting enzyme in the endosperm starch synthesis pathway.Storage material analysis of mature kernel showed that hundred-kernel weight and starch content of mutant kernel were significantly reduced,while the soluble sugar content was significantly increased.Using a confirmed Bt2 gene mutant 1774 and 5601Q heterozygous mutants to perform an allelic test,it was found that the segregation ratio of normal to shrunken kernel in the hybrid offspring was in line with 3:1.By sequencing and analyzing the genome sequence within the region,it was found that the Bt2 gene in the 5601Q mutant harbored a Mutator 19 transposon on the second exon（1022 bp downstream of ATG）.The above experimental evidences showed that 5601Q is a new allelic mutant of Bt2 gene.1.Fine mapping of kernel mutants 1748 and et2 The kernel mutants 1748 and et2 were obtained from the Maize Genetics Cooperation Stock Center.Both of these materials were shrunken kernel mutants which were generated by a random transposon insertion.After continuous self-crossing for multiple generations,the kernel shrinkage of these two mutants could be inherited stably and separation ratio of WT and mutant kernel followed the Mendelian heredity law,indicating this phenotype was controlled by recessive single gene.The F₂ segregating population was constructed by crossing 1748 and et2 into Z58 inbred line respectively.The target gene of 1748 was located in the 1.90 Mb region of 219.10 Mb ～ 221.00 Mb on chromosome 3 of maize by map-based cloning.By analyzing the gene annotation information in the region,we found a gene named Shrunken2（Sh2）and the kernel phenotype of Sh2 mutant was similar to the mutant 1748.The Shrunken2（Sh2）gene has been reported to encode the large subunit of adenosine diphosphate glucose pyrophosphorylase（AGPase）.The results of real-time quantitative PCR showed that the expression level of Sh2 gene in the kernels of the 1748 mutant was significantly lower than that of the wild type.It is speculated that 1748 may be a new mutant of the Sh2 gene.In addition,using 730 kernels（230 mutant and 500 wild-type）of the seed mutant et2 F₂ population,the target gene was mapped to the maize chromosome 2 between molecular marker xnk109（15.67 Mb）and Y15（15.97 Mb）about 300 kb by map-based cloning,of which there were 2 exchange events at the left marker xnk109 and 1exchange event at the right marker Y15.These results will lay the foundation for cloning et2 gene and analyzing its function.