Gene Mapping And Analysis Of Candidate Gene Of Maize Kernel Mutant Suk1

Maize is an important food and feed crop,and its kernel size is an important factor affecting the yield.Maize kernels are endosperm-type seeds,and starch accounts for 90%of the weight of the endosperm.The degree of starch filling directly affects the weight of the kernels.In this study,a natural mutant of maize kernel suk1（sunken kernel 1）obtained by the research group during the breeding process was subjected to phenotypic analysis,physiological and biochemical analysis,genetic analysis,tissue section observation,gene mapping,and expression analysis of mutant genes.The main results are as follows:（1）Trait analysis of mutants:The difference between suk1 and wild-type kernels began to appear at 20 d after pollination.The tops of suk1 kernels were swollen and plump,and the tops of wild-type kernels began to sag,while suk1 matured kernels showed deep sag at the top,and wild-type kernels had a shallower sag.Both the embryo and endosperm of suk1 developed normally,but compared with the wild type,the 100-kernel weight was 15.8%lower and the germination rate was 5%lower.The fresh kernel weight and volume of suk1 were significantly higher than those of the wild type at 20 d,25 d and 30 d after pollination,but significantly lower than those of the wild type at 45 d after pollination.Endosperm cells in mature kernels of suk1were smaller than those of wild type,and the surface of starch kernels was shriveled and concave.Starch formation and accumulation were hindered,and starch content was significantly reduced.（2）Mutant gene mapping and variant analysis:F₂ populations with three different genetic backgrounds of suk1 and B73,W22 and Mo17 were constructed,and the phenotype of suk1was controlled by a single recessive gene through genetic analysis.B73,suk1 and F₂ recessive pools were used for BSA sequencing analysis,and the mutant gene was initially located between 0～30 Mb of chromosome 9,and the Indel marker was developed for further fine mapping.The mutant gene was located between the markers In10.86 and In10.96 100 Kb range,including 5 candidate genes.Through candidate gene function analysis,results and analysis of RNA-seq,results and analysis of q RT-PCR and allele verification,it was proved that Zm00001d045042 was the target gene and the mutated gene was a new allele of Sh1.The mutation occurred in the deletion of 4 bases（ATTC）in exon 8 of Zm00001d045042,which resulted in the premature appearance of the stop codon and ultimately reduced sucrose synthase activity.（3）Expression levels of Mn1,Sus1,Su1,Sbe3,Bt2 and Sh2 in suk1 kernels by q RT-PCR analysis of the expression levels of Mn1,Sus1,Su1,Sbe3,Bt2 and Sh2 in wild-type and suk1kernels at different days after pollination were significantly increased.Sh1 mutation leads to the decrease of sucrose synthase activity and the increase of sucrose content,which affects starch synthesis.However,the up-regulated expression of genes related to starch synthesis compensates for some of the starch synthesis defects caused by Sh1 mutation.