Map-based cloning of ZmSS5, construction and validation of its regulatory pathway for maize kernel weight.

key messageMap-based cloning of ZmSS5. ZmSS5 pathway was constructed and validated. The 'ZmSS5 hypothesis' was proposed. Kernel weight (KW) is an important determinant of maize yield. Despite the identification of numerous quantitative trait loci (QTL) for KW, research into the cloning and molecular regulation of these loci is limited, limiting progress in genetic improvement and molecular breeding for KW. In this study, linkage analysis by chromosomal segment introgression lines, marker development, high-throughput sequencing, amplification and sequence alignment of genome and cDNA, analysis of gene expression and variable splicing, detection of sucrose content and starch synthase activity, the AtSS5 T-DNA insertion mutant and the ZmSS5 EMS mutant were used, and Zm00001d051976 was finally confirmed as the functional gene of the QTL for KW (qKW4a) encoding starch synthase 5 (ZmSS5). Based on RNA-seq from two early stages of grain development, a weighted correlation network analysis was constructed to identify and verify the ZmSS5 pathway, which regulates starch metabolism and KW formation through C/N balance. We also proposed the 'ZmSS5 hypothesis', which suggests that proliferation of starch granules and an increase in ZmSS5 levels are accompanied by cell proliferation, which affects starch unit storage and KW formation by C/N balance. This will provide new ideas for the molecular regulation of KW formation in maize, as well as gene and marker resources for KW molecular breeding.