Apple sweetness is primarily attributed to the high content and perceived sweet taste of fructose. A previous study used an F1 hybrid population of Malus × domestica [‘Honeycrisp’ (HC) × ‘Qinguan’ (QG) (2n = 34)] to identify quantitative trait loci (QTLs) for fructose content in fruit, revealing a stable QTL on linkage group (LG) 03 in the HC genetic map. In this study, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses of genes within this interval in combination with RNA-sequencing identified a cell wall invertase gene MdCWINV1, whose expression was highly associated with the dynamic changes in fructose content in parental fruits. The coding sequences were conserved between the two cultivars, while the promoters carried 73 single nucleotide polymorphisms (SNPs). Based on transcriptional regulatory element prediction, a unique SNP, CWINV1pro-1080 (A/C), located at −1080 bp upstream of the ATG start codon in the HC-P1 haplotype, was identified and predicted to affect the binding of the transcription factor MdWRKY20. β-glucuronidase (GUS) assays, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR), dual-luciferase assays, and genetic transformation confirmed that MdWRKY20 specifically binds to the CWINV1pro-1080 (A) haplotype and significantly suppresses MdCWINV1 expression, reduces CWINV activity, and consequently decreases fructose accumulation. This study elucidated the functional role of MdCWINV1 as a key gene regulating fructose content and clarified how natural mutations in its promoter influence gene expression and sugar composition.

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