Natural variation in MdMYB31 underlies volatile ester accumulation and fruit aroma evolution during apple domestication

Volatile organic compounds (VOCs) produced by fruits are the defining elements of their distinctive aromas, and understanding the genetic basis for natural variation in fruit VOC profiles can help breeders to develop high-quality fruit varieties. Here, we identify a natural variation responsible for the diversity of VOC profiles among apple species. By quantifying fruit VOCs in 110 Malus accessions from 19 species, we demonstrated that fruit odor became more pronounced during domestication and that VOC esters (VOC-ESTs) impart a characteristic fruity odor that distinguishes the ripe fruit of cultivated apple (Malus × domestica Borkh.) from those of wild apples. Quantitative trait locus (QTL) mapping using a segregating population derived from a cross between 'Honeycrisp' (low VOC-ESTs) and 'Qinguan' (high VOC-ESTs) revealed a stable QTL (EST-LG06) on linkage group 06 that is associated with VOC-ESTs. This locus contains a MYB transcription factor gene (MdMYB31) that is tightly associated with VOC-EST accumulation and whose encoded protein upregulates the key alcohol acyl transferase genes MdAAT1 and MdAAT2. Further analyses showed that natural variation in the coding region of MdMYB31 affects its transcriptional regulatory activity, giving rise to distinct VOC-EST profiles in different apple germplasms. Importantly, the MdMYB31 genotype with stronger transcriptional regulatory activity appears to have been selected during apple domestication, thereby enhancing fruit odor. Collectively, these findings constitute a major advance in determining the genetic basis of variation in apple VOC-EST levels and set the stage for the development of aromatic apple cultivars through molecular breeding.