Regulatory structural variations upstream of a NAC transcription factor control fruit maturity timing in peach

Fruit maturity date is a key developmental trait in fruit trees, as it determines the timing of when fruits enter the ripening phase, thereby shaping the harvest calendar and fruit quality. In peach, ripening time varies over several months, yet the genetic and regulatory basis underlying this extensive phenological diversity has remained largely unresolved. Here, we dissect the long-standing chromosome 4 maturity date locus (qMD4.1) and show that a multi-allelic series of structural variants in the upstream regulatory region of the transcription factor NAC1 explains most of the observed variation in ripening time in cultivated peach. Across multiple independent populations and diverse genetic backgrounds, these variants (collectively termed Md) stratify maturity date from ultra-early to late cultivars with largely additive and dosage-dependent effects. Using recombinant-based genetic dissection, long-read sequencing, transcriptomic analyses, and epigenomic data integration, we show that Md alleles are consistently associated with allele-dependent shifts in the temporal dynamics of NAC1 transcript accumulation, and that the affected region overlaps with chromatin features characteristic of regulatory activity. By resolving the genetic architecture of a major phenological locus, our results support a central role for cis-regulatory structural variation in modulating developmental timing in peach and provide biologically interpretable markers for predicting maturity date. More broadly, this work illustrates how non-coding structural variation can contribute to variation in fruit developmental trajectories, with implications for breeding strategies aimed at modulating harvest windows in peach and other stone fruit species.