Natural variation in MdNAC5 contributes to fruit firmness and ripening divergence in apple

Fruit firmness is an important trait for characterizing the quality and value of apple. It is also serves as an indicator of fruit maturity, as it is a complex trait regulated by multiple genes. Re-sequencing techniques can be employed to elucidate variations in such complex fruit traits. Here, the whole genomes of 294 F1 hybrids of ‘Fuji’ and ‘Cripp's Pink’ were re-sequenced, and a high-density bin-map was constructed using 5,014 bin markers with a total map distance of 2,213.23 cM and an average map distance of 0.44 cM. Quantitative trait loci (QTLs) of traits related to fruit were mapped, and an A-T allele variant identified in the coding region of MdNAC5 was found to potentially regulate fruit firmness and ripening. The overexpression of MdNAC5A resulted in higher production of methionine and 1-aminocyclopropanecarboxylic acid compared to MdNAC5T, leading to reduced fruit firmness and accelerated ripening in apples and tomatoes. Furthermore, the activities of MdNAC5A and MdNAC5T were enhanced through their differential binding to the promoter regions of MdACS1 and MdERF3. Spatial variations in MdNAC5A and MdNAC5T caused changes in MdACS1 expression following their interaction with MdERF3. Ultimately, utilizing different MdNAC5 alleles offers a strategy to manipulate fruit firmness in apple breeding.