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 also serves as an indicator of fruit maturity, as it is a complex trait regulated by multiple genes. Resequencing techniques can be employed to elucidate variations in such complex fruit traits. Here, the whole genomes of 294 F _<b>1</b> hybrids of 'Fuji' and 'Cripp's Pink' were resequenced, and a high-density binmap was constructed using 5014 bin markers with a total map distance of 2213.23 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 <i>MdNAC5</i> was found to potentially regulate fruit firmness and ripening. The overexpression of <i>MdNAC5</i> ^{<b><i>A</i></b>} resulted in higher production of methionine and 1-aminocyclopropanecarboxylic acid compared to <i>MdNAC5</i> ^{<b><i>T</i></b>} , leading to reduced fruit firmness and accelerated ripening in apples and tomatoes. Furthermore, the activities of MdNAC5 ^<b>A</b> and MdNAC5 ^<b>T</b> were enhanced through their differential binding to the promoter regions of <i>MdACS1</i> and <i>MdERF3</i>. Spatial variations in MdNAC5 ^<b>A</b> and MdNAC5 ^<b>T</b> caused changes in <i>MdACS1</i> expression following their interaction with MdERF3. Ultimately, utilizing different <i>MdNAC5</i> alleles offers a strategy to manipulate fruit firmness in apple breeding.