Omics-assisted genetic dissection and coordinated improvement of apple flesh color, flavor, and fruit size

Breeding red-fleshed apples with enhanced health benefits is a primary objective of modern apple improvement, yet high acidity and small fruit size limit their commercial potential. Resolving trade-offs among flesh color, flavor, and appearance and dissecting their underlying genetic relationships remain major challenges. In this study, using the red-fleshed 'CSR6R6' (Malus sieversii) and the cultivated 'Royal Gala' (M. domestica) as parents, we developed an F₁ population comprising 140 individuals and performed integrative large-scale multi-omics analyses, identifying 13,331,096 SNPs and 2,134 metabolites. Red flesh correlated positively with malic acid but negatively with fruit weight. A major QTL linked to red flesh was mapped, and six genes regulating anthocyanin and proanthocyanidin biosynthesis were functionally validated, including MdUGT89A2.1, MdUGT89A2.2, MdALMT4, MdALMT4-like, MdTT1, and MdLAR1. Notably, MdALMT4 and MdALMT4-like encode malate transporters that coordinate malate accumulation and anthocyanin biosynthesis, suggesting a genetic link between acidity and pigmentation. Integrative multi-omics analysis revealed a large-scale regulatory network, revealing gene-metabolite-phenotype interactions and accurately predicting pathways involving the anthocyanin regulator MdTT1 and the fruit shape gene MdMADS13. Leveraging this network, we identified opposing effects of red-flesh QTL haplotypes across traits, defining the genetic basis of phenotypic antagonism, and resolved 11 positive and 13 negative haplotypes affecting red flesh, malic acid, and fruit weight. Pseudo-backcrossing further demonstrated that aggregating positive haplotypes substantially mitigates trade-offs among flesh color, flavor, and appearance, enabling their concurrent improvement. These findings will advance omics-assisted apple breeding, offering potential strategies for genetic improvement of other perennial fruit trees.