Extreme-phenotype GWAS unravels a complex nexus between apple (Malus domestica) red-flesh colour and internal flesh browning

The genetic link between apple red flesh (RF) coloration and the internal flesh browning disorder (FBD) is a major challenge when breeding high fruit quality RF apple cultivars. A genome-wide association study (GWAS) was conducted in a population of about 900 red-leaved seedlings to identify genomic regions and putative candidate genes using whole genome sequencing of the pools of extreme phenotypes (XP) for the RF colour coverage (using the weighted cortex index (WCI)) and FBD. This study identified novel genomic regions contributing to WCI and FBD variation in the red-leaved seedlings. The FBD-associated regions were enriched for genes regulating senescence, heat shock proteins, cytochrome P450, ascorbate metabolism and pectin methyl esterases. Although there were no significant regions in common for WCI and FBD, there were several genes (e.g. MYB85, MYB66, ethylene insensitive 3, DNAJ heat shock protein, WRKY7, and NAC42) enriched commonly between the genomic regions associated with these traits, potentially underpinning the genetic connection between WCI and FBD. Some of the differentially expressed genes between the R6:MdMYB10 and white-fleshed ‘control’ apples resided within the GWAS hotspot for WCI (e.g. chalcone synthase, UDP-Glycosyl transferase) and FBD (e.g. Rho GTPase activating protein, lipoxygenase 1, phytoene synthase) – validating the XP-GWAS findings. Paralogs of several genes resided in the trait-associated genomic regions, suggesting that whole genome duplication plays an important role in the regulation of these traits. Adverse genetic correlations between WCI and sensory traits were observed, and strategies to develop FBD-free high fruit quality RF cultivars are discussed.