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During analysis of kiwifruit derived from hybrids between the high vitamin C (ascorbic acid; AsA) species <i>Actinidia eriantha</i> and <i>A. chinensis</i>, we observed bimodal segregation of fruit AsA concentration suggesting major gene segregation. To test this hypothesis, we performed whole-genome sequencing on pools of hybrid genotypes with either high or low AsA fruit. Pool-GWAS (genome-wide association study) revealed a single Quantitative Trait Locus (QTL) spanning more than 5 Mbp on chromosome 26, which we denote as qAsA26.1. A co-dominant PCR marker was used to validate this association in four diploid (<i>A. chinensis</i> &#215; <i>A. eriantha</i>) &#215; <i>A. chinensis</i> backcross families, showing that the <i>A. eriantha</i> allele at this locus increases fruit AsA levels by 250 mg/100 g fresh weight. Inspection of genome composition and recombination in other <i>A. chinensis</i> genetic maps confirmed that the qAsA26.1 region bears hallmarks of suppressed recombination. The molecular fingerprint of this locus was examined in leaves of backcross validation families by RNA sequencing (RNASEQ). This confirmed strong allelic expression bias across this region as well as differential expression of transcripts on other chromosomes. This evidence suggests that the region harbouring qAsA26.1 constitutes a supergene, which may condition multiple pleiotropic effects on metabolism.