Traditionally, Sicilian blood oranges (Citrus sinensis) have been associated with cardiovascular health, and consumption has been shown to prevent obesity in mice fed a high-fat diet. Despite increasing consumer interest in these healthpromoting attributes, production of blood oranges remains unreliable due largely to a dependency on cold for full color formation. We show that Sicilian blood orange arose by insertion of a Copia-like retrotransposon adjacent to a gene encoding Ruby, a MYB transcriptional activator of anthocyanin production. The retrotransposon controls Ruby expression, and cold dependency reflects the induction of the retroelement by stress. A blood orange of Chinese origin results from an independent insertion of a similar retrotransposon, and color formation in its fruit is also cold dependent. Our results suggest that transposition and recombination of retroelements are likely important sources of variation in Citrus.

Mandarin (Citrus reticulata), citron (Citrus medica), and pummelo (Citrus maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of Citrus: sweet orange, sour orange, clementine, lemon, lime, and grapefruit. Citron produces anthocyanins in its young leaves and flowers, as do species in genera closely related to Citrus, but mandarins do not, and pummelo varieties that produce anthocyanins have not been reported. We investigated the activity of the Ruby gene, which encodes a MYB transcription factor controlling anthocyanin biosynthesis, in different accessions of a range of Citrus species and in domesticated cultivars. A white mutant of lemon lacks functional alleles of Ruby, demonstrating that Ruby plays an essential role in anthocyanin production in Citrus. Almost all the natural variation in pigmentation by anthocyanins in Citrus species can be explained by differences in activity of the Ruby gene, caused by point mutations and deletions and insertions of transposable elements. Comparison of the allelic constitution of Ruby in different species and cultivars also helps to clarify many of the taxonomic relationships in different species of Citrus, confirms the derivation of commercial varieties during domestication, elucidates the relationships within the subgenus Papeda, and allows a new genetic classification of mandarins.

Highlights d Noemi is essential for the production of flavonoid pigments in citrus d Noemi is essential for the regulation of fruit acidity in citrus d Retrotransposons are associated with the acidless phenotype in commercial varieties d A specific ancient mutation retraces the steps of citrus history and cultivation

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