Comparing Two Varieties of Blood Orange: A Differential Methylation Region Within the Specific Encoding Sequence of a Retrotransposon Adjacent to the Ruby Locus

The blood orange arose from the insertion of a retrotransposon adjacent to the Ruby gene, an MYB-type transcriptional activator of anthocyanin production, as reported previously. However, the intricate process of anthocyanin regulation among different varieties of blood orange remains incompletely understood. In this study, mRNA levels of the transcription factors Ruby and TT8 were found to be upregulated in the juice vesicle tissues of a variety with higher concentrations of anthocyanins in the pulp compared with another variety with a lower anthocyanin content. In contrast, comparative analysis of the two varieties using two-dimensional electrophoresis and mass spectrometry did not identify differentially expressed proteins related to anthocyanin biosynthesis in the juice vesicle tissues. Furthermore, higher anthocyanin contents were observed in various tissues of transgenic Arabidopsis thaliana overexpressing the Ruby gene from blood orange compared with the wildtype plant. Moreover, the long terminal repeat (LTR) region of a retrotransposon inserted upstream of the Ruby locus exhibited the ability to drive reporter expression through histochemical assay in a transgenic seedling. Thus, a PCR-based molecular marker was developed, targeting the upstream sequence of the Ruby locus to identify Citrus hybrids with the unique trait of red-fleshed fruit. Intriguingly, bisulfite sequencing revealed differentially methylated regions within a Gag-Pol polyprotein-encoding sequence of a retrotransposon adjacent to Ruby locus when comparing two varieties with different anthocyanin contents. A higher average level of methylation status was observed in the fruit with a lower anthocyanin content. In conclusion, methylation modifications at specific upstream positions on the Ruby locus may influence anthocyanin production in blood oranges.