Development of molecular markers for genotyping of Ruby, a locus controlling anthocyanin pigment production in Citrus with its functional analysis

Abstract

Anthocyanins, one of the major plant pigments, are responsible for the coloration of various plant tissues and organs, including fruits. They are known to have significant health benefits. Anthocyanin biosynthesis is influenced by environmental factors such as cold temperature and is transcriptionally regulated by MYB/bHLH/WD40 complexes composed of R2R3-MYB, basic helix-loop-helix (bHLH), and WD40-type transcription factors (TFs). The Ruby locus, which encodes a MYB TF, controls anthocyanin production in the fruit of blood orange varieties in a dominant manner. For the marker-assisted selection of citrus breeding lines with high anthocyanin production, we established PCR-based molecular markers for robust genotyping of Ruby locus alleles. Two dominant Ruby alleles, RD-1 and RD-2, were specifically detected in the blood orange varieties with high anthocyanin contents in their fruit flesh, but not in the varieties rich in lycopene. For functional analysis of Ruby alleles, we generated transgenic Arabidopsis expressing a functional wild-type R, a non-functional r-1, or a dominant RD-2 allele. Expression levels of flavonoid/anthocyanin biosynthetic genes and anthocyanin production were highly increased in the transgenic plants expressing the dominant Ruby allele by its retrotransposon-mediated native promoter under cold treatment, suggesting that the retrotransposon may contain cis-regulatory elements that respond to cold temperature. Our results also support that the MYB TF encoded by Ruby could be a key component of regulatory complexes that control flavonoid/anthocyanin biosynthesis in citrus.