Analysis of flower color diversity revealed the co-regulation of cyanidin and peonidin in the red petals coloration of Rosa rugosa

Rosa rugosa is limited in landscaping applications due to its monotonous color, especially the lack of red-flowered varieties. Comprehensive assessment of petal color diversity in R. rugosa could promote to explore the mechanism of flower color formation. In this study, the variation and diversity of petal coloring of 193 R. rugosa germplasms were assessed by chromatic values (L∗, a∗, and b∗), and then divided into seven clusters belonging to three groups with pinkish-purple (185 individuals), white (6), and red (2) petals, respectively. Total anthocyanin content was the most important factor affecting flower color diversity and red hue formation of R. rugosa petals. There were significant correlations between petal color chromatic indexes and the sum content and the ratio of two major anthocyanin, namely cyanidin 3,5-O-diglucoside (Cy3G5G), peonidin 3,5-O-diglucoside (Pn3G5G). Both high levels of Cy3G5G + Pn3G5G and Cy3G5G/Pn3G5G were necessary conditions for red phenotype formation. Five cyanidin up-stream structural genes (RrF3'H1, RrDFR1, RrANS1, RrUF3GT1, RrUF35GT1) and one cyanidin down-stream structural gene (RrCCoAOMT1) were the key indicators which contributed to Cy3G5G + Pn3G5G and Cy3G5G/Pn3G5G, respectively. Functional verification showed that overexpression of RrDFR1, combined with silent expression of RrCCoAOMT1, could make R. rugosa petals redder by increasing the levels of Cy3G5G + Pn3G5G and Cy3G5G/Pn3G5G. These results provided a robust theoretical basis for further revealing the molecular mechanism of red petals coloration in R. rugosa.