A Retrotransposon Insertion in the Promoter Causes Harlequin/Black Flowers in Orchids.

The harlequin/black flowers in orchids contain dark purple spots and various pigmentation patterns, which appeared as a new color in 1996. We analyzed this phenotype by microscopy, HPLC, gene functional characterization, genome structure analysis, and transient overexpression system to obtain a better understanding of the black color formation in orchids. Most mesophyll cells of harlequin flowers showed extremely high accumulation of anthocyanins as well as a high expression of MYB11 () as the major regulatory R2R3-MYB transcription factor for regulating the production of the black color. In addition, we analyzed the expression of basic helix-loop-helix factors, WD40 repeat proteins, and - and -like repressors for their association with the spot pattern formation. To understand the high expression of in harlequin flowers, we isolated the promoter sequences of from red and harlequin flowers. A retrotransposon, named (), was identified and inserted in the upstream regulatory region of The insertion resulted in strong expression of and thus extremely high accumulation of anthocyanins in the harlequin flowers of the Yushan Little Pearl variety. A dual luciferase assay showed that the insertion of enhanced expression by at least 2-fold compared with plants not carrying the insertion. Furthermore, the presence of explains the high mutation rates resulting in many variations of pigmentation patterning in harlequin flowers of orchids.