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Pink-flowered gentian plants (Gentiana scabra) have been bred from spontaneous mutations of blue-flowered gentian plants, but the formation mechanism(s) is unknown so far. To investigate the process, two independent pink-flowered gentian plant lines were analyzed by a molecular biological approach. HPLC analysis showed that petals of the blue-flowered cultivar contained a small amount of cyanidin derivatives and major delphinidin derivatives, whereas pink petals had only a small amount of cyanidin derivatives. To find the causal factor(s) of this change, we focused on flavonoid 3′,5′-hydroxylase (F3′,5′H), which is a key enzyme for delphinidin biosynthesis in the flavonoid biosynthetic pathway. Molecular analyses confirmed that the loss of delphinidin synthesis could be attributed to the insertions of different transposable elements in the F3′,5′H gene in each independent pink-flowered gentian plant. Sequence analysis showed that these transposable elements were classified into an hAT superfamily and terminal-repeat retrotransposon in miniature (TRIM), by which normal F3′,5′H transcripts were interrupted. Southern blot analysis indicated that they belong to high copy number elements and are also found in a related gentian species (G. triflora). These results suggest that the transposable elements inserted in F3′,5′H are the source of the mutations and may also play a substantial role in the genomic evolution of the genus Gentiana. [ABSTRACT FROM AUTHOR]