Isolation and characterization of the C-class MADS-box gene involved in the formation of double flowers in Japanese gentian

BACKGROUND: Generally, double-flowered varieties are more attractive than single-flowered varieties in ornamental plants. Japanese gentian is one of the most popular floricultural plants in Japan, and it is desirable to breed elite double-flowered cultivars. In this study, we attempted to characterize a doubled-flower mutant of Japanese gentian. To identify the gene that causes the double-flowered phenotype in Japanese gentian, we isolated and characterized MADS-box genes. RESULTS: Fourteen MADS-box genes were isolated, and two of them were C-class MADS-box genes (GsAG1 and GsAG2). Both GsAG1 and GsAG2 were categorized into the PLE/SHP subgroup, rather than the AG/FAR subgroup. In expression analyses, GsAG1 transcripts were detected in the second to fourth floral whorls, while GsAG2 transcripts were detected in only the inner two whorls. Transgenic Arabidopsis expressing GsAG1 lacked petals and formed carpeloid organs instead of sepals. Compared with a single-flowered gentian cultivar, a double-flowered gentian mutant showed decreased expression of GsAG1 but unchanged expression of GsAG2. An analysis of the genomic structure of GsAG1 revealed that the gene had nine exons and eight introns, and that a 5,150-bp additional sequence was inserted into the sixth intron of GsAG1 in the double-flowered mutant. This insert had typical features of a Ty3/gypsy-type LTR-retrotransposon, and was designated as Tgs1. Virus-induced gene silencing of GsAG1 by the Apple latent spherical virus vector resulted in the conversion of the stamen to petaloid organs in early flowering transgenic gentian plants expressing an Arabidopsis FT gene. CONCLUSIONS: These results revealed that GsAG1 plays a key role as a C-functional gene in stamen organ identity. The identification of the gene responsible for the double-flowered phenotype will be useful in further research on the floral morphogenesis of Japanese gentian. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-015-0569-3) contains supplementary material, which is available to authorized users.