Development of DNA markers that discriminate between white- and blue-flowers in Japanese gentian plants

Abstract

We developed molecular markers for discrimination of white and blue flower color in Japanese gentian plants. White-flowered gentians can be classified into two types, based on genetic and physiological features. One type includes four allelic variations (gtmyb3-1, gtmyb3-2, gtmyb3-3, and gtmyb3-4) of an anthocyanin biosynthetic regulator gene (GtMYB3), distinguished by three PCR-based molecular markers. The other type contains a newly identified inactive allele (ans1) of the anthocyanidin synthase (ANS) gene with a premature stop codon generated from a 4-bp deletion in the second exon. The ans1 allele was distinguished from the active ANS allele by a cleaved amplified polymorphism sequence (CAPS) marker. The genotypes of 12 white-flowered gentian cultivars/lines could be identified and classified as either ans1 or gtmyb3 using these four molecular markers. No white-flowered gentians contained ans1 and gtmyb3 alleles simultaneously. The mutated ANS gene co-segregated with white flower color in an F 2 population, demonstrating that the CAPS marker is useful to discriminate between white and blue flowers in gentian. Markers to discriminate flower color in Japanese gentian will be useful for early selection of progeny and for breeding management.

Construction of the first genetic linkage map of Japanese gentian (Gentianaceae)

“…Comparative sequencing analysis identified significant in/del polymorphisms with the intron regions of CHS and FT1, and marker primer sets were designed in the proximity of the in/dels of each gene (Table 6). For chalcone isomerase ( CHI ), flavanone 3-hydroxylase ( FHT ), flavonoid 3 ′ ,5 ′ -hydroxylase ( F3 ′ 5 ′ H ), anthocyanidin synthase ( ANS ), MYB3 , basic helix loop helix 1 ( GtbHLH1 ), terminal flowering 1 ( TFL1 ) and W14/15 genes, we used the primer sets reported in previous studies [11,12,14,16,17]. No in/dels were detected in the genomic sequences of FT2 and TFL1 genes, but some SNPs were detected using restriction enzymes.…”

“…Our previous research revealed that the intron lengths of several flavonoid biosynthetic genes, including CHS , CHI , FHT , F3 ′ 5 ′ H , ANS, and the transcriptional factors GtMYB3 and GtbHLH1 exhibited significant polymorphisms among Japanese gentian cultivars [10,16,17]. Primers for GtFT1 , GtFT2, and GtTFL1 , which are regulator genes of flowering time [12], were also designed from the difference in the genomic sequences between Aki6PS and SP6A1.…”

“…Based on these molecular studies, several genetic markers have been developed. For example, we developed genetic markers that discriminate among blue, pink, and white flower color [15,16]. To protect breeders’ rights, markers based on fragment length polymorphisms [sequence characterized amplified region (SCAR) and SSR markers] were developed to identify Japanese gentian cultivars [17,18].…”

The Gentianaceae - Volume 2: Biotechnology and Applications

“…Genotyping using genetic markers co-segregated with blue-, white-or pink-flower colors in an F 2 population bred from flower color mutant lines Nakatsuka et al 2012). Detailed analysis showed that Japanese gentians contained two F3′5′H loci, designated as F3′5′H1 and F3′5′H2.…”

Transcriptional regulators of flavonoid biosynthesis and their application to flower color modification in Japanese gentians

“…In addition, the white-flowered cultivar 'Polarno-White' has been characterized as a GtMYB3-deficient mutant derived from the insertion of a transposable element into the coding region of the GtMYB3 gene. Furthermore, this gene undoubtedly contributes to anthocyanin pigmentation in gentian flowers, as evidenced by the fact that other whiteflowered gentian cultivars/lines also contain mutations, such as In/Dels, in the GtMYB3 gene (Nakatsuka et al 2011a) and that suppression of the GtMYB3 gene by a chimeric repressor can reduce flower pigmentation (see below). Yeast two-hybrid analysis showed that GtMYB3 interacted strongly with both GtbHLH1 and GtWDR1 (Nakatsuka et al 2008, unpublished data).…”