Excision of Transposable Elements from the Chalcone Isomerase and Dihydroflavonol 4-Reductase Genes May Contribute to the Variegation of the Yellow-Flowered Carnation (Dianthus caryophyllus)

Abstract

In the "Rhapsody" cultivar of the carnation, which bears white flowers variegated with red flecks and sectors, a transposable element, dTdic1, belonging to the Ac/Ds superfamily, was found within the dihydroflavonol 4-reductase (DFR) gene. The red flecks and sectors of "Rhapsody" may be attributable to a reversion to DFR activity after the excision of dTdic1. The yellow color of the carnation petals is attributed to the synthesis and accumulation of chalcone 2'-glucoside. In several of the carnation cultivars that bear yellow flowers variegated with white flecks and sectors, both the chalcone isomerase (CHI) and DFR genes are disrupted by dTdic1.

Cloning and heterologous expression of cDNAs encoding flavonoid glucosyltransferases from Dianthus caryophyllus

“…The primer pairs for amplification of cDNA fragments for semi-quantitative PCR were as follows; phenylalanine ammonia-lyase (PAL) (Yoshimoto et al 2000), 5Ј-CTTCTACAAGGGTACTCCGGTATTCGT-3Ј and 5Ј-ACTGAAGCTCAGAACAGTAAGATGCC-3Ј; CHS (AF267173), 5Ј-GGAGAAATTTCGACGCATGTGCGA-T-3Ј and 5Ј-CAATCGAGCCCTTCACCTGTTGTG-3Ј; chalcone isomerase (CHI) (Itoh et al 2002), 5Ј-GGATGGAGATTGGAGGCAGATTC-3Ј and 5Ј-CATC-TTGTGGAATCGACTCGTGCT-3Ј; flavanone 3-b-hydroxylase (F3H) (X72592), 5Ј-GAACGTCCGAAAG-TGGCGTATAATG-3Ј and 5Ј-TTAGCAAGCCTCTTAT-GACGGGCGA-3Ј; dihydroflavonol 4-reductase (DFR) (Itoh et al 2002), 5Ј-CATTTGTAACAAAAAAAG-ATGGTTTC-3Ј and 5Ј-GGAAAACACAAATCTGAT-AACTAAGCAGA-3Ј; anthocyanidin synthase (ANS) (U82432), 5Ј-TGAGGATCCAGAGGTTCGAGCCA-AG-3Ј and 5Ј-CGGAAGACTTATGTTGAATATGT-3Ј; D. caryophyllus UDP-Glc: flavonoid glucosyltransferase (designated "DicGT1" as follows), 5Ј-AGCT-CCGGGGATCCACACAATGTCAGCAAATTCTAACT ACATGAACA-3Ј and 5Ј-ACTAGAGCGGCCGCAGT-TTAGTTAGAAGTGACGATCATGTCG-3Ј; DicGT2, 5Ј-A G C T C C G G G G AT C C A AT T C C TA G C C AT -TCCTCCAAAATTAC-3Ј and 5Ј-ACTAGAGCGGCCG-CATAAATGATGATGATTTATTGAAGAACATC-3Ј; DicGT3, 5Ј-AGCTCCGGGGATCCATCTTCTATTAAC-AATCAGAAACGATA-3Ј and 5Ј-ACTAGAGCGGCC-GCTACTATTATGAACTTGTGATTAGTTCA-3Ј; DicGT4, 5Ј-ATGGATATCATGAGAAAATCATACATA-TACACAAACAATA-3Ј and 5Ј-GACGGAGCTCTTTT-ATCAAACTATATTTTTGTACTAGGA-3Ј; DicGT5, 5Ј-GATCGATATCATTCATATTTCCGCCAACATCAATT GAGA-3Ј and 5Ј-ATTGCTGCAGCTATGGGTAATAC-AACACTTTGATTCTAAAG-3Ј; ubiquitin (UBQ), 5Ј-ATATTGCAGTTCTAGCATTCCCTTTCGGCGGA-3Ј and 5Ј-CGTATTTCGCTCTATCCGCCTTGTACATGT-GAG-3Ј. The LA-PCR mixture (10 ml) was prepared according to the supplier's recommendations using 1 ml of the cDNA mixture and 2 pmol of each primer.…”

“…Carnation cultivars bearing yellow flowers have CHI and DFR genes disrupted by the transposable element dTdic1s (Itoh et al 2002). The insertion event of dTdic1 likely occurred due to the breeding process within the past several centuries, because all carnation lines bearing yellow flowers have identical insertion sites of dTdic1 in the CHI and DFR genes (Itoh et al 2002).…”

“…The insertion event of dTdic1 likely occurred due to the breeding process within the past several centuries, because all carnation lines bearing yellow flowers have identical insertion sites of dTdic1 in the CHI and DFR genes (Itoh et al 2002). It is likely then that one mutant carnation harboring both disrupted CHI and DFR genes bearing yellow flowers emerged in the way of breeding has spread worldwide by breeders.…”

“…Since then, biochemical and molecular biological techniques have allowed the identification of pathway enzymes and their corresponding genetic loci (Forkmann and Dangelmayr 1980;Itoh et al 2002). Currently, most of the genes encoded the enzymes involved in flavonoid and anthocyanin synthesis have been characterized in other plant species.…”

Sequence variations in the flavonoid 3^|^#8242;,5^|^#8242;-hydroxylase gene associated with reddish flower phenotypes in three delphinium varieties

“…Although introns are important for gene expression in many eukaryotes (reviewed by Hir et al 2003), the chalcone isomerase (CHI) gene offers an example of an intron-less gene that can be expressed. In some species, the CHI gene has four exons and three introns (Itoh et al 2002;Kuittinen and Aguadé 2000;Shimada et al 2003). In P. hybrida, CHI gene B has three introns and is expressed in immature anthers, however, the CHI gene A has no introns and is expressed in the corolla (van Tunen et al 1988).…”