Genetic Relationships Among Hirado azalea Cultivars and Their Putative Parents Inferred from Flavonoid 3', 5' Hydroxylase Gene Sequences

Abstract

The putative parents of the Hirado azalea (Rhododendron × pulchrum) are R. scabrum, R. ripense, R. × mucronatum, and other related cultivars. Hirado azalea shows a wide range of flower color variation, but the genetic basis for this color variation is not well understood. In this study, we investigated the anthocyanin pathway gene, flavonoid 3', 5' hydroxylase (F3'5'H), by genomic DNA analysis, cDNA sequence analysis, and deduced amino acid sequences to assess the genetic relationships between these taxa, as well as investigating the genetic basis of color variation in this group. In R. scabrum and red and pink flowered Hirado azalea cultivars, in which delphinidin (Dp) derivatives are absent, only the exon 2 region was amplified using specific primers in a coding region (CDS) of the F3'5'H gene, except for exon 1, suggesting that the DNA structure of exon 1 is defective in these plants that lack Dp derivatives. On the other hand, R. ripense, R. macrosepalum, and R. yedoense var. poukhanense and R. × mucronatum ‘Shiro-ryūkyū’, with Dp derivatives have normal F3'5'H DNA gene structure. The lengths of the F3'5'H cDNA nucleotide sequences of these wild species were 1533 bp (510 AA), whereas in R. × mucronatum ‘Shiro-ryūkyū’, two different sequence lengths were observed—1533 and 1551 bp (510 and 516 AA). R. ripense, R. × mucronatum ‘Shiro-ryūkyū’, and four purple and white flowered Hirado azalea cultivars were grouped in the same cluster in the F3'5'H gene phylogeny. Among the four Hirado azalea cultivars, the lengths of F3'5'H in CDS were 1551 bp, which included a 5 bp insertion adjacent to the stop codon in ‘Ademurasaki’ and ‘Hakuhō’. However, ‘Hirado-no-homare’ and ‘Shirokujyaku’ lacked this insertion and had 1533 bp CDS. When PCR was performed to distinguish the 5 bp insertion, the amplified product was found in some R. ripense individuals and R. × mucronatum ‘Shiro-ryūkyū’, but not in R. scabrum or R. macrosepalum. These results suggest that the wide range of flower color in Hirado azalea cultivars is caused by variation in the F3'5'H genotype derived from hybridization between R. scabrum and either R. ripense or R. × mucronatum ‘Shiro-ryūkyū’.