Isolation and analysis of the key enzyme genes in the flavonoid biosynthesis pathway in chrysanthemum

Flower color is an important factor for customer consideration, and one of the most important pigments in flowers is anthocyanin. Therefore, this study aims to determine the anthocyanin biosynthesis-related genes in the pink chrysanthemum (Chrysanthemum morifolium) cultivar 'Pelican'. One each of CmplCHI and CmplANS, and two each of CmplCHS, CmplF3H, and CmplDFR were isolated and showed almost the same known nucleotide sequences. CmplDFR1 and 2 showed different amino acid sequence lengths at the stop codon position because of a 7 bp nucleotide sequence insertion in CmplDFR1; CmplDFR1 is 375 aa long and CmplDFR2 is 374 aa long. The amino acids of all the genes contained a specific conserved region as the known anthocyanin biosynthesis gene. CmplCHSs and CmplCHI showed a high gene expression level from stage 1 having unpigmented petals, and CmplDFRs and CmplANS showed a high gene expression level from stage 2 having pale pink petals. However, CmplDFR2 showed constant gene expression from stage 2 to 6, unlike the other isolated genes. The present results suggest that CmplCHS1 and CmplCHI function as early biosynthetic genes, and CmplDFR1 and CmplANS function as late biosynthetic genes. CmplDFR2 might not function as a key enzyme in the anthocyanin biosynthesis pathway of 'Pelican'.