Evolutionary relationships of bHLHs and validation of CmbHLHs. A, Evolutionary relationships of 149 AtbHLHs and 25 CmbHLHs. Full-length amino acid sequences of bHLHs in the chrysanthemum transcriptome dataset and arabidopsis genome were first aligned using ClustalW in MEGA5. The phylogenetic tree was constructed according to the neighbour-joining method. Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates were collapsed. The evolutionary distances were computed using the p-distance method. All ambiguous positions were removed for each sequence pair. Evolutionary analyses were conducted in MEGA5. The black circle represents 25 CmbHLHs. B, Phylogenetic tree of bHLHs and conserved motifs of chrysanthemum and other plants in subfamilies 2, 5 and 24. The black circle represents CmbHLHs. Conserved motifs are listed behind each subfamily. C, Validation of CmbHLHs in subfamilies 2, 5 and 24. The x-axis and y-axis represent the three developmental stages of the capitulum and the FPKM values, respectively. Red and black lines indicate the light and dark treatments, respectively.