Aquilegia (Ranunculaceae), a genus of perennial herbs characterized by elegant leaves and unique flowers, exhibits promising application prospects in Northeast China. Chalcone synthase participates in the first enzymatic reaction of the anthocyanin biosynthesis pathway and plays a crucial role in flower color formation. In the present study, eight AoCHSs were identified and a comprehensive analysis of AoCHSs was then carried out, considering physical and chemical properties, conservative motifs, phylogenetic relationships, and cis-acting elements. The expression patterns of the AoCHSs were analyzed based on the transcriptome of A. oxysepala, which differs in sepal color between two species, to identify the candidate genes involved in flower color variation. AoCHS2/3/5 expression levels were found to be upregulated at PrA stage in A. oxysepala with dark purple sepals, while remaining consistently low in the pale-yellow species. Combining KEGG annotations and expression patterns, AoCHS5 was identified as a key gene for anthocyanin biosynthesis in Aquilegia that could play a role in the variation in flower color. The results of correlation network analysis showed that AoCHS5 was highly associated with MYB, bHLH, and WRKY. These results provided genetic resources for accelerating the molecular breeding of innovative Aquilegia flower colors.

Aquilegia oxysepala is a characteristic wildflower of Northeast China, which has great application potential owing to its unique floral morphology and strong adaptability. The R2R3-MYB is one of the largest transcription factor families and has important roles in anthocyanin biosynthesis. A total of 90 AoMYBs were identified from A. oxysepala and divided into 29 subfamilies via phylogenetic analysis. Using bioinformatics methods, a systematic analysis was conducted on the physicochemical properties, gene structures, conserved motifs, and other characteristics of these genes. Based on the transcriptomic data of A. oxysepala and A. oxysepala f. pallidiflora across different floral developmental stages, 32 differentially expressed genes (DEGs) belonging to the R2R3-MYB family were screened, and their expression patterns were analyzed. The results indicated that AoMYB32 and AoMYB34 were significantly upregulated only at the pre-anthesis stage (PrA) in A. oxysepala, whereas their expression levels remained relatively low in A. oxysepala f. pallidiflora during the same stage. Correlation network analysis indicated that AoMYB34 was positively correlated with six functional genes associated with anthocyanin synthesis, namely PAL-2, C4H, 4CL, CHS, F3′5′H, and ANS. Reverse transcription quantitative PCR (RT-qPCR) validation revealed that AoMYB32 and AoMYB34 exhibited significant upregulation at the PrA stage in A. oxysepala, while their expression showed no obvious fluctuations across the entire flowering period in A. oxysepala f. pallidiflora. In summary, it is postulated that AoMYB32 and AoMYB34 are candidate genes associated with flower color variation formation of A. oxysepala. This study provides a theoretical basis and genetic resources for deciphering the mechanism of flower color formation and facilitating flower color improvement breeding in A. oxysepala.