did not result in a reciprocal best hit to any known myb transcription factor controlling flower color. Given that changes in expression are likely causes of many of the shifts in flower color in Aquilegia, identifying the transregulators of the pathway is particularly important. Using RT-PCR and 3′ and 5′ RACE we identified 2 novel myb sequences, AqMyb12 and AqMyb17 (GenBank accession nos. FJ908090 and FJ908091) expressed in sepals of A. formosa. After alignment with the inferred amino acid sequences of other myb proteins (Nakatsuka et al., 2008a) we found that AqMyb12 and AqMyb17 clustered with other known regulators of floral anthocyanins (e.g., Antirrhinum ROSEA1, ROSEA2, and VENOSA and Petunia AN2; Fig. 2.4D) suggesting that these 2 genes are strong candidates as key regulators of the ABP in Aquilegia.
Our analysis revealed 27 candidates for enzymatic genes in the flavonoid pathway and 7 candidates for transcription factors regulating the core anthocyanin pathway. Interestingly, 4 genes of the core ABP (CHI, F3H, DFR, and ANS) appear to exist as single copies. Loss of enzymatic function of any of these genes would cause the loss of anthocyanin production. However, if these genes are truly single copy then such mutations would also eliminate the products of the side-branch pathways dependent on each enzyme and these losses would occur in all tissues (Fig. 2.3). These potentially detrimental pleiotropic effects likely favor mutations that only change the expression of these genes in floral tissues. In fact, down-regulation of DFR and ANS has been correlated with loss of anthocyanin production in most white/yellow-flowered species of Aquilegia, suggesting that mutations in a common transregulator are responsible for these color shifts (Whittall et al., 2006b). Loss of expression of these 2 enzymes would enable the continued production of most other products of the flavonoid pathway such as flavones and flavonols in floral tissue and may be particularly favored (Whittall et al., 2006b). We also found single copies of F3′H and F3′5′H and thus changes in expression patterns of these genes is most likely responsible for shifts between blue and red flowers. As described above, loss of enzymatic function in these genes in red-flowered species would make future transitions to blue flowers especially unlikely. Because red to blue transitions have apparently occurred in Aquilegia (Fig. 2.2) we would predict that F3′H and F3′5′H have likely retained enzymatic function but lost expression in red-flowered Aquilegia species.
In addition to identified candidates for nearly all major genes in the flavonoid pathway, 2 features of Aquilegia make it a particularly powerful system for identifying the molecular basis of flower color evolution. First