Molecular evolution of the chalcone synthase gene family and identification of the expressed copy in flower petal tissue of Viola cornuta

Chalcone synthase (CHS), the first committed enzyme in the flavonoid biosynthetic pathway, is commonly encoded by multi-gene families with select members of these families accounting for the majority of expression. We have examined the CHS gene family in Viola cornuta, a plant whose flowers undergo ontogenetic color change. Using both RNA and RNA/DNA samples isolated from floral tissues at different pigment stages, we obtained 14 unique sequences from 60 total clones of a 288 bp fragment from the catalytic region of CHS. The V. cornuta sequences were monophyletic when compared to CHS orthologs from other taxa. Substitution models generally indicated unequal rates of transition and transversion as well as significant rate variation among sites. With a Tamura-Nei correction, nucleotide divergence ranged from 0.3 to 10.6% with the vast majority as synonymous changes. The nucleotide divergence pattern suggests designation of three V. cornuta CHS clades; based on divergence of CHS orthologs, these clades are consistent with three CHS orthologs in V. cornuta. Sequences from only a single clade were found to be expressed in all three floral pigment stages.