Co-expression network analyses of anthocyanin biosynthesis genes in Ruellia (Wild Petunias; Acanthaceae)

Anthocyanins are major pigments contributing to flower coloration and as such knowledge of molecular architecture underlying the anthocyanin biosynthetic pathway (ABP) is key to understanding flower color diversification. To identify ABP structural genes and associated regulatory networks, we sequenced 16 transcriptomes generated from 10 species of Ruellia and then conducted co-expression analyses among resulting data.

Complete coding sequences for 12 candidate structural loci representing eight genes plus nine candidate regulatory loci were assembled. Analysis of non-synonymous/synonymous (dn/ds) mutation rates indicated all identified loci are under purifying selection, suggesting overall selection to prevent the accumulation of deleterious mutations. Additionally, upstream enzymes have lower rates of molecular evolution compared to downstream enzymes. However, site-specific tests of selection yielded evidence for positive selection at several sites, including four in F3'H2 and five in DFR3, and these sites are located in protein binding regions. A species-level phylogenetic tree constructed using a newly implemented hybrid transcriptome-RADseq approach implicates several flower color transitions among the 10 species. We found evidence of both regulatory and structural mutations to F3'5'H in helping to explain the evolution of red flowers from purple-flowered ancestors.

Sequence comparisons and co-expression analyses of ABP loci revealed that mutations in regulatory loci are likely to play a greater role in flower color transitions in Ruellia compared to mutations in underlying structural genes.