Methods: First, studies of codon usage in monocots were reviewed. The current information was then extended regarding codon usage, as well as codon-pair context bias, using four completely sequenced non-grass monocot genomes (Musa acuminata, Musa balbisiana, Phoenix dactylifera and Spirodela polyrhiza) for which comparable transcriptome datasets are available. Measurements were taken regarding relative synonymous codon usage, effective number of codons, derived optimal codon and GC content and then the relationships investigated to infer the underlying evolutionary forces.
Key Results: The research identified optimal codons, rare codons and preferred codon-pair context in the non-grass monocot species studied. In contrast to the bimodal distribution of GC3 (GC content in third codon position) in grasses, non-grass monocots showed a unimodal distribution. Disproportionate use of G and C (and of A and T) in two- and four-codon amino acids detected in the analysis rules out the mutational bias hypothesis as an explanation of genomic variation in GC content. There was found to be a positive relationship between CAI (codon adaptation index; predicts the level of expression of a gene) and GC3. In addition, a strong correlation was observed between coding and genomic GC content and negative correlation of GC3 with gene length, indicating a strong impact of GC-biased gene conversion (gBGC) in shaping codon usage and nucleotide composition in non-grass monocots.
Conclusion: Optimal codons in these non-grass monocots show a preference for G/C in the third codon position. These results support the concept that codon usage and nucleotide composition in non-grass monocots are mainly driven by gBGC.
METHODS: Phylogenetic analysis was used to identify candidate Dendrobium catenatum R2R3-MYB (DcaMYB) sequences associated with pigment and cell shape development. Gene silencing of candidate DhMYBs in Dendrobium hybrid by direct application of dsRNA to developing flowers was followed by observation of gene expression level and flower phenotypes. Silencing of the structural gene chalcone synthase was used as a comparative control.
KEY RESULTS: Ten candidate flower-associated DcaMYBs were identified. Flowers treated with dsRNA of DhMYB22 and DhMYB60 sequences were less pigmented and had relatively low expression of anthocyanin biosynthetic genes (F3'H and DFR), lower total anthocyanin concentration and markedly lower levels of cyanidin-3-glucoside and cyanidin-3-rutinoside. Petals of DhMYB22-treated flowers and sepals of DhMYB60-treated flowers showed the greatest colour difference relative to the same organs in untreated flowers. DhMYB22-treated flowers had relatively narrow and constricted lips, while DhMYB60-treated flowers had narrow and constricted sepals. No significant difference in shape was observed for DhCHS-treated or untreated flowers.
CONCLUSIONS: Our results demonstrate that DhMYB22 and DhMYB60 regulate pigment intensity and floral organ shape in Dendrobium. This is a first report of MYB regulation of floral organ shape in orchids.