Codon usage bias predicts the functional MYB10 gene in Populus

Analysis of codon usage bias (CUB) in different species can reveal the patterns of genetic information transfer across those species. To better understand the characteristics of MYB10-a key regulator of anthocyanin biosynthesis-and identify the true (functional) MYB10 gene among the two candidates in Populus, we analysed the coding sequences of MYB10 genes in 10 different species using Codon W, CHIPS, CUSP, and CAI. Majority of the optimal amino acid codons of MYB10 genes ended with A/U, and GGA, UCA, GCA, AGA, and CCA were over-represented in all plant species studied. Among the two most promising MYB10 gene candidates in Populus, Potri.17G125700 shared a higher similarity of codon usage with MYB10 genes from other plant species, suggesting that it encodes the functional MYB10 in Populus. We verified this speculation by cloning both candidate MYB10 genes from Populus into vectors to produce transiently transformed seedlings. Colour phenotypes and anthocyanin content of the transiently transformed seedlings indicated that Potri.17G125700 encodes the true MYB10 transcription factor, which positively regulates anthocyanin accumulation in Populus. Furthermore, CUB analysis was used to select the most promising MYB12 candidate in Malus sp. (crabapple). Our results demonstrate the effectiveness of CUB analysis as a promising method to identify the functional gene from a set of candidates in long-living plants with complex genetics.