Comprehensive analysis of chloroplast codon usage patterns in the important medicinal genus Panax

The genus Panax comprises highly valued medicinal plants, yet codon usage patterns in its chloroplast genomes remain insufficiently characterized at the genus level. Here, we systematically investigated chloroplast codon usage bias (CUB) across nine Panax species and evaluated the potential factors shaping these patterns.

Filtered coding sequences from the complete chloroplast genomes of nine Panax species were analyzed for nucleotide composition, relative synonymous codon usage, relative synonymous codon frequency, high-frequency codons, and candidate optimal codons. ENC-plot, PR2-plot, neutrality-plot, and correspondence analyses were performed to assess the contributions of compositional constraints, mutational bias, and possible selective effects. Codon-frequency patterns were also compared with those of four model organisms. Chloroplast genes exhibited moderate and highly conserved CUB across the genus, with a pronounced preference for A/T-ending codons. GC content at the third codon position ranged from 30.15% to 30.52%. Eleven candidate optimal codons were shared by all nine species, and all terminated in A or T. The combined ENC, PR2, neutrality, and correspondence analyses suggested that Panax chloroplast CUB was shaped by multiple factors rather than by GC-related mutational pressure alone. Nicotiana tabacum showed the greatest codon-usage similarity among the tested organisms.

The highly conserved A/T-ending codon preference provides a comparative reference for chloroplast genome evolution and codon optimization in Panax. However, the analytical approaches used here are indirect, and the candidate optimal codons should not be regarded as experimentally validated indicators of translational efficiency. Codon-usage similarity alone is also insufficient to predict heterologous expression efficiency. Nevertheless, these findings provide a useful resource for future chloroplast genetic-engineering studies.