Codon Usage Bias Analysis of Chloroplast Genomes in Six Cucurbitaceae Species

Background and Objectives: Codon usage bias (CUB), the non-uniform usage of synonymous codons, is prevalent in plant chloroplast genomes and plays important roles in gene expression and genome evolution. However, systematic comparisons of CUB patterns across genera in the Cucurbitaceae family remain limited. This study aimed to characterize CUB patterns and identify their driving forces in the chloroplast genomes of six Cucurbitaceae species. Methods: We analyzed the complete chloroplast genomes of six Cucurbitaceae species, watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (Cucumis sativus), pumpkin (Cucurbita moschata), wax gourd (Benincasa hispida), and bitter gourd (Momordica charantia). CUB patterns were assessed using ENC-plot, neutrality plot, and PR2-plot analyses, all implemented through a custom, reproducible Python-based workflow. Results: The overall codon usage bias was weak across all six species, with a clear preference for A/U-ending codons. Candidate preferred codons (hereafter referred to as optimal codons for brevity) identified independently within each species were largely shared across the six species, although species-specific codons were also detected. Combined ENC-plot, neutrality plot, and PR2-plot analyses suggest that natural selection, rather than mutation pressure, is the dominant force shaping CUB in these genomes, although these approaches provide indirect evidence. Conclusions: These findings provide a reference for codon optimization of exogenous genes in chloroplast genetic engineering of Cucurbitaceae crops. The Python-based workflow developed in this study also offers a transparent, reproducible alternative to conventional CUB analysis approaches.