Objective To clarify the codon usage bias in the chloroplast genome of the medicinal plant Chlorophytum laxum, analyze the impacts of natural selection and mutation pressure on the formation of codon bias, and screen the optimal codons, so as to provide a theoretical basis for subsequent chloroplast genetic engineering and phylogenetic studies.

Method The complete chloroplast genome sequence of C. laxum (OR078538) was obtained from the NCBI database. After strict filtering to exclude short sequences (< 300 bp) and sequences containing internal stop codons, 49 valid protein-coding sequences (CDS) were obtained. Bioinformatics programs including CUSP and CodonW 1.4.2 were used to calculate the GC content at different codon positions (GC₁, GC₂, GC₃, and GC_all), the effective number of codons (ENC), and the relative synonymous codon usage (RSCU). Subsequently, combined with correlation analysis models such as neutrality plot, ENC-plot, and PR2-plot, the relative contributions of mutation pressure and natural selection to the formation of codon usage bias were comprehensively evaluated. Finally, the optimal codons of this species were identified by constructing high- and low-expression gene libraries.

Result The average overall GC content (GC_all) of the 49 chloroplast CDSs of C. laxum was 38.10%, and the GC content at different codon positions exhibited significant imbalance: GC₁ (46.92%) > GC₂ (39.21%) > GC₃ (28.17%). The ENC values ranged from 37.26 to 60.01, with an average of 46.70 (genes with ENC > 45 accounted for 65.31%), indicating a weak overall codon usage bias. Correlation and neutrality plot analyses revealed no significant correlation between GC₁₂ and GC₃ (R² = -0.011). The ENC-plot and PR2-plot indicated that the vast majority of genes deviated from the standard expected curve, and the usage frequencies of G and T at the third base position were higher than those of C and A, respectively. Through comprehensive screening, 17 optimal codons (e.g., UUU, UUA, AUU) were identified, all of which end in A or U.

Conclusion The overall codon usage bias of the C. laxum chloroplast genome is relatively weak, with a strong preference for synonymous codons ending in A or U. This bias pattern is primarily driven by natural selection pressure, while the influence of gene mutation pressure is relatively weak. The 17 identified optimal codons can provide a reference for codon modification in the chloroplast genetic engineering of C. laxum and related Liliaceae plants.