Characterization of Codon Bias in the Chloroplast Genome of Salix matsudana

To uncover the codon usage bias (CUB) Salix matsudana and its driving factors in the chloroplast genome of Sal: X CodonW and CUSP software were used to analyze the codon usage patterns of 51 filtered protein-coding sequences. The results showed that the GC content of the GC₁, GC₂, GC₃and the GC_all content of the codons in the chloroplast genome of S. matsudana were 45.85%, 37.77%, 29.41%, and 37.67%, respectively. The effective number of codons (ENC) values ranged from 33.59 to 57.39, with an average of 45.49, and 33 genes had ENC values exceeding 45, indicating weak codon usage bias in most protein-coding sequences. Additionally, 32 codons had relative synonymous codon usage (RSCU) values ≥1, and these codons were more likely to end with A or U. Further neutrality plot, ENC-plot, and PR2-plot analyses suggested that mutation and natural selection commonly influenced codon usage bias, with natural selection being the predominant factor. Through the analysis, 13 optimal codons were identified in the chloroplast genome of S. matsudana, providing theoretical support for its genetic improvement and gene engineering applications.