Codon usage bias analysis of the chloroplast genome of cassava

Chloroplast codon preference affects gene expression efficiency and is important for chloroplast genetic engineering applications. Despite being the third most important food crop, cassava (Manihot esculenta) has received little attention for its chloroplast codon usage patterns. The analysis of cassava chloroplast genomic codon usage and preferences was conducted using CodonW, CUSP, and R software programs. The codons of cassava chloroplast genes had a GC content of 37.90%, whereas codon positions 1 to 3 had GC contents of 46.64%, 39.19%, and 27.58%, respectively, suggesting a codon preference ending in A or U. A weak codon bias was observed, with the effective number of codons (ENC) ranging from 36.55 to 61.29. There was a significant correlation between GC1 and GC2, but not between GC3 and GC1, or GC3 and GC2, indicating that the base composition of the first two codon positions was similar but significantly different from that of the third codon position. Relative synonymous codon usage (RSCU) analysis showed that of the 30 codons with RSCU greater than 1, 13 ended in A, 16 ended in U, and 1 ended in G. This further demonstrated that the codons in the cassava chloroplast genome preferentially ended in A or U. The correlation and regression coefficients for GC12 and GC3 were 0.228 and 0.351, respectively, and the correlations were not significant, suggesting that natural selection and mutational pressure worked together to shape the codon preference in cassava chloroplasts. Furthermore, the ENC plot and ENC ratio analysis revealed that natural selection rather than mutational pressure significantly influenced codon preferences in cassava chloroplasts. In the PR2-plot analysis, most of the genes were located in the lower half of the plot, particularly in the lower right corner, indicating that the third base of the synonymous codon was preferred to end in U/G, especially U. Finally, a two-way Chi-squared contingency test was performed to identify the optimal codons of UUC, UCU, CUA, CCU, GCU, AAC, and GGU. These results can be used as a scientific basis to improve the expression of exogenous genes in cassava chloroplasts by optimizing their codons.