Codon usage bias and its influencing factors in the chloroplast genome of Macadamia integrifolia Maiden & Betche

Codon usage bias of 51 genes from the chloroplast genome of Macadamia integrifolia Maiden & Betche was analyzed to determine the main factors affecting the formation of codon bias. The GC content at different codon positions in the chloroplast genes was GC1 > GC2 > GC3, and GC3 was significantly correlated with effective number of codons (ENC) (mean value 48.80 > threshold 45), indicating that the third codon position had direct impact on weak codon bias in the chloroplast genome of M. integrifolia. Neutral-plot analysis showed that there was no significant correlation between GC12 and GC3, with a correlation coefficient and regression slope of 0.186 and 0.265, respectively. ENC-plot analysis showed that most genes were located around the standard curve, and their ENC ratios were distributed between -0.05 and 0.05, suggesting that the differences between actual and expected ENC were small. PR2-plot analysis revealed that the third codon position was biased, where pyrimidine T/C was used more frequently than purine A/G. Relative synonymous codon usage (RSCU) analysis showed that most of the 29 high-frequency codons, 19 high-expression codons, and 16 optimal codons preferentially ended with A or U. Phylogenetic analysis showed that the relationship between M. integrifolia and M. ternifolia was similar. These results suggest that natural selection and base mutation are the main factors influencing weak codon bias in the chloroplast genome of M. integrifolia, and the third codon base, as another influencing factor, prefers to use A or U.