Natural Selection as the Primary Driver of Codon Usage Bias in the Mitochondrial Genomes of Three Medicago Species

Abstract

Objectives: Codon usage bias is a fundamental feature of gene expression that can influence evolutionary processes and genetic diversity. This study aimed to investigate the mitochondrial codon usage characteristics and their driving forces in three Medicago species: Medicago polymorpha, Medicago sativa, and Medicago truncatula.

Methods: The complete mitochondrial genome sequences of the three species were downloaded from GenBank, and 21 shared coding sequences were screened. Codon usage patterns were analyzed using CodonW 1.4.2 and CUSP software. Key parameters, including the relative synonymous codon usage (RSCU), effective number of codons (ENC), codon adaptation index (CAI), codon bias index (CBI), and frequency of optimal codons (Fop), were calculated. Phylogenetic trees and RSCU clustering maps were constructed to explore evolutionary relationships.

Results: The GC contents of the mitochondrial genomes followed the order of GC1 > GC2 > GC3. ENC values averaged above 35, while CAI, CBI, and Fop values ranged from 0.160 to 0.161, -0.078 to -0.076, and 0.362 to 0.363, respectively, indicating a weak preference for codons ending with A/U. Correlation and neutrality analyses suggested that codon usage bias was influenced by both mutation pressure and natural selection, with natural selection being the dominant factor. Fifteen optimal codons, predominantly ending with A/U, were identified. Phylogenetic analysis confirmed the close relationship among the three Medicago species, consistent with traditional taxonomy, whereas the RSCU clustering did not align with the phylogenetic relationships.

Conclusions: This study provides insights into the mitochondrial codon usage patterns and their evolutionary determinants in Medicago species, highlighting the predominant role of natural selection in shaping codon usage bias. The findings offer a foundation for comparative genomic studies and evolutionary analyses and may be beneficial for improving genetic engineering and breeding programs of Medicago species.

Keywords: Medicago; codon usage bias; mitochondrial genome; natural selection; phylogenetic analysis.

Figure 1

Heatmap of nucleotide composition at the third codon position in the mitochondrial genomes of three Medicago species.

Figure 2

Base composition analysis of codons in the mitochondrial genomes of three Medicago species.

Figure 3

Correlation heatmap of codon usage indices in three Medicago species. codon No: codon number; GC1: GC content of codon base 1; GC2: GC content of codon base 2; GC3: GC content of codon base 3; GC_all: total codon GC content; GC3s: GC content of mitochondrial genome synonymous codon base 3; CAI: codon adaptation index; CBI: codon preference index; Fop: frequency of optimal codon usage; ENC: effective codon count; **: significant correlation at the 0.01 level; *: significant correlation at the 0.05 level. In the upper-right section, numerical values quantify the correlation coefficients between the indices. The lower-left section uses colored blocks to visually depict these correlations, where red signifies a positive correlation and blue indicates a negative one. The intensity of the color reflects the strength of the correlation, with darker shades representing stronger associations.

Figure 4

RSCU of codons in the CDSs of the mitochondrial genomes of three Medicago species. Each color in the stacked bars corresponds to a specific synonymous codon for the amino acids. The same colors are used in the RSCU bar chart above to represent the RSCU values of the respective codons. * indicates the stop codons.

Figure 5

Neutrality plots of mitochondrial genomes in three Medicago species. Equations y′, y″, and y‴ represent M. polymorpha, M. sativa, and M. truncatula, respectively.

Figure 6

ENC plot analysis of mitochondrial genomes in three Medicago species.

Figure 7

PR2 plot analysis of mitochondrial genomes in three Medicago species. The PR2 plot is divided into four quadrants by the central intersection point (G=C and A=T). Quadrant I (top-right) indicates an excess of A and G at the third codon position, while Quadrant III (bottom-left) indicates an excess of T and C. Quadrant II (top-left) indicates an excess of G and T, while Quadrant IV (bottom-right) indicates an excess of C and A.

Figure 8

Phylogenetic tree of mitochondrial CDSs from 10 species.

Figure 9

Dendrogram heatmap of RSCU-based clustering for mitochondrial CDSs from 10 species. The heatmap displays the RSCU values of the codons for each gene, with the color intensity reflecting the usage frequency of each codon.