Natural Selection as the Primary Driver of Codon Usage Bias in the Mitochondrial Genomes of Three Medicago Species
Abstract
1. Introduction
2. Materials and Methods
2.1. Acquisition of CDS from Mitochondrial Genomes of Three Medicago Species
2.2. Codon Usage Parameter Analysis
- Nucleotide frequencies at the third codon position: A3s, T3s, C3s, and G3s (frequency of adenine, thymine, cytosine, and guanine at the third synonymous codon position, respectively).
- GC content: GC3s (mean GC content at the third synonymous codon position), ATall/GCall (total AT/GC content), and GC1all/GC2all/GC3all (GC content at codon positions 1, 2, and 3).
- Codon bias indices: ENC (effective number of codons), CAI (codon adaptation index), CBI (codon bias index), RSCU (relative synonymous codon usage), and Fop (frequency of optimal codons).
2.3. Neutrality Plot Analysis
2.4. ENC Plot Analysis
2.5. PR2 Plot Analysis
2.6. Identification of Optimal Codons
2.7. Phylogenetic and RSCU Clustering Analysis
3. Results
3.1. Annotation and Analysis of Protein-Coding Genes
3.2. Analysis of Codon Usage Bias-Related Indices
3.3. Results of Neutrality Plot Analysis
3.4. Results of ENC Plot Analysis
3.5. Results of PR2 Plot Analysis
3.6. Optimal Codons
3.7. Phylogenetic Relationships and RSCU Clustering Patterns
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
CAI | Codon adaptation index |
CBI | Codon bias index |
CDS | Coding sequences |
CUB | Codon usage bias |
ENC | Effective number of codons |
GRAVY | Grand Average of Hydropathicity |
ML | Maximum likelihood |
RSCU | Relative synonymous codon usage |
SCUB | Synonymous codon usage bias |
MP | Medicago polymorpha |
MS | Medicago sativa |
MT | Medicago truncatula |
GC1 | GC content at codon positions 1 |
GC2 | GC content at codon positions 2 |
GC3 | GC content at codon positions 3 |
GC12 | Average GC1 and GC2 |
GCall | Total GC content |
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Tribe | Genus | Species | Genbank Accession Number |
---|---|---|---|
Phaseoleae | Glycine | Glycine max | OR687435.1 |
Phaseoleae | Glycine | Glycine soja | MF955859.1 |
Trifolieae | Medicago | Medicago polymorpha | MW971562.1 |
Trifolieae | Medicago | Medicago sativa | ON782580.1 |
Trifolieae | Medicago | Medicago truncatula | KT971339.1 |
Trifolieae | Trifolium | Trifolium grandiflorum | MT039391.1 |
Trifolieae | Trifolium | Trifolium aureum | MT039392.1 |
Trifolieae | Trifolium | Trifolium meduseum | MT039390.1 |
Trifolieae | Trifolium | Trifolium pratense | MT039389.1 |
Trifolieae | Trigonella | Trigonella foenum-graecum | OP605625.1 |
Gene Group | Gene Names |
---|---|
ATP synthase | atp1, atp4, atp8 |
Cytochrome c biogenesis | ccmB, ccmC, ccmFn |
Ubiquinol cytochrome c reductase | cob |
Cytochrome c oxidase | cox1 |
Maturases | matR |
NADH dehydrogenase | nad2, nad3, nad4, nad5, nad7, nad9 |
Ribosomal protein large subunit | rpl16, rpl5 |
Ribosomal protein small subunit | rps12, rps14, rps3, rps4 |
Genes | M. polymorpha | M. sativa | M. truncatula | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
CAI | CBI | Fop | ENC | CAI | CBI | Fop | ENC | CAI | CBI | Fop | ENC | |
atp1 | 0.172 | −0.082 | 0.362 | 52.73 | 0.172 | −0.082 | 0.362 | 52.73 | 0.172 | −0.082 | 0.362 | 52.73 |
atp4 | 0.154 | −0.044 | 0.376 | 61 | 0.154 | −0.044 | 0.376 | 61 | 0.154 | −0.044 | 0.376 | 61 |
atp8 | 0.158 | 0.006 | 0.425 | 55.47 | 0.157 | 0 | 0.419 | 55.89 | 0.158 | 0.006 | 0.425 | 55.47 |
ccmB | 0.158 | −0.092 | 0.335 | 55.25 | 0.158 | −0.092 | 0.335 | 55.25 | 0.158 | −0.092 | 0.335 | 55.25 |
ccmC | 0.145 | −0.048 | 0.356 | 48.53 | 0.145 | −0.048 | 0.356 | 48.53 | 0.145 | −0.048 | 0.356 | 48.53 |
ccmFn | 0.169 | −0.026 | 0.391 | 57.95 | 0.169 | −0.026 | 0.391 | 58.06 | 0.169 | −0.026 | 0.391 | 57.95 |
cob | 0.159 | −0.157 | 0.3 | 52.58 | 0.161 | −0.153 | 0.303 | 53.1 | 0.159 | −0.157 | 0.3 | 52.87 |
cox1 | 0.176 | −0.063 | 0.369 | 54.87 | 0.176 | −0.063 | 0.369 | 54.87 | 0.176 | −0.063 | 0.369 | 54.87 |
matR | 0.146 | −0.001 | 0.41 | 56.98 | 0.145 | −0.001 | 0.41 | 56.72 | 0.146 | −0.001 | 0.41 | 57.02 |
nad2 | 0.18 | −0.119 | 0.342 | 52.59 | 0.188 | −0.077 | 0.374 | 52.39 | 0.18 | −0.119 | 0.342 | 52.59 |
nad3 | 0.18 | −0.129 | 0.321 | 53.28 | 0.18 | −0.129 | 0.321 | 53.28 | 0.18 | −0.129 | 0.321 | 53.28 |
nad4 | 0.163 | −0.071 | 0.353 | 52.15 | 0.163 | −0.071 | 0.353 | 52.15 | 0.163 | −0.071 | 0.353 | 52.15 |
nad5 | 0.17 | −0.116 | 0.345 | 52.47 | 0.165 | −0.109 | 0.347 | 53.16 | 0.17 | −0.116 | 0.345 | 52.36 |
nad7 | 0.185 | −0.102 | 0.359 | 49.55 | 0.185 | −0.102 | 0.359 | 49.55 | 0.185 | −0.102 | 0.359 | 49.55 |
nad9 | 0.168 | −0.098 | 0.354 | 58.42 | 0.168 | −0.098 | 0.354 | 58.42 | 0.168 | −0.098 | 0.354 | 58.42 |
rpl16 | 0.16 | −0.094 | 0.372 | 57.92 | 0.16 | −0.094 | 0.372 | 57.92 | 0.16 | −0.096 | 0.376 | 57.33 |
rpl5 | 0.167 | −0.077 | 0.376 | 57.63 | 0.167 | −0.077 | 0.376 | 57.63 | 0.167 | −0.073 | 0.376 | 58.05 |
rps12 | 0.137 | −0.098 | 0.355 | 47.11 | 0.137 | −0.098 | 0.355 | 47.11 | 0.137 | −0.098 | 0.355 | 47.11 |
rps14 | 0.133 | −0.121 | 0.333 | 52.43 | 0.133 | −0.121 | 0.333 | 52.43 | 0.133 | −0.121 | 0.333 | 52.43 |
rps3 | 0.162 | −0.056 | 0.387 | 59.61 | 0.161 | −0.059 | 0.386 | 59.38 | 0.159 | −0.069 | 0.379 | 59.24 |
rps4 | 0.127 | −0.047 | 0.379 | 54.08 | 0.127 | −0.047 | 0.379 | 54.33 | 0.127 | −0.047 | 0.379 | 54.33 |
Amino Acid | M. polymorpha | M. sativa | M. truncatula | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Codon | RSCU-H | RSCU-L | △RSCU | Codon | RSCU-H | RSCU-L | △RSCU | Codon | RSCU-H | RSCU-L | △RSCU | |
Ala | GCA | 0.7619 | 1.0256 | −0.2637 | GCA | 0.7619 | 1.0256 | −0.2637 | GCA | 0.7619 | 1.0526 | −0.2907 |
GCC | 0.1905 | 0.5128 | −0.3223 | GCC | 0.1905 | 0.5128 | −0.3223 | GCC | 0.1905 | 0.5263 | −0.3358 | |
GCG | 0.5714 | 0.9231 | −0.3517 | GCG | 0.5714 | 0.9231 | −0.3517 | GCG | 0.5714 | 0.9474 | −0.376 | |
GCU | 2.4762 | 1.5385 | 0.9377 | GCU | 2.4762 | 1.5385 | 0.9377 | GCU | 2.4762 | 1.4737 | 1.0025 | |
Cys | UGC | 0 | 0.4 | −0.4 | UGC | 0 | 0.4 | −0.4 | UGC | 0 | 0.4 | −0.4 |
UGU | 2 | 1.6 | 0.4 | UGU | 2 | 1.6 | 0.4 | UGU | 2 | 1.6 | 0.4 | |
Asp | GAC | 0.2222 | 0.9231 | −0.7009 | GAC | 0.2222 | 0.9231 | −0.7009 | GAC | 0.2222 | 0.9231 | −0.7009 |
GAU | 1.7778 | 1.0769 | 0.7009 | GAU | 1.7778 | 1.0769 | 0.7009 | GAU | 1.7778 | 1.0769 | 0.7009 | |
Glu | GAA | 2 | 1.4359 | 0.5641 | GAA | 2 | 1.4359 | 0.5641 | GAA | 2 | 1.4054 | 0.5946 |
GAG | 0 | 0.5641 | −0.5641 | GAG | 0 | 0.5641 | −0.5641 | GAG | 0 | 0.5946 | −0.5946 | |
Phe | UUC | 0.4444 | 0.8182 | −0.3738 | UUC | 0.4444 | 0.8182 | −0.3738 | UUC | 0.4444 | 0.8182 | −0.3738 |
UUU | 1.5556 | 1.1818 | 0.3738 | UUU | 1.5556 | 1.1818 | 0.3738 | UUU | 1.5556 | 1.1818 | 0.3738 | |
Gly | GGA | 1.5385 | 1.0667 | 0.4718 | GGA | 1.5385 | 1.0667 | 0.4718 | GGA | 1.5385 | 1 | 0.5385 |
GGC | 0.4615 | 0.8 | −0.3385 | GGC | 0.4615 | 0.8 | −0.3385 | GGC | 0.4615 | 0.8182 | −0.3567 | |
GGG | 0.4615 | 1.1556 | −0.6941 | GGG | 0.4615 | 1.1556 | −0.6941 | GGG | 0.4615 | 1.1818 | −0.7203 | |
GGU | 1.5385 | 0.9778 | 0.5607 | GGU | 1.5385 | 0.9778 | 0.5607 | GGU | 1.5385 | 1 | 0.5385 | |
His | CAC | 0.1818 | 0.4 | −0.2182 | CAC | 0.1818 | 0.4 | −0.2182 | CAC | 0.1818 | 0.4 | −0.2182 |
CAU | 1.8182 | 1.6 | 0.2182 | CAU | 1.8182 | 1.6 | 0.2182 | CAU | 1.8182 | 1.6 | 0.2182 | |
Ile | AUA | 1.0645 | 1.1695 | −0.105 | AUA | 1.0645 | 1.1695 | −0.105 | AUA | 1.0645 | 1.1579 | −0.0934 |
Ile | AUC | 0.5806 | 0.8644 | −0.2838 | AUC | 0.5806 | 0.8644 | −0.2838 | AUC | 0.5806 | 0.8947 | −0.3141 |
AUU | 1.3548 | 0.9661 | 0.3887 | AUU | 1.3548 | 0.9661 | 0.3887 | AUU | 1.3548 | 0.9474 | 0.4074 | |
Lys | AAA | 1.2222 | 1.0769 | 0.1616 | AAA | 1.2222 | 1.0606 | 0.1616 | AAA | 1.2222 | 1.0159 | 0.2063 |
AAG | 0.7778 | 0.9231 | −0.1616 | AAG | 0.7778 | 0.9394 | −0.1616 | AAG | 0.7778 | 0.9841 | −0.2063 | |
Leu | CUA | 0.3913 | 0.6885 | −0.2972 | CUA | 0.3913 | 0.6885 | −0.2972 | CUA | 0.3913 | 0.6885 | −0.2972 |
CUC | 0.3913 | 0.9836 | −0.5923 | CUC | 0.3913 | 0.9836 | −0.5923 | CUC | 0.3913 | 0.9836 | −0.5923 | |
CUG | 0.3913 | 0.5902 | −0.1989 | CUG | 0.3913 | 0.5902 | −0.1989 | CUG | 0.3913 | 0.5902 | −0.1989 | |
CUU | 1.4348 | 1.082 | 0.3528 | CUU | 1.4348 | 1.082 | 0.3528 | CUU | 1.4348 | 1.082 | 0.3528 | |
UUA | 1.9565 | 1.4754 | 0.4811 | UUA | 1.9565 | 1.4754 | 0.4811 | UUA | 1.9565 | 1.4754 | 0.4811 | |
UUG | 1.4348 | 1.1803 | 0.2545 | UUG | 1.4348 | 1.1803 | 0.2545 | UUG | 1.4348 | 1.1803 | 0.2545 | |
Met | AUG | 1 | 1 | 0 | AUG | 1 | 1 | 0 | AUG | 1 | 1 | 0 |
Asn | AAC | 0.9091 | 0.7429 | 0.1662 | AAC | 0.9091 | 0.7429 | 0.1662 | AAC | 0.9091 | 0.7429 | 0.1662 |
AAU | 1.0909 | 1.2571 | −0.1662 | AAU | 1.0909 | 1.2571 | −0.1662 | AAU | 1.0909 | 1.2571 | −0.1662 | |
Pro | CCA | 0.7143 | 1.2121 | −0.4978 | CCA | 0.7143 | 1.2121 | −0.4978 | CCA | 0.7143 | 1.2121 | −0.4978 |
CCC | 0.8571 | 0.9697 | −0.1126 | CCC | 0.8571 | 0.9697 | −0.1126 | CCC | 0.8571 | 0.9697 | −0.1126 | |
CCG | 0.8571 | 1.0909 | −0.2338 | CCG | 0.8571 | 1.0909 | −0.2338 | CCG | 0.8571 | 1.0909 | −0.2338 | |
CCU | 1.5714 | 0.7273 | 0.8441 | CCU | 1.5714 | 0.7273 | 0.8441 | CCU | 1.5714 | 0.7273 | 0.8441 | |
Gln | CAA | 1.6364 | 1.2727 | 0.3031 | CAA | 1.6364 | 1.3333 | 0.3031 | CAA | 1.6364 | 1.3333 | 0.3031 |
CAG | 0.3636 | 0.7273 | −0.3031 | CAG | 0.3636 | 0.6667 | −0.3031 | CAG | 0.3636 | 0.6667 | −0.3031 | |
Arg | AGA | 1.5429 | 1.7468 | −0.2039 | AGA | 1.5429 | 1.7468 | −0.2039 | AGA | 1.5429 | 1.7692 | −0.2263 |
AGG | 0.1714 | 0.9873 | −0.8159 | AGG | 0.1714 | 0.9873 | −0.8159 | AGG | 0.1714 | 1 | −0.8286 | |
CGA | 1.2 | 0.9873 | 0.2127 | CGA | 1.2 | 0.9873 | 0.2127 | CGA | 1.2 | 1 | 0.2 | |
CGC | 0.5143 | 0.6835 | −0.1692 | CGC | 0.5143 | 0.6835 | −0.1692 | CGC | 0.5143 | 0.6923 | −0.178 | |
CGG | 0.8571 | 0.6076 | 0.2495 | CGG | 0.8571 | 0.6076 | 0.2495 | CGG | 0.8571 | 0.6154 | 0.2417 | |
Arg | CGU | 1.7143 | 0.9873 | 0.727 | CGU | 1.7143 | 0.9873 | 0.727 | CGU | 1.7143 | 0.9231 | 0.7912 |
Ser | AGC | 0.8571 | 0.5373 | 0.3198 | AGC | 0.8571 | 0.5373 | 0.3198 | AGC | 0.8571 | 0.5538 | 0.3033 |
AGU | 0.5143 | 1.0746 | −0.5603 | AGU | 0.5143 | 1.0746 | −0.5603 | AGU | 0.5143 | 1.0154 | −0.5011 | |
UCA | 1.3714 | 1.0746 | 0.2968 | UCA | 1.3714 | 1.0746 | 0.2968 | UCA | 1.3714 | 1.1077 | 0.2637 | |
UCC | 0.5143 | 0.806 | −0.2917 | UCC | 0.5143 | 0.806 | −0.2917 | UCC | 0.5143 | 0.7385 | −0.2242 | |
UCG | 1.0286 | 0.9851 | 0.0435 | UCG | 1.0286 | 0.9851 | 0.0435 | UCG | 1.0286 | 1.0154 | 0.0132 | |
UCU | 1.7143 | 1.5224 | 0.1919 | UCU | 1.7143 | 1.5224 | 0.1919 | UCU | 1.7143 | 1.5692 | 0.1451 | |
Thr | ACA | 1.5238 | 1.2414 | 0.2824 | ACA | 1.5238 | 1.2414 | 0.2824 | ACA | 1.5238 | 1.2857 | 0.2381 |
ACC | 1.1429 | 1.2414 | −0.0985 | ACC | 1.1429 | 1.2414 | −0.0985 | ACC | 1.1429 | 1.2857 | −0.1428 | |
ACG | 0.7619 | 0.6897 | 0.0722 | ACG | 0.7619 | 0.6897 | 0.0722 | ACG | 0.7619 | 0.5714 | 0.1905 | |
ACU | 0.5714 | 0.8276 | −0.2562 | ACU | 0.5714 | 0.8276 | −0.2562 | ACU | 0.5714 | 0.8571 | −0.2857 | |
Val | GUA | 1.1111 | 1.2683 | −0.1572 | GUA | 1.1111 | 1.2683 | −0.1572 | GUA | 1.1111 | 1.2683 | −0.1572 |
GUC | 0.8889 | 0.878 | 0.0109 | GUC | 0.8889 | 0.878 | 0.0109 | GUC | 0.8889 | 0.878 | 0.0109 | |
GUG | 0.4444 | 0.9756 | −0.5312 | GUG | 0.4444 | 0.9756 | −0.5312 | GUG | 0.4444 | 0.9756 | −0.5312 | |
GUU | 1.5556 | 0.878 | 0.6776 | GUU | 1.5556 | 0.878 | 0.6776 | GUU | 1.5556 | 0.878 | 0.6776 | |
Trp | UGG | 1 | 1 | 0 | UGG | 1 | 1 | 0 | UGG | 1 | 1 | 0 |
Tyr | UAC | 0.3333 | 0.7273 | −0.394 | UAC | 0.3333 | 0.7273 | −0.394 | UAC | 0.3333 | 0.6667 | −0.3334 |
UAU | 1.6667 | 1.2727 | 0.394 | UAU | 1.6667 | 1.2727 | 0.394 | UAU | 1.6667 | 1.3333 | 0.3334 |
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Shen, Y.; Qi, L.; Yang, L.; Lu, X.; Liu, J.; Wang, J. Natural Selection as the Primary Driver of Codon Usage Bias in the Mitochondrial Genomes of Three Medicago Species. Genes 2025, 16, 673. https://doi.org/10.3390/genes16060673
Shen Y, Qi L, Yang L, Lu X, Liu J, Wang J. Natural Selection as the Primary Driver of Codon Usage Bias in the Mitochondrial Genomes of Three Medicago Species. Genes. 2025; 16(6):673. https://doi.org/10.3390/genes16060673
Chicago/Turabian StyleShen, Yingfang, Leping Qi, Lijuan Yang, Xingxing Lu, Jiaqian Liu, and Jiuli Wang. 2025. "Natural Selection as the Primary Driver of Codon Usage Bias in the Mitochondrial Genomes of Three Medicago Species" Genes 16, no. 6: 673. https://doi.org/10.3390/genes16060673
APA StyleShen, Y., Qi, L., Yang, L., Lu, X., Liu, J., & Wang, J. (2025). Natural Selection as the Primary Driver of Codon Usage Bias in the Mitochondrial Genomes of Three Medicago Species. Genes, 16(6), 673. https://doi.org/10.3390/genes16060673