Comparative Analysis of Mitochondrial and Chloroplast Genomes in Alfalfa (Medicago sativa L.)

Codon usage bias is a widespread phenomenon in many organisms, including higher plants and plays an important role in regulating gene expression efficiency and accuracy. As an important forage species with high nutritional value, Medicago sativa (alfalfa) has been extensively utilized not only in traditional breeding but also in molecular genetic improvement. Understanding the characteristics of codon preference in its organelle genomes can provide valuable guidance for optimizing gene design and enhancing the expression of exogenous genes in alfalfa or related species.

A total of 30 mitochondrial and 51 chloroplast high-quality coding sequences were obtained from the NCBI database. Codon usage patterns and preferences were analyzed using CodonW, R, MEGA and Excel.

The effective number of codons (ENC) in mitochondria ranged from 44.73 to 61.0, with an average GC content of 0.42 and GC content at the third codon position (GC3s) of 0.37. In chloroplasts, the ENC ranged from 38.90 to 57.76, with an average GC content of 0.37 and GC3s of 0.27. Analyses using ENC-plot, neutrality plot and PR2 bias analysis suggested that codon usage bias in Medicago sativa is influenced by both natural selection and mutation pressure. Twelve optimal codons were identified in mitochondria (mostly ending in ‘A’ and ‘U’) and fourteen in chloroplasts, also showing a preference for ‘A/U’-ending codons.