Comparative Genomics of Triticum, Secale, and Triticale: Codon Usage Bias in Chloroplast Genomes and Its Implications for Evolution and Genetic Engineering

Chloroplast codon usage bias (CUB) records both maternal phylogeny and selection intensity. Characterizing CUB in the synthetic cereal × <i>Triticosecale</i> and its Triticum and Secale parents is therefore a prerequisite for plastid-based engineering and for tracing the evolutionary consequences of recent allopolyploidy. Complete plastome sequences of five taxa-Triticum monococcum, T. turgidum, T. aestivum, Secale cereale and × Triticosecale sp.-were downloaded. Protein-coding genes were extracted to calculate overall GC, GC1-GC3, SCUO, RSCU, ENC-GC3s, neutrality, and PR2 plots. Optimal codons were defined as RSCU ≥ 1 and △RSCU ≥ 0.8. The results showed that the chloroplast genomes of these five species are low in GC content for the third base of codons, suggesting an end preference for A or U bases. The SCUO values ranged from 0.22 to 0.23, suggesting no significant codon usage bias. GC content was relatively low (38.78-39.16%), with the order GC1 > GC2 > GC3. RSCU analysis indicated that codons ending with A/T are more commonly used. Neutral mapping, ENC-GC3s, and the PR2 plot all showed that the preference of codon usage for the majority of functional genes was influenced by a combination of mutation and natural selection pressure, and the influence of natural selection was predominant. RSCU clustering recovers the expected maternal tree (Triticum clade + triticale). All optimal codons terminate with A or U, yielding identical plastid translation tables for the five species. Despite its recent hybrid origin, triticale plastid CUB is indistinguishable from its wheat maternal ancestor and is governed mainly by selection. The compiled optimal codon set provides an immediate reference for chloroplast transformation and for dissecting selection relaxation in newly synthesized triticale combinations.