Codon Usage Bias Pattern Difference Among the Triticeae Chloroplast Genomes Provides Insights into Lineage Evolution and Plastid Genome Engineering: A Meta- Analysis Perspective

The Triticeae tribe (Poaceae) comprises the world's most economically significant industrial and food crops, including wheat, barley, rye, and high-yield forage grasses. However, the mechanisms by which codon optimization guides production in the chloroplast genomes of the entire Triticeae tribe and the entire Poaceae family have not yet been elucidated in current industrial production. In this study, we performed the first systematic analysis of chloroplast codon usage bias and its driving mechanisms using 76 species covering all 20 recognized genera of the Triticeae, and we reconstructed the phylogenetic relationships within the tribe. To validate our findings across taxonomic scales, we further integrated a meta‑analysis of 285 species from 12 Poaceae subfamilies. Our results show that chloroplast genomes of the Triticeae exhibit a weak overall AT‑biased codon usage, with natural selection as the dominant evolutionary driver and mutation pressure playing a synergistic role, except for divergent patterns observed only in Aegilops and Eremopyrum . The phylogeny corroborates Leymus as a distinct clade and confirms the polyphyletic origin of Elymus . The Poaceae‑wide meta‑analysis confirmed similar patterns to the Triticeae, but with a larger number of stably preferred codons (30 vs. 6) and a significantly higher GC3 content in C4 subfamilies than in basal C3 subfamilies. Notably, we constructed a complete functional pathway spanning codons, genes, proteins and traits: 23 preferred‑codon‑enriched gene sets in the Triticeae are mainly involved in chloroplast photosynthesis and protein translation, whereas 18 unpreferred‑codon‑enriched gene sets are predominantly associated with respiratory chain electron transport and material transport. The present study provides guidelines for the optimisation of the chloroplast genomes of the Triticeae tribe and industrial crops in the Poaceae family. This theoretical foundation and scalable methodological framework will accelerate the development of high-biomass, stress-tolerant varieties.