A Comparative Analysis of the Codon Usage Bias of HSP90 Genes in Six Poaceae Forages

Codon usage bias is important for regulating protein translation efficiency and accuracy. The HSP90 gene, a pivotal gene in plants, maintains homeostasis in plant protein stress responses and organelle immune defense functions. We systematically examine codon usage preferences in six forage grass species and the regulatory mechanisms of the HSP90 gene in governing codon preference. A set of metrics is evaluated, including effective codon number (ENC), codon adaptation index, and relative synonymous codon usage. Neutral evolutionary trajectories reveal usage preferences for six plant codons, with natural selection serving as the primary driving factor. The correlation between the ENC–GC3 curve (ENC relative to third-position GC content in synonymous codons) and codon bias index reveals these genes to exhibit moderate codon bias. The phenomenon of evolutionary constraints is exemplified by a propensity for C/G-terminating codons, concomitant with a suppression of NUA/NCG codons (NUA is an abbreviation for UA dinucleotide, and NCG is an abbreviation for CG dinucleotide). Phylogenomic reconstruction reveals a conserved diversification pathway, positioning P. giganteum A. Rich. at the basal node of the evolutionary framework. This study identified through systematic assessment that natural selection is the primary evolutionary force driving the biased use of codons in grass HSP90 genes. This finding provides actionable insights for enhancing abiotic stress tolerance in forage germplasm through precise codon engineering.