Chloroplast genome sequencing of Poa pratensis and comparative chloroplast genomics analysis with other 29 grass species.

Grass species of the Poaceae family play central roles in terrestrial ecosystems and agriculture, and land management. Despite their ecosystem and economic importance, comprehensive comparative studies of the chloroplast genome characteristics and evolutionary patterns across Poaceae remain limited. In this study, we sequenced and assembled the complete chloroplast genome of Poa pratensis and conducted a comparative analysis with 29 additional Poaceae grass species. All 30 species exhibited the canonical quadripartite structure, with genome lengths ranging from 133,331 to 140,622 bp and GC content between ranging from 38.10% to 38.82%. Codon usage analyses revealed a consistent bias toward A/T-ending codons, with 29-30 codons per species exhibiting relative synonymous codon usage (RSCU) values > 1, while effective number of codons (ENC) values indicated relatively weak codon usage bias (CUB) across all taxa. Simple-sequence-repeat (SSR) analyses identified 58-98 SSRs per species, predominantly mononucleotides and octanucleotides predominantly localized mainly non-coding regions. Integration of neutrality plots, PR2 analysis, and ENC-GC3s plots, indicated that natural selection, rather than mutation pressure, was the dominant force shaping codon usage patterns. Ka/Ks analysis demonstrated pervasive strong purifying selection (Ka/Ks < 0.5) across most genes, with only a few, exceptions, clpP, matK, infA, and select hypothetical genes exhibiting relaxed constraints. Phylogenetic and collinearity analyses revealed well-resolved relationships, confirming that Poa pratensis and Poa supina share the closest genetic affinity, and supporting established taxonomic classifications. Despite overall structural conservation, functional constraints preserved genome integrity while permitting adaptive diversification. Strong purifying selection observed across chloroplast genes underscores their essential roles in photosynthesis and other core cellular processes. The identified molecular markers and resolved phylogenetic relationships provide valuable resources for grass breeding, genetic diversity assessment, and conservation. Collectively, this study establishes a comprehensive chloroplast genomic framework for elucidating evolutionary dynamics in Poaceae and informs future molecular breeding and biotechnological applications.