Analysis of chloroplast genomes and SSR classification and identification of Agropyron species and closely related species.

To thoroughly analyze the characteristics of the chloroplast genome in the genus Agropyron Gaertn and provide a theoretical basis for its evolutionary biology and the molecular marker-assisted taxonomic identification of the broad-spike and narrow spike clades. This study conducted a systematic analysis of the chloroplast genomes from seven Agropyron species and two closely related species (Elymus trachycaulus and Elytrigia elongata). The chloroplast genome of Agropyron Gaertn exhibits a typical quadripartite structure, ranging in size from 135 to 137 kb and containing 131 genes. The rps12 and ycf3 genes show high variability, while the intergenic regions (IR) exhibit high GC content of 43.91% to 44.01%. SSR and scattered repetitive sequences exhibit significant interspecific differences among the Agropyron Gaertn and the closely related species. For instance, Elymus trachycaulus and Elytrigia elongata share the locus (CCATA)3, while simultaneously retaining genus-specific markers. For instance, locus (ATATA)3 is unique to Elytrigia elongata, and Elymus trachycaulus lacks the rps12 gene intron variation, forming a sequence profile that combines both conservation and differentiation. Based on the distribution patterns of SSR loci and nucleotide diversity analysis in this study, the combination patterns of SSR loci in the chloroplast genome can serve as candidate basis for the molecular-assisted taxonomic identification of the broad-spike/narrow-spike clades. Phylogenetic analysis revealed that most species within the genus Agropyron form monophyletic clades, while Elymus trachycaulus clusters closely with Elytrigia elongata due to shared characteristics such as high GC content (44.01%) in the intercalary region of the chloroplast genome and with LSC length (80,642 bp). Combined with its narrow-spike morphology, this result supports the molecular marker-assisted taxonomic identification to identify Elymus trachycaulus into the narrow-spike clade, demonstrating the synergy between morphological identification and molecular evidence in auxiliary taxonomy. This study lays the foundation for characterizing the chloroplast genomes of Agropyron species and developing molecular markers for the identification of the broad-spike/narrow-spike clades. Further multidisciplinary research is needed to explore the potential applications of these molecular markers and the mechanisms underlying the species' adaption.