Chloroplast Genome Evolution, Heteroplasmy, and Inverted Repeat Dynamics in the Elymus Complex (Triticeae, Poaceae): Insights from Single-Molecule Sequencing of Elymus ciliaris and Comparative Analysis of St-Genome Lineages

ABSTRACT Background/Objectives Elymus sensu lato (Poaceae) is arguably the largest and most complex genus in the tribe Triticeae. It includes hybrids and polyploids based on x=7 chromosomes, all including the St genome, forming a valuable genepool for forage grass and cereal breeding. Analysis of chloroplast genome diversity and structural dynamics is critical for resolving maternal lineages, reticulate evolution and biodiversity across this agronomically important complex, refining their taxonomy, conservation and exploitation. Methods We sequenced the complete chloroplast genome (plastome) of Elymus ciliaris (4x=2n=28; StStYY) using ultra-long Oxford Nanopore single-molecule reads and compared it to 77 additional chloroplast genomes representing major St-genome lineages in Elymus s.l. (Pseudoroegneria St; Elymus s.s. StH, StY; Thinopyrum StJ/E; Campeiostachys StYH; Kengyilia StYP). We analyzed structure, nucleotide diversity, inverted repeat (IR) dynamics, and phylogenetic signal. Results The E. ciliaris chloroplast genome was 135,008 bp long (38.3% GC) with a canonical quadripartite structure. Single-molecule reads (n=81) revealed heteroplasmy: two Small-Single-Copy (SSC) orientations at 30%:70% frequency, indicating an inversion polymorphism. Across the Elymus group, comparative analysis of chloroplast assemblies showed high structural conservation but lineage-specific IR-boundary shifts. Kengyilia exhibits exceptional IR expansion. Nucleotide diversity hotspots localize to the large single-copy region, especially in StY lineages. Phylogenies recover a monophyletic St-containing clade but do not delineate genera, reflecting reticulate evolution. Conclusions Single-molecule sequencing uncovered heteroplasmy with an inversion polymorphism in a single plant of Elymus ciliaris, hidden in short read assemblies. There were no other polymorphisms, as expected for chloroplast sequences. Our analyses showed that a Pseudoroegneria-like St chloroplast genome predominates as the maternal donor across Elymus polyploids. Variable regions and IR dynamics offer strong models for chloroplast genome evolution in reticulate lineages and suggest exploiting plastome variation to complement nuclear biodiversity studies.