Abstract
The unambiguous identification of individual chromosomes is fundamental to studying genome organisation and evolution. Sheepgrass (Leymus chinensis (Trin.) Tzvel), a perennial tetraploid grass closely related to wheat, is a valuable genetic resource for crop improvement. Here, we employed low-coverage sequencing and bioinformatics analysis to isolate and characterise a set of species-characteristic repetitive sequences in sheepgrass, including CL6, CL95, CL121, CL151, CL162, CL163, CL239, 5 S ribosomal DNA (rDNA), and 26 S rDNA. A multicolour fluorescence in situ hybridisation (FISH) assay using a probe cocktail targeting CL6, CL121, CL162, 5 S rDNA, and 26 S rDNA successfully distinguished all 28 sheepgrass chromosomes. On the basis of the close evolutionary relationship between wheat and sheepgrass, single-copy genes with known linkage map locations for wheat were applied to sheepgrass chromosomes as probes to identify homologous/homeologous chromosomes 1–7. We found that the distributions and abundances of repetitive sequences, as well as the length and arm ratio, varied among homologous/homeologous chromosomes, providing direct cytogenetic evidence for the allotetraploid nature of sheepgrass. FISH-based karyotyping of different geographical accessions revealed substantial intraspecific variations in the chromosomal locations of repetitive DNA. Moreover, single-copy gene amplification was detected in sheepgrass, and chromosomal translocations were detected among the accessions. This study advances our understanding of sheepgrass genome structure and evolution through the establishment of a chromosome identification system integrated with wheat genetic maps. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-026-08628-1.