Unambiguous chromosome identification by multicolour fluorescence in situ hybridisation reveals the genome organisation and evolution of sheepgrass (Leymus chinensis)

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.