| Summary: | As a perennial herb in Triticeae, <i>Elymus dahuricus</i> is widely distributed in Qinghai–Tibetan Plateau and Central Asia. It has been used as high-quality fodders for improving degraded grassland. The genomic constitution of <i>E. dahuricus</i> (2n = 6x = 42) has been revealed as StStHHYY by cytological approaches. However, the universal karyotyping nomenclature system of <i>E. dahuricus</i> is not fully established by traditional fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH). In this study, the non-denaturing fluorescent in situ hybridization (ND-FISH) using 14 tandem-repeat oligos could effectively distinguish the entire <i>E. dahuricus</i> chromosomes pairs, while Oligo-FISH painting by bulked oligo pools based on wheat-barley collinear regions combined with GISH analysis, is able to precisely determine the linkage group and sub-genomes of the individual <i>E. dahuricus</i> chromosomes. We subsequently established the 42-chromosome karyotype of <i>E. dahuricus</i> with distinctive chromosomal FISH signals, and characterized a new type of intergenomic rearrangement between 2H and 5Y. Furthermore, the comparative chromosomal localization of the centromeric tandem repeats and immunostaining by anti-CENH3 between cultivated barley (<i>Hordeum vulgare</i> L.) and <i>E. dahuricus</i> suggests that centromere-associated sequences in H subgenomes were continuously changing during the process of polyploidization. The precise karyotyping system based on ND-FISH and Oligo-FISH painting methods will be efficient for describing chromosomal rearrangements and evolutionary networks for polyploid <i>Elymus</i> and their related species.
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