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Genetic resistance to Wheat streak mosaic virus (WSMV) offers the most attractive and environmentally safe strategy for disease control. While effective resistance in hexaploid bread wheat (Triticum aestivum) has recently been described in only one case, the Wsm2 gene, more successful resistance has been introgressed from the related hexaploid wheatgrass, Thinopyrum intermedium, as the Wsm1 and Wsm3 genes. In the current study, fluorescent in situ hybridization (FISH) with genomic DNA from Th. intermedium, Aegilops tauschii and repetitive DNA probes was applied to four breeding populations of wheat-Th. intermedium, previously tested for WSMV-resistance. Three different wheat-Th. intermedium recombinant chromosomes, the well-known 4Ai#2S.4DL and two novel, 1B and 3D, were identified to be associated with WSMV-resistance. These novel introgressed genes from Th. intermedium were designated as Wsm4 and Wsm5 respectively. The Wsm4 gene was pinpointed to a 6% interstitial map region on the 1BS flanked by Xgwm4144 and Xgwm1100 markers. Six new PCRmarkers, five linked to Wsm1 and one to Wsm4 were identified. Molecular markers now provide a good coverage of the 4Ai#2S arm for effective marker assisted selection and the new genes increase our arsenal to combat the disease. Two highly repetitive satellite DNA families, Afa and pSc119.2, were isolated for the first time from Th. intermedium and their diversity in respect to copies from other Triticeae species were investigated. They showed contrasting evolutionary dynamics leading to time dependent or independent homogenization of Afa and pSc119.2 sequences. Both repeats are excellent cytological markers and characterized the 4Ai#2S chromosomal arm, in the alien wheat lines and the Th. intermedium genome. Southern hybridization, with methylation sensitive and insensitive restriction enzymes and immunostaining with anti-5-methyl-cytosine antibodies were employed to assess DNA methylation. Overall, no massive changes were evident in the wheat genome, however the alien arm showed reduced cytosine methylation which is characteristic for actively transcribing chromatin.