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Wheat cultivation in many regions faces threats by devastating fungal infections. However, wheat cultivar 92R137 shows resistance to Puccinia striiformis infection. To isolate the stripe rust resistance gene Yr26, an integrated transcriptomic and comparative genomics approach was undertaken. Near-isogenic lines of wheat (carrying Yr26 or not) infected with two Puccinia striiformis f. sp. tritici (Pst) (Virulence or avirulence to Yr26) were analysed in a dual detailed time series RNA-seq study. The emerging IWGSC refseq v1.0 genome assembly sequence serves as a valuable template and was also used for comparative genomics studies of the gene candidate region with the genome sequences of close relatives and wheat progenitors. Using bulked segregant analysis (BSA) to identify polymorphic SNPs between parent and resistant DNA (R-bulk) and susceptible DNA (S-bulk), flanking markers for Yr26 were identified. These two markers were mapped to the Chinese spring reference genome sequence, spanning a region of about 250 kb. The synteny analysis of this candidate region in CS chr1B with chr1A, chr1D, Wild Emmer Wheat (Td_chr1A and Td_chr1B) and Barley (chr1H) identified three candidate Yr26 genes. To detect gene candidates a dual time series RNA-seq analysis was performed. Genes differently expressed between rust susceptible (NIL-S) host lines and rust resistant (NIL-R) lines, carrying the Yr26 candidate gene were analysed. Both lines were inoculated with Pst carrying different avirulence factors (Pst-CYR32 and Pst-V26), compatible or incompatible with the corresponding wheat lines. Differential gene expression analysis (DEG) between compatible and incompatible interaction revealed DEGs in the wheat genome and in the Pst genome. From a network analysis of both wheat and Pst genes, we inferred connected co-expressed modules. Resulting modules showed particular enrichments for disease resistance, defense response to fungus and cell wall components.