A reference-grade chromosome-level genome assembly of a historically important U.S. bread wheat cultivar Timstein.

We report a near telomere-to-telomere high quality genome assembly of the historically important spring wheat cultivar, Timstein, generated using PacBio HiFi long-read sequencing data followed by Hi-C scaffolding. The assembly spanned 14.76 Gb, accounting for all 21 chromosomes of the A, B, and D subgenomes. Gene annotations identified around 105 K high-confidence (HC) gene models. The genome was comprised of ~85% transposable elements, primarily from the Gypsy, Copia, and CACTA families. For each subgenome, the BUSCO completeness score ranging from 97.4 to 99.6% and LTR Assembly Index (LAI) values surpassing 13 indicated the assembly quality was reference grade. Synteny analysis with IWGSC Chinese Spring (CS) RefSeq v2.1 revealed strong chromosomal collinearity between two genomes. Timstein has been extensively studied in classical genetics research for stem and leaf rust resistance and septoria nodorum blotch (SNB) susceptibility. This high-quality genome assembly provides cultivar-resolved references that can expand the wheat pangenome, supports structural and functional genomics studies, and enables fine mapping of disease resistance/susceptibility loci for their utilization in wheat genetics and breeding programs.