GBS-based identification of Triticum timopheevii introgressions in a wheat pre-breeding population.

Triticum timopheevii (2n = 4x = 28, GGAtAt) is a valuable wild wheat relative for improving disease resistance and agronomic performance in bread wheat, although the detection of introgressed chromosomal segments remains challenging due to close genomic similarity. A wheat × T. timopheevii pre-breeding population was analyzed using Genotyping-by-sequencing (GBS) combined with a skim-seq pipeline to identify and characterize T. timopheevii introgressions. Read coverage analysis based on a combined T. aestivum-T. timopheevii reference genome enabled high-resolution detection of major chromosomal introgressions and copy-number changes. Multiple large-scale chromosomal rearrangements were identified, most frequently involving wheat chromosome 2B and T. timopheevii chromosome 2G, involving the replacement of a ~625 Mb wheat segment with a ~526 Mb T. timopheevii segment. Additional rearrangements included a 6A-6At translocation, a nullisomic 6B genotype, and widespread 1B.1R rye introgressions. These findings were confirmed by fluorescence in situ hybridization. Phenotyping of plant-pathogen interactions showed that some lines carrying 2G introgressions displayed reduced disease symptoms against individual leaf or yellow rust isolates. Several genotypes also showed favorable yield-component traits, including increased spikelet and grain number. Our results demonstrate that GBS-based read coverage analysis combined with cytogenetic validation is an effective approach for dissecting genome structure and identifying candidate introgressions with potential breeding relevance. The recurrent involvement of specific chromosomes highlights preferential homoeologous recombination patterns and supports the targeted utilization of T. timopheevii genetic diversity for wheat improvement.