Wheat (Triticum aestivum) is one of the most important food crops with an urgent need for increase in its production to feed the growing world. Wheat’s wild relative species provide a hugely untapped reservoir of allelic and genetic diversity for wheat improvement. At the Nottingham Wheat Research Centre we are sequencing the genomes of wild relatives of wheat from its secondary and tertiary gene pools to speed up the identification of causal genes of beneficial traits and also to develop diagnostic markers for tracking in marker assisted backcross programmes.

Triticum timopheevii (2n = 4x = 28) is a tetraploid wheat wild relative species containing the At and G genomes that has been exploited in many wheat pre-breeding programmes over the last few decades. In this study, we report the generation of a chromosome-scale reference genome assembly of T. timopheevii accession PI 94760 based on PacBio HiFi reads and chromosome conformation capture (Hi-C).

The assembly comprised a total size of 9.35 Gb, featuring a contig N50 of 42.4 Mb, and 166,325 predicted gene models. Comparative genome analysis confirmed previously known chromosomal translocations and indicated new chromosome rearrangements. Analysis of the genomic distribution of DNA methylation showed that the G genome had on average more methylated bases than the At genome. The G genome was also more closely related to the S genome of Aegilops speltoides than to the B genome of hexaploid or tetraploid wheat.

The assembly enabled the characterisation of pre-known and identification of new T. timopheevii introgressions in the 10+ wheat genomes and 760 Genebank accessions. We show the utility of this resource in our pre-breeding programme for the characterisation of wheat-T. timopheevii introgression lines.

In addition, the genome assembly has been crucial in work being carried out with collaborators aimed at finding new disease resistance alleles/genes for Fusarium Head Blight and leaf rust found in the wheat-T. timopheevii introgression lines.

In summary, the T. timopheevii genome assembly provides a valuable resource for genome-informed discovery and cloning of agronomically important genes for future food security.