The 3G introgressions were generated from a programme designed to transfer large numbers of chromosome segments from the T. timopheevii A^t and G genomes into wheat (Devi et al., 2019). In summary, T. timopheevii was used to pollinate the variety Paragon, which carried the ph1 mutation (Devi et al., 2019). The resulting F₁ interspecific hybrids were re-pollinated with Paragon to generate backcross progeny (BC₁, BC₂, BC₃, etc) which were self-pollinated. The backcross progeny were initially screened using the Axiom® Wild-Relative Genotyping Array (Devi et al., 2019) to identify introgressions. However, later generations were reanalysed using chromosome-specific KASP markers (Grewal et al., 2020a) to detect lines of wheat homozygous for T. timopheevii A^t and G genome introgressions (King et al., 2022). The T. timopheevii accession (accession PI 94760, obtained from the United States Department of Agriculture, USDA) used in this work had previously been screened at the John Innes Centre (Steed et al., 2022) and identified as carrying resistance to FHB. To determine if any of the homozygous T. timopheevii introgressions generated carried the resistance to FHB a series of them were screened (Steed et al., 2022). Two of the lines, designated as Tim5 and Tim6, with introgressions derived from the same region of Chr3G, were identified as carrying strong FHB resistance. KASP markers originally determined that Tim6 carries the largest introgression from 3G while Tim5 carries a much smaller introgression from the same region as Tim6. Skim-sequencing was used to establish that the Tim5 3G introgression was 60 Mbp in size and the Tim6 introgression 615 Mbp (Steed et al., 2025). Both Tim5 and Tim6 carry an additional homozygous introgression from 2G of T. timopheevii (King et al., 2022).

The hexaploid introgression lines Tim5 and Tim6 were each pollinated with 4 different durum genotypes, two provided by Karim Ammar (CIMMYT, Mexico); CIMMYT durum 3 (CD3) and CIMMYT durum 4 (CD4), and two provided by Curtis Pozniak (University of Saskatchewan, Canada); Canadian DT (CDT) and Canadian CD (CCD). The resulting F₁ hybrids were then backcrossed twice with the same durum genotype from which they were initially derived to produce BC₂ populations. As the FHB resistance had previously been located to the 3G introgressions (Steed et al., 2022), the presence or absence of the 2G and 3G introgressions in plants in the BC₂ generation was determined using KASP analysis and plants both lacking the 3G introgression and retaining the 2G introgressions were discarded. Each of the BC₂ plants generated was then allowed to self-pollinate to produce a BC₂F₁ generation which were also genotyped with KASP markers to identify plants homozygous for the 3G introgression (Fig. 1).

The KASP markers used to detect the presence of the 2G and 3G introgressions in durum were originally designed to detect the introgressions in a hexaploid background (King et al., 2022). These 68 markers were therefore screened in this programme, and only the markers polymorphic between T. timopheevii and all durum backgrounds were selected for genotyping of the introgression lines. The genotyping protocol was as described in Grewal et al. (2020b). Briefly, an automated PIPETMAX 268 (Gilson) was used to set up the genotyping reactions (1 ng genomic DNA, 2.5 µl KASP reaction mix, 0.068 µl primer mix and 2.43 µl nuclease-free water in a final volume of 5 µl) and performed in a ProFlex PCR system (Applied Biosystems by Life Technology). Polymerase chain reaction conditions were: 15 min at 94 ^oC; 10 touchdown cycles of 10 s at 94 ^oC, 1 min at 65 − 57 ^oC (dropping 0.8 ^oC per cycle); 35 cycles of 15 s at 94 ^oC, 1 min at 57 ^oC. Fluorescence detection of the reaction s was performed using a QantStudio 5 (Applied Biosystems) and the data analyzed using the QantStudio™ Design and Analysis Software V1.5.0 (Applied Biosystems).

Multi-colour GISH was used to check the wheat chromosome complement of all the durum BC₂F₁ plants identified as having a homozygous 3G introgression via KASP genotyping. Multi-colour GISH cannot identify the T. timopheevii 3G introgression as it is unable to distinguish between the G genome of T. timopheevii and the B genome of wheat.

Preparation of the root-tip metaphase chromosome spreads, the protocol for multi-colour GISH and the image capture was as described in Grewal et al. (2020b). Briefly, genomic DNA was extracted fromTriticum urartu (for detection of the A-genome), Aegilops speltoides Tausch (for detection of the B-genome) and Aegilops tauschii Coss. (for detection of the D-genome) as described above and labelled by nick translation with ChromaTide™ Alexa Fluor™ 488-5-dUTP (Invitrogen; C11397; coloured green), DEAC-dUTP (Jena Bioscience; NU-803-DEAC; coloured blueish purple) and ChromaTide™ AlexaFluor™ 594-5-dUTP (Invitrogen; C11400; coloured red) respectively. Slides were probed using 150 ng of T. urartu, 150 ng of Ae. speltoides and 300 ng of Ae. tauschii, in the ratio 3:3:6. Slides were counterstained using 4’,6-diamidino-2-phenylindole,dihydrochloride (DAPI) and analysed using a fully-automated Zeiss Axio ImagerZ2 upright epifluorescence microscope (Carl Zeiss Ltd). Image capture was performed using a MetaSystems Coolcube 1-m CCD camera and image analysis was carried out using Metafer4 (automated metaphase image capture) and ISIS (image processing) software (Metasystems GmbH).

Genomic DNA was extracted from the original hexaploid Tim5 and Tim6 homozygous introgression lines and for the homozygous durum lines as described for KASP genotyping. The DNA was sent to Novogene UK Ltd for library preparation and sequencing. Randon shearing of the genomic DNA obtained smaller fragments which were end-repaired, A-tailed and ligated with Illumina adapters. Purification of the fragments took place after size selection and PCR amplification after which the libraries were quantified via Qubit and qPCR and the size distribution again checked with a fragment analyser. Libraries were pooled and sequenced on Illumina NovaSeq 6000 S4 flowcells generating 150bp paired-end reads with an average coverage of 0.05x per library. A custom introgression mapping pipeline (King et al., 2025) was used to characterise introgression lines, where the sequence reads were mapped to a combined reference sequence consisting of concatenated assemblies of wheat cv. Chinese Spring RefSeqv2.1 (Zhu et al., (2021) and the T. timopheevii genome (Grewal et al., 2024).

Type 2 FHB resistance was assessed for the durum wheats CD4 and CDT and the introgression lines Tim5CD4 and Tim6CDC along with Paragon and the Tim5 and Tim6 hexaploid introgression lines in a polytunnel experiment at JIC in the summer of 2024. Inoculum (10µl) of a DON producing F. graminearum isolate (1x10⁶ conidia ml^− 1) was introduced directly into the central spikelet for between 10 to 27 individual spikes per line from multiple plants by point inoculation at mid-anthesis. Disease was assessed at 21dpi and the number of infected spikelets above and below the point of inoculation (POI) recorded.

Grain from the inoculated spikes from five plants of each line was harvested at maturity from both above and below the point of POI, and the ‘above’ and ‘below’ seed from each plant combined to produce each sample with plants acting as replicates. Four non-inoculated ears were also sampled in the same way for each line. Grain number and weight were determined for above and below the POI for both treated and non-treated plants. The hundred grain weight of treated spikes was calculated as a percentage of the control for each sampled plant to generate the relative grain weight (RGW).

Grain from above and below the POI was ground to a fine powder in a pestle and mortar and DON quantified using AgraStrip® Pro WATEX® lateral flow devices according to the manufacturer’s recommendations. All procedures were adjusted to account for the weight of sample relative to that used in routine analysis. 1g of flour of each sample was added to a 15 ml Falcon centrifuge tube. 5 ml of the AgraStrip® Pro WATEX® test kit extraction buffer was added to each tube, the tubes shaken for 2 mins and then centrifuged at 1902 x g for 1 min. 1 ml of AgraStrip® Pro WATEX® test kit dilution buffer was added to an Eppendorf tube, 100 µl supernatant of the sample extract added and mixed and centrifuged at 2000 x g for 30 secs. The AgraStrip® Pro Deoxynivalenol WATEX® lateral flow cartridge was inserted into the port of the AgraVision™ Pro reader, 200 µl diluted extract added to the lateral flow cartridge and results read from the machine following analysis.

Hexaploid wheat-T. timopheevii introgression lines Tim5 and Tim6, found to have FHB resistance (King et al. 2022; Steed et al., 2022), were crossed to four durum wheat genotypes to transfer the two 3G T. timopheevii introgressions from hexaploid wheat into durum wheat for FHB trait analysis (Fig. 1). Seed numbers at each generation were low (Table 1) which would have affected the number of plants recovered in the later generations that retained the required 3G introgressions. However, KASP analysis of BC₂F₁ plants generated, from crosses between Tim5 and Tim6 and the 4 durum wheats, suggested the durum background might have influenced fertility, e.g., no BC₂ seed with durum CCD as the background germinated (Table 1).

BC₂F₁ plants generated from crosses between Tim5, Tim6 and the four durum genotypes identified individuals derived from CD3 and CD4 homozygous for the T. timopheevii 3G introgression in Tim5. Further plants derived from crosses involving CDT were identified as being homozygous for the introgression carried in Tim6. Thus, while both the introgressions from Tim5 and Tim6 were successfully transferred to a durum background (successful combinations are shown in red in Table 1), they were not transferred to all the backgrounds used in the study.

Screening of the 68 KASP markers used to detect the 2G and 3G introgressions in hexaploid wheat, identified six markers able to detect the 2G introgressions in all durum backgrounds and four markers able to detect the 3G introgressions (Fig. 2 and Tables S1 and S2).

Multi-colour GISH, carried out on all the BC₂F₁ durum plants identified with KASP genotyping as homozygous for the 3G introgressions, confirmed that the durum chromosome complement had been restored, i.e. all plants were found to contain 28 chromosomes with 14 A-genome chromosomes and 14 B-genome chromosomes (Fig. 3).

Hexaploid Tim5 and Tim6 have been characterised by skim-sequencing previously (Steed et al. 2025). Skim-sequencing of their durum derivatives, Tim5CD3, Tim5CD4 and Tim6CDT, confirmed the presence of the homozygous 3G introgressions and that neither had been reduced in size compared to the original hexaploid lines, i.e., the larger 3G introgression from Tim6 was 615 Mbp and the smaller 3G introgression from Tim5 was 60Mbp in the durum lines (Fig. 4 and Table S3). Skim sequencing also confirmed the loss of the 2G segments in Tim5CD3, Tim5CD4 and Tim6CDT.

Point inoculation was used to assess Type 2 FHB resistance in the durum wheats CD4 and CDT and their derived durum introgression lines Tim5CD4 and Tim6CDT along with hexaploid wheat cv. Paragon and theTim5 and Tim6 hexaploid introgression lines in a polytunnel experiment at JIC in summer of 2024. The number of spikelets with FHB symptoms was assessed both above and below the POI. Representative spikes of each line are shown in Fig. 5. Tim6 had significantly less infected spikelets than Paragon both above and below the POI while Tim5 exhibited an intermediate level of resistance (Fig. 6). Both durum lines carrying 3G introgressions were significantly more resistant than their respective durum parents both below and above the POI (Fig. 6).

The 100-grain weight, both above and below the POI, as a percentage of the grain from the equivalent region of uninoculated controls was calculated for each line (Fig. 7). Relative grain weight above the POI was much less than that below for all lines. Both Tim5 and Tim6 had relatively greater grain weights than Paragon both above and below the POI. Relative grain weight in inoculated spikes of durum CD4 was much less than that in other lines, (29% below POI and 3% above POI) including CDT, and this broadly reflects the higher level of FHB symptoms observed in this line (Fig. 6). Relative grain weight of Tim5CD4 (carrying the small segment of 3G) was much greater than that of CD4, being almost 96% for spikelets below POI (Fig. 7).

The level of DON was assessed for the grain below the POI. The DON level in CDT (27 mg/kg) was similar to that in Paragon while the level of DON (2.5 mg/kg) in line Tim6CDT that contains the Tim6 introgression was significantly less (Fig. 8). The DON level in the line CD4 was very high (307 mg/kg) (Fig. 8) but the DON level in grain of its equivalent line carrying the Tim5 introgression was over ten-fold less (29 mg/kg) (Fig. 8). The DON level in Tim6CDT was also reduced by a similar amount relative to the CDT parent line. This is more evident in Fig. 8b where CD4 and Tim5CD4 have been removed to aid interpretation.