Transfer of wild relative introgressions into durum from hexaploid wheat for exploitation in research and breeding

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Two wheat- Triticum timopheevii introgression lines generated at the Wheat Research Centre (WRC) at the University of Nottingham were previously reported to carry Type 1 resistance to Fusarium head blight (FHB), with the resistance located to an introgression from chromosome 3G of T. timopheevii . Further analyses of these introgression lines, however, has recently shown that the 3G segment confers a potent type II resistance in hexaploid wheat, leading to reduced loss of grain weight and reduced deoxynivalenol (DON) accumulation in grain. Resistance levels to FHB are particularly low in durum wheat where resistance genes identified in hexaploid wheat have had little or no effect when transferred into durum wheat. Here we demonstrate that the 3G introgression is effective against FHB infection and DON accumulation in grain when transferred to a durum wheat background. durum wheat introgression Fusarium head blight breeding plant pathology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key message Introgressions from T. timopheevii have been successfully transferred from hexaploid wheat to durum demonstrating the feasibility of this approach. Initial analyses also showed the transfer of FHB resistance into durum. 1. Introduction Introgressions from wild relatives have had a major impact on wheat production. The spontaneous 1B/1R translocation from rye, which resulted in increased production and several diseases resistances (Crespo-Herrera et al., 2017), was present in many of the top wheat varieties in the 1980’s and is still present in some varieties today. However, while it was previously thought that only a handful of wheat varieties carried introgressions from wild relatives, recent sequence analysis has revealed that their presence is widespread (Przewieslik-Allen et al., 2021 ; Keilwagen et al., 2022 ; Schulthess et al., 2022 ; Heuberger et al., 2024 ). This observation raises the obvious question of why introgressions are so prevalent in elite wheat varieties, the most likely explanation being that they have been unconsciously selected for over time in breeding programmes as they confer selective advantages (He et al., 2019 ; Zhao et al., 2023 ). An example of this is the Aegilops ventricosa 2NS introgression, which confers a yield advantage, as well as the major source of wheat blast resistance. 2NS is present in over 85% of CIMMYT wheat varieties (Kishii, 2019 ). Wild relative introgression’s influence on production is not surprising since they provide a vast reservoir of genetic variation far above anything available in wheat. Many of the wild relatives have been subject to millions of years of evolution as compared to bread wheat which evolved once or twice only 10,000 years ago. Conventional breeding via the inter-crossing of different wheat genotypes has led to steady incremental improvements overtime, introgressions from wild relatives often result in jumps in improvement, e.g., the short arm of chromosome 1R from rye translocated to the long arm of chromosome 1B of wheat (Worland and Snape, 2014; Schlegel and Meinel, 1994 ; Villareal et al., 1991 ), T. dicoccoides introgressions in wheat variety Robigus and its progeny and present in 50% of the UK recommended list varieties since 2014 (Przewieslik-Allen et al., 2021 ). While the wild relatives have the potential to transform wheat improvement on a global scale only a tiny fraction of the available diversity has been exploited. The main reason for this is, that until recently, the technology required to routinely transfer variation into wheat and detect and characterise new wheat/wild relative introgressions on a large scale has been unavailable. With the advent of new sequencing technologies, coupled with marker development and improved strategies to induce recombination between the chromosomes of wheat and those of wild relatives, it is now possible to routinely transfer the genomes of wild relatives from a range of different species into tetraploid and hexaploid wheat. For example, 98.8% of the Aegilops mutica (King et al., 2025 ) and 98.9% of the T. timopheevii genomes (King et al., 2022 ) have been transferred into wheat backgrounds. In total, a series of lines carrying 135 wheat/ Ae. mutica and 204 wheat/ T. timopheevii introgressions have been generated (King et al., 2022 , 2019 , 2017 ; Devi et al., 2019 ). These lines can now be used to screen for genetic variation, present in the genomes of the accessions of Ae. mutica and T. timopheevii used to generate the introgressions (it is important to note that the type of genetic variation carried by different accessions is often not the same, particularly in outbreeding species). While trait analysis on the Ae. mutica and T. timopheevii introgression lines (King et.al. 2022 ; King et al., 2025 ) is still at an early-stage, genetic variation for a range of traits has already been identified, e.g. resistance to rust diseases (Fellers et al., 2020 ), resistance to Fusarium head blight (FHB), (Steed et al., 2022 ), higher grain zinc and iron concentrations (Guwela et al., 2024 ), and floral morphology for hybrid wheat production (Othmeni et al., 2025 ). FHB is a major disease of both hexaploid and tetraploid wheat with infection generally occurring during flowering when plants are most susceptible. While FHB is of major importance in bread wheat, it is even more of a problem in durum where existing resistance genes identified in bread wheat, e.g., Fhb1 located on chromosome 3B, may have little or no effect (Prat et al., 2014 ). Furthermore, while more than 250 QTL for FHB resistance have been identified in hexaploid wheat (Jia et al., 2018 ), only a small number have been identified in durum wheat (Zhao et al., 2018 ). Fusarium graminearum sensu stricto , Fusarium culmorum and Fusarium asiaticum can all produce trichothecene mycotoxins, e.g., deoxynivalenol (DON) and nivalenol which accumulate in the grain. These toxins are harmful to both humans and animals, and their accumulation is one of the most important factors in FHB disease (Amarasinghe et al., 2019 ). Resistance to FHB is generally classified into two broad types: resistance to initial infection (type 1) and resistance to disease spread in the spike (type 2) (Schroeder and Christensen 1963 ). Steed et al. ( 2022 ) reported the identification of two introgressions, differing in size but derived from the same region of T. timopheevii chromosome 3G, conferring strong FHB resistance in bread wheat. Recent experiments demonstrated that introgression of the segment of 3G confers potent type 2 resistance in hexaploid wheat, alongside reduced loss of grain weight and reduced DON accumulation in grain (Steed et al., 2025 ). In this paper we describe the transfer of these 3G introgressions into durum wheat and the initial analysis of their effectiveness against FHB infection and DON accumulation in the grain. 2. Materials and Methods 2.1. Generation of wheat lines carrying 3G introgressions 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 F 1 interspecific hybrids were re-pollinated with Paragon to generate backcross progeny (BC 1 , BC 2 , BC 3 , 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 ). 2.2. Transfer of the 3G introgressions into durum wheat 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 F 1 hybrids were then backcrossed twice with the same durum genotype from which they were initially derived to produce BC 2 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 BC 2 generation was determined using KASP analysis and plants both lacking the 3G introgression and retaining the 2G introgressions were discarded. Each of the BC 2 plants generated was then allowed to self-pollinate to produce a BC 2 F 1 generation which were also genotyped with KASP markers to identify plants homozygous for the 3G introgression (Fig. 1 ). 2.3. KASP genotyping 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 o C; 10 touchdown cycles of 10 s at 94 o C, 1 min at 65 − 57 o C (dropping 0.8 o C per cycle); 35 cycles of 15 s at 94 o C, 1 min at 57 o C. 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). 2.4. Multi-colour genomic in situ hybridisation (GISH) analysis Multi-colour GISH was used to check the wheat chromosome complement of all the durum BC 2 F 1 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 from Triticum 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). 2.5. Skim-sequencing 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 ). 2.6. FHB disease assessment by point inoculation 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 (1x10 6 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. 2.7. Grain weight analysis 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). 2.8. DON analysis methodology 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. 3. Results 3.1. Transfer of the 3G introgressions into durum wheat 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 BC 2 F 1 plants generated, from crosses between Tim5 and Tim6 and the 4 durum wheats, suggested the durum background might have influenced fertility, e.g., no BC 2 seed with durum CCD as the background germinated (Table 1 ). Table 1 Transfer of the 3G introgressions into durum wheat with the number of seed sown and germinated at each generation. Also shown are the number of plants that KASP markers identified with a heterozygous 3G introgression in the BC 2 and as homozygous in the BC 2 F 1 Crosses in red show successful combinations, i.e., durum wheats with homozygous 3G introgressions from Tim5 and Tim6. Cross No. of crosses F 1 germ/sown BC 1 germ/sown BC 2 germ/sown No. BC 2 3G het. BC 2 F 1 germ/sown No. BC 2 F 1 3G hom. Tim5 x CD3 6 5/19 8/13 14/18 4/14 25/53 4/25 Tim5 x CD4 6 1/2 3/6 7/15 4/7 33/55 9/33 Tim5 x CDT 5 1/18 4/5 17/17 0 - - Tim5 x CCD 6 4/8 4/5 0/10 - - - Tim6 x CD3 7 11/16 7/8 12/17 2/12 5/26 0 Tim6 x CD4 2 7/8 3/7 9/25 0 - - Tim6 x CDT 7 6/15 1/3 15/16 7/15 25/68 5/25 Tim6 x CCD 6 11/20 4/4 0/14 - - - BC 2 F 1 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. 3.2. KASP analysis 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 ). 3.3. Multi-colour GISH characterisation of durum introgression lines Multi-colour GISH, carried out on all the BC 2 F 1 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 ). 3.4. Skim-sequencing of Tim5 and Tim6 durum introgression lines 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. 3.4. FHB resistance assessment 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. 8 b where CD4 and Tim5CD4 have been removed to aid interpretation. 4. Discussion In the work described here, we demonstrate the feasibility of transferring Triticum timopheevii -derived 3G introgressions, initially developed in hexaploid wheat, into a durum background. The resulting plants possessed 28 chromosomes comprising 14A chromosomes, 12B chromosomes and two recombinant 3B/3G chromosomes at the BC 2 F 1 generation. This confirms that introgressions generated in a hexaploid context can be stabilised in tetraploid backgrounds through targeted backcrossing and marker-assisted selection. The two T. timopheevii 3G introgressions from Tim5 and Tim6 were not successfully transferred to all the durum genotypes, possibly reflecting background effects on fertility. However, given the low seed numbers recovered at each generation, it is more likely that limited plant numbers rather than true incompatibility restricted recovery. Increasing population size should enable successful transfer of both introgressions to all four durum genotypes, work that is currently ongoing at the Nottingham Wheat Research Centre (WRC). Combining KASP genotyping, multi-colour GISH and skim-sequencing proved essential for confirming the identity and integrity of the introgressions. KASP markers allowed precise tracking of both 3G and 2G introgressions from Tim5 and Tim6 across successive generations. GISH validated the restoration of the durum chromosome complement, confirming 14 A- and 14 B-genome chromosomes (including the 3B/3G recombinants), and absence of D-genome chromosomes. Skim-sequencing of the durum lines corroborated that the introgression segments in durum were identical in size to those in the original hexaploid lines. Together, these complementary tools provided robust confirmation of successful introgression transfer of the 3G region. The pilot analyses demonstrate that transferring wild relative introgressions from hexaploid wheat into durum could be an effective strategy for improving FHB resistance in durum wheat. Earlier attempts to introduce resistance loci from bread wheat into durum have often resulted in diminished expression of resistance (Zhu et al., 2022 ; Prat et al., 2017 ). In contrast, both durum lines carrying the T. timopheevii 3G introgressions exhibited strong type II resistance and markedly reduced DON accumulation compared with their recurrent durum parents. This reduction is particularly valuable because durum grain typically accumulates higher DON levels than bread wheat with comparable FHB severity (Gaikpa et al., 2019 ). Reducing DON contamination has both health and economic implications, as mycotoxin levels determine grain marketability. The estimated cost of downgrading wheat from food to feed due to excessive mycotoxins in Europe alone exceeds three billion euros over the past decade (Johns et al., 2022 ). Hence, the observed ten-fold reduction in DON in the durum introgression lines highlights the potential value of T. timopheevii -derived resistance for breeding safer and more resilient durum cultivars. While further analyses need to be undertaken both in the glasshouse and the field, the results described here are consistent with those described for hexaploid wheat (Steed et al., 2025 ). This work provides a further demonstration of the potentially critical role diversity from the wild relatives can play in strategic wheat breeding programmes. Technological advances in sequencing, marker development and recombination induction now make it possible to systematically transfer genetic variation from ancient wild relatives to both bread and durum wheat (Othmeni et al., 2019 ). The work described here provides a proof of concept for scaling such transfers, and large-scale programmes are now underway at the WRC to introduce hundreds of hexaploid-derived introgressions into durum backgrounds. Once fully characterised, these lines will be made freely available through collaborations with both public and private breeding partners to maximise their use in future wheat improvement. Declarations 7. Funding This research was funded by the Leverhulme Trust (Grant No. RPG-2022-044) and the Biotechnology and Biological Sciences Research Council [grant number BB/P016855/1] as part of the Designing Future Wheat (DFW) Programme. 8. Competing interests The authors have no relevant financial or non-financial interests to disclose. 9. Author contribution MO, CY, DS, SA, ASG, SG, IPK, JK: germplasm generation; MO, CY, DS, SA, ASG, KH: DNA extraction, KASP genotyping and GISH; SG: sequencing data analysis; PN, AS, RB: FHB screening and DON analysis; JK, IPK, SG, PN: conceptualisation and manuscript writing. JK, IPK, SG, PN: funding acquisition. All authors have read and approved the final version of the manuscript. 10. Data Availability The raw skim-sequence reads for all wheat- T. timopheevii introgression lines have been deposited at the European Nucleotide Archive (ENA) under project accession PRJEB89936. The raw genotyping data is available from the authors on request. 11. Code Availability The custom scripts for the combined introgression mapping pipeline are available via https://github.com/Surbhigrewal/Introgression_mapping. References Adhikari L, Shrestha S, Wu S, Crain J, Gao L, Evers B, Wilson D, Ju Y, Koo DH, Hucl P, Pozniak C, Walkowiak S, Wang X, Wu J, Glaubitz JC, DeHaan L, Friebe B, Poland J (2022). 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TPG 15: e20183. https://doi.org/10.1002/tpg2.20183 Zhu T, Wang L, Rimbert H, Rodriguez JC, Deal KR, De Oliveira R, Choulet F, Keeble-Gagnère G, Tibbits J, Rogers J, Eversole K, Appels R, Gu YQ, Mascher M, Dvorak J, Luo M-C (2021). Optical maps refine the bread wheat Triticum aestivum cv. Chinese Spring genome assembly. Plant J. 107: 303-314. https://doi.org/10.1111/tpj.15289 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTables.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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17:54:05","extension":"html","order_by":51,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154518,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/8e4b2ec77ff1a0de0f1febbb.html"},{"id":98342294,"identity":"49425fe5-41a3-4e8c-ad82-80ab9d9550d7","added_by":"auto","created_at":"2025-12-16 17:54:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":171562,"visible":true,"origin":"","legend":"\u003cp\u003eCrossing scheme for the transfer of the \u003cem\u003eT. timopheevii\u003c/em\u003e 3G introgressions, carrying FHB resistance, from hexaploid wheat lines Tim5 and Tim6 to durum wheat genotypes CD3, CD4, CDT and CCD.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/d58c5bb1ee0efdb96bc8aab3.png"},{"id":98342335,"identity":"69bb5c34-1793-4cdd-9868-8a47234d6f94","added_by":"auto","created_at":"2025-12-16 17:54:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":268001,"visible":true,"origin":"","legend":"\u003cp\u003eKASP genotyping of the \u003cem\u003eT. timopheevii\u003c/em\u003e introgressions in the durum wheat backgrounds CD3, CD4 and CDT derived from parental introgression lines Tim5 and Tim6 in bread wheat background cv. Paragon. A call of ‘Wh’ (coloured blue) shows a homozygous wheat call and a call of ‘Tt’ (coloured green) shows a homozygous \u003cem\u003eT. timopheevii\u003c/em\u003e call.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/eb21eccf31717773c38e504f.png"},{"id":98342352,"identity":"e0b84f23-70e2-4bfb-aa07-d7b98a6f5c0d","added_by":"auto","created_at":"2025-12-16 17:54:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2503937,"visible":true,"origin":"","legend":"\u003cp\u003eMulti-colour GISH of metaphase spreads from BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e plants shown to be homozygous for the 3G introgressions. (a) Tim5CD3 (b) Tim5CD4 (c) Tim6CDT. The A-genome chromosomes are shown in green and the B-genome chromosomes in purple.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/50b21d62ff9ee490a1a4ac1d.png"},{"id":98342245,"identity":"3e5b59c9-b10c-4cf1-8fa5-eee27e06521e","added_by":"auto","created_at":"2025-12-16 17:54:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1495960,"visible":true,"origin":"","legend":"\u003cp\u003eSkim-sequencing of the homozygous durum introgression lines. (a) Tim5CD3, (b) Tim5CD4 and (c) Tim6CDT. Red points indicate regions with abnormal coverage. Coverage deviation towards 1 in the \u003cem\u003eT. timopheevii\u003c/em\u003e Chr3G indicates the presence of that region of the chromosome in the introgression line. Coverage deviation towards 0 on Chr3B indicates the equivalent loss of that region of the chromosome in the introgression line.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/3759a0ca60822e2b7807c652.png"},{"id":98342244,"identity":"991c9e00-32fc-418e-a958-99dd4a9726ab","added_by":"auto","created_at":"2025-12-16 17:54:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":700192,"visible":true,"origin":"","legend":"\u003cp\u003ePoint Inoculated spikes of (a) Hexaploid lines Paragon, Tim5 and Tim6, (b) CD3 and its derived introgression line Tim5CD3, (c) CDT and two spikes of its derived introgression line Tim6CDT and (d) CD4 and its derived introgression line Tim5CD4.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/022654c1d746725cd385ae1a.png"},{"id":98342325,"identity":"34c78fa1-3179-4156-9c14-9c460c979bc1","added_by":"auto","created_at":"2025-12-16 17:54:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":31849,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of infected spikelets (a) above and (b) below the POI at 21 dpi in Paragon, Tim5 and Tim6 hexaploid introgression lines, CD4, CDT and Tim5CD4 and Tim6CDT introgression lines.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/d0bd9dbd408ccf128a7513ed.png"},{"id":98439502,"identity":"de6e2176-68d5-4f95-97b3-a90f5ed88dd6","added_by":"auto","created_at":"2025-12-17 17:02:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":33290,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted mean for calculated 100 grain weight as a percentage of the grain from uninoculated control plants (a) above and (b) below POI in Paragon, Tim5 and Tim6 hexaploid introgression lines, CD4, CDT and Tim5CD4 and Tim6CDT introgression lines.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/e0912f0774b41f13127eeb6c.png"},{"id":98438995,"identity":"9e9e5877-d437-49b0-a070-5dba050b64d9","added_by":"auto","created_at":"2025-12-17 17:00:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":30464,"visible":true,"origin":"","legend":"\u003cp\u003eDON content of grain below the POI in (a) Paragon, Tim5 and Tim6 hexaploid introgression lines, CD4, CDT and Tim5CD4 and Tim6CDT introgression lines and in\u003cstrong\u003e \u003c/strong\u003e(b) the same lines as (a) except for CD4 and Tim5CD4, to reveal the differences between DON levels in other lines.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/1eda5b676a6d27def19d4263.png"},{"id":104552348,"identity":"0f8c7868-d9df-4ad0-8417-7cb28d2fcc20","added_by":"auto","created_at":"2026-03-13 08:27:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5995821,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/0b58358b-bf08-4332-bd7f-1197b79d3f2a.pdf"},{"id":98342344,"identity":"4aec7155-1fdc-43e8-a50f-935ec09ca7e2","added_by":"auto","created_at":"2025-12-16 17:54:06","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14511,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8261341/v1/da12df907b5a31cd9853a549.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transfer of wild relative introgressions into durum from hexaploid wheat for exploitation in research and breeding","fulltext":[{"header":"Key message","content":"\u003cp\u003eIntrogressions from \u003cem\u003eT. timopheevii\u003c/em\u003e have been successfully transferred from hexaploid wheat to durum demonstrating the feasibility of this approach. Initial analyses also showed the transfer of FHB resistance into durum.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eIntrogressions from wild relatives have had a major impact on wheat production. The spontaneous 1B/1R translocation from rye, which resulted in increased production and several diseases resistances (Crespo-Herrera et al., 2017), was present in many of the top wheat varieties in the 1980\u0026rsquo;s and is still present in some varieties today. However, while it was previously thought that only a handful of wheat varieties carried introgressions from wild relatives, recent sequence analysis has revealed that their presence is widespread (Przewieslik-Allen et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Keilwagen et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Schulthess et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Heuberger et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This observation raises the obvious question of why introgressions are so prevalent in elite wheat varieties, the most likely explanation being that they have been unconsciously selected for over time in breeding programmes as they confer selective advantages (He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). An example of this is the \u003cem\u003eAegilops ventricosa\u003c/em\u003e 2NS introgression, which confers a yield advantage, as well as the major source of wheat blast resistance. 2NS is present in over 85% of CIMMYT wheat varieties (Kishii, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWild relative introgression\u0026rsquo;s influence on production is not surprising since they provide a vast reservoir of genetic variation far above anything available in wheat. Many of the wild relatives have been subject to millions of years of evolution as compared to bread wheat which evolved once or twice only 10,000 years ago. Conventional breeding via the inter-crossing of different wheat genotypes has led to steady incremental improvements overtime, introgressions from wild relatives often result in jumps in improvement, e.g., the short arm of chromosome 1R from rye translocated to the long arm of chromosome 1B of wheat (Worland and Snape, 2014; Schlegel and Meinel, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Villareal et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), T. \u003cem\u003edicoccoides\u003c/em\u003e introgressions in wheat variety Robigus and its progeny and present in 50% of the UK recommended list varieties since 2014 (Przewieslik-Allen et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile the wild relatives have the potential to transform wheat improvement on a global scale only a tiny fraction of the available diversity has been exploited. The main reason for this is, that until recently, the technology required to routinely transfer variation into wheat and detect and characterise new wheat/wild relative introgressions on a large scale has been unavailable. With the advent of new sequencing technologies, coupled with marker development and improved strategies to induce recombination between the chromosomes of wheat and those of wild relatives, it is now possible to routinely transfer the genomes of wild relatives from a range of different species into tetraploid and hexaploid wheat. For example, 98.8% of the \u003cem\u003eAegilops mutica\u003c/em\u003e (King et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and 98.9% of the \u003cem\u003eT. timopheevii\u003c/em\u003e genomes (King et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) have been transferred into wheat backgrounds. In total, a series of lines carrying 135 wheat/\u003cem\u003eAe. mutica\u003c/em\u003e and 204 wheat/\u003cem\u003eT. timopheevii\u003c/em\u003e introgressions have been generated (King et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Devi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These lines can now be used to screen for genetic variation, present in the genomes of the accessions of \u003cem\u003eAe. mutica and T. timopheevii\u003c/em\u003e used to generate the introgressions (it is important to note that the type of genetic variation carried by different accessions is often not the same, particularly in outbreeding species).\u003c/p\u003e \u003cp\u003eWhile trait analysis on the \u003cem\u003eAe. mutica\u003c/em\u003e and \u003cem\u003eT. timopheevii\u003c/em\u003e introgression lines (King et.al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; King et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) is still at an early-stage, genetic variation for a range of traits has already been identified, e.g. resistance to rust diseases (Fellers et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), resistance to Fusarium head blight (FHB), (Steed et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), higher grain zinc and iron concentrations (Guwela et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and floral morphology for hybrid wheat production (Othmeni et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFHB is a major disease of both hexaploid and tetraploid wheat with infection generally occurring during flowering when plants are most susceptible. While FHB is of major importance in bread wheat, it is even more of a problem in durum where existing resistance genes identified in bread wheat, e.g., \u003cem\u003eFhb1\u003c/em\u003e located on chromosome 3B, may have little or no effect (Prat et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Furthermore, while more than 250 QTL for FHB resistance have been identified in hexaploid wheat (Jia et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), only a small number have been identified in durum wheat (Zhao et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eFusarium graminearum sensu stricto\u003c/em\u003e, \u003cem\u003eFusarium culmorum\u003c/em\u003e and \u003cem\u003eFusarium asiaticum\u003c/em\u003e can all produce trichothecene mycotoxins, e.g., deoxynivalenol (DON) and nivalenol which accumulate in the grain. These toxins are harmful to both humans and animals, and their accumulation is one of the most important factors in FHB disease (Amarasinghe et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Resistance to FHB is generally classified into two broad types: resistance to initial infection (type 1) and resistance to disease spread in the spike (type 2) (Schroeder and Christensen \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1963\u003c/span\u003e). Steed et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported the identification of two introgressions, differing in size but derived from the same region of \u003cem\u003eT. timopheevii\u003c/em\u003e chromosome 3G, conferring strong FHB resistance in bread wheat. Recent experiments demonstrated that introgression of the segment of 3G confers potent type 2 resistance in hexaploid wheat, alongside reduced loss of grain weight and reduced DON accumulation in grain (Steed et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this paper we describe the transfer of these 3G introgressions into durum wheat and the initial analysis of their effectiveness against FHB infection and DON accumulation in the grain.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Generation of wheat lines carrying 3G introgressions\u003c/h2\u003e \u003cp\u003eThe 3G introgressions were generated from a programme designed to transfer large numbers of chromosome segments from the \u003cem\u003eT. timopheevii\u003c/em\u003e A\u003csup\u003et\u003c/sup\u003e and G genomes into wheat (Devi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In summary, \u003cem\u003eT. timopheevii\u003c/em\u003e was used to pollinate the variety Paragon, which carried the \u003cem\u003eph1\u003c/em\u003e mutation (Devi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The resulting F\u003csub\u003e1\u003c/sub\u003e interspecific hybrids were re-pollinated with Paragon to generate backcross progeny (BC\u003csub\u003e1\u003c/sub\u003e, BC\u003csub\u003e2\u003c/sub\u003e, BC\u003csub\u003e3\u003c/sub\u003e, etc) which were self-pollinated. The backcross progeny were initially screened using the Axiom\u0026reg; Wild-Relative Genotyping Array (Devi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) to identify introgressions. However, later generations were reanalysed using chromosome-specific KASP markers (Grewal et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e) to detect lines of wheat homozygous for \u003cem\u003eT. timopheevii\u003c/em\u003e A\u003csup\u003et\u003c/sup\u003e and G genome introgressions (King et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The \u003cem\u003eT. timopheevii\u003c/em\u003e 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., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and identified as carrying resistance to FHB. To determine if any of the homozygous \u003cem\u003eT. timopheevii\u003c/em\u003e introgressions generated carried the resistance to FHB a series of them were screened (Steed et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Both Tim5 and Tim6 carry an additional homozygous introgression from 2G of \u003cem\u003eT. timopheevii\u003c/em\u003e (King et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Transfer of the 3G introgressions into durum wheat\u003c/h2\u003e \u003cp\u003eThe 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 F\u003csub\u003e1\u003c/sub\u003e hybrids were then backcrossed twice with the same durum genotype from which they were initially derived to produce BC\u003csub\u003e2\u003c/sub\u003e populations. As the FHB resistance had previously been located to the 3G introgressions (Steed et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the presence or absence of the 2G and 3G introgressions in plants in the BC\u003csub\u003e2\u003c/sub\u003e generation was determined using KASP analysis and plants both lacking the 3G introgression and retaining the 2G introgressions were discarded. Each of the BC\u003csub\u003e2\u003c/sub\u003e plants generated was then allowed to self-pollinate to produce a BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e generation which were also genotyped with KASP markers to identify plants homozygous for the 3G introgression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. KASP genotyping\u003c/h2\u003e \u003cp\u003eThe 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., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These 68 markers were therefore screened in this programme, and only the markers polymorphic between \u003cem\u003eT. timopheevii\u003c/em\u003e and all durum backgrounds were selected for genotyping of the introgression lines. The genotyping protocol was as described in Grewal et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). Briefly, an automated PIPETMAX 268 (Gilson) was used to set up the genotyping reactions (1 ng genomic DNA, 2.5 \u0026micro;l KASP reaction mix, 0.068 \u0026micro;l primer mix and 2.43 \u0026micro;l nuclease-free water in a final volume of 5 \u0026micro;l) and performed in a ProFlex PCR system (Applied Biosystems by Life Technology). Polymerase chain reaction conditions were: 15 min at 94 \u003csup\u003eo\u003c/sup\u003eC; 10 touchdown cycles of 10 s at 94 \u003csup\u003eo\u003c/sup\u003eC, 1 min at 65\u0026thinsp;\u0026minus;\u0026thinsp;57 \u003csup\u003eo\u003c/sup\u003eC (dropping 0.8 \u003csup\u003eo\u003c/sup\u003eC per cycle); 35 cycles of 15 s at 94 \u003csup\u003eo\u003c/sup\u003eC, 1 min at 57 \u003csup\u003eo\u003c/sup\u003eC. Fluorescence detection of the reaction s was performed using a QantStudio 5 (Applied Biosystems) and the data analyzed using the QantStudio\u0026trade; Design and Analysis Software V1.5.0 (Applied Biosystems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Multi-colour genomic \u003cem\u003ein situ\u003c/em\u003e hybridisation (GISH) analysis\u003c/h2\u003e \u003cp\u003eMulti-colour GISH was used to check the wheat chromosome complement of all the durum BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e plants identified as having a homozygous 3G introgression via KASP genotyping. Multi-colour GISH cannot identify the \u003cem\u003eT. timopheevii\u003c/em\u003e 3G introgression as it is unable to distinguish between the G genome of \u003cem\u003eT. timopheevii\u003c/em\u003e and the B genome of wheat.\u003c/p\u003e \u003cp\u003ePreparation of the root-tip metaphase chromosome spreads, the protocol for multi-colour GISH and the image capture was as described in Grewal et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). Briefly, genomic DNA was extracted from\u003cem\u003eTriticum urartu\u003c/em\u003e (for detection of the A-genome), \u003cem\u003eAegilops speltoides\u003c/em\u003e Tausch (for detection of the B-genome) and \u003cem\u003eAegilops tauschii\u003c/em\u003e Coss. (for detection of the D-genome) as described above and labelled by nick translation with ChromaTide\u0026trade; Alexa Fluor\u0026trade; 488-5-dUTP (Invitrogen; C11397; coloured green), DEAC-dUTP (Jena Bioscience; NU-803-DEAC; coloured blueish purple) and ChromaTide\u0026trade; AlexaFluor\u0026trade; 594-5-dUTP (Invitrogen; C11400; coloured red) respectively. Slides were probed using 150 ng of \u003cem\u003eT. urartu\u003c/em\u003e, 150 ng of \u003cem\u003eAe. speltoides\u003c/em\u003e and 300 ng of \u003cem\u003eAe. tauschii\u003c/em\u003e, in the ratio 3:3:6. Slides were counterstained using 4\u0026rsquo;,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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Skim-sequencing\u003c/h2\u003e \u003cp\u003eGenomic 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., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) 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., (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and the \u003cem\u003eT. timopheevii\u003c/em\u003e genome (Grewal et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. FHB disease assessment by point inoculation\u003c/h2\u003e \u003cp\u003eType 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 (10\u0026micro;l) of a DON producing \u003cem\u003eF. graminearum\u003c/em\u003e isolate (1x10\u003csup\u003e6\u003c/sup\u003e conidia ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Grain weight analysis\u003c/h2\u003e \u003cp\u003eGrain 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 \u0026lsquo;above\u0026rsquo; and \u0026lsquo;below\u0026rsquo; 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. DON analysis methodology\u003c/h2\u003e \u003cp\u003eGrain from above and below the POI was ground to a fine powder in a pestle and mortar and DON quantified using AgraStrip\u0026reg; Pro WATEX\u0026reg; lateral flow devices according to the manufacturer\u0026rsquo;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\u0026reg; Pro WATEX\u0026reg; 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\u0026reg; Pro WATEX\u0026reg; test kit dilution buffer was added to an Eppendorf tube, 100 \u0026micro;l supernatant of the sample extract added and mixed and centrifuged at 2000 x g for 30 secs. The AgraStrip\u0026reg; Pro Deoxynivalenol WATEX\u0026reg; lateral flow cartridge was inserted into the port of the AgraVision\u0026trade; Pro reader, 200 \u0026micro;l diluted extract added to the lateral flow cartridge and results read from the machine following analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Transfer of the 3G introgressions into durum wheat\u003c/h2\u003e \u003cp\u003eHexaploid wheat-\u003cem\u003eT. timopheevii\u003c/em\u003e introgression lines Tim5 and Tim6, found to have FHB resistance (King et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Steed et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), were crossed to four durum wheat genotypes to transfer the two 3G \u003cem\u003eT. timopheevii\u003c/em\u003e introgressions from hexaploid wheat into durum wheat for FHB trait analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Seed numbers at each generation were low (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) which would have affected the number of plants recovered in the later generations that retained the required 3G introgressions. However, KASP analysis of BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e plants generated, from crosses between Tim5 and Tim6 and the 4 durum wheats, suggested the durum background might have influenced fertility, e.g., no BC\u003csub\u003e2\u003c/sub\u003e seed with durum CCD as the background germinated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTransfer of the 3G introgressions into durum wheat with the number of seed sown and germinated at each generation. Also shown are the number of plants that KASP markers identified with a heterozygous 3G introgression in the BC\u003csub\u003e2\u003c/sub\u003e and as homozygous in the BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e Crosses in red show successful combinations, i.e., durum wheats with homozygous 3G introgressions from Tim5 and Tim6.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCross\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of crosses\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003cp\u003egerm/sown\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBC\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003cp\u003egerm/sown\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBC\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003cp\u003egerm/sown\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo. BC\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e3G het.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003cp\u003egerm/sown\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo. BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e 3G hom.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTim5 x CD3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5/19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8/13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14/18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25/53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4/25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTim5 x CD4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1/2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3/6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7/15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33/55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9/33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTim5 x CDT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1/18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17/17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTim5 x CCD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTim6 x CD3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11/16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12/17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2/12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5/26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTim6 x CD4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3/7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9/25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTim6 x CDT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6/15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15/16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7/15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25/68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5/25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTim6 x CCD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11/20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e plants generated from crosses between Tim5, Tim6 and the four durum genotypes identified individuals derived from CD3 and CD4 homozygous for the \u003cem\u003eT. timopheevii\u003c/em\u003e 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\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), they were not transferred to all the backgrounds used in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. KASP analysis\u003c/h2\u003e \u003cp\u003eScreening 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand Tables S1 and S2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Multi-colour GISH characterisation of durum introgression lines\u003c/h2\u003e \u003cp\u003eMulti-colour GISH, carried out on all the BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Skim-sequencing of Tim5 and Tim6 durum introgression lines\u003c/h2\u003e \u003cp\u003eHexaploid Tim5 and Tim6 have been characterised by skim-sequencing previously (Steed et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003eand Table S3\u003c/b\u003e). Skim sequencing also confirmed the loss of the 2G segments in Tim5CD3, Tim5CD4 and Tim6CDT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. FHB resistance assessment\u003c/h2\u003e \u003cp\u003ePoint 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Tim6 had significantly less infected spikelets than Paragon both above and below the POI while Tim5 exhibited an intermediate level of resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Both durum lines carrying 3G introgressions were significantly more resistant than their respective durum parents both below and above the POI (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The DON level in the line CD4 was very high (307 mg/kg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) but the DON level in grain of its equivalent line carrying the Tim5 introgression was over ten-fold less (29 mg/kg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The DON level in Tim6CDT was also reduced by a similar amount relative to the CDT parent line. This is more evident in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb where CD4 and Tim5CD4 have been removed to aid interpretation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the work described here, we demonstrate the feasibility of transferring \u003cem\u003eTriticum timopheevii\u003c/em\u003e-derived 3G introgressions, initially developed in hexaploid wheat, into a durum background. The resulting plants possessed 28 chromosomes comprising 14A chromosomes, 12B chromosomes and two recombinant 3B/3G chromosomes at the BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e generation. This confirms that introgressions generated in a hexaploid context can be stabilised in tetraploid backgrounds through targeted backcrossing and marker-assisted selection.\u003c/p\u003e \u003cp\u003eThe two \u003cem\u003eT. timopheevii\u003c/em\u003e 3G introgressions from Tim5 and Tim6 were not successfully transferred to all the durum genotypes, possibly reflecting background effects on fertility. However, given the low seed numbers recovered at each generation, it is more likely that limited plant numbers rather than true incompatibility restricted recovery. Increasing population size should enable successful transfer of both introgressions to all four durum genotypes, work that is currently ongoing at the Nottingham Wheat Research Centre (WRC).\u003c/p\u003e \u003cp\u003eCombining KASP genotyping, multi-colour GISH and skim-sequencing proved essential for confirming the identity and integrity of the introgressions. KASP markers allowed precise tracking of both 3G and 2G introgressions from Tim5 and Tim6 across successive generations. GISH validated the restoration of the durum chromosome complement, confirming 14 A- and 14 B-genome chromosomes (including the 3B/3G recombinants), and absence of D-genome chromosomes. Skim-sequencing of the durum lines corroborated that the introgression segments in durum were identical in size to those in the original hexaploid lines. Together, these complementary tools provided robust confirmation of successful introgression transfer of the 3G region.\u003c/p\u003e \u003cp\u003eThe pilot analyses demonstrate that transferring wild relative introgressions from hexaploid wheat into durum could be an effective strategy for improving FHB resistance in durum wheat. Earlier attempts to introduce resistance loci from bread wheat into durum have often resulted in diminished expression of resistance (Zhu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Prat et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In contrast, both durum lines carrying the \u003cem\u003eT. timopheevii\u003c/em\u003e 3G introgressions exhibited strong type II resistance and markedly reduced DON accumulation compared with their recurrent durum parents. This reduction is particularly valuable because durum grain typically accumulates higher DON levels than bread wheat with comparable FHB severity (Gaikpa et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReducing DON contamination has both health and economic implications, as mycotoxin levels determine grain marketability. The estimated cost of downgrading wheat from food to feed due to excessive mycotoxins in Europe alone exceeds three billion euros over the past decade (Johns et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Hence, the observed ten-fold reduction in DON in the durum introgression lines highlights the potential value of \u003cem\u003eT. timopheevii\u003c/em\u003e-derived resistance for breeding safer and more resilient durum cultivars. While further analyses need to be undertaken both in the glasshouse and the field, the results described here are consistent with those described for hexaploid wheat (Steed et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis work provides a further demonstration of the potentially critical role diversity from the wild relatives can play in strategic wheat breeding programmes. Technological advances in sequencing, marker development and recombination induction now make it possible to systematically transfer genetic variation from ancient wild relatives to both bread and durum wheat (Othmeni et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The work described here provides a proof of concept for scaling such transfers, and large-scale programmes are now underway at the WRC to introduce hundreds of hexaploid-derived introgressions into durum backgrounds. Once fully characterised, these lines will be made freely available through collaborations with both public and private breeding partners to maximise their use in future wheat improvement.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e7. Funding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Leverhulme Trust (Grant No. RPG-2022-044) and the Biotechnology and Biological Sciences Research Council [grant number\u0026nbsp;BB/P016855/1] as part of the Designing Future Wheat (DFW) Programme.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Competing interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Author contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMO, CY, DS, SA, ASG, SG, IPK, JK: germplasm generation; MO, CY, DS, SA, ASG, KH: DNA extraction, KASP genotyping and GISH; SG: sequencing data analysis; PN, AS, RB: FHB screening and DON analysis; JK, IPK, SG, PN: conceptualisation and manuscript writing. JK, IPK, SG, PN: funding acquisition. All authors have read and approved the final version of the manuscript. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10. Data Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw skim-sequence reads for all wheat-\u003cem\u003eT. timopheevii\u003c/em\u003e introgression lines have been deposited at the European Nucleotide Archive (ENA) under project accession PRJEB89936. The raw genotyping data is available from the authors on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e11. Code Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe custom scripts for the combined introgression mapping pipeline are available via https://github.com/Surbhigrewal/Introgression_mapping.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdhikari L, Shrestha S, Wu S, Crain J, Gao L, Evers B, Wilson D, Ju Y, Koo DH, Hucl P, Pozniak C, Walkowiak S, Wang X, Wu J, Glaubitz JC, DeHaan L, Friebe B, Poland J (2022). A high-throughput skim-sequencing approach for genotyping, dosage estimation and identifying translocations. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e12:\u003c/strong\u003e 17583. https://doi.org/10.1038/s41598-022-19858-2\u003c/li\u003e\n\u003cli\u003eAmarasinghe C, Sharanowski B, Fernando WGD (2019). 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Chinese Spring genome assembly. \u003cem\u003ePlant J.\u003c/em\u003e \u003cstrong\u003e107:\u003c/strong\u003e 303-314. https://doi.org/10.1111/tpj.15289\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"durum wheat, introgression, Fusarium head blight, breeding, plant pathology","lastPublishedDoi":"10.21203/rs.3.rs-8261341/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8261341/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntrogressions of wild relative chromosome segments into the genome of wheat provide an almost untapped source of genetic variation. Two wheat-\u003cem\u003eTriticum timopheevii\u003c/em\u003e introgression lines generated at the Wheat Research Centre (WRC) at the University of Nottingham were previously reported to carry Type 1 resistance to Fusarium head blight (FHB), with the resistance located to an introgression from chromosome 3G of \u003cem\u003eT. timopheevii\u003c/em\u003e. Further analyses of these introgression lines, however, has recently shown that the 3G segment confers a potent type II resistance in hexaploid wheat, leading to reduced loss of grain weight and reduced deoxynivalenol (DON) accumulation in grain. Resistance levels to FHB are particularly low in durum wheat where resistance genes identified in hexaploid wheat have had little or no effect when transferred into durum wheat. Here we demonstrate that the 3G introgression is effective against FHB infection and DON accumulation in grain when transferred to a durum wheat background.\u003c/p\u003e","manuscriptTitle":"Transfer of wild relative introgressions into durum from hexaploid wheat for exploitation in research and breeding","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 17:53:28","doi":"10.21203/rs.3.rs-8261341/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"17af3bd7-f6ff-4bb5-9ff3-371699338d0c","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-13T08:27:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-16 17:53:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8261341","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8261341","identity":"rs-8261341","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}