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Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars., Comparison of multiple genome assemblies from wheat reveals extensive diversity that results from the complex breeding history of wheat and provides a basis for further potential improvements to this important food crop.

Wheat remains a critical global food source, pressured by climate change and the need to maximize yield, improve processing and nutritional quality and ensure safety. An enormous amount of research has been conducted to understand gluten protein composition and structure in relation to end-use quality, yet progress has become stagnant. This is mainly due to the need and inability to biochemically characterize the intact functional glutenin polymer in order to correlate to quality, necessitating reduction to monomeric subunits and a loss of contextual information. While some individual gluten proteins might have a positive or negative influence on gluten quality, it is the sum total of these proteins, their relative and absolute expression, their sub-cellular trafficking, the amount and size of glutenin polymers, and ratios between gluten protein classes that define viscoelasticity of gluten. The sub-cellular trafficking of gluten proteins during seed maturation is still not completely clear and there is evidence of dual pathways and therefore different destinations for proteins, either constitutively or temporally. The trafficking of proteins is also unclear in endosperm cells as they undergo programmed cell death; Golgi disappear around 12 DPA but protein filling continues at least to 25 DPA. Modulation of the timing of cellular events will invariably affect protein deposition and therefore gluten strength and function. Existing and emerging proteomics technologies such as proteoform profiling and top-down proteomics offer new tools to study gluten protein composition as a whole system and identify compositional patterns that can modify gluten structure with improved functionality.

Fusarium infection is a worldwide agricultural problem of billion dollar proportions globally, and it has increasingly threatened entire regional food supplies. In addition to the toxin deoxynivalenol (DON), Fusarium species express digestive enzymes that degrade starch and protein, affecting the quality of infected grains, especially wheat processing performance which depends largely on gluten proteins. In this study, the impact of Fusarium protease on the functionality of Canada Western Red Spring (CWRS) wheat was assessed by adding Fusarium-damaged kernels (FDK) to a FDK-free base wheat sample. Digestion of beta-casein by extracts of flours, milled from sound and FDK-spiked wheat samples, demonstrated elevated cleavage in FDK-spiked flour extracts as follows: N-terminal to lysine (eight-fold), N- and C-terminal to isoleucine (four-fold and three-fold, respectively), N-terminal to tyrosine (three-fold) and C-terminal to arginine at P1′ (five-fold). Comparison of abbreviated (45 min) and standard (135 min) extensigraph test results indicated that desirable increases in dough resistance to extension (Rmax) due to gluten re-polymerization after longer resting were partially to completely counteracted in FDK-spiked flours in a dose-dependent manner. Baking tests confirmed that while loaf volume is similar, proofed dough from FDK-spiked samples caused detectable loaf collapse at 3% FDK. Extensigraph Rmax and Fusarium protease levels were inversely related, and effected by both the extent and severity of infection. While the current FDK tolerances for grading Canadian wheat can effectively control protease damage, prevalence of deoxynivalenol (DON) weak- and non-producing Fusarium strains/species (e.g., F. avenaceum) in some growing regions must be considered to protect functionality if grading is solely based on DON content.

In this study, changes in hydrophobic interactions among gluten proteins were analyzed during dough mixing. Size-exclusion high-performance chromatography and two-dimensional fluorescence difference gel electrophoresis were performed on proteins extracted with 1-propanol by weakening the hydrophobic interaction. The amount of proteins extracted with 30% 1-propanol increased from the start of mixing to peak consistency, suggesting that the hydrophobic interactions among the strongly aggregated proteins weakened and resulted in disaggregation. The amount of proteins extracted with 10% 1-propanol decreased during hydration, indicating that these proteins aggregated through relatively weak hydrophobic interactions. The proteins that extractability decreased were mainly low molecular weight glutenin, α-gliadin, and γ-gliadin. The amount of monomeric proteins extracted with 30% 1-propanol decreased after peak consistency. The decreased protein was mainly ω-gliadin, indicating that ω-gliadin aggregated with other proteins through hydrophobic interactions. A front-face fluorescence analysis was performed on the dough with the addition of 8-anilino-1-naphthalenesulfonic acid or thioflavin T. The fluorescence intensity increased as a result of exposure to the hydrophobic groups of the gluten proteins and the formation of protein aggregates during dough mixing. These results indicate the importance of hydrophobic interactions in dough formation.

Fusarium infection is a worldwide agricultural problem of billion dollar proportions globally, and it has increasingly threatened entire regional food supplies. In addition to the toxin deoxynivalenol (DON), Fusarium species express digestive enzymes that degrade starch and protein, affecting the quality of infected grains, especially wheat processing performance which depends largely on gluten proteins. In this study, the impact of Fusarium protease on the functionality of Canada Western Red Spring (CWRS) wheat was assessed by adding Fusarium-damaged kernels (FDK) to a FDK-free base wheat sample. Digestion of beta-casein by extracts of flours, milled from sound and FDK-spiked wheat samples, demonstrated elevated cleavage in FDK-spiked flour extracts as follows: N-terminal to lysine (eight-fold), N- and C-terminal to isoleucine (four-fold and three-fold, respectively), N-terminal to tyrosine (three-fold) and C-terminal to arginine at P1′ (five-fold). Comparison of abbreviated (45 min) and standard (135 min) extensigraph test results indicated that desirable increases in dough resistance to extension (Rmax) due to gluten re-polymerization after longer resting were partially to completely counteracted in FDK-spiked flours in a dose-dependent manner. Baking tests confirmed that while loaf volume is similar, proofed dough from FDK-spiked samples caused detectable loaf collapse at 3% FDK. Extensigraph Rmax and Fusarium protease levels were inversely related, and effected by both the extent and severity of infection. While the current FDK tolerances for grading Canadian wheat can effectively control protease damage, prevalence of deoxynivalenol (DON) weak- and non-producing Fusarium strains/species (e.g., F. avenaceum) in some growing regions must be considered to protect functionality if grading is solely based on DON content.

Grain protein concentration (GPC) is an important trait in durum cultivar development as a major determinant of the nutritional value of grain and end-use product quality. However, it is challenging to simultaneously select both GPC and grain yield (GY) due to the negative correlation between them. To characterize quantitative trait loci (QTL) for GPC and understand the genetic relationship between GPC and GY in Canadian durum wheat, we performed both traditional and conditional QTL mapping using a doubled haploid (DH) population of 162 lines derived from Pelissier × Strongfield. The population was grown in the field over 5 years and GPC was measured. QTL contributing to GPC were detected on chromosome 1B, 2B, 3A, 5B, 7A, and 7B using traditional mapping. One major QTL on 3A (QGpc.spa-3A.3) was consistently detected over 3 years accounting for 9.4–18.1% of the phenotypic variance, with the favorable allele derived from Pelissier. Another major QTL on 7A (QGpc.spa-7A) detected in 3 years explained 6.9–14.8% of the phenotypic variance, with the beneficial allele derived from Strongfield. Comparison of the QTL described here with the results previously reported led to the identification of one novel major QTL on 3A (QGpc.spa-3A.3) and five novel minor QTL on 1B, 2B and 3A. Four QTL were common between traditional and conditional mapping, with QGpc.spa-3A.3 and QGpc.spa-7A detected in multiple environments. The QTL identified by conditional mapping were independent or partially independent of GY, making them of great importance for development of high GPC and high yielding durum.

Although most of the durum wheat produced in the Canadian prairies in 2017 and 2018 met the test weight (TW) requirements for the top grades of Canada Western Amber Durum (CWAD), some samples of top grades were inferior in milling quality. To understand the abnormality, this study was conducted to investigate TW, thousand kernel weight (TKW) and kernel size distribution (KSD) in relation to durum milling potential, semolina composition and pasta quality. With reduction of kernel size, semolina and total milling yields decreased progressively, and kernels passing through no.6 slotted sieve had detrimental impact on milling. The overall relationship between TW and milling yields appeared to be genotype dependent. At similar TW, variety showed lower milling yields had greater proportion of smaller kernels. By account for the difference in KSD, greater relationships (R2 &gt, 0.91, p &lt, 0.001) were found for TKW and proportion of kernels passing No.6 slotted sieve with milling yields than TW (R2 = 0.75, p &lt, 0.001). This infers potential use of small kernels (passing No.6 slotted sieve) as a new objective grading factor for rapid prediction of milling quality of CWAD. Although small kernels exhibited much higher yellow pigment than the larger ones, pasta made from small kernels was duller, redder and less yellow, likely due to the higher semolina ash and protein contents, which adversely affected pasta color.

The fate of ergot alkaloids during the milling of durum and subsequent production and cooking of pasta was examined. Durum samples containing varying amounts of ergot sclerotia (0.01&ndash, 0.1% by mass) were milled, and all milling product was analyzed for 10 ergot alkaloids using liquid chromatography with tandem mass spectrometry. Spaghetti was prepared from the semolina obtained during milling. Ergocristine, ergocristinine, and ergotamine were the predominant ergot alkaloids observed in the milling fractions and spaghetti. Approximately 84% of the total ergot alkaloid mass of the whole grain durum resided in the milling product fractions associated with the outer kernel layers (bran, shorts, feeds). No consistent loss of ergot alkaloids was observed during the production or cooking of spaghetti. However, changes in the ratio of R- to S-enantiomers occurred during the milling and cooking of spaghetti. Products containing bran, shorts, and feeds, as well as cooked spaghetti, contained a higher proportion of the less biologically active S-enantiomers. The results of this study emphasize the need to monitor R- and S-enantiomers, and to consider food and feed products, as opposed to whole grain, when assessing any exposure of consumers to ergot alkaloids.

This research investigated changes in the amounts and sizes of monomeric proteins and protein aggregates during dough mixing, with a focus on the contribution of non-covalent bonds in the aggregation of gluten proteins. High protein flour (HF) and low protein flour (LF) were used in this study. As dough mixing progressed from flour to overmixed dough, the total amount of protein aggregates increased while the amount of monomeric protein decreased. Omega-gliadin was the major monomeric protein that decreased in quantity. Interestingly, the amount of larger-sized protein aggregates decreased and that of smaller-sized protein aggregates increased. The amount of gluten protein macro-polymer aggregated through strong non-covalent bonds decreased whereas aggregates formed with weaker non-covalent bonds increased. LF dough behaved similar to HF dough. Large-sized gluten protein aggregates disaggregated due to the weakening of non-covalent bonds and became smaller. Omega-gliadin was incorporated into gluten protein aggregates during dough mixing.

By applying a high-resolution LC-MS method, proteoform profiling of wheat glutenin proteins has revealed numerous protein pairs differing by an average mass of 163 ± 1 Da. The most abundant of 21 such pairs, 40 344 Da and 40 181 Da (coded P40344 and P40181, respectively), were purified and subjected to peptide mapping. P40344 matched the calculated mature protein mass for Uniprot B2Y2Q7, an s-type low-molecular weight glutenin subunit (LMW-GS). The P40344 and P40181 digests differed by a single digest peptide, unambiguously matched to the C-terminus of the protein and differing by the loss of a tyrosine. Of 327 unique mature wheat LMW-GS sequences from 369 UNIPROT accessions, tyrosine is at the C-terminus in all but one accession with evidence of transcription. The C-terminal LMW-GS sequences are also highly conserved, with only 10 variants of the last six amino acids. This is the first observation of cleavage of a C-terminal tyrosine other than alpha-tubulin, and the first evidence of this post-translational modification in plants. The identification of this novel post-translational modifications in gluten proteins may provide new insights on the differential modulation of biological processes involved in seed maturation and their effect on functional properties.

Methodology for the quantitative analysis of intact glutenin proteins by ESI-LC-MS was developed with 1 Da mass resolution, constituting the first published application of proteoform profiling to plant biology. Two parent lines and 28 F5 crosses were analyzed in two blocks, 14 months apart. Control sample data were used to align retention times and normalize abundance between sample sets. A total of 4622 observations of 347 distinct proteoforms between 17 899 and 88 744 Da were observed. Proteoform abundances spanned a 1000-fold range and were linear (r2 > 0.990) with dilution. A novel method for the objective elimination of low intensity, noise-dominated data using replicate variability within the dataset is presented. Two abundant PTMs were detected; one known but uncharacterized Bx and Dy high-molecular-weight glutenin subunit (HMWGS) PTM and the other in 24 low molecular weight proteoform pairs. Finally, 16 abundant proteoforms were detected in progeny but not in either parent. This application should increase the statistical power of correlations between gluten complement and functional data and drive the detection of novel PTMs that may indicate differential regulation of the cellular processes related to quality.

The aim of this study was to investigate the distribution of total yellow pigments, total phenolic compounds, and their antioxidant activities in various durum wheat milling fractions. Carotenoid composition of yellow pigment extract was also examined using UPLC. The ABTS radical scavenging activity of the milling fractions decreased in the order of short bran/bran > feed flour > flour/semolina in both total phenolic and total yellow pigment extracts. Yellow pigments extracts from bran, short bran, and feed flour exhibited 5.6–15.4% higher antioxidant activity than those of total phenolic extracts from the corresponding milling fractions. The UPLC results showed a non-carotenoid peak at Rt 0.47 min which was present in fractions of the grain outer layers but absent in semolina and flour. This peak absorbed in the UV range of 271 to 327 nm. These observations suggest that the unknown peak could be composed of phenolic compounds co-extracted in their free form with carotenoids in the polar water-saturated butanol solvent. The compounds in this peak could result in overestimation of carotenoid content and antioxidant activity in bran, short bran and feed flour as the peak contributed to 18.3–26.0% of total carotenoids if it was taken into account.

Fu, B. X., Hatcher, D. W. and Schlichting, L. 2014. Effects of sprout damage on durum wheat milling and pasta processing quality. Can. J. Plant Sci. 94: 545–553. Due to concerns over the unusual sprouting problem observed in the 2010 harvest of Canada Western Amber Durum Wheat (CWAD), it was deemed necessary by the industry to investigate and determine if appropriate tolerances for sprout damage were in place, particularly for No. 3 CWAD. More information on the impact of visually assessed sprout damage on durum quality is needed to better define the associated acceptable level of falling number (FN). To this end, two different samples of CWAD were sourced for this project: a No. 1 CWAD (FN 479 s) and a No. 5 CWAD (FN 68 s) degraded primarily due to sprout damage. A total of 19 samples were used in the study, i.e., a series of eight composite samples prepared by blending the No. 1 with increasing amounts of No. 5, as well as the two control extremes. The FNs of the blends were well characterized, displaying an incremental decrease of ∼50 s with increasing sprout damage. Each wheat sample was milled in duplicate. The resulting semolina was analyzed for ash, pigment, pigment loss, yellowness (b*), and speckiness. Protein content, gluten index and alveograph parameters were also evaluated. The semolina was made into spaghetti for colour measurement and texture evaluation. Results indicated that there was no change in ash content, pigment or semolina b* value even at 50% blend (FN 101 s). However, a noticeable increase in total speck count and the number of dark specks in the semolina were detected once the blending ratio reached the 35% level (FN 208 s). The increase in speck count was largely due to mildew associated with the No. 5 CWAD sample. The influence of sprout damage on gluten strength was minimal at all levels of blending. A significant increase in spaghetti redness (a*) was detected in blends with 25% (FN 152 s) or more of No. 5 CWAD. A decline in spaghetti brightness (L*) was also observed when transitioning from the 15% blend (FN 204 s) and very evident at the 35% blend (FN 123 s) level. No discernible differences due to sprout damage were noticed within the composite blends in terms of processing properties, firmness and cooking loss of the cooked pasta, although spaghetti made from the No. 5 sample showed slight checking, higher cooking loss and lower firmness.