The accumulation of plant hormones in different tissues leads to transcriptional reprogramming that guides the downstream phenotypic response. Current models are largely derived from Arabidopsis, and increasing evidence indicates that monocots only partially conform to these canonical pathways, with additional variability even within the clade. In cereals, salicylate (SA), jasmonate (JA), and ethylene (ET) signaling play important roles in both inflorescence development and pathogen defense. This communication focuses on the transcriptional responses of wheat inflorescence tissue in three genotypes 8 h following exogenous applications of salicylic acid (SA), methyl jasmonate (MeJA), and the ethylene-releasing compound, ethephon (ETp). Nearly 8000 differentially expressed genes (DEGs) were detected in response to MeJA, approximately half of which were conserved across all three genotypes. In contrast, SA and ETp elicited limited responses, each inducing fewer than 100 DEGs with little to no overlap among genotypes. The comparatively feeble response to SA and ETp suggests a delayed response compared to MeJA. Other studies have assessed differential transcriptomes at 24 h or later. Notably, other studies in cereals, including wheat, have also reported less pronounced transcriptional changes to salicylate and ethylene signaling. These findings demonstrate both conservation and genotype-specific variation in the transcriptional response to plant hormones.

In the face of evolving agricultural practices and climate change, tools towards an integrated biovigilance platform to combat crop diseases, spore sampling, DNA diagnostics and predictive trajectory modelling were optimized. These tools revealed microbial dynamics and were validated by monitoring cereal rust fungal pathogens affecting wheat, oats, barley and rye across four growing seasons (2015-2018) in British Columbia and during the 2018 season in southern Alberta. ITS2 metabarcoding revealed disparity in aeromycobiota diversity and compositional structure across the Canadian Rocky Mountains, suggesting a barrier effect on air flow and pathogen dispersal. A novel bioinformatics classifier and curated cereal rust fungal ITS2 database, corroborated by real-time PCR, enhanced the precision of cereal rust fungal species identification. Random Forest modelling identified crop and land-use diversification as well as atmospheric pressure and moisture as key factors in rust distribution. As a valuable addition to explain observed differences and patterns in rust fungus distribution, trajectory HYSPLIT modelling tracked rust fungal urediniospores' northeastward dispersal from the Pacific Northwest towards southern British Columbia and Alberta, indicating multiple potential origins. Our Canadian case study exemplifies the power of an advanced biovigilance toolbox towards developing an early-warning system for farmers to detect and mitigate impending disease outbreaks.

Our research focused on developing a highly sensitive whole-spore real-time immuno-PCR (RT-iPCR) assay for the detection of three wheat fungal pathogens: Pyrenophora tritici-repentis (Ptr), Fusarium graminearum (Fg), and Puccinia striiformis forma specialis (f. sp.) tritici (Pst). RT-iPCR measurements were compared to more well-established quantitative PCR (qPCR) assays to compare their performance. While specificity remained a challenge for RT-iPCR, the direct spore measurements negate the need for DNA extraction, making RT-iPCR a potentially valuable technique that warrants further research. An alternative approach was developed to determine DNA extraction efficiency and quantification of spore numbers by qPCR, which is currently a methodological gap in most qPCR spore measurements. DNA extraction efficiency determined for Fg, Pst, and Ptr spores were 5.0 +/- 0.1, 7.0 +/- 0.4, and 290 +/- 36%, respectively, demonstrating important implications for the accuracy of these techniques when DNA recovery is not considered.

Cereal rust diseases are caused by Puccinia spp. urediniospores that travel by air currents to cause losses in wheat, oat, barley, and rye. In western Canada, inoculum can arrive from the USA through the Great Plains or via a Pacific corridor west of the Rocky Mountains. Moreover, climate and land use are known factors in Puccinia dissemination, but their relative effects remain poorly understood. We investigated aeromycobiota in western Canada by weekly air-sampling over four growing seasons (2015-2018) three mixed-crop sites in British Columbia (BC) and one season (2018) at five sites in southern Alberta (AB). ITS2-based metabarcoding and novel bioinformatic comparative analyses to known, especially curated cereal rust fungal sequences, was used for species identification. The overall aeromycobiota and rust fungal community diversity was higher west than east of the Canadian Rockies. This mountain range delineates climate and land use and also creates a barrier to wind flow to prevent the spread of rusts and other plant pathogens (e.g., Bipolaris, Blumeria). We recovered seven major cereal rusts and revealed their geographic and seasonal dynamics at the eight sampling sites. Forward and reverse trajectory HYSPLIT model simulations predicted the potential sources of rust urediniospores and their pathways of movement modulated by air currents, through which some pronounced changes in the abundance of wheat rust pathogens were explained. This study paves the way for a potential application for pathogen monitoring and disease risk forecasting as part of the design and development of an early-warning system for enhanced biovigilance against crop diseases.

We are reporting on the utilization of high-throughput sequencing and different sequencing analysis tools to delineate identification of different isolates of the stripe rust fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Different approaches are shown: utilization of rDNA sequences and random sequences that may be very useful to make sure that isolates belong to Pst and to distinguished closely related isolates. Identification of unique/lost sequences could lead to the identification of effectors associated with specific isolates.

Stripe rust (Puccinia striiformis f. sp. tritici (Pst)) and powdery mildew (Blumeria graminis f. sp. tritici (Bgt)) are important diseases of wheat in Canada and worldwide. Molecular detection methods permit spore detection of few spores; therefore, there is a need to determine initial inoculum thresholds for pathogens to cause disease under both controlled environments and in the field. Susceptible wheat cultivars 'Avocet' and 'AC Barrie' were inoculated with different quantities of spores (0, 10(3), 10(4), 10(5), 10(6), and 10(7)) of Pst and Bgt. Disease incidence, severity and infection type were evaluated. Results of controlled environment studies showed that the minimum number of spores necessary to cause appreciable incidence and severity for Pst was at higher spore concentrations of 10(5)-10(6) spores. Conversely, low incidence and severity levels were observed at 10(3)-10(4) spores for Bgt. Despite occurrence of natural Pst infection, results of field studies in 2016 and 2017 in Southern Alberta demonstrated that significant increases in severity levels were observed following application of 1.2 x 10(7) spores. Collectively, these results demonstrated that stripe rust severities increased with increasing spore concentration only at high spore levels. In contrast, Bgt severity increased with spore concentration from 10(3) to 10(7) spores mL(-1). In vitro and in vivo spore germination tests demonstrated germination rates of Pst spores were reduced at lower spore concentrations compared to germination rates at the higher concentrations. Understanding of minimum spore numbers required for disease development will be a prerequisite for predicting epidemics and devising fungicide control measures for future sustainable agricultural systems.

Bread wheat is a staple crop for human consumption. Recent technological advances have not only helped in availability of a high quality and annotated reference genome of wheat (IWGSC RefSeq v2.1.) but also in pangenomic sequencing of cultivars of different origin including stress tolerant and susceptible cultivars, and transcriptomic sequencing of lines resistant/tolerant to different biotic and abiotic stresses. However, despite the vast progress made in sequencing the genomes and transcriptomes, functional annotation of wheat genes is still lagging behind those of model plants. The host defense response to viruses, known as virus-induced gene silencing (VIGS), has been effectively manipulated as a tool to study gene function in model and crop plants. VIGS allows researchers to generate and screen large loss-of-function phenotypic data with less efforts and no requirements of stable transformation in a relatively short span of time as compared to other methods such as TILLING, EcoTILLING, and RNAi. In this chapter, we discussed the basics of posttranscriptional gene silencing and described step-by-step protocols for functional annotation of wheat genes using the barley stripe mosaic virus-based VIGS system.

Winter field survival (WFS) in autumn-seeded winter cereals is a complex trait associated with low temperature tolerance (LTT), prostrate growth habit (PGH), and final leaf number (FLN). WFS and the three sub-traits were analyzed by a genome-wide association study of 96 rye ( Secale cereal L.) genotypes of different origins and winter-hardiness levels. A total of 10,244 single nucleotide polymorphism (SNP) markers were identified by genotyping by sequencing and 259 marker-trait-associations (MTAs; p < 0.01) were revealed by association mapping. The ten most significant SNPs ( p < 1.49e−04) associated with WFS corresponded to nine strong candidate genes: Inducer of CBF Expression 1 ( ICE1 ), Cold-regulated 413-Plasma Membrane Protein 1 ( COR413-PM1 ), Ice Recrystallization Inhibition Protein 1 ( IRIP1 ), Jasmonate-resistant 1 ( JAR1 ), BIPP2C1 -like protein phosphatase, Chloroplast Unusual Positioning Protein-1 ( CHUP1 ), FRIGIDA -like 4 ( FRL4-like ) protein, Chalcone Synthase 2 ( CHS2 ), and Phenylalanine Ammonia-lyase 8 ( PAL8 ). Seven of the candidate genes were also significant for one or several of the sub-traits supporting the hypothesis that WFS, LTT, FLN, and PGH are genetically interlinked. The winter-hardy rye genotypes generally carried additional allele variants for the strong candidate genes, which suggested allele diversity was a major contributor to cold acclimation efficiency and consistent high WFS under varying field conditions.

We present a chromosome-scale annotated assembly of the rye ( Secale cereale L. inbred line ‘Lo7’) genome, which we use to explore Triticeae genomic evolution, and rye’s superior disease and stress tolerance. The rye genome shares chromosome-level organization with other Triticeae cereals, but exhibits unique retrotransposon dynamics and structural features. Crop improvement in rye, as well as in wheat and triticale, will profit from investigations of rye gene families implicated in pathogen resistance, low temperature tolerance, and fertility control systems for hybrid breeding. We show that rye introgressions in wheat breeding panels can be characterised in high-throughput to predict the yield effects and trade-offs of rye chromatin.

The plasticity of plant development allows acclimation to environmental stresses such as heat and drought. For example, increased vein density with more vein meetings is proposed to provide more efficient and robust water delivery when water availability is reduced. We demonstrate that Arabidopsis leaves respond to short term heat or drought by increasing leaf vein density, which in heat includes a higher density of vein meetings. We find that a set of genes, UNHINGED, FORKED1, FORKED-LIKE1, 2 and 3, which influence PINFORMED1 localization and control vein meeting in developing leaves, is required for the vein pattern response to heat or drought. Comparison of delta C-13 (parts per thousand) and nitrogen (%) under different conditions suggests that while wild type and mutant genotypes enriched delta C-13 (parts per thousand) under drought conditions, only wild type increased total shoot nitrogen suggesting increased photosynthetic assimilation. One explanation is that the mutants' open vein pattern fails to supply sufficient water to support increased photosynthesis. Finally, treatment with ABA caused changes to leaf vein pattern similar to heat or drought treatment, a response that was eliminated in the abscisic acid insensitive1 but not the forked1 genotype. This suggests that the vein pattern response to reduced water availability may in part be controlled by ABA.

The advent of high-throughput next-generation sequencing technologies in the last decade, coupled with its substantial decrease in cost in the recent years and development of complementary array-based genotyping platforms, has revolutionized the generation of genome-wide markers and propelled several statistical methods for unearthing marker-phenotype association. This chapter provides an outline of the conceptual principles and steps of methods widely used for genome-wide association studies (GWAS) in cereals. Specifically, we focussed on presenting practical steps, starting from assembling populations suitable for phenotyping, genotyping platforms, estimation of population structure from genome-wide markers, estimation of linkage disequilibrium (LD), and methods of GWAS. We also highlighted the available sources of cereal genome assemblies and major software packages used for GWAS, namely, TASSEL-5.0, PLINK, and GAPIT-3.0.

Overwintering cereals accumulate low temperature tolerance (LTT) during cold acclimation in the autumn. Simultaneously, the plants adjust to the colder season by making developmental changes at the shoot apical meristem. These processes lead to higher winter hardiness in winter rye varieties (Secale cereale L.) adapted to Northern latitudes as compared to other cereal crops. To dissect the winter-hardiness trait in rye, a panel of 96 genotypes of different origins and growth habits was assessed for winter field survival (WFS), LTT, and six developmental traits. Best Linear Unbiased Estimates for WFS determined from five field trials correlated strongly with LTT (r = 0.90, p < 0.001); thus, cold acclimation efficiency was the major contributor to WFS. WFS also correlated strongly (p < 0.001) with final leaf number (r = 0.80), prostrate growth habit (r = 0.61), plant height (r = 0.34), but showed weaker associations with top internode length (r = 0.30, p < 0.01) and days to anthesis (r = 0.25, p < 0.05). The heritability estimates (h2) for WFS-associated traits ranged from 0.45 (prostrate growth habit) to 0.81 (final leaf number) and were overall higher than for WFS (h2 = 0.48). All developmental traits associated with WFS and LTT are postulated to be regulated by phytohormone levels at shoot apical meristem.

AAC Network is a semi-dwarf hard red winter wheat (Triticum aestivum L.) cultivar that is well adapted across western Canada and eligible for grades of Canada Western Red Winter (CWRW) wheat. It was developed using wheat × maize pollen doubled haploid methodology. AAC Network was evaluated in the Western Canadian Winter Wheat Cooperative registration trials relative to CDC Buteo, Emerson, Moats, and AAC Elevate for 4 yr (2016–2019). Based on 44 replicated trials, AAC Network produced grain yield similar to AAC Elevate, the highest yielding check, with a protein concentration 0.9 units higher. AAC Network had fair to good winter survival, relatively late maturity, short straw with excellent lodging resistance, and high test weight. AAC Network expressed resistance to stem and stripe rust, moderate resistance to leaf rust and common bunt, and intermediate resistance to Fusarium head blight. In addition to increased grain protein concentration, AAC Network showed improvements in gluten strength and flour water absorption, and it maintained the excellent milling yield and low flour ash attributes of the CWRW wheat class.

Wheat is affected by many fungal diseases that can cause severe yield and quality losses. Disease prediction models generally employ weather data to estimate potential for infection to determine timing for fungicide applications, but these models fail to account for the presence and quantity of pathogen inoculum. This study adapted highly specific qPCR primers to identify and quantify, in real-time, inoculum present in air for the six most important wheat pathogens in Canada. Fungal spores were collected using either Burkard spore collectors and quantified using qPCR or microscope slides covered with adhesive tape and identified and quantified using microscopy. Samples were collected from seven different sites in southern Alberta throughout the 2015-2017 growing seasons. The results demonstrated that qPCR can reliably identify and quantify spores from Puccinia striiformis f. sp. tritici, P. triticina, P. graminis f. sp. tritici, Blumeria graminis f. sp. tritici, Pyrenophora tritici-repentis, and Fusarium graminearum. The limits of detection of DNA for primer pairs in singleplex tests ranged from 0.0001 ng for P. graminis to 0.001 ng for P. tritici-repentis, which corresponded to approximately 3 spores for P. tritici-repentis and F. graminearum and 1 spore for the other pathogens. Conversely, microscopy permitted identification of rusts to the genus but not to the species level and was ineffective in quantification of the remainder of the wheat pathogens. This study will contribute to the development of a fast and reliable forecasting system that will enable identification and quantification of airborne pathogens in real-time before initial disease symptoms appear.

As one of the largest protein families in plants, F-box proteins are involved in many important cellular processes. Until now, a limited number of investigations have been conducted on wheat F-box genes due to its variable structure and large and polyploid genome. Classification, identification, structural analysis, evolutionary relationship, and chromosomal distribution of some wheat F-box genes are described in the present study. A total number of 1013 potential F-box proteins which are encoded by 409 genes was identified in wheat, and classified into 12 subfamilies based on their C-terminal domain structures. Furthermore, proteins with identical or similar C-terminal domain were clustered together. Location of 409 F-box genes was identified on all 21 wheat chromosomes but showed an uneven distribution. Segmental duplication was the main reason for the increase in the number of wheat F-box genes. Gene expression analysis based on digital PCR showed that most of the F-box genes were highly expressed in the later development stages of wheat, including the formation of spike, grain, flag leaf, and participated in drought stress (DS), heat stress (HS), and their combination (HD). Of the nine F-box genes we investigated using quantitative PCR (qPCR) following fungal pathogen infection, five were involved in wheat resistance to the infection by leaf rust pathogen and one in the susceptible response. These results provide important information on wheat F-box proteins for further functional studies, especially the proteins that played roles in response to heat and drought stresses and leaf rust pathogen infection.

A Triticeae type III non-specific lipid transfer protein (nsLTP) was shown for the first time to be translocated from the anther tapetum to the pollen cell wall. Two anther-expressed non-specific lipid transfer proteins (nsLTPs) were identified in triticale (× Triticosecale Wittmack). LTPc3a and LTPc3b contain a putative signal peptide sequence and eight cysteine residues in a C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C pattern. These proteins belong to the type III class of nsLTPs which are expressed exclusively in the inflorescence of angiosperms. The level of LTPc3 transcript in the anther was highest at the tetrad and uninucleate microspore stages, and absent in mature pollen. In situ hybridization showed that LTPc3 was expressed in the tapetal layer of the developing triticale anther. The expression of the LTPc3 protein peaked at the uninucleate microspore stage, but was also found to be associated with the mature pollen. Accordingly, an LTPc3a::GFP translational fusion expressed in transgenic Brachypodium distachyon first showed activity in the tapetum, then in the anther locule, and later on the mature pollen grain. Altogether, these results represent the first detailed characterization of a Triticeae anther-expressed type III nsLTP with possible roles in pollen cell wall formation.

F-box proteins constitute one of the largest protein families and play many important roles in plant. To date, less is known about functions of wheat F-box protein. A Kelch-type F-box gene TaFBK was isolated from wheat TcLr15. TaFBK transcripts were more abundant in pistil and leaf tissues, and accumulated more in the susceptible combination than that in the resistant one. The treatment of SA, MeJA, NaCl and PEG caused a slight up-regulation of TaFBK. GFP localization studies showed that TaFBK accumulated in the nucleus and cytoplasm. Moreover, TaFBK bound TaSkp1 by the F-box domain and recognized TaSLY1 via the Kelch domain.

Fall-seeded rye improve their frost resistance during a cold acclimation period in the fall, which is often associated with an accumulation of red/purple-colored anthocyanins in plant tissues. The pigments provide antioxidant activity and are proposed to scavenge excess reactive oxygen species (ROS) and mitigate light stress occurring during cold exposure. The relationship between anthocyanin and winter hardiness in rye (Secale cereale L.) was assessed by analyzing 96 genotypes with varying levels of winter survival. HPLC-QTOF MS/MS analyses of tissue extracts prepared from cold-acclimated plants revealed presence of accumulated anthocyanins in leaves of 74 genotypes and 51 of these showed low levels of anthocyanins presence in crown tissues. An overall higher abundance and diversity of anthocyanins was noted for the most cold-hardy as compared to the less hardy genotypes. A total of 18 anthocyanins originating from the pelargonidin, cyanidin, and delphinidin precursors were identified, of which the glycosylated cyanidins were accumulated in 37 of the 39 most winter hardy genotypes, but only detected in six of the 38 most tender genotypes. The link between anthocyanin concentration and profiles with winter-hardiness levels for the 96 rye genotypes suggested a role for cyanidin-derived anthocyanins in mediating enhanced winter hardiness in rye.

We report on pyramiding different disease resistance genes against fungal pathogens in Canadian winter wheat germplasm based on available DNA markers and gene sequences. Genetic resistance represents a safe, economical and ecological method for protecting plants, growers and the health of consumers. Major diseases of wheat on the Canadian Prairies are common bunt, rusts (leaf, stem and stripe) and Fusarium head blight. Over the years markers for resistance genes against these diseases have been identified and used by the international wheat community. We describe markers that we have used to pyramid different resistance genes and indicate their presence in Canadian winter wheat cultivars issued from the winter wheat breeding program at the Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre, the only winter wheat breeding program in Western Canada actively delivering new varieties for all regions of the Canadian Prairies. The sources of resistance and identities of PCR primers and amplification conditions are indicated to enable the transfer and pyramiding of different resistance (R) genes to breeding lines. We conclude by reviewing new tools for identifying R genes in wheat and indicate how mutagenesis and gene editing can help future efforts to extend the protection offered by known R genes.