Determining the Impact of Genotype × Environment on Oat Protein Isolate Composition Using HPLC and LC-MS Techniques.

The effect of genotype and environment on oat protein composition was analyzed through size exclusion-high-performance liquid chromatography (SE-HPLC) and liquid chromatography-mass spectrometry (LC-MS) to characterize oat protein isolate (OPI) extracted from three genotypes grown at three locations in the Canadian Prairies. SE-HPLC identified four fractions in OPI, including polymeric globulins, avenins, glutelins, and albumins, and smaller proteins. The protein composition was dependent on the environment, rather than the genotype. The proteins identified through LC-MS were grouped into eight categories, including globulins, prolamins/avenins, glutelins, enzymes/albumins, enzyme inhibitors, heat shock proteins, grain softness proteins, and allergenic proteins. Three main globulin protein types were also identified, including the P14812|SSG2-12S seed storage globulin, the Q6UJY8_TRITU-globulin, and the M7ZQM3_TRIUA-Globulin-1 S. Principal component analysis indicated that samples from Manitoba showed a positive association with the M7ZQM3_TRIUA-Globulin-1 S allele and Q6UJY8_TRITU-globulin, while samples from Alberta and Saskatchewan had a negative association with them. The results show that the influence of G × E on oat protein fractions and their relative composition is crucial to understanding genotypes' behavior in response to different environments.

Unraveling Plant-Pathogen Interactions in Cereals Using RNA-seq.

Over the past two decades, there have been significant advancements in the realm of transcriptomics, or the study of genes and their expression. Modern RNA sequencing technologies and high-performance computing are creating a "big data" revolution that provides new opportunities to explore the interactions between cereals and pathogens that affect grain yield and food safety. These data are being used to annotate genes and gene variants, as well as identify differentially expressed genes and create global gene co-expression networks. Moreover, these data can unravel the complex interactions between pathogen and host and identify genes and pathways involved in these interactions. This information can then be used for disease mitigation and the development of crops with superior resistance.

Evolutionary divergence in embryo and seed coat development of U's Triangle Brassica species illustrated by a spatiotemporal transcriptome atlas.

The economically valuable Brassica species include the six related members of U's Triangle. Despite the agronomic and economic importance of these Brassicas, the impacts of evolution and relatively recent domestication events on the genetic landscape of seed development have not been comprehensively examined in these species. Here we present a 3D transcriptome atlas for the six species of U's Triangle, producing a unique resource that captures gene expression data for the major subcompartments of the seed, from the unfertilized ovule to the mature embryo and seed coat. This comprehensive dataset for seed development in tetraploid and ancestral diploid Brassicas provides new insights into evolutionary divergence and expression bias at the gene and subgenome levels during the domestication of these valued crop species. Comparisons of gene expression associated with regulatory networks and metabolic pathways operating in the embryo and seed coat during seed development reveal differences in storage reserve accumulation and fatty acid metabolism among the six Brassica species. This study illustrates the genetic underpinnings of seed traits and the selective pressures placed on seed production, providing an immense resource for continued investigation of Brassica polyploid biology, genomics and evolution.

Alternative splicing dynamics and evolutionary divergence during embryogenesis in wheat species.

Among polyploid species with complex genomic architecture, variations in the regulation of alternative splicing (AS) provide opportunities for transcriptional and proteomic plasticity and the potential for generating trait diversities. However, the evolution of AS and its influence on grain development in diploid grass and valuable polyploid wheat crops are poorly understood. To address this knowledge gap, we developed a pipeline for the analysis of alternatively spliced transcript isoforms, which takes the high sequence similarity among polyploid wheat subgenomes into account. Through analysis of synteny and detection of collinearity of homoeologous subgenomes, conserved and specific AS events across five wheat and grass species were identified. A global analysis of the regulation of AS in diploid grass and polyploid wheat grains revealed diversity in AS events not only between the endosperm, pericarp and embryo overdevelopment, but also between subgenomes. Analysis of AS in homoeologous triads of polyploid wheats revealed evolutionary divergence between gene-level and transcript-level regulation of embryogenesis. Evolutionary age analysis indicated that the generation of novel transcript isoforms has occurred in young genes at a more rapid rate than in ancient genes. These findings, together with the development of comprehensive AS resources for wheat and grass species, advance understanding of the evolution of regulatory features of AS during embryogenesis and grain development in wheat.

Multiple wheat genomes reveal global variation in modern breeding.

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.

Machine learning analyses of methylation profiles uncovers tissue-specific gene expression patterns in wheat.

DNA methylation is a mechanism of epigenetic modification in eukaryotic organisms. Generally, methylation within genes promoter inhibits regulatory protein binding and represses transcription, whereas gene body methylation is associated with actively transcribed genes. However, it remains unclear whether there is interaction between methylation levels across genic regions and which site has the biggest impact on gene regulation. We investigated and used the methylation patterns of the bread wheat cultivar Chinese Spring to uncover differentially expressed genes (DEGs) between roots and leaves, using six machine learning algorithms and a deep neural network. As anticipated, genes with higher expression in leaves were mainly involved in photosynthesis and pigment biosynthesis processes whereas genes that were not differentially expressed between roots and leaves were involved in protein processes and membrane structures. Methylation occurred preponderantly (60%) in the CG context, whereas 35 and 5% of methylation occurred in CHG and CHH contexts, respectively. Methylation levels were highly correlated (r = 0.7 to 0.9) between all genic regions, except within the promoter (r = 0.4 to 0.5). Machine learning models gave a high (0.81) prediction accuracy of DEGs. There was a strong correlation (p-value = 9.20×10-10 ) between all features and gene expression, suggesting that methylation across all genic regions contribute to gene regulation. However, the methylation of the promoter, the CDS and the exon in CG context was the most impactful. Our study provides more insights into the interplay between DNA methylation and gene expression and paves the way for identifying tissue-specific genes using methylation profiles.

Copy number variation of TdDof controls solid-stemmed architecture in wheat.

Stem solidness is an important agronomic trait of durum (Triticum turgidum L. var. durum) and bread (Triticum aestivum L.) wheat that provides resistance to the wheat stem sawfly. This dominant trait is conferred by the SSt1 locus on chromosome 3B. However, the molecular identity and mechanisms underpinning stem solidness have not been identified. Here, we demonstrate that copy number variation of TdDof, a gene encoding a putative DNA binding with one finger protein, controls the stem solidness trait in wheat. Using map-based cloning, we localized TdDof to within a physical interval of 2.1 Mb inside the SSt1 locus. Molecular analysis revealed that hollow-stemmed wheat cultivars such as Kronos carry a single copy of TdDof, whereas solid-stemmed cultivars such as CDC Fortitude carry multiple identical copies of the gene. Deletion of all TdDof copies from CDC Fortitude resulted in the loss of stem solidness, whereas the transgenic overexpression of TdDof restored stem solidness in the TdDof deletion mutant pithless1 and conferred stem solidness in Kronos. In solid-stemmed cultivars, increased TdDof expression was correlated with the down-regulation of genes whose orthologs have been implicated in programmed cell death (PCD) in other species. Anatomical and histochemical analyses revealed that hollow-stemmed lines had stronger PCD-associated signals in the pith cells compared to solid-stemmed lines, which suggests copy number-dependent expression of TdDof could be directly or indirectly involved in the negative regulation of PCD. These findings provide opportunities to manipulate stem development in wheat and other monocots for agricultural or industrial purposes.

Histology and RNA Sequencing Provide Insights Into Fusarium Head Blight Resistance in AAC Tenacious.

Fusarium head blight (FHB) is a serious fungal disease affecting wheat and other cereals worldwide. This fungus causes severe yield and quality losses from a reduction in grain quality and contamination of grain with mycotoxins. Intensive breeding efforts led to the release of AAC Tenacious, which was the first spring wheat cultivar registered in Canada with a resistant (R) rating to FHB. To elucidate the physiological mechanisms of resistance, we performed histological and transcriptomic analyses of AAC Tenacious and a susceptible control Roblin after inoculation with Fusarium graminearum (Fg). The spikelet and rachis of infected wheat spikes were hand sectioned and monitored by confocal and fluorescent microscopy. Visible hyphae were observed within the inoculated spikelets for AAC Tenacious; however, the infection was largely restricted to the point of inoculation (POI), whereas the adjacent florets in Roblin were heavily infected. Significant cell wall thickening within the rachis node below the POI was evident in AAC Tenacious compared to Roblin in response to Fg inoculation. Rachis node and rachilla tissues from the POI and the rachis node below the POI were collected at 5 days post inoculation for RNAseq. Significant changes in gene expression were detected in both cultivars in response to infection. The rachis node below the POI in AAC Tenacious had fewer differentially expressed genes (DEGs) when compared to the uninoculated control, likely due to its increased disease resistance. Analysis of DEGs in Roblin and AAC Tenacious revealed the activation of genes and pathways in response to infection, including those putatively involved in cell wall modification and defense response.

The Transcriptional Landscape of Polyploid Wheats and Their Diploid Ancestors during Embryogenesis and Grain Development.

Modern wheat production comes from two polyploid species, Triticum aestivum and Triticum turgidum (var durum), which putatively arose from diploid ancestors Triticum urartu, Aegilops speltoides, and Aegilops tauschii How gene expression during embryogenesis and grain development in wheats has been shaped by the differing contributions of diploid genomes through hybridization, polyploidization, and breeding selection is not well understood. This study describes the global landscape of gene activities during wheat embryogenesis and grain development. Using comprehensive transcriptomic analyses of two wheat cultivars and three diploid grasses, we investigated gene expression at seven stages of embryo development, two endosperm stages, and one pericarp stage. We identified transcriptional signatures and developmental similarities and differences among the five species, revealing the evolutionary divergence of gene expression programs and the contributions of A, B, and D subgenomes to grain development in polyploid wheats. The characterization of embryonic transcriptional programming in hexaploid wheat, tetraploid wheat, and diploid grass species provides insight into the landscape of gene expression in modern wheat and its ancestral species. This study presents a framework for understanding the evolution of domesticated wheat and the selective pressures placed on grain production, with important implications for future performance and yield improvements.plantcell;31/12/2888/FX1F1fx1.

Mapping of Genetic Loci Conferring Resistance to Leaf Rust From Three Globally Resistant Durum Wheat Sources.

Genetic resistance in the host plant is the most economical and environmentally friendly strategy for controlling wheat leaf rust, caused by Puccinia triticina Eriks. The durum wheat lines Gaza (Middle East), Arnacoris (France) and Saragolla (Italy) express high levels of resistance to the Mexican races of P. triticina. Three recombinant inbred line (RIL) populations, derived from crosses of each of these resistance sources to the susceptible line ATRED #2, were evaluated for leaf rust reactions at CIMMYT's leaf rust nurseries in Mexico. Genetic analyses of host reactions suggested oligogenic control of resistance in all populations. The F8 RILs from each cross were genotyped using the Illumina iSelect 90K array, and high-density genetic maps were constructed for each population. Using composite interval mapping, a total of seven quantitative trait loci (QTL) that provide resistance to leaf rust were identified. Two QTL designated as QLr.usw-6BS and QLr.usw-6BL were identified on chromosome 6B in Gaza, which explained up to 78.5% and 21.3% of the observed leaf rust severity variance, respectively. A major QTL designated as QLr.usw-7BL was detected on the long arm of chromosome 7B in Arnacoris, which accounted for up to 65.9% of the disease severity variance. Arnacoris also carried a minor QTL on chromosome 1BL, designated as QLr.usw-1BL.1 that explained up to 17.7% of the phenotypic variance. Three QTL conferred leaf rust resistance in Saragolla, namely QLr.usw-2BS, QLr.usw-3B, and QLr.usw-1BL.2, which accounted for up to 42.3, 9.4, and 7.1% of the phenotypic variance, respectively. Markers flanking each QTL were physically mapped against the durum wheat reference sequence and candidate genes involved in disease resistance were identified within the QTL intervals. The QTL identified in this study and their closely linked markers are useful resources for gene pyramiding and breeding for durable leaf rust resistance in durum wheat.

Fusarium Head Blight in Durum Wheat: Recent Status, Breeding Directions, and Future Research Prospects.

Fusarium head blight (FHB) is a major fungal disease affecting wheat production worldwide. Since the early 1990s, FHB, caused primarily by Fusarium graminearum, has become one of the most significant diseases faced by wheat producers in Canada and the United States. The increasing FHB problem is likely due to the increased adoption of conservation tillage practices, expansion of maize production, use of susceptible wheat varieties in rotation, and climate variability. Durum wheat (Triticum turgidum sp. durum) is notorious for its extreme susceptibility to FHB and breeding for resistance is complicated because sources of FHB resistance are rare in the primary gene pool of tetraploid wheat. Losses due to this disease include yield, test weight, seed quality, food and feed quality, and when severe, market access. More importantly, it is the contamination with mycotoxins, such as deoxynivalenol, in Fusarium-infected durum kernels that causes the most serious economic as well as food and feed safety concerns. Several studies and thorough reviews have been published on germplasm development and breeding for FHB resistance and the genetics and genomics of FHB resistance in bread or common wheat (T. aestivum); however, similar reviews have not been conducted in durum wheat. Thus, the aim of this review is to summarize and discuss the recent research efforts to mitigate FHB in durum wheat, including quantitative trait locus mapping, genome-wide association studies, genomic prediction, mutagenesis and characterization of genes and pathways involved in FHB resistance. It also highlights future directions, FHB-resistant germplasm, and the potential role of morphological traits to enhance FHB resistance in durum wheat.

Haplotype Loci Under Selection in Canadian Durum Wheat Germplasm Over 60 Years of Breeding: Association With Grain Yield, Quality Traits, Protein Loss, and Plant Height.

Durum wheat was introduced in the southern prairies of western Canada in the late nineteenth century. Breeding efforts have mainly focused on improving quality traits to meet the pasta industry demands. For this study, 192 durum wheat lines were genotyped using the Illumina 90K Infinium iSelect assay, and resulted in a total of 14,324 polymorphic SNPs. Genetic diversity changed over time, declining during the first 20 years of breeding in Canada, then increased in the late 1980s and early 1990s. We scanned the genome for signatures of selection, using the total variance Fst-based outlier detection method (Lositan), the hierarchical island model (Arlequin) and the Bayesian genome scan method (BayeScan). A total of 407 outliers were identified and clustered into 84 LD-based haplotype loci, spanning all 14 chromosomes of the durum wheat genome. The association analysis detected 54 haplotype loci, of which 39% contained markers with a complete reversal of allelic state. This tendency to fixation of favorable alleles corroborates the success of the Canadian durum wheat breeding programs over time. Twenty-one haplotype loci were associated with multiple traits. In particular, hap_4B_1 explained 20.6, 17.9 and 16.6% of the phenotypic variance of pigment loss, pasta b∗ and dough extensibility, respectively. The locus hap_2B_9 explained 15.9 and 17.8% of the variation of protein content and protein loss, respectively. All these pleiotropic haplotype loci offer breeders the unique opportunity for further improving multiple traits, facilitating marker-assisted selection in durum wheat, and could help in identifying genes as functional annotations of the wheat genome become available.

Genetic analysis of resistance to stripe rust in durum wheat (Triticum turgidum L. var. durum).

Stripe rust, caused by the fungal pathogen Puccinia striiformis Westend. f. sp. tritici Eriks, is an important disease of bread wheat (Triticum aestivum L.) worldwide and there is an indication that it may also become a serious disease of durum wheat (T. turgidum L. var. durum). Therefore, we investigated the genetic architecture underlying resistance to stripe rust in adapted durum wheat germplasm. Wheat infection assays were conducted under controlled conditions in Canada and under field conditions in Mexico. Disease assessments were performed on a population of 155 doubled haploid (DH) lines derived from the cross of Kofa (susceptible) and W9262-260D3 (moderately resistant) and on a breeding panel that consisted of 92 diverse cultivars and breeding lines. Both populations were genotyped using the 90K single-nucleotide polymorphism (SNP) iSelect assay. In the DH population, QTL for stripe rust resistance were identified on chromosome 7B (LOD 6.87-11.47) and chromosome 5B (LOD 3.88-9.17). The QTL for stripe rust resistance on chromosome 7B was supported in the breeding panel. Both QTL were anchored to the genome sequence of wild emmer wheat, which identified gene candidates involved in disease resistance. Exome capture sequencing identified variation in the candidate genes between Kofa and W9262-260D3. These genetic insights will be useful in durum breeding to enhance resistance to stripe rust.

Characterization and mapping of leaf rust resistance in four durum wheat cultivars.

Widening the genetic basis of leaf rust resistance is a primary objective of the global durum wheat breeding effort at the International Wheat and Maize Improvement Center (CIMMYT). Breeding programs in North America are following suit, especially after the emergence of new races of Puccinia triticina such as BBG/BP and BBBQD in Mexico and the United States, respectively. This study was conducted to characterize and map previously undescribed genes for leaf rust resistance in durum wheat and to develop reliable molecular markers for marker-assisted breeding. Four recombinant inbred line (RIL) mapping populations derived from the resistance sources Amria, Byblos, Geromtel_3 and Tunsyr_2, which were crossed to the susceptible line ATRED #2, were evaluated for their reaction to the Mexican race BBG/BP of P. triticina. Genetic analyses of host reactions indicated that leaf rust resistance in these genotypes was based on major seedling resistance genes. Allelism tests among resistant parents supported that Amria and Byblos carried allelic or closely linked genes. The resistance in Geromtel_3 and Tunsyr_2 also appeared to be allelic. Bulked segregant analysis using the Infinium iSelect 90K single nucleotide polymorphism (SNP) array identified two genomic regions for leaf rust resistance; one on chromosome 6BS for Geromtel_3 and Tunsyr_2 and the other on chromosome 7BL for Amria and Byblos. Polymorphic SNPs identified within these regions were converted to kompetitive allele-specific PCR (KASP) assays and used to genotype the RIL populations. KASP markers usw215 and usw218 were the closest to the resistance genes in Geromtel_3 and Tunsyr_2, while usw260 was closely linked to the resistance genes in Amria and Byblos. DNA sequences associated with these SNP markers were anchored to the wild emmer wheat (WEW) reference sequence, which identified several candidate resistance genes. The molecular markers reported herein will be useful to effectively pyramid these resistance genes with other previously marked genes into adapted, elite durum wheat genotypes.

High density mapping and haplotype analysis of the major stem-solidness locus SSt1 in durum and common wheat.

Breeding for solid-stemmed durum (Triticum turgidum L. var durum) and common wheat (Triticum aestivum L.) cultivars is one strategy to minimize yield losses caused by the wheat stem sawfly (Cephus cinctus Norton). Major stem-solidness QTL have been localized to the long arm of chromosome 3B in both wheat species, but it is unclear if these QTL span a common genetic interval. In this study, we have improved the resolution of the QTL on chromosome 3B in a durum (Kofa/W9262-260D3) and common wheat (Lillian/Vesper) mapping population. Coincident QTL (LOD = 94-127, R2 = 78-92%) were localized near the telomere of chromosome 3BL in both mapping populations, which we designate SSt1. We further examined the SSt1 interval by using available consensus maps for durum and common wheat and compared genetic to physical intervals by anchoring markers to the current version of the wild emmer wheat (WEW) reference sequence. These results suggest that the SSt1 interval spans a physical distance of 1.6 Mb in WEW (positions 833.4-835.0 Mb). In addition, minor QTL were identified on chromosomes 2A, 2D, 4A, and 5A that were found to synergistically enhance expression of SSt1 to increase stem-solidness. These results suggest that developing new wheat cultivars with improved stem-solidness is possible by combining SSt1 with favorable alleles at minor loci within both wheat species.