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Gliadin sequence coverage profile and Acid-PAGE of Paragon and two Paragon γ-irradiated lines. Panel a) and b) display superimposed sequence coverage profiles of Paragon (grey) and Paragon γ-irradiated lines P3-75 (red) and P5-20 (dotted green line) for chromosome 1B and 6A, respectively. The absence of green peaks in 1A and red peaks on 6A indicates the absence of coverage of gluten genes and α-gliadin genes in lines P5-20 and P3-75, respectively. Panel c) and d) show the Acid-PAGE gliadin protein profiles of Paragon (outer lanes) in comparison to the Paragon mutant (central lanes, in duplicate). The deletion lines display protein bands missing in corresponding gliadin families, as indicated with red arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Source publication
p>We developed an in-solution gluten exome capture system called GlutEnSeq (Gluten gene Enrichment and Sequencing), covering the sequence variation of thousands of prolamin genes from various Triticeae species and cultivars. We assessed the efficacy of this capture system in hexaploid wheat (Triticum aestivum L.) using Illumina sequencing. On-targe...
Contexts in source publication
Context 1
... the data) resulted in what we refer to as condensed on-target coverage profiles or, for short, "coverage profiles" for each of the accessions under study. Below, these coverage profiles are compared to each other in various combinations, as appropriate -CS versus CS deletion line 1BS-19/6DS-4 (Fig. 2), Paragon versus Paragon γ-irradiated lines (Fig. 3) and Fielder versus Fielder CRISPR gliadin-edited lines (Fig. ...
Context 2
... Paragon γ-irradiated lines were selected as they were previously found to lack some gliadin bands on Acid-PAGE ( Jouanin et al., 2019). Line P5-20 lacks bands associated to γ-gliadins (which are located on the short arms of the group 1 chromosomes) while line P3-75 lacks bands related to α-gliadins (located on the short arms of chromosome 6; Fig. 3b and d). A coverage profile of Paragon together with the two Paragon γ-irradiated mutants P5-20 and P3-75 of chromosomes 1B and 6A is shown (Fig. 3a and ...
Context 3
... lacks bands associated to γ-gliadins (which are located on the short arms of the group 1 chromosomes) while line P3-75 lacks bands related to α-gliadins (located on the short arms of chromosome 6; Fig. 3b and d). A coverage profile of Paragon together with the two Paragon γ-irradiated mutants P5-20 and P3-75 of chromosomes 1B and 6A is shown (Fig. 3a and ...
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... comparison between sequence coverage profiles of Paragon and mutants shows the absence of coverage across a large part of the gluten locus on chromosome 1B for the P5-20 mutant line (Fig. 3a). For line P3-75, there is no coverage for the complete Gli-2 locus on chromosome 6A for α-gliadins (Fig. 3c). The other loci (on 1A, 1D, 6B and 6D) are unchanged compared to CS (not shown). These results correlate with the protein bands missing on the Acid-PAGE ...
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... comparison between sequence coverage profiles of Paragon and mutants shows the absence of coverage across a large part of the gluten locus on chromosome 1B for the P5-20 mutant line (Fig. 3a). For line P3-75, there is no coverage for the complete Gli-2 locus on chromosome 6A for α-gliadins (Fig. 3c). The other loci (on 1A, 1D, 6B and 6D) are unchanged compared to CS (not shown). These results correlate with the protein bands missing on the Acid-PAGE ...
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... comparison between coverage profiles of Fielder and the geneedited mutants shows the absence of coverage in a large part of the gluten locus on chromosome 1B in line α2γ3-39_G7 (Fig. 4a), which was similar to the CS deletion line (Fig. 2a) and P5-20 Paragon γ-irradiated line (Fig. 3a). The Acid-PAGE profile was in accordance with this result (Fig. 4b) and was similar to that of P5-20 (Fig. 3b) where a γ-gliadin band was ...
Context 7
... Fielder and the geneedited mutants shows the absence of coverage in a large part of the gluten locus on chromosome 1B in line α2γ3-39_G7 (Fig. 4a), which was similar to the CS deletion line (Fig. 2a) and P5-20 Paragon γ-irradiated line (Fig. 3a). The Acid-PAGE profile was in accordance with this result (Fig. 4b) and was similar to that of P5-20 (Fig. 3b) where a γ-gliadin band was ...
Similar publications
Starch and prolamin composition and content are important indexes for determining the processing and nutritional quality of wheat (Triticum aestivum L.) grains. Several transcription factors (TFs) regulate gene expression during starch and protein biosynthesis in wheat. Storage protein activator (TaSPA), a member of the basic leucine zipper (bZIP)...
Citations
... Using γ-irradiation, large chromosomal deletions have been induced in two mutant lines of the wheat variety Paragon. Both mutants revealed a lack of sequence coverage in a large part of the gluten loci [25]. However, a clear need for more sophisticated approaches remains. ...
... Some gluten genes were deleted, as evidenced by droplet digital PCR (ddPCR) results for alphagliadins [27]. Evidence that in some regenerated plants blocks of genes had been deleted came from mapping of sequence data obtained with GlutEnSeq, a variant to RenSeq that targets gluten genes, to the Chinese Spring reference genome [25]. This is presumably the result of double-strand breaks in different gliadin genes that occur in tandem arrays in the wheat genome, leading to the deletion of tandem gene blocks in between the genes with double strand breaks. ...
By using gene editing technologies such as CRISPR/Cas, precise modifications can be made in the genome. CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as it can induce mutations of multiple alleles simultaneously, to obtain regenerants that are homozygous for the desired mutation. A range of gene-edited traits have been developed in hexaploid bread wheat, including various nutrition and health-related traits, plant architecture, pest and disease resistance, tolerance to abiotic stress, and traits that enable more efficient breeding. Wheat is also known as a cause of some human diseases, particularly coeliac disease (CD), with a prevalence of 1–2% of the population. In the EU alone, at least 4.5 million people suffer from it. CD is a chronic inflammation of the small intestine, induced and maintained in genetically predisposed individuals by the consumption of gluten proteins from wheat, barley and rye. As there is no cure, patients must follow a life-long gluten-free diet. The dominant epitopes in gluten proteins that trigger the disease, have been characterized, but they cannot be removed by classical breeding without affecting baking quality, as it concerns over 100 gluten genes that occur partly as blocks of genes in the genome of wheat. Using gene editing, two studies have shown that it is possible to modify the epitopes in several alpha- and gamma-gliadins simultaneously, while deleting some of the genes completely. In some lines more than 80% of the alpha-gliadin genes were modified. These proof-of-principle studies show that it is feasible to use gene editing, along with other breeding approaches, to completely remove the CD epitopes from bread wheat. Gene-edited coeliac-safe wheat will have economic, social and environmental impact on food security, nutrition and public health, but the realisation will (partially) depend on new European legislation for plants produced by gene editing.
... Previously, characterization of mutations in the gliadin genes was performed by targeted capture (Jouanin et al., 2019) or targeted amplification (S anchez-Le on et al., 2018). While these approaches proved to be effective for detecting the presence of edited alleles, they had reduced ability to discern which of the specific gene copies was modified and to accurately assess offtarget editing activities. ...
Wheat immunotoxicity is associated with abnormal reaction to gluten‐derived peptides. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality. Here, the multiplexed CRISPR‐Cas9 editing of cultivar Fielder was used to modify gluten‐encoding genes, specifically focusing on ω‐ and γ‐gliadin gene copies, which were identified to be abundant in immunoreactive peptides based on the analysis of wheat genomes assembled using the long‐read sequencing technologies. The whole‐genome sequencing of an edited line showed mutation or deletion of nearly all ω‐gliadin and half of the γ‐gliadin gene copies and confirmed the lack of editing in the α/β‐gliadin genes. The estimated 75% and 64% reduction in ω‐ and γ‐gliadin content, respectively, had no negative impact on the end‐use quality characteristics of grain protein and dough. A 47‐fold immunoreactivity reduction compared to a non‐edited line was demonstrated using antibodies against immunotoxic peptides. Our results indicate that the targeted CRISPR‐based modification of the ω‐ and γ‐gliadin gene copies determined to be abundant in immunoreactive peptides by analysing high‐quality genome assemblies is an effective mean for reducing immunotoxicity of wheat cultivars while minimizing the impact of editing on protein quality.
... An examination of the sequences of T0 plants revealed the deletion of 1-23 nt and even an addition of 1 nt in different lines. Conventional breeding methods cannot lower gluten concentration in bread wheat due to 100 genes and pseudogenes placed in tandem at the Gli-2 loci of 6A, -B, and -D chromosomes for α-gliadins (Jouanin et al. 2019). Using the CRISPR/Cas9 technique to target a conserved section of the α-gliadin genes, the researchers designed two sgRNAs and obtained wheat lines with low-gluten (Sánchez-León et al. 2018). ...
Main conclusion
This review provides a comprehensive overview of the CRISPR/Cas9 technique and the research areas of this gene editing tool in improving wheat quality.
Abstract
Wheat (Triticum aestivum L.), the basic nutrition for most of the human population, contributes 20% of the daily energy needed because of its, carbohydrate, essential amino acids, minerals, protein, and vitamin content. Wheat varieties that produce high yields and have enhanced nutritional quality will be required to fulfill future demands. Hexaploid wheat has A, B, and D genomes and includes three like but not identical copies of genes that influence important yield and quality. CRISPR/Cas9, which allows multiplex genome editing provides major opportunities in genome editing studies of plants, especially complicated genomes such as wheat. In this overview, we discuss the CRISPR/Cas9 technique, which is credited with bringing about a paradigm shift in genome editing studies. We also provide a summary of recent research utilizing CRISPR/Cas9 to investigate yield, quality, resistance to biotic/abiotic stress, and hybrid seed production. In addition, we provide a synopsis of the laboratory experience-based solution alternatives as well as the potential obstacles for wheat CRISPR studies. Although wheat’s extensive genome and complicated polyploid structure previously slowed wheat genetic engineering and breeding progress, effective CRISPR/Cas9 systems are now successfully used to boost wheat development.
... The method is a gluten exome capture system based on thousands of prolamin genes from Triticeae species and cultivars. Gluten gene sequences were enriched around 10,000-fold in a wheat variety (Jouanin et al. 2019a). Targeted gene capture methods with long-read sequencing make it possible to obtain complete sequences of highly repetitive gluten genes and provide precise information on which immunogenic gluten genes are missing in specific wheat lines such as the eleven selected wheat lines in this study. ...
Key message
Eleven wheat lines that are missing genes for the 1D-encoded omega-5 gliadins will facilitate breeding efforts to reduce the immunogenic potential of wheat flour for patients susceptible to wheat allergy.
Abstract
Efforts to reduce the levels of allergens in wheat flour that cause wheat-dependent exercise-induced anaphylaxis are complicated by the presence of genes encoding omega-5 gliadins on both chromosomes 1B and 1D of hexaploid wheat. In this study, we screened 665 wheat germplasm samples using gene specific DNA markers for omega-5 gliadins encoded by the genes on 1D chromosome that were obtained from the reference wheat Chinese Spring. Eleven wheat lines missing the PCR product corresponding to 1D omega-5 gliadin gene sequences were identified. Two of the lines contained the 1BL·1RS translocation. Relative quantification of gene copy numbers by qPCR revealed that copy numbers of 1D omega-5 gliadins in the other nine lines were comparable to those in 1D null lines of Chinese Spring, while copy numbers of 1B omega-5 gliadins were like those of Chinese Spring. 2-D immunoblot analysis of total flour proteins from the selected lines using a specific monoclonal antibody against the N-terminal sequence of omega-5 gliadin showed no reactivity in regions of the blots containing previously identified 1D omega-5 gliadins. Interestingly, RP-UPLC analysis of the gliadin fractions of the selected lines indicated that the expression of omega-1,2 gliadins was also significantly reduced in seven of the lines, implying that 1D omega-5 gliadin and 1D omega-1,2 gliadin genes are tightly linked on the Gli-D1 loci of chromosome 1D. Wheat lines missing the omega-5 gliadins encoded by the genes on 1D chromosome should be useful in future breeding efforts to reduce the immunogenic potential of wheat flour.
... We used the coding sequences of gliadin genes from the wheat variety 'Xiaoyan81' (Wang et al., 2017) as queries to identify gliadin genes in the genome sequence of the cultivar Fielder (Sato et al., 2021) and identified 44 gliadin genes, 28 encoding a-gliadins, 11 encoding c-gliadins, and 5 encoding x-gliadins (Dataset S2). In agreement with previous reports (Sabelli & Shewry, 1991;Anderson et al., 1997;Ozuna et al., 2015;Clavijo et al., 2017;Jouanin et al., 2019a), all c-gliadin genes were located in the Gli-1 locus on the short arm of each of the group 1 chromosomes (Fig. 1c). ...
Gluten is composed of glutenins and gliadins and determines the viscoelastic properties of dough and end‐use quality in wheat (Triticum aestivum L.). Gliadins are important for wheat end‐use traits, but the contribution of individual gliadin genes is unclear, since gliadins are encoded by a complex, multigenic family, including many pseudogenes.
We used CRISPR/Cas9‐mediated gene editing and map‐based cloning to investigate the contribution of the γ‐gliadin genes annotated in the wheat cultivar ‘Fielder’, showing that Gli‐γ1‐1D and Gli‐γ2‐1B account for most of the γ‐gliadin accumulation.
The impaired activity of only two γ‐gliadin genes in knockout mutants improved end‐use quality and reduced gluten epitopes associated with celiac disease (CD). Furthermore, we identified an elite haplotype of Gli‐γ1‐1D linked to higher end‐use quality in a wheat germplasm collection and developed a molecular marker for this allele for marker‐assisted selection.
Our findings provide information and tools for biotechnology‐based and classical breeding programs aimed at improving wheat end‐use quality.
... The CRISPR/Cas9 gene editing is in development to produce wheat lines with fewer gluten genes. This technology has been used to edit α-gliadin and γgliadin genes [58][59][60][61]. However, although they represent beneficial progress for subjects suffering from CD, the public acceptance of these transgenic foods and food products still remains a puzzling problem. ...
Celiac peptide-generating α- and γ-gliadins consist of a disordered N-terminal domain extended by an α-helical-folded C-terminal domain. Celiac peptides, primarily located along the disordered part of α- and γ-gliadin molecules, are nicely exposed and directly accessible to proteolytic enzymes occurring in the gastric (pepsin) and intestinal (trypsin, chymotrypsin) fluids. More than half of the potential celiac peptides identified so far in gliadins exhibit cleavage sites for pepsin. However, celiac peptides proteolytically truncated by one or two amino acid residues could apparently retain some activity toward HLA-DQ2 and HLA-DQ8 receptors in docking experiments. Together with the uncleaved peptides, these still active partially degraded CD peptides account for the incapacity of the digestion process to inactivate CD peptides from gluten proteins. In contrast, sourdough fermentation processes involve other proteolytic enzymes susceptible to the deep degradation of celiac peptides. In particular, sourdough supplemented by fungal prolyl endoproteases enhances the degrading capacities of the sourdough fermentation process toward celiac peptides. Nevertheless, since tiny amounts of celiac peptides sufficient to trigger deleterious effects on CD people can persist in sourdough-treated bread and food products, it is advisable to avoid consumption of sourdough-treated food products for people suffering from celiac disease. As an alternative, applying the supplemented sourdough process to genetically modified low gluten or celiac-safe wheat lines should result in food products that are safer for susceptible and CD people.
... In those species which lack a reference genome, it is even more challenging to perform a genome-wide NLR survey. Exon capture enrichment allows the selected sequencing of an exome [54], or a specific gene family [32]. The highly conserved domains shared by different NLRs provides perfect targets for enrichment. ...
Background
Nucleotide-binding and leucine-rich repeat (NLR) genes have attracted wide attention due to their crucial role in protecting plants from pathogens. SMRT-RenSeq, combining PacBio sequencing after resistance gene enrichment sequencing (RenSeq), is a powerful method for selectively capturing and sequencing full-length NLRs. Haynaldia villosa, a wild grass species with a proven potential for wheat improvement, confers resistance to multiple diseases. So, genome-wide identification of the NLR gene family in Haynaldia villosa by SMRT-RenSeq can facilitate disease resistance genes exploration.
Results
In this study, SMRT-RenSeq was performed to identify the genome-wide NLR complement of H. villosa. In total, 1320 NLRs were annotated in 1169 contigs, including 772 complete NLRs. All the complete NLRs were phylogenetically analyzed and 11 main clades with special characteristics were derived. NLRs could be captured with high efficiency when aligned with cloned R genes, and cluster expansion in some specific gene loci was observed. The physical location of NLRs to individual chromosomes in H. villosa showed a perfect homoeologous relationship with group 1, 2, 3, 5 and 6 of other Triticeae species, however, NLRs physically located on 4VL were largely in silico predicted to be located on the homoeologous group 7. Fifteen types of integrated domains (IDs) were integrated in 52 NLRs, and Kelch and B3 NLR-IDs were found to have expanded in H. villosa, while DUF948, NAM-associated and PRT_C were detected as unique integrated domains implying the new emergence of NLR-IDs after H. villosa diverged from other species.
Conclusion
SMRT-RenSeq is a powerful tool to identify NLR genes from wild species using the baits of the evolutionary related species with reference sequences. The availability of the NLRs from H. villosa provide a valuable library for R gene mining and transfer of disease resistance into wheat.
... However, the authors also stated the need to further perform deep sequencing to characterize these mutations. For this purpose, they developed GlutEnSeq; an in-solution gluten exome capture system [46]. In this approach, captured gluten sequence reads were mapped to the Chinese Spring reference genome. ...
... As reported previously, the presence of spaced site-specific double-strand breaks (DSBs) could be associated with large deletions in rice by CRISPR/Cas9 [56]. The presence of large deletions in α-gliadin genes, as these are arranged in tandem, was also reported in wheat CRISPR lines [46]. However, the mechanism and the extent of such rearrangements need to be studied in more detail in future works. ...
The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family.
... A common phenotype is that downregulation of one or a subset of gluten protein encoding genes is accompanied by the compensatory upregulation of alternate storage proteins (30)(31)(32)(33)(34), with a change in technological properties (35). However, technology to characterize gluten in wheat products is the subject of ongoing research (36), as is the targeted removal of CD reactive epitopes from wheat (37). ...
Gluten related disorders, such as coeliac disease, wheat allergy and baker's asthma are triggered by proteins present in food products made from wheat and related cereal species. The only treatment of these medical illnesses is a strict gluten-free diet; however, gluten-free products that are currently available in the market can have lower nutritional quality and are more expensive than traditional gluten containing cereal products. These constraints have led to the development of gluten-free or gluten-reduced ingredients. In this vein, a non-GMO wheat flour that purports to contain “65% less allergenic gluten” was recently brought to market. The present study aims to understand the alteration of the proteome profile of this wheat flour material. Liquid chromatography-mass spectrometry was used to investigate the proteome profile of the novel wheat flour, which was contrasted to a wheat flour control. Using both trypsin and chymotrypsin digests and a combined database search, 564 unique proteins were identified with 99% confidence. These proteins and the specific peptides used to identify them were mapped to the wheat genome to reveal the associated chromosomal regions in the novel wheat flour and the mixed wheat control. Of note, several ω- and γ-gliadins, and low-molecular weight glutenins mapping to the short arm of chromosome 1, as well as α-gliadins from the chromosome 6 short arm were absent or expressed at lower levels in the novel wheat variety. In contrast, the high-molecular weight glutenins and α-amylase/trypsin inhibitors were notably more abundant in this variety. A targeted quantitation experiment was developed using multiple reaction monitoring assays to quantify 359 tryptic and chymotryptic peptides from gluten and related allergenic proteins revealing a 33% decrease of gluten protein content in the novel wheat flour sample in comparison to mixed wheat control. However, additional mapping of known allergenic epitopes showed the presence of 53% higher allergenic peptides. Overall, the current study highlights the importance of proteomic analyses especially when complemented by sequence analysis and epitope mapping for monitoring immunostimulatory proteins.
... The editing efficiency in T1 individuals varied between cultivars and gRNA used (highest up to 75% edited reads in one cultivar, others between 1,5%-14,7%). A second study aiming to establish low gluten wheat lines targeted alpha-and gamma-gliadin loci using a multiplexing approach involving five gRNAs, two targeting alpha-and three gamma-gliadin genes, in separate cassettes on a vector (Fig. 3v) (Jouanin et al. 2019). Finally, fragment deletions can be engineered also in wheat targeting a pair of sgRNAs to the same gene (Cui et al. 2019). ...
... IV Using a single sgRNA to target multiple copies of alphagliadin gene copies (Sánchez-León et al. 2018). and V Multiplexing using five sgRNAs driven as separate cassettes and targeting alpha-and gamma-gliadin genes (Jouanin et al. 2019). ...
Genome-editing technologies offer unprecedented opportunities for crop improvement with superior precision and speed. This review presents an analysis of the current state of genome editing in the major cereal crops- rice, maize, wheat and barley. Genome editing has been used to achieve important agronomic and quality traits in cereals. These include adaptive traits to mitigate the effects of climate change, tolerance to biotic stresses, higher yields, more optimal plant architecture, improved grain quality and nutritional content, and safer products. Not all traits can be achieved through genome editing, and several technical and regulatory challenges need to be overcome for the technology to realize its full potential. Genome editing, however, has already revolutionized cereal crop improvement and is poised to shape future agricultural practices in conjunction with other breeding innovations.
