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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. 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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Although the economic value of wheat flour is determined by the complement of gluten proteins, these proteins have been challenging to study because of the complexity of the major protein groups and the tremendous sequence diversity among wheat cultivars. The completion of a high-quality wheat genome sequence from the reference wheat Chinese Spring recently facilitated the assembly and annotation of a complete set of gluten protein genes from a single cultivar, making it possible to link individual proteins in the flour to specific gene sequences. In a proteomic analysis of total wheat flour protein from Chinese Spring using quantitative two-dimensional gel electrophoresis combined with tandem mass spectrometry, gliadins or low-molecular-weight glutenin subunits were identified as the predominant proteins in 72 protein spots. Individual spots were associated with 40 of 56 Chinese Spring gene sequences, including 16 of 26 alpha gliadins, 10 of 11 gamma gliadins, six of seven omega gliadins, one of two delta gliadins, and nine of ten LMW-GS. Most genes that were not associated with protein spots were either expressed at low levels in endosperm or encoded proteins with high similarity to other proteins. A wide range of protein accumulation levels were observed and discrepancies between transcript levels and protein levels were noted. This work together with similar studies using other commercial cultivars should provide new insight into the molecular basis of wheat flour quality and allergenic potential.

α-Gliadins are a major group of gluten proteins in wheat flour that contribute to the end-use properties for food processing and contain major immunogenic epitopes that can cause serious health-related issues including celiac disease (CD). α-Gliadins are also the youngest group of gluten proteins and are encoded by a large gene family. The majority of the gene family members evolved independently in the A, B, and D genomes of different wheat species after their separation from a common ancestral species. To gain insights into the origin and evolution of these complex genes, the genomic regions of the Gli-2 loci encoding α-gliadins were characterized from the tetraploid wild emmer, a progenitor of hexaploid bread wheat that contributed the AABB genomes. Genomic sequences of Gli-2 locus regions for the wild emmer A and B genomes were first reconstructed using the genome sequence scaffolds along with optical genome maps. A total of 24 and 16 α-gliadin genes were identified for the A and B genome regions, respectively. α-Gliadin pseudogene frequencies of 86% for the A genome and 69% for the B genome were primarily caused by C to T substitutions in the highly abundant glutamine codons, resulting in the generation of premature stop codons. Comparison with the homologous regions from the hexaploid wheat cv. Chinese Spring indicated considerable sequence divergence of the two A genomes at the genomic level. In comparison, conserved regions between the two B genomes were identified that included α-gliadin pseudogenes containing shared nested TE insertions. Analyses of the genomic organization and phylogenetic tree reconstruction indicate that although orthologous gene pairs derived from speciation were present, large portions of α-gliadin genes were likely derived from differential gene duplications or deletions after the separation of the homologous wheat genomes ~ 0.5 MYA. The higher number of full-length intact α-gliadin genes in hexaploid wheat than that in wild emmer suggests that human selection through domestication might have an impact on α-gliadin evolution. Our study provides insights into the rapid and dynamic evolution of genomic regions harboring the α-gliadin genes in wheat.

Background: Omega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour., Results: A mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes., Conclusions: While breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

Gliadins are a major component of wheat seed proteins. However, the complex homoeologous Gli-2 loci (Gli-A2, -B2 and -D2) that encode the α-gliadins in commercial wheat are still poorly understood. Here we analyzed the Gli-D2 locus of Xiaoyan 81 (Xy81), a winter wheat cultivar. A total of 421.091 kb of the Gli-D2 sequence was assembled from sequencing multiple bacterial artificial clones, and 10 α-gliadin genes were annotated. Comparative genomic analysis showed that Xy81 carried only eight of the α-gliadin genes of the D genome donor Aegilops tauschii, with two of them each experiencing a tandem duplication. A mutant line lacking Gli-D2 (DLGliD2) consistently exhibited better breadmaking quality and dough functionalities than its progenitor Xy81, but without penalties in other agronomic traits. It also had an elevated lysine content in the grains. Transcriptome analysis verified the lack of Gli-D2 α-gliadin gene expression in DLGliD2. Furthermore, the transcript and protein levels of protein disulfide isomerase were both upregulated in DLGliD2 grains. Consistent with this finding, DLGliD2 had increased disulfide content in the flour. Our work sheds light on the structure and function of Gli-D2 in commercial wheat, and suggests that the removal of Gli-D2 and the gliadins specified by it is likely to be useful for simultaneously enhancing the end-use and health-related traits of common wheat. Because gliadins and homologous proteins are widely present in grass species, the strategy and information reported here may be broadly useful for improving the quality traits of diverse cereal crops., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Wheat Gli-2 loci encode complex groups of α-gliadin prolamins that are important for breadmaking, but also major triggers of celiac disease (CD). Elucidation of α-gliadin evolution provides knowledge to produce wheat with better end-use properties and reduced immunogenic potential. The Gli-2 loci contain a large number of tandemly duplicated genes and highly repetitive DNA, making sequence assembly of their genomic regions challenging. Here, we constructed high-quality sequences spanning the three wheat homeologous α-gliadin loci by aligning PacBio-based sequence contigs with BioNano genome maps. A total of 47 α-gliadin genes were identified with only 26 encoding intact full-length protein products. Analyses of α-gliadin loci and phylogenetic tree reconstruction indicate significant duplications of α-gliadin genes in the last ~2.5 million years after the divergence of the A, B and D genomes, supporting its rapid lineage-independent expansion in different Triticeae genomes. We showed that dramatic divergence in expression of α-gliadin genes could not be attributed to sequence variations in the promoter regions. The study also provided insights into the evolution of CD epitopes and identified a single indel event in the hexaploid wheat D genome that likely resulted in the generation of the highly toxic 33-mer CD epitope.

Among the wheat prolamins important for its end-use traits, α-gliadins are the most abundant, and are also a major cause of food-related allergies and intolerances. Previous studies of various wheat species estimated that between 25 and 150 α-gliadin genes reside in the Gli-2 locus regions. To better understand the evolution of this complex gene family, the DNA sequence of a 1.75-Mb genomic region spanning the Gli-2 locus was analyzed in the diploid grass, Aegilops tauschii, the ancestral source of D genome in hexaploid bread wheat. Comparison with orthologous regions from rice, sorghum, and Brachypodium revealed rapid and dynamic changes only occurring to the Ae. tauschii Gli-2 region, including insertions of high numbers of non-syntenic genes and a high rate of tandem gene duplications, the latter of which have given rise to 12 copies of α-gliadin genes clustered within a 550-kb region. Among them, five copies have undergone pseudogenization by various mutation events. Insights into the evolutionary relationship of the duplicated α-gliadin genes were obtained from their genomic organization, transcription patterns, transposable element insertions and phylogenetic analyses. An ancestral glutamate-like receptor (GLR) gene encoding putative amino acid sensor in all four grass species has duplicated only in Ae. tauschii and generated three more copies that are interspersed with the α-gliadin genes. Phylogenetic inference and different gene expression patterns support functional divergence of the Ae. tauschii GLR copies after duplication. Our results suggest that the duplicates of α-gliadin and GLR genes have likely taken different evolutionary paths; conservation for the former and neofunctionalization for the latter., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

The complete set of unique γ-gliadin genes is described for the wheat cultivar Chinese Spring using a combination of expressed sequence tag (EST) and Roche 454 DNA sequences. Assemblies of Chinese Spring ESTs yielded 11 different γ-gliadin gene sequences. Two of the sequences encode identical polypeptides and are assumed to be the result of a recent gene duplication. One gene has a 3' coding mutation that changes the reading frame in the final eight codons. A second assembly of Chinese Spring γ-gliadin sequences was generated using Roche 454 total genomic DNA sequences. The 454 assembly confirmed the same 11 active genes as the EST assembly plus two pseudogenes not represented by ESTs. These 13 γ-gliadin sequences represent the complete unique set of γ-gliadin genes for cv Chinese Spring, although not ruled out are additional genes that are exact duplications of these 13 genes. A comparison with the ESTs of two other hexaploid cultivars (Butte 86 and Recital) finds that the most active genes are present in all three cultivars, with exceptions likely due to too few ESTs for detection in Butte 86 and Recital. A comparison of the numbers of ESTs per gene indicates differential levels of expression within the γ-gliadin gene family. Genome assignments were made for 6 of the 13 Chinese Spring γ-gliadin genes, i.e., one assignment from a match to two γ-gliadin genes found within a tetraploid wheat A genome BAC and four genes that match four distinct γ-gliadin sequences assembled from Roche 454 sequences from Aegilops tauschii, the hexaploid wheat D-genome ancestor.

The utility of mining DNA sequence data to understand the structure and expression of cereal prolamin genes is demonstrated by the identification of a new class of wheat prolamins. This previously unrecognized wheat prolamin class, given the name δ-gliadins, is the most direct ortholog of barley γ3-hordeins. Phylogenetic analysis shows that the orthologous δ-gliadins and γ3-hordeins form a distinct prolamin branch that existed separate from the γ-gliadins and γ-hordeins in an ancestral Triticeae prior to the branching of wheat and barley. The expressed δ-gliadins are encoded by a single gene in each of the hexaploid wheat genomes. This single δ-gliadin/γ3-hordein ortholog may be a general feature of the Triticeae tribe since examination of ESTs from three barley cultivars also confirms a single γ3-hordein gene. Analysis of ESTs and cDNAs shows that the genes are expressed in at least five hexaploid wheat cultivars in addition to diploids Triticum monococcum and Aegilops tauschii. The latter two sequences also allow assignment of the δ-gliadin genes to the A and D genomes, respectively, with the third sequence type assumed to be from the B genome. Two wheat cultivars for which there are sufficient ESTs show different patterns of expression, i.e., with cv Chinese Spring expressing the genes from the A and B genomes, while cv Recital has ESTs from the A and D genomes. Genomic sequences of Chinese Spring show that the D genome gene is inactivated by tandem premature stop codons. A fourth δ-gliadin sequence occurs in the D genome of both Chinese Spring and Ae. tauschii, but no ESTs match this sequence and limited genomic sequences indicates a pseudogene containing frame shifts and premature stop codons. Sequencing of BACs covering a 3 Mb region from Ae. tauschii locates the δ-gliadin gene to the complex Gli-1 plus Glu-3 region on chromosome 1.

A survey and analysis is made of all available omega-gliadin DNA sequences including omega-gliadin genes within a large genomic clone, previously reported gene sequences, and ESTs identified from the large wheat EST collection. A contiguous portion of the Gli-B3 locus is shown to contain two apparently active omega-gliadin genes, two pseudogenes, and four fragments of the 3' portion of omega-gliadin sequences. Comparison of omega-gliadin sequences allows a phylogenetic picture of their relationships and genomes of origin. Results show three groupings of omega-gliadin active gene sequences assigned to each of the three hexaploid wheat genomes, and a fourth group thus far consisting of pseudogenes assigned to the A-genome. Analysis of omega-gliadin ESTs allows reconstruction of two full-length model sequences encoding the AREL- and ARQL-type proteins from the Gli-A3 and Gli-D3 loci, respectively. There is no DNA evidence of multiple active genes from these two loci. In contrast, ESTs allow identification of at least three to four distinct active genes at the Gli-B3 locus of some cultivars. Additional results include more information on the position of cysteines in some omega-gliadin genes and discussion of problems in studying the omega-gliadin gene family.

To better understand the molecular evolution of the large alpha-gliadin gene family, a half-million bacterial artificial chromosome (BAC) library clones from tetraploid durum wheat, Triticum turgidum ssp. durum (2n = 4x = 28, genome AB), were screened for large genomic segments carrying the alpha-gliadin genes of the Gli-2 loci on the group 6 homoeologous chromosomes. The resulting 220 positive BAC clones--each containing between one and four copies of alpha-gliadin sequences--were fingerprinted for contig assembly to produce contiguous chromosomal regions covering the Gli-2 loci. While contigs consisting of as many as 21 BAC clones and containing up to 17 alpha-gliadin genes were formed, many BAC clones remained as singletons. The accuracy of the order of BAC clones in the contigs was verified by Southern hybridization analysis of the BAC fingerprints using an alpha-gliadin probe. These results indicate that alpha-gliadin genes are not evenly dispersed in the Gli-2 locus regions. Hybridization of these BACs with probes for long terminal repeat retrotransposons was used to determine the abundance and distribution of repetitive DNA in this region. Sequencing of BAC ends indicated that 70% of the sequences were significantly similar to different classes of retrotransposons, suggesting that these elements are abundant in this region. Several mechanisms underlying the dynamic evolution of the Gli-2 loci are discussed., (Copyright 2004 Springer-Verlag)