SUMMARYThe gluten proteins from wheat, barley and rye are responsible both for celiac disease (CD) and for nonceliac gluten sensitivity, two pathologies affecting up to 6-8% of the human population worldwide. The wheat a-gliadin proteins contain three major CD immunogenic peptides: p31-43, which induces the innate immune response; the 33-mer, formed by six overlapping copies of three highly stimulatory epitopes; and an additional DQ2.5-glia-a3 epitope which partially overlaps with the 33-mer. Next-generation sequencing (NGS) and Sanger sequencing of a-gliadin genes from diploid and polyploid wheat provided six types of a-gliadins (named 1-6) with strong differences in their frequencies in diploid and polyploid wheat, and in the presence and abundance of these CD immunogenic peptides. Immunogenic variants of the p31-43 peptide were found in most of the a-gliadins. Variants of the DQ2.5-glia-a3 epitope were associated with specific types of a-gliadins. Remarkably, only type 1 a-gliadins contained 33-mer epitopes. Moreover, the full immunodominant 33-mer fragment was only present in hexaploid wheat at low abundance, probably as the result of allohexaploidization events from subtype 1.2 a-gliadins found only in Aegilops tauschii, the D-genome donor of hexaploid wheat. Type 3 a-gliadins seem to be the ancestral type as they are found in most of the a-gliadin-expressing Triticeae species. These findings are important for reducing the incidence of CD by the breeding/selection of wheat varieties with low stimulatory capacity of T cells. Moreover, advanced genome-editing techniques (TALENs, CRISPR) will be easier to implement on the small group of a-gliadins containing only immunogenic peptides.

The awn is a long needle-like structure formed at the tip of the lemma in the florets of some grass species. It plays a role in seed dispersal and protection against animals, and can contribute to the photosynthetic activity of spikes. Three main dominant inhibitors of awn development (Hd, B1 and B2) are known in hexaploid wheat, but the causal genes have not been cloned yet and a genetic association with awn length diversity has been found only for the B1 allele. To analyze the prevalence of these three awning inhibitors, we attempted to predict the genotypes of 189 hexaploid wheat varieties collected worldwide using markers tightly linked to these loci. Using recombinant inbred lines derived from two common wheat cultivars, Chinese Spring and Mironovskaya 808, both with short awns, and a high-density linkage map, we performed quantitative trait locus analysis to identify tightly linked markers. Because this linkage map was constructed with abundant array-based markers, we converted the linked markers to PCR-based markers and determined the genotypes of 189 hexaploids. A significant genotype-phenotype correlation was observed at the Hd and B1 regions. We also found that interaction among these three awning inhibitors is involved in development of a membranous outgrowth at the base of awn resembling the Hooded mutants of barley. For the hooded awn phenotype, presence of the Hd dominant allele was essential but not sufficient, so B2 and other factors appear to act epistatically to produce the ectopic tissue. On the other hand, the dominant B1 allele acted as a suppressor of the hooded phenotype. These three awning inhibitors largely contribute to the genetic variation in awn length and shape of common wheat.

SummaryGluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten-free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from a-, c-, x-gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP-HPLC and mass spectrometry (LC-MS/ MS), whereas gluten immunogenicity was assayed by an anti-gliadin 33-mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only c-and a-gliadins, respectively. Four plasmid combinations were highly effective in the silencing of x-gliadins and c-gliadins, and three of these also silenced a-gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC-MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic a-and x-gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars.

Synthetic hexaploid wheat is an effective genetic resource for transferring agronomically important genes from Aegilops tauschii to common wheat. Wide variation in grain size and shape, one of the main targets for wheat breeding, has been observed among Ae. tauschii accessions. To identify the quantitative trait loci (QTLs) responsible for grain size and shape variation in the wheat D genome under a hexaploid genetic background, six parameters related to grain size and shape were measured using SmartGrain digital image software and QTL analysis was conducted using four F2 mapping populations of wheat synthetic hexaploids. In total, 18 QTLs for the six parameters were found on five of the seven D-genome chromosomes. The identified QTLs significantly contributed to the variation in grain size and shape among the synthetic wheat lines, implying that the D-genome QTLs might be at least partly functional in hexaploid wheat. Thus, synthetic wheat lines with diverse D genomes from Ae. tauschii are useful resources for the identification of agronomically important loci that function in hexaploid wheat.

13,347 high-confidence SNPs were discovered through transcriptome sequencing of Aegilops tauschii, which are useful for genomic analysis and molecular breeding of hexaploid wheat. In organisms with large and complex genomes, such as wheat, RNA-seq analysis is cost-effective for discovery of genome-wide single nucleotide polymorphisms (SNPs). In this study, deep sequencing of the spike transcriptome from two Aegilops tauschii accessions representing two major lineages led to the discovery of 13,347 high-confidence (HC) SNPs in 4,872 contigs. After removing redundant SNPs detected in the leaf transcriptome from the same accessions in an earlier study, 10,589 new SNPs were discovered. In total, 5,642 out of 5,808 contigs with HC SNPs were assigned to the Ae. tauschii draft genome sequence. On average, 732 HC polymorphic contigs were mapped in silico to each Ae. tauschii chromosome. Based on the polymorphic data, we developed markers to target the short arm of chromosome 2D and validated the polymorphisms using 20 Ae. tauschii accessions. Of the 29 polymorphic markers, 28 were successfully mapped to 2DS in the diploid F2 population of Ae. tauschii. Among ten hexaploid wheat lines, which included wheat synthetics and common wheat cultivars, 25 of the 43 markers were polymorphic. In the hexaploid F2 population between a common wheat cultivar and a synthetic wheat line, 23 of the 25 polymorphic markers between the parents were available for genotyping of the F2 plants and 22 markers mapped to chromosome 2DS. These results indicate that molecular markers that developed from polymorphisms between two distinct lineages of Ae. tauschii might be useful for analysis not only of the diploid, but also of the hexaploid wheat genome.

Construction of high-resolution genetic maps is important for genetic and genomic research, as well as for molecular breeding. Single nucleotide polymorphisms (SNPs) are the predominant class of genetic variation and can be used as molecular markers. Aegilops tauschii, the D-genome donor of common wheat, is considered a valuable genetic resource for wheat improvement. Our previous study implied that Ae. tauschii accessions can be genealogically divided into two major lineages. In this study, the transcriptome of two Ae. tauschii accessions from each lineage, lineage 1 (L1) and 2 (L2), was sequenced, yielding 9435 SNPs and 739 insertion/deletion polymorphisms (indels) after de novo assembly of the reads. Based on 36 contig sequences, 31 SNPs and six indels were validated on 20 diverse Ae. tauschii accessions. Because almost all of the SNP markers were polymorphic between L1 and L2, and the D-genome donor of common wheat is presumed to belong to L2, these markers are available for D-genome typing in crosses between common wheat varieties and L1-derived synthetic wheat. Due to the conserved synteny between wheat and barley chromosomes, the high-density expressed sequence tag barley map and the hypothetical gene order in barley can be applied to develop markers on target chromosomal regions in wheat.

BackgroundCuticular wax production on plant surfaces confers a glaucous appearance and plays important roles in plant stress tolerance. Most common wheat cultivars, which are hexaploid, and most tetraploid wheat cultivars are glaucous; in contrast, a wild wheat progenitor, Aegilops tauschii, can be glaucous or non-glaucous. A dominant non-glaucous allele, Iw2, resides on the short arm of chromosome 2D, which was inherited from Ae. tauschii through polyploidization. Iw2 is one of the major causal genes related to variation in glaucousness among hexaploid wheat. Detailed genetic and phylogeographic knowledge of the Iw2 locus in Ae. tauschii may provide important information and lead to a better understanding of the evolution of common wheat.ResultsGlaucous Ae. tauschii accessions were collected from a broad area ranging from Armenia to the southwestern coastal part of the Caspian Sea. Linkage analyses with five mapping populations showed that the glaucous versus non-glaucous difference was mainly controlled by the Iw2 locus in Ae. tauschii. Comparative genomic analysis of barley and Ae. tauschii was then used to develop molecular markers tightly linked with Ae. tauschii Iw2. Chromosomal synteny around the orthologous Iw2 regions indicated that some chromosomal rearrangement had occurred during the genetic divergence leading to Ae. tauschii, barley, and Brachypodium. Genetic associations between specific Iw2-linked markers and respective glaucous phenotypes in Ae. tauschii indicated that at least two non-glaucous accessions might carry other glaucousness-determining loci outside of the Iw2 locus.ConclusionAllelic differences at the Iw2 locus were the main contributors to the phenotypic difference between the glaucous and non-glaucous accessions of Ae. tauschii. Our results supported the previous assumption that the D-genome donor of common wheat could have been any Ae. tauschii variant that carried the recessive iw2 allele.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0246-y) contains supplementary material, which is available to authorized users.

The large genome and allohexaploidy of common wheat have complicated construction of a high-density genetic map. Although improvements in the throughput of next-generation sequencing (NGS) technologies have made it possible to obtain a large amount of genotyping data for an entire mapping population by direct sequencing, including hexaploid wheat, a significant number of missing data points are often apparent due to the low coverage of sequencing. In the present study, a microarray-based polymorphism detection system was developed using NGS data obtained from complexity-reduced genomic DNA of two common wheat cultivars, Chinese Spring (CS) and Mironovskaya 808. After design and selection of polymorphic probes, 13,056 new markers were added to the linkage map of a recombinant inbred mapping population between CS and Mironovskaya 808. On average, 2.49 missing data points per marker were observed in the 201 recombinant inbred lines, with a maximum of 42. Around 40% of the new markers were derived from genic regions and 11% from repetitive regions. The low number of retroelements indicated that the new polymorphic markers were mainly derived from the less repetitive region of the wheat genome. Around 25% of the mapped sequences were useful for alignment with the physical map of barley. Quantitative trait locus (QTL) analyses of 14 agronomically important traits related to flowering, spikes, and seeds demonstrated that the new high-density map showed improved QTL detection, resolution, and accuracy over the original simple sequence repeat map.