Comparative Study of Plastomes in Solanum tuberosum with Different Cytoplasm Types.

The potato is one of the most important food crops in the world. Improving the efficiency of potato breeding is of great importance for solving the global food problem. Today, researchers distinguish between six potato cytoplasm types: A, M, P, T, W, D. In the current study, the complete chloroplast genomes of Solanum tuberosum accessions with five out of the six major cytoplasmic genome types were sequenced (T-, W-, D-, A-, and P-genomes). A comparative analysis of the plastomes in potato accessions with different cytoplasm types was carried out for the first time. The time of origin of the different cytoplasm types was estimated. The presence of two main groups of chloroplast genomes among cultivated potato was confirmed. Based on the phylogenetic analysis of the complete plastome sequences, five main evolutionary branches of chloroplast genomes can be distinguished within the Petota section. Samples with A- and P- cytoplasm formed isolated and distant groups within a large and polymorphic group of samples with M-type cytoplasm, suggesting that A and P genomes arose independently. The findings suggest that the diversity of the T-genome in S. tuberosum Group Tuberosum could be initially low due to a bottle neck already existing at the origin of the Chilean clade. Differences in the rbcL gene sequence may be one of the factors causing differences in economically important traits in species with A and T-type cytoplasm. The data obtained will contribute to the development of methods for molecular marking of cytoplasm types and increase knowledge about the evolution and diversity of potato.

QTL mapping of oleic acid content in modern VNIIMK sunflower (Helianthus annuus L.) lines by using GBS-based SNP map.

Oleic acid is a monounsaturated fatty acid increasing oil oxidative stability. High content of oleic acid is thus a valuable trait in oilseed crops. Sunflower (Helianthus annuus L.) normally accumulates linoleic acid as a major fatty acid, but a mutant expressing a high oleic phenotype form was previously obtained by chemical mutagenesis and mapped on the sunflower genome. Several studies suggest the presence of additional genes involved in the control of the high content of oleic acid, with their expression possibly depending on the genetic background. To test this hypothesis, we performed a QTL mapping of the high oleic acid trait within two independent F2 crosses involving lines with contrasting oleic acid content from the Pustovoit All-Russia Research Institute of Oil Crops (VNIIMK) collection. We applied genotyping-by-sequencing (GBS) to construct single nucleotide polymorphism-based genetic maps and performed QTL mapping using quantitative and qualitative encoding for oleic acid content. Our results support the finding that the oleic acid content in the assessed crosses is controlled by one major effect locus. However, different dominant/recessive effects of the major locus were reported for both crosses. Additionally, a possible translocation between chromosome 7 and 14 was reported in one assessed cross. We defined a set of single nucleotide polymorphism markers for each cross which could be used for marker-assisted selection.

Refinement of Rf1-gene localization and development of the new molecular markers for fertility restoration in sunflower.

BACKGROUND: Ability to restore male fertility is important trait for sunflower breeding. The most commonly used fertility restoration gene in the production of sunflower hybrids is Rf1. The localization of Rf1 on the linkage group 13 has been previously shown, however, its exact position, its sequence and molecular mechanism for fertility restoration remain unknown. Therefore, several markers linked to Rf1 gene, commonly used for MAS, don't always allow to identify the genotype of plants. For this reason, the search for new markers and precise localization of the Rf1 gene is an urgent task. METHODS AND RESULTS: Based on previously identified single nucleotide polymorphisms (SNPs) at LG13, significantly associated with the ability to restore male fertility, two markers have been developed that have performed well after careful evaluation. These markers, together with other Rf1 markers, were applied for genotyping 72 diversity panel accessions and 291 individuals of F2 segregating population, obtained from crossing the cytoplasmic male sterility (CMS) AHO33 and restorer RT085HO lines. The analysis revealed no recombinants between Rf1 gene and SRF833 marker, the distance between Rf1 and SRF122 marker was 1.0 cM. CONCLUSIONS: Data obtained made it possible to specify the localization of the Rf1 gene and reduce the list of candidate genes to the 3 closely linked PPR-genes spanning a total of 59 Kb. However, it cannot be ruled out that analysis of the candidate region in the genome of fertility restorer lines can reveal new candidate genes in this locus that are absent in the cytoplasmic male sterility maintainer reference sequence.

Influence of Increased Radiation Background on Antioxidative Responses of Helianthus tuberosus L.

As a result of the accident at the Chornobyl Nuclear Power Plant, significant territories were exposed to ionizing radiation. Some isotopes, such as 137Cs, are capable of making a significant impact on living organisms in the long-term perspective. The generation of reactive oxygen species is one mechanism by which ionizing radiation affects living organisms, initiating mechanisms of antioxidant protection. In this article, the effect of increased ionizing radiation on the content of non-enzymatic antioxidants and the activity of antioxidant defense enzymes of Helianthus tuberosum L. was studied. This plant is widely distributed in Europe and characterized by high adaptability to abiotic factors. We found that the activity of antioxidant defense enzymes, such as catalase and peroxidase, weakly correlated with radiation exposure. The activity of ascorbate peroxidase, on the contrary, is strongly positively correlated with radiation exposure. The samples growing on the territory with constant low exposure to ionizing radiation were also characterized by an increased concentration of ascorbic acid and water-soluble phenolic compounds compared to the controls. This study may be useful for understanding the mechanisms underlying the adaptive reactions of plants under prolonged exposure to ionizing radiation.

Genetic Diversity and Pedigree Analysis of Red Currant Germplasm.

This represents the first report on the genetic diversity of red currant germplasm collections based on genotyping-by-sequencing (GBS) data. Genotypes of 75 individuals of different origin were assessed in more than 7.5K genome positions. Multidimensional scaling (MDS) analysis has been performed. There are five accessions that are significantly isolated from each other and from the rest of the analyzed cultivars. F1 offspring of R. petraeum Wulf (Rote Hollandische) and Gondouin, as well as Rote Spatlese (F2 of R. petraeum and F2 of R. multiflorum Kit.), are the most genetically isolated on the MDS plot. Ribes multiflorum is closer to the rest of cultivars than the three abovementioned accessions. Purpurnaya cultivar (F1 of Rote Spatlese) is located between Rote Hollandische and R. multiflorum. Other genotypes, mostly represented by varieties having several species in a pedigree, occupied the rest of MDS plot relatively evenly. Descendants of R. multiflorum have been placed in the left part of MDS plot, which underlines their genetic diversity from other accessions. White- and pink-fruited cultivars were clustered together, underlining genetic relatedness. Admixture analysis of GBS data reveals six clusters (K = 6). Presumably, clustering reflects relatedness to R. petraeum, R. rubrum, R. vulgare var macrocarpum, R. multiflorum, R. vulgare, and Jonker van Tets. Based on genotyping data, F1 offspring of R. warscewiczs Jancz (cultivar Viksne), R. altissimum Turcz (Cirald), and R. palczewskii (Jancz.) Pojark (Skorospelaya) have not exhibited strict separation and were placed in a pool with other varieties. This supports modern taxonomic classifications that do not consider R. altissimum and R. palczewskii as independent species.

Genetic mapping of loci involved in oil tocopherol composition control in Russian sunflower (Helianthus annuus L.) lines.

Tocopherols are antioxidants that preserve oil lipids against oxidation and serve as a natural source of vitamin E in the human diet. Compared with other major oilseeds like rapeseed and soybean, sunflower (Helianthus annuus L.) exhibits low phenotypic diversity of tocopherol composition, both in wild and cultivated accessions from germplasm collections. Two major mutations that alter tocopherol composition were identified in genetic collections, and several studies suggested additional loci controlling tocopherol composition, with their expression possibly depending on the genetic background. In the present study, we performed QTL mapping of tocopherol composition in two independent F2 crosses between lines with contrasting tocopherol composition from the Pustovoit All-Russia Research Institute of Oil Crops (VNIIMK) collection. We used genotyping-bysequencing (GBS) to construct single nucleotide polymorphism-based genetic maps, and performed QTL mapping using quantitative and qualitative encoding for phenotypic traits. Our results support the notion that the tocopherol composition in the assessed crosses is controlled by two loci. We additionally selected and validated two single nucleotide polymorphism markers for each cross which could be used for marker-assisted selection.

Genotyping and lipid profiling of 601 cultivated sunflower lines reveals novel genetic determinants of oil fatty acid content.

BACKGROUND: Sunflower is an important oilseed crop domesticated in North America approximately 4000 years ago. During the last century, oil content in sunflower was under strong selection. Further improvement of oil properties achieved by modulating its fatty acid composition is one of the main directions in modern oilseed crop breeding. RESULTS: We searched for the genetic basis of fatty acid content variation by genotyping 601 inbred sunflower lines and assessing their lipid and fatty acid composition. Our genome-wide association analysis based on the genotypes for 15,483 SNPs and the concentrations of 23 fatty acids, including minor fatty acids, revealed significant genetic associations for eleven of them. Identified genomic regions included the loci involved in rare fatty acids variation on chromosomes 3 and 14, explaining up to 34.5% of the total variation of docosanoic acid (22:0) in sunflower oil. CONCLUSIONS: This is the first large scale implementation of high-throughput lipidomic profiling to sunflower germplasm characterization. This study contributes to the genetic characterization of Russian sunflower collections, which made a substantial contribution to the development of sunflower as the oilseed crop worldwide, and provides new insights into the genetic control of oil composition that can be implemented in future studies.

Exploring the alpha-gliadin locus: the 33-mer peptide with six overlapping coeliac disease epitopes in Triticum aestivum is derived from a subgroup of Aegilops tauschii.

Most alpha-gliadin genes of the Gli-D2 locus on the D genome of hexaploid bread wheat (Triticum aestivum) encode for proteins with epitopes that can trigger coeliac disease (CD), and several contain a 33-mer peptide with six partly overlapping copies of three epitopes, which is regarded as a remarkably potent T-cell stimulator. To increase genetic diversity in the D genome, synthetic hexaploid wheat lines are being made by hybridising accessions of Triticum turgidum (AB genome) and Aegilops tauschii (the progenitor of the D genome). The diversity of alpha-gliadins in A. tauschii has not been studied extensively. We analysed the alpha-gliadin transcriptome of 51 A. tauschii accessions representative of the diversity in A. tauschii. We extracted RNA from developing seeds and performed 454 amplicon sequencing of the first part of the alpha-gliadin genes. The expression profile of allelic variants of the alpha-gliadins was different between accessions, and also between accessions of the Western and Eastern clades of A. tauschii. Generally, both clades expressed many allelic variants not found in bread wheat. In contrast to earlier studies, we detected the 33-mer peptide in some A. tauschii accessions, indicating that it was introduced along with the D genome into bread wheat. In these accessions, transcripts with the 33-mer peptide were present at lower frequencies than in bread wheat varieties. In most A. tauschii accessions, however, the alpha-gliadins do not contain the epitope, and this may be exploited, through synthetic hexaploid wheats, to breed bread wheat varieties with fewer or no coeliac disease epitopes.

Genetic Characterization of Russian Rapeseed Collection and Association Mapping of Novel Loci Affecting Glucosinolate Content.

Rapeseed is the second most common oilseed crop worldwide. While the start of rapeseed breeding in Russia dates back to the middle of the 20th century, its widespread cultivation began only recently. In contrast to the world's rapeseed genetic variation, the genetic composition of Russian rapeseed lines remained unexplored. We have addressed this question by performing genome-wide genotyping of 90 advanced rapeseed accessions provided by the All-Russian Research Institute of Oil Crops (VNIIMK). Genome-wide genetic analysis demonstrated a clear difference between Russian rapeseed varieties and the rapeseed varieties from the rest of the world, including the European ones, indicating that rapeseed breeding in Russia proceeded in its own independent direction. Hence, genetic determinants of agronomical traits might also be different in Russian rapeseed lines. To assess it, we collected the glucosinolate content data for the same 90 genotyped accessions obtained during three years and performed an association mapping of this trait. We indeed found that the loci significantly associated with glucosinolate content variation in the Russian rapeseed collection differ from those previously reported for the non-Russian rapeseed lines.

Ultra-performance liquid chromatography-mass spectrometry for precise fatty acid profiling of oilseed crops.

Oilseed crops are one of the most important sources of vegetable oils for food and industry. Nutritional and technical properties of vegetable oil are primarily determined by its fatty acid (FA) composition. The content and composition of FAs in plants are commonly determined using gas chromatography-mass spectrometry (GS-MS) or gas chromatography-flame ionization detection (GC-FID) techniques. In the present work, we applied ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) technique to FA profiling of sunflower and rapeseed seeds and compared this method with the GC-FID technique. GC-FID detected 11 FAs in sunflower and 13 FAs in rapeseed, while UPLC-MS appeared to be more sensitive, detecting about 2.5 times higher numbers of FAs in both plants. In addition to even-chain FAs, UPLC-MS was able to detect odd-chain FAs. The longest FA detected using GC-FID was an FA with 24 carbon atoms, whereas UPLC-MS could reveal the presence of longer FAs with the tails of up to 28 carbon atoms. Based on our results, we may conclude that UPLC-MS has great potential to be used for the assessment of FA profiles of oil crops.

UPLC⁻MS Triglyceride Profiling in Sunflower and Rapeseed Seeds.

Sunflower and rapeseed are among the most important sources of vegetable oil for food and industry. The main components of vegetable oil are triglycerides (TAGs) (about 97%). Ultra- performance liquid chromatography coupled with mass spectrometry (UPLC⁻MS) profiling of TAGs in sunflower and rapeseed has been performed and the TAG profiles obtained for these species have been compared. It has been identified that 34 TAGs are shared by sunflower and rapeseed. It was demonstrated that TAGs 52:2, 52:5, 52:6, 54:3; 54:4, 54:7, 56:3, 56:4, and 56:5 had the highest variability levels between sunflower and rapeseed with the higher presence in rapeseed. TAGs 50:2, 52:3, 52:4, 54:5, and 54:6 also showed high variability, but were the most abundant in sunflower. Moreover, the differences in TAG composition between the winter-type and spring-type rapeseed have been revealed, which may be associated with freezing tolerance. It was shown that winter-type rapeseed seeds contain TAGs with a lower degree of saturation, while in spring-type rapeseed highly saturated lipids are the most abundant. These findings may give new insights into the cold resistance mechanisms in plants the understanding of which is especially important in terms of global climate changes.

Correction: Complete mitochondrial genome sequence of the "copper moss" Mielichhoferia elongata reveals independent nad7 gene functionality loss.

[This corrects the article DOI: 10.7717/peerj.4350.].

Complete mitochondrial genome sequence of the "copper moss" Mielichhoferia elongata reveals independent nad7 gene functionality loss.

The mitochondrial genome of moss Mielichhoferia elongata has been sequenced and assembled with Spades genome assembler. It consists of 100,342 base pairs and has practically the same gene set and order as in other known bryophyte chondriomes. The genome contains 66 genes including three rRNAs, 24 tRNAs, and 40 conserved mitochondrial proteins genes. Unlike the majority of previously sequenced bryophyte mitogenomes, it lacks the functional nad7 gene. The phylogenetic reconstruction and scrutiny analysis of the primary structure of nad7 gene carried out in this study suggest its independent pseudogenization in different bryophyte lineages. Evaluation of the microsatellite (simple sequence repeat) content of the M. elongata mitochondrial genome indicates that it could be used as a tool in further studies as a phylogenetic marker. The strongly supported phylogenetic tree presented here, derived from 33 protein coding sequences of 40 bryophyte species, is consistent with other reconstructions based on a number of different data sets.

Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplicon sequencing.

BACKGROUND: Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD. RESULTS: A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 'unique deduced protein fragments' of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes. CONCLUSIONS: The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains.

Expansion of the gamma-gliadin gene family in Aegilops and Triticum.

BACKGROUND: The gamma-gliadins are considered to be the oldest of the gliadin family of storage proteins in Aegilops/Triticum. However, the expansion of this multigene family has not been studied in an evolutionary perspective. RESULTS: We have cloned 59 gamma-gliadin genes from Aegilops and Triticum species (Aegilops caudata L., Aegilops comosa Sm. in Sibth. & Sm., Aegilops mutica Boiss., Aegilops speltoides Tausch, Aegilops tauschii Coss., Aegilops umbellulata Zhuk., Aegilops uniaristata Vis., and Triticum monococcum L.) representing eight different genomes: Am, B/S, C, D, M, N, T and U. Overall, 15% of the sequences contained internal stop codons resulting in pseudogenes, but this percentage was variable among genomes, up to over 50% in Ae. umbellulata. The most common length of the deduced protein, including the signal peptide, was 302 amino acids, but the length varied from 215 to 362 amino acids, both obtained from Ae. speltoides. Most genes encoded proteins with eight cysteines. However, all Aegilops species had genes that encoded a gamma-gliadin protein of 302 amino acids with an additional cysteine. These conserved nine-cysteine gamma-gliadins may perform a specific function, possibly as chain terminators in gluten network formation in protein bodies during endosperm development. A phylogenetic analysis of gamma-gliadins derived from Aegilops and Triticum species and the related genera Lophopyrum, Crithopsis, and Dasypyrum showed six groups of genes. Most Aegilops species contained gamma-gliadin genes from several of these groups, which also included sequences from the genera Lophopyrum, Crithopsis, and Dasypyrum. Hordein and secalin sequences formed separate groups. CONCLUSIONS: We present a model for the evolution of the gamma-gliadins from which we deduce that the most recent common ancestor (MRCA) of Aegilops/Triticum-Dasypyrum-Lophopyrum-Crithopsis already had four groups of gamma-gliadin sequences, presumably the result of two rounds of duplication of the locus.

Celiac disease T-cell epitopes from gamma-gliadins: immunoreactivity depends on the genome of origin, transcript frequency, and flanking protein variation.

BACKGROUND: Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the α-gliadin fraction of gluten, γ-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic γ-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in γ-gliadin transcripts of developing bread wheat grains. RESULTS: A detailed analysis of the genetic variation present in γ-gliadin transcripts of bread wheat (T. aestivum, allo-hexaploid, carrying the A, B and D genome), together with genomic γ-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic γ-gliadin loci, Gli-A1, Gli-B1 or Gli-D1. Almost half of the γ-gliadin transcripts of bread wheat (49%) was assigned to locus Gli-D1. Transcripts from each locus differed in CD epitope content and composition. The Gli-D1 transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the Gli-A1 transcripts (8.6) and the Gli-B1 transcripts (5.4). The natural variants of the major CD epitope from γ-gliadins, DQ2-γ-I, showed variation in their capacity to induce in vitro proliferation of a DQ2-γ-I specific and HLA-DQ2 restricted T-cell clone. CONCLUSIONS: Evaluating the CD epitopes derived from γ-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat cultivars directly from their transcripts (cDNAs).

A universal approach to eliminate antigenic properties of alpha-gliadin peptides in celiac disease.

Celiac disease is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins, including the α-gliadins. It has been shown that α-gliadins harbor several major epitopes involved in the disease pathogenesis. A major step towards elimination of gluten toxicity for celiac disease patients would thus be the elimination of such epitopes from α-gliadins. We have analyzed over 3,000 expressed α-gliadin sequences from 11 bread wheat cultivars to determine whether they encode for peptides potentially involved in celiac disease. All identified epitope variants were synthesized as peptides and tested for binding to the disease-associated HLA-DQ2 and HLA-DQ8 molecules and for recognition by patient-derived α-gliadin specific T cell clones. Several specific naturally occurring amino acid substitutions were identified for each of the α-gliadin derived peptides involved in celiac disease that eliminate the antigenic properties of the epitope variants. Finally, we provide proof of principle at the peptide level that through the systematic introduction of such naturally occurring variations α-gliadins genes can be generated that no longer encode antigenic peptides. This forms a crucial step in the development of strategies to modify gluten genes in wheat so that it becomes safe for celiac disease patients. It also provides the information to design and introduce safe gluten genes in other cereals, which would exhibit improved quality while remaining safe for consumption by celiac disease patients.

Molecular markers based on LTR retrotransposons BARE-1 and Jeli uncover different strata of evolutionary relationships in diploid wheats.

Molecular markers based on retrotransposon insertions are widely used for various applications including phylogenetic analysis. Multiple cases were described where retrotransposon-based markers, namely sequence-specific amplification polymorphism (SSAP), were superior to other marker types in resolving the phylogenetic relationships due to their higher variability and informativeness. However, the patterns of evolutionary relationships revealed by SSAP may be dependent on the underlying retrotransposon activity in different periods of time. Hence, the proper choice of retrotransposon family is essential for obtaining significant results. We compared the phylogenetic trees for a diverse set of diploid A-genome wheat species (Triticum boeoticum, T. urartu and T. monococcum) based on two unrelated retrotransposon families, BARE-1 and Jeli. BARE-1 belongs to Copia class and has a uniform distribution between common wheat (T. aestivum) genomes of different origin (A, B and D), indicating similar activity in the respective diploid genome donors. Gypsy-class family Jeli was found by us to be an A-genome retrotransposon with >70% copies residing in A genome of hexaploid common wheat, suggesting a burst of transposition in the history of A-genome progenitors. The results indicate that a higher Jeli transpositional activity was associated with T. urartu versus T. boeoticum speciation, while BARE-1 produced more polymorphic insertions during subsequent intraspecific diversification; as an outcome, each retrotransposon provides more informative markers at the corresponding level of phylogenetic relationships. We conclude that multiple retroelement families should be analyzed for an image of evolutionary relationships to be solid and comprehensive.

Tetraploid and hexaploid wheat varieties reveal large differences in expression of alpha-gliadins from homoeologous Gli-2 loci.

BACKGROUND: Alpha-gliadins form a multigene protein family encoded by multiple alpha-gliadin (Gli-2) genes at three genomic loci, Gli-A2, Gli-B2 and Gli-D2, respectively located on the homoeologous wheat chromosomes 6AS, 6BS, and 6DS. These proteins contain a number of important celiac disease (CD)-immunogenic domains. The alpha-gliadins expressed from the Gli-B2 locus harbour fewer conserved CD-epitopes than those from Gli-A2, whereas the Gli-D2 gliadins have the highest CD-immunogenic potential. In order to detect differences in the highly CD-immunogenic alpha-gliadin fraction we determined the relative expression level from the homoeologous Gli-2 loci in various tetraploid and hexaploid wheat genotypes by using a quantitative pyrosequencing method and by analyzing expressed sequence tag (EST) sequences. RESULTS: We detected large differences in relative expression levels of alpha-gliadin genes from the three homoeologous loci among wheat genotypes, both as relative numbers of expressed sequence tag (EST) sequences from specific varieties and when using a quantitative pyrosequencing assay specific for Gli-A2 genes. The relative Gli-A2 expression level in a tetraploid durum wheat cultivar ('Probstdorfer Pandur') was 41%. In genotypes derived from landraces, the Gli-A2 frequency varied between 12% and 58%. In some advanced hexaploid bread wheat cultivars the genes from locus Gli-B2 were hardly expressed (e.g., less than 5% in 'Lavett') but in others they made up more than 40% (e.g., in 'Baldus'). CONCLUSION: Here, we have shown that large differences exist in relative expression levels of alpha-gliadins from the homoeologous Gli-2 loci among wheat genotypes. Since the homoelogous genes differ in the amount of conserved CD-epitopes, screening for differential expression from the homoeologous Gli-2 loci can be employed for the pre-selection of wheat varieties in the search for varieties with very low CD-immunogenic potential. Pyrosequencing is a method that can be employed for such a 'gene family-specific quantitative transcriptome profiling'.

Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes.

BACKGROUND: Bread wheat (Triticum aestivum) is an important staple food. However, wheat gluten proteins cause celiac disease (CD) in 0.5 to 1% of the general population. Among these proteins, the alpha-gliadins contain several peptides that are associated to the disease. RESULTS: We obtained 230 distinct alpha-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87%) contained an internal stop codon. All alpha-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, alpha-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome) sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-alpha9 and glia-alpha20, but never the intact epitopes glia-alpha and glia-alpha2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome), as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific. CONCLUSION: Our analysis shows that alpha-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic analysis of all known epitopes in gliadins and glutenins will lead to better understanding of the differences in toxicity among wheat varieties. On the basis of such insight, breeding strategies can be designed to generate less toxic varieties of wheat which may be tolerated by at least part of the CD patient population.