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

Salentijn EM, Goryunova SV, Bas N, van der Meer IM, van den Broeck HC, Bastien T, Gilissen LJ, Smulders MJ - BMC Genomics (2009)

Bottom Line: 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').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'.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Research International, Wageningen UR, Wageningen, the Netherlands. elma.salentijn@wur.nl

ABSTRACT

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'.

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Deduced α-gliadin isoforms and CD-epitope frequencies of cultivars. Amino acid sequences of the N-terminal part of α-gliadins as deduced from α-gliadin transcripts present in developing grains at 14 days post anthesis (14DPA) of single genotypes derived from the respective cultivars 'Lavett' (3A), 'Baldus' (3B), This N-terminal locus contains several CD-epitopes: p31-49 (PGQQQPFPPQQPYPQPQPF, in blue), triggering the innate immune response; and the HLA-DQ2+ restricted T-cell epitopes Glia-α9(αI) (PFPQPQLPY, in red); Glia-α9(αIII) (PYPQPQLPY); Glia-α2(αII) (PQPQLPYPQ) and Glia-α20 (FRPQQPYPQ, in green). The HLA-DQ2+ epitope frequency calculated as the number of conserved (ESTs) for 'Lavett', 'Baldus' is respectively 2.7 and 1.6. The consensus nucleotide sequences of the EST contigs were assigned to a predicted genomic location by clustering (Clustal W) with genomic sequences from diploid species with genome AA (T. monococcum), BB (SS) (Aegilops speltoides) and DD (Aegilops tauschii), as in Van Herpen et al. [14]. GenBank accession numbers of the ESTs: 'Lavett' GH160345-GH160365, 'Baldus' GH162284-GH160344.
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Figure 3: Deduced α-gliadin isoforms and CD-epitope frequencies of cultivars. Amino acid sequences of the N-terminal part of α-gliadins as deduced from α-gliadin transcripts present in developing grains at 14 days post anthesis (14DPA) of single genotypes derived from the respective cultivars 'Lavett' (3A), 'Baldus' (3B), This N-terminal locus contains several CD-epitopes: p31-49 (PGQQQPFPPQQPYPQPQPF, in blue), triggering the innate immune response; and the HLA-DQ2+ restricted T-cell epitopes Glia-α9(αI) (PFPQPQLPY, in red); Glia-α9(αIII) (PYPQPQLPY); Glia-α2(αII) (PQPQLPYPQ) and Glia-α20 (FRPQQPYPQ, in green). The HLA-DQ2+ epitope frequency calculated as the number of conserved (ESTs) for 'Lavett', 'Baldus' is respectively 2.7 and 1.6. The consensus nucleotide sequences of the EST contigs were assigned to a predicted genomic location by clustering (Clustal W) with genomic sequences from diploid species with genome AA (T. monococcum), BB (SS) (Aegilops speltoides) and DD (Aegilops tauschii), as in Van Herpen et al. [14]. GenBank accession numbers of the ESTs: 'Lavett' GH160345-GH160365, 'Baldus' GH162284-GH160344.

Mentions: By studying the deduced amino acid sequences the number of different isoforms and the frequency Gli-2 transcripts encoding conserved CD-epitopes were determined. In the 5' region (135 to 216 base pairs in size) of the Gli-2 transcripts analyzed here a total of 37 different α-gliadin isoforms were evident (Figure 3 and 4). The transcripts derived from the hexaploid genotypes derived from the cultivars 'Lavett' (Figure 3A) and 'Baldus' (Figure 3B) and the tetraploid genotypes derived from the landraces 'Tripshiro' (Figure 4A) and 'Sinde' (Figure 4B) encoded 12, 17, 8 and 8 different isoforms, respectively. These numbers are reflecting the minimal variation in α-gliadin isoforms expected to be present in the gluten fraction of the genotypes analyzed. Several isoforms were rare and only encoded by a single transcript, but some were highly expressed, which reflects their relative frequency in the gluten transcriptome. For instance, almost 25% of the α-gliadin transcripts (15 out of 61 ESTs) from 'Baldus' were encoding one isoform (no. A1, Figure 3B).


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

Salentijn EM, Goryunova SV, Bas N, van der Meer IM, van den Broeck HC, Bastien T, Gilissen LJ, Smulders MJ - BMC Genomics (2009)

Deduced α-gliadin isoforms and CD-epitope frequencies of cultivars. Amino acid sequences of the N-terminal part of α-gliadins as deduced from α-gliadin transcripts present in developing grains at 14 days post anthesis (14DPA) of single genotypes derived from the respective cultivars 'Lavett' (3A), 'Baldus' (3B), This N-terminal locus contains several CD-epitopes: p31-49 (PGQQQPFPPQQPYPQPQPF, in blue), triggering the innate immune response; and the HLA-DQ2+ restricted T-cell epitopes Glia-α9(αI) (PFPQPQLPY, in red); Glia-α9(αIII) (PYPQPQLPY); Glia-α2(αII) (PQPQLPYPQ) and Glia-α20 (FRPQQPYPQ, in green). The HLA-DQ2+ epitope frequency calculated as the number of conserved (ESTs) for 'Lavett', 'Baldus' is respectively 2.7 and 1.6. The consensus nucleotide sequences of the EST contigs were assigned to a predicted genomic location by clustering (Clustal W) with genomic sequences from diploid species with genome AA (T. monococcum), BB (SS) (Aegilops speltoides) and DD (Aegilops tauschii), as in Van Herpen et al. [14]. GenBank accession numbers of the ESTs: 'Lavett' GH160345-GH160365, 'Baldus' GH162284-GH160344.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2636828&req=5

Figure 3: Deduced α-gliadin isoforms and CD-epitope frequencies of cultivars. Amino acid sequences of the N-terminal part of α-gliadins as deduced from α-gliadin transcripts present in developing grains at 14 days post anthesis (14DPA) of single genotypes derived from the respective cultivars 'Lavett' (3A), 'Baldus' (3B), This N-terminal locus contains several CD-epitopes: p31-49 (PGQQQPFPPQQPYPQPQPF, in blue), triggering the innate immune response; and the HLA-DQ2+ restricted T-cell epitopes Glia-α9(αI) (PFPQPQLPY, in red); Glia-α9(αIII) (PYPQPQLPY); Glia-α2(αII) (PQPQLPYPQ) and Glia-α20 (FRPQQPYPQ, in green). The HLA-DQ2+ epitope frequency calculated as the number of conserved (ESTs) for 'Lavett', 'Baldus' is respectively 2.7 and 1.6. The consensus nucleotide sequences of the EST contigs were assigned to a predicted genomic location by clustering (Clustal W) with genomic sequences from diploid species with genome AA (T. monococcum), BB (SS) (Aegilops speltoides) and DD (Aegilops tauschii), as in Van Herpen et al. [14]. GenBank accession numbers of the ESTs: 'Lavett' GH160345-GH160365, 'Baldus' GH162284-GH160344.
Mentions: By studying the deduced amino acid sequences the number of different isoforms and the frequency Gli-2 transcripts encoding conserved CD-epitopes were determined. In the 5' region (135 to 216 base pairs in size) of the Gli-2 transcripts analyzed here a total of 37 different α-gliadin isoforms were evident (Figure 3 and 4). The transcripts derived from the hexaploid genotypes derived from the cultivars 'Lavett' (Figure 3A) and 'Baldus' (Figure 3B) and the tetraploid genotypes derived from the landraces 'Tripshiro' (Figure 4A) and 'Sinde' (Figure 4B) encoded 12, 17, 8 and 8 different isoforms, respectively. These numbers are reflecting the minimal variation in α-gliadin isoforms expected to be present in the gluten fraction of the genotypes analyzed. Several isoforms were rare and only encoded by a single transcript, but some were highly expressed, which reflects their relative frequency in the gluten transcriptome. For instance, almost 25% of the α-gliadin transcripts (15 out of 61 ESTs) from 'Baldus' were encoding one isoform (no. A1, Figure 3B).

Bottom Line: 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').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'.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Research International, Wageningen UR, Wageningen, the Netherlands. elma.salentijn@wur.nl

ABSTRACT

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'.

Show MeSH
Related in: MedlinePlus