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

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'.

SUBMITTER: Salentijn EM

PROVIDER: S-EPMC2636828 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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

Salentijn Elma Mj EM Goryunova Svetlana V SV Bas Noor N van der Meer Ingrid M IM van den Broeck Hetty C HC Bastien Thomas T Gilissen Luud J W J LJ Smulders Marinus J M MJ

BMC genomics 20090126

<h4>Background</h4>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 p ...[more]

PMID: 19171027

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Project description:Wheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources. TILLING is a powerful reverse genetics approach that combines chemical mutagenesis with a high-throughput screen for mutations. Wheat is specially well-suited for TILLING due to the high mutation densities tolerated by polyploids, which allow for very efficient screens. Despite this, few TILLING populations are currently available. In addition, current TILLING screening protocols require high-throughput genotyping platforms, limiting their use.We developed mutant populations of pasta and common wheat and organized them for TILLING. To simplify and decrease costs, we developed a non-denaturing polyacrylamide gel set-up that uses ethidium bromide to detect fragments generated by crude celery juice extract digestion of heteroduplexes. This detection method had similar sensitivity as traditional LI-COR screens, suggesting that it represents a valid alternative. We developed genome-specific primers to circumvent the presence of multiple homoeologous copies of our target genes. Each mutant library was characterized by TILLING multiple genes, revealing high mutation densities in both the hexaploid (~1/38 kb) and tetraploid (~1/51 kb) populations for 50% GC targets. These mutation frequencies predict that screening 1,536 lines for an effective target region of 1.3 kb with 50% GC content will result in ~52 hexaploid and ~39 tetraploid mutant alleles. This implies a high probability of obtaining knock-out alleles (P = 0.91 for hexaploid, P = 0.84 for tetraploid), in addition to multiple missense mutations. In total, we identified over 275 novel alleles in eleven targeted gene/genome combinations in hexaploid and tetraploid wheat and have validated the presence of a subset of them in our seed stock.We have generated reverse genetics TILLING resources for pasta and bread wheat and achieved a high mutation density in both populations. We also developed a modified screening method that will lower barriers to adopt this promising technology. We hope that the use of this reverse genetics resource will enable more researchers to pursue wheat functional genomics and provide novel allelic diversity for wheat improvement.

Dataset Information

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

Publications

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

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