Development of the GlutEnSeq capture system for sequencing gluten gene families in hexaploid bread wheat with deletions or mutations induced by γ-irradiation or CRISPR/Cas9

Jouanin, Aurélie; Borm, Theo; Boyd, Lesley A.; Cockram, James; Leigh, Fiona; Santos, Bruno; Visser, Richard G. F.; Smulders, M.J.M.

doi:10.1016/j.jcs.2019.04.008

Cited by 23 publications

(20 citation statements)

References 32 publications

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“…This might be accomplished using a capture system that includes baits for all high-confidence exons from the International Wheat Genome Sequencing Consortium (IWGSC) genome assembly of Chinese Spring that is commercially available from Arbor Biosciences (Ann Arbor, MI). Alternately, baits for the capture system may be specific for genomic regions encoding the major gluten proteins in Chinese Spring as annotated by Huo et al (2018a;2018b) or based on the sequences of all known gluten genes from various Triticeae species and cultivars (Jouanin et al, 2019). However, even with complete sequence information it will be very challenging to identify regions that can be used as triggers in RNAi constructs given the similarities in the sequences of the different genes.…”

Section: Discussionmentioning

confidence: 99%

Reducing the Immunogenic Potential of Wheat Flour: Silencing of Alpha Gliadin Genes in a U.S. Wheat Cultivar

et al. 2020

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The alpha gliadins are a group of more than 20 proteins with very similar sequences that comprise about 15%-20% of the total flour protein and contribute to the functional properties of wheat flour dough. Some alpha gliadins also contain immunodominant epitopes that trigger celiac disease, a chronic autoimmune disease that affects approximately 1% of the worldwide population. In an attempt to reduce the immunogenic potential of wheat flour from the U.S. spring wheat cultivar Butte 86, RNA interference was used to silence a subset of alpha gliadin genes encoding proteins containing celiac disease epitopes. Two of the resulting transgenic lines were analyzed in detail by quantitative twodimensional gel electrophoresis combined with tandem mass spectrometry. Although the RNA interference construct was designed to target only some alpha gliadin genes, all alpha gliadins were effectively silenced in the transgenic plants. In addition, some off-target silencing of high molecular weight glutenin subunits was detected in both transgenic lines. Compensatory effects were not observed within other gluten protein classes. Reactivities of IgG and IgA antibodies from a cohort of patients with celiac disease toward proteins from the transgenic lines were reduced significantly relative to the nontransgenic line. Both mixing properties and SDS sedimentation volumes suggested a decrease in dough strength in the transgenic lines when compared to the control. The data suggest that it will be difficult to selectively silence specific genes within families as complex as the wheat alpha gliadins. Nonetheless, it may be possible to reduce the immunogenic potential of the flour and still retain many of the functional properties essential for the utilization of wheat.

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Section: Discussionmentioning

confidence: 99%

Reducing the Immunogenic Potential of Wheat Flour: Silencing of Alpha Gliadin Genes in a U.S. Wheat Cultivar

et al. 2020

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“…Using chemical treatments such as ethyl-methane sulfonate (EMS) or ionizing irradiation, random mutations can be generated in the plant genome. These may result in removal of gliadin loci, as has been shown for bread wheat (23,50). Wheat deletion lines produced with irradiation, e.g., those lacking the 6D alpha-gliadin locus with many CD epitopes (50) may be used in a breeding program, but only together with other approaches, as combining multiple chromosomal deletions is often lethal.…”

Section: Breeding Genomics Biotechnology and Gene Editing Of Glutementioning

confidence: 99%

“…Application of CRISPR/Cas9 has been successful for targeted mutagenesis of single copy genes across all three homoeologous loci in hexaploid wheat (55). However, mutating target-specific sequences in large gene families in a polyploid species, such as gluten-encoding genes in wheat, has only recently been reported (21)(22)(23).…”

Section: Breeding Genomics Biotechnology and Gene Editing Of Glutementioning

confidence: 99%

“…During repair, the innate DNA repair mechanism of the plant can generate mistakes, usually resulting in small deletions of one or a few nucleotides. In the case of the tandemly repeated gliadin genes in wheat, simultaneous double-strand breaks may occur in consecutive genes, which can result in deletion of large DNA fragments carrying one or more gliadin genes (23). Large deletions between multiple target sites have also been obtained, for example in rice (56).…”

Section: Breeding Genomics Biotechnology and Gene Editing Of Glutementioning

confidence: 99%

“…With the ultimate goal of removing the immunogenic gluten epitopes from the human diet, this technology is being used in the development of wheat lines with fewer gluten genes and/or gluten genes with inactivated CD epitopes. As proof of concept, CRISPR/Cas9 technology has been used to edit α-gliadin genes (21) as well as both α-and γ-gliadin genes (22)(23)(24) in bread wheat. Along with ωgliadins, these gliadin types rank highest in abundance and overall immunogenicity compared with the low molecular weight (LMW) and high molecular weight (HMW) glutenins (11,12).…”

Section: Introductionmentioning

confidence: 99%

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