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. (July 2019)

• Record Type: Journal Article
• Authors: Jouanin, Aurélie; Borm, Theo; Boyd, Lesley A.; Cockram, James; Leigh, Fiona; Santos, Bruno A.C.M.; Visser, Richard G.F.; Smulders, Marinus J.M.
• Is Part Of: Journal of cereal science. Volume 88(2019)
• Page Start: 157
• Page End: 166
• Publication Date: 2019-07
• Subjects: Gene -- Editing -- Mutation -- Breeding -- Gliadin -- Coeliac -- Disease; Grain -- Periodicals; Cereal products -- Periodicals; Céréales -- Périodiques; Produits céréaliers -- Périodiques; Cereal products; Grain; Periodicals; 664.705
• DOI: 10.1016/j.jcs.2019.04.008
• Languages: English
• ISSNs: 0733-5210

Abstract

Abstract: We developed an in-solution gluten exome capture system called GlutEnSeq (Gluten gene Enrichment and Sequencing), covering the sequence variation of thousands of prolamin genes from various Triticeae species and cultivars. We assessed the efficacy of this capture system in hexaploid wheat ( Triticum aestivum L.) using Illumina sequencing. On-target regions were determined based on the Chinese Spring (CS) reference genome sequence. Gluten gene sequences were generally enriched around 10, 000-fold. The loss of gluten genes in CS deletion line 1BS-19/6DS-4 was detected as absence of gluten gene coverage on chromosomes 1B and Un (containing the Unmapped α-gliadin genes of chromosome 6D). Two γ-irradiated lines of cultivar Paragon, shown to be affected in their gliadin protein profile, were found to contain homozygous deletions for the α-gliadins on 6A and the γ-gliadins on 1B. Four Fielder CRISPR/Cas9 gliadin gene-edited lines revealed homozygous deletions of the γ-gliadins on 1B and heterozygous deletions for the α-gliadins on 6A. We also detected a decrease of gluten gene coverage within some gluten genes. The bioinformatics pipeline developed here will be further optimised to enable characterisation of small indels within individual gluten genes, in order to fully analyse CRISPR/Cas9 mutant lines for their decrease in immunogenicity for Coeliac patients. Highlights: GlutEnSeq is a gluten exome capture system. Based on thousands of prolamin genes from Triticeae species and cultivars. Gluten gene sequences were enriched around 10, 000-fold. Absence of α- and γ-gliadin loci was shown in chromosome deletion and γ-irradiated bread wheat lines. In CRISPR/Cas9 gliadin gene-edited bread wheat lines the gene coverage was decreased.