Optimisation of droplet digital PCR for determining copy number variation of α-gliadin genes in mutant and gene-edited polyploid bread wheat. (March 2020)

• Record Type: Journal Article
• Authors: Jouanin, Aurélie; Tenorio-Berrio, Rubén; Schaart, Jan G.; Leigh, Fiona; Visser, Richard G.F.; Smulders, Marinus J.M.
• Is Part Of: Journal of cereal science. Volume 92(2020)
• Publication Date: 2020-03
• Subjects: ddPCR -- CNV -- CRISPR/Cas9 -- Coeliac disease; Grain -- Periodicals; Cereal products -- Periodicals; Céréales -- Périodiques; Produits céréaliers -- Périodiques; Cereal products; Grain; Periodicals; 664.705
• DOI: 10.1016/j.jcs.2019.102903
• Languages: English
• ISSNs: 0733-5210

Abstract

Abstract: Targeted and random mutagenesis of gene families require accurate quantification. Droplet digital PCR (ddPCR) enables high-throughput screening of copy number variation (CNV). We tested the accuracy of ddPCR for CNV analysis in the large α-gliadin gene family, using degenerate primers. First, duplex ddPCR assays measured α-gliadins in diploid (15–17 copies) and tetraploid (70–76 copies) wheat accessions and a corresponding number in resulting Synthetic Hexaploid Wheat, demonstrating linear amplification up to 86–95 genes. Second, we amplified 61 α-gliadin genes in Chinese Spring. Most α-gliadins of the homoeologous chromosomes 6A and 6D were correctly amplified, as corroborated using deletion and nullisomic-tetrasomic lines, but one group of genes from 6B were not amplified with these primers. Third, in Paragon we amplified 61 α-gliadin genes while selected γ-irradiated mutant lines revealed reductions of 25–50%. Finally, using two duplex ddPCR assays, we showed that CRISPR/Cas9-targeting of α-gliadins in Fielder produced indels (1–50 bp) in up to 10 α-gliadin genes plus large deletions (>300 bp) in 20 of 87 amplified α-gliadin genes. ddPCR is suitable for high-throughput screening of CNV and gene-editing-induced mutations in large gene families, in polyploids. In wheat, ddPCR enables screening of gliadins in breeding programs towards hypoimmunogenic gluten for coeliac patients. Graphical abstract: Image 1 Highlights: ddPCR enables high-throughput screening of copy number variation (CNV). We demonstrate linear detection up to 95 alpha-gliadin genes in single a bread wheat variety. Our dual-assay ddPCR approach distinguished CRISPR/Cas-induced small indels in alpha-gliadins from large deletions.