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

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.

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  • Name: Jouanin, Aurélie, Type: Corresponding Author,
  • Name: Tenorio-Berrio, Rubén, Type: Author,
  • Name: Schaart, Jan G., Type: Author,
  • Name: Leigh, Fiona, Type: Author,
  • Name: Visser, Richard G.F., Type: Author,
  • Name: Smulders, Marinus J.M., Type: Author,
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Citation Report https://scite.ai/reports/10.1016/j.jcs.2019.102903
DFW Organisation NIAB
DFW Work Package 3
DOI 10.1016/j.jcs.2019.102903
Date Last Updated 2020-11-30T15:26:13.063289
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Open Access Status hybrid
PDF URL https://www.sciencedirect.com/science/article/pii/S0733521019305843/pdfft?md5=cd485e0918d744db2716db9a979f8ab5&pid=1-s2.0-S0733521019305843-main.pdf
Publisher URL https://doi.org/10.1016/j.jcs.2019.102903