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

Jouanin, Aurélie; Tenorio-Berrio, Rubén; Schaart, Jan G.; Leigh, Fiona; Visser, Richard G. F.; Smulders, M.J.M.

doi:10.1016/j.jcs.2019.102903

Cited by 23 publications

(7 citation statements)

References 39 publications

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“…(2019) used gene‐specific primers and found that 26 alpha‐gliadins were expressed in the developing endosperm of CS. Note that the number of genes varies between wheat varieties, and that expression levels vary among genes and between varieties (Shewry and Lookhart, 2003; Salentijn et al ., 2009; Noma et al ., 2019; Jouanin et al ., 2020a).…”

Section: Introductionmentioning

confidence: 99%

Exploring the alpha‐gliadin locus: the 33‐mer peptide with six overlapping coeliac disease epitopes in Triticum aestivum is derived from a subgroup of Aegilops tauschii

et al. 2021

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Summary Most alpha‐gliadin genes of the Gli‐D2 locus on the D genome of hexaploid bread wheat (Triticum aestivum) encode for proteins with epitopes that can trigger coeliac disease (CD), and several contain a 33‐mer peptide with six partly overlapping copies of three epitopes, which is regarded as a remarkably potent T‐cell stimulator. To increase genetic diversity in the D genome, synthetic hexaploid wheat lines are being made by hybridising accessions of Triticum turgidum (AB genome) and Aegilops tauschii (the progenitor of the D genome). The diversity of alpha‐gliadins in A. tauschii has not been studied extensively. We analysed the alpha‐gliadin transcriptome of 51 A. tauschii accessions representative of the diversity in A. tauschii. We extracted RNA from developing seeds and performed 454 amplicon sequencing of the first part of the alpha‐gliadin genes. The expression profile of allelic variants of the alpha‐gliadins was different between accessions, and also between accessions of the Western and Eastern clades of A. tauschii. Generally, both clades expressed many allelic variants not found in bread wheat. In contrast to earlier studies, we detected the 33‐mer peptide in some A. tauschii accessions, indicating that it was introduced along with the D genome into bread wheat. In these accessions, transcripts with the 33‐mer peptide were present at lower frequencies than in bread wheat varieties. In most A. tauschii accessions, however, the alpha‐gliadins do not contain the epitope, and this may be exploited, through synthetic hexaploid wheats, to breed bread wheat varieties with fewer or no coeliac disease epitopes.

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Section: Introductionmentioning

confidence: 99%

Exploring the alpha‐gliadin locus: the 33‐mer peptide with six overlapping coeliac disease epitopes in Triticum aestivum is derived from a subgroup of Aegilops tauschii

et al. 2021

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“…An approximation proposed by Jouanin et al uses droplet digital PCR (ddPCR) to identify small (1–50 bp) and large (>300 bp) InDels in wheat α-gliadin genes targeted with CRISPR/Cas [ 45 ]. However, the authors also stated the need to further perform deep sequencing to characterize these mutations.…”

Section: Discussionmentioning

confidence: 99%

A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9

Guzmán-López

Marín-Sanz

Sánchez-León

et al. 2021

IJMS

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The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family.

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“…Thus, an accurate and precise CN measurement is critical for lines selection, being single or low CN generally desired [38]. Many studies have been carried out to compare the performances of qPCR and ddPCR in view of improving detection and quantification methods intended for those laboratories committed in official GMO control [15,[39][40][41][42][43][44]. In general, they argued in favor to ddPCR, being this technique insensitive to PCR inhibiting components often present in complex matrices and not dependent on calibration with standard curves obtained from certified reference materials for quantification.…”

Section: Discussionmentioning

confidence: 99%

Integrated approach for the molecular characterization of edited plants obtained via Agrobacterium tumefaciens-mediated gene transfer

Costa

Vinciguerra

Giacomelli

et al. 2021

Eur Food Res Technol

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Agrobacterium tumefaciens-mediated gene transfer—actually the most used method to engineer plants—may lead to integration of multiple copies of T-DNA in the plant genome, as well as to chimeric tissues composed of modified cells and wild type cells. A molecular characterization of the transformed lines is thus a good practice to select the best ones for further investigation. Nowadays, several quantitative and semi-quantitative techniques are available to estimate the copy number (CN) of the T-DNA in genetically modified plants. In this study, we compared three methods based on (1) real-time polymerase chain reaction (qPCR), (2) droplet digital PCR (ddPCR), and (3) next generation sequencing (NGS), to carry out a molecular characterization of grapevine edited lines. These lines contain a knock-out mutation, obtained via CRISPR/Cas9 technology, in genes involved in plant susceptibility to two important mildew diseases of grapevine. According to our results, qPCR and ddPCR outputs are largely in agreement in terms of accuracy, especially for low CN values, while ddPCR resulted more precise than qPCR. With regard to the NGS analysis, the CNs detected with this method were often not consistent with those calculated by qPCR and ddPCR, and NGS was not able to discriminate the integration points in three out of ten lines. Nevertheless, the NGS method can positively identify T-DNA truncations or the presence of tandem/inverted repeats, providing distinct and relevant information about the transgene integration asset. Moreover, the expression analysis of Cas9 and single guide RNA (sgRNA), and the sequencing of the target site added new information to be related to CN data. This work, by reporting a practical case-study on grapevine edited lines, explores pros and cons of the most advanced diagnostic techniques available for the precocious selection of the proper transgenic material. The results may be of interest both to scientists developing new transgenic lines, and to laboratories in charge of GMO control.

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

Cited by 23 publications

References 39 publications

Exploring the alpha‐gliadin locus: the 33‐mer peptide with six overlapping coeliac disease epitopes in Triticum aestivum is derived from a subgroup of Aegilops tauschii

Exploring the alpha‐gliadin locus: the 33‐mer peptide with six overlapping coeliac disease epitopes in Triticum aestivum is derived from a subgroup of Aegilops tauschii

A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9

Integrated approach for the molecular characterization of edited plants obtained via Agrobacterium tumefaciens-mediated gene transfer

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