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A proposal for standardization of transgenic reference sequences used in food forensics

  • Filipa Moreira
    Affiliations
    Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208, Matosinhos, Portugal
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  • João Carneiro
    Affiliations
    Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208, Matosinhos, Portugal
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  • Filipe Pereira
    Correspondence
    Corresponding author.
    Affiliations
    Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208, Matosinhos, Portugal
    Search for articles by this author
      The need for standardized procedures is of paramount importance in the highly regulated and often controversial field of genetically modified organisms (GMOs) and food forensics [
      • Caldwell J.M.
      Food analysis using organelle DNA and the effects of processing on assays.
      ,
      • Woolfe M.
      • Primrose S.
      Food forensics: using DNA technology to combat misdescription and fraud.
      ,
      • Araújo R.
      • Pereira F.
      • Asch B.v.
      Applications of DNA-based methods in food forensics.
      ]. A genetically modified (GM) or transgenic plant contains a gene or genes that have been introduced artificially into their genome in order to improve agronomic traits [
      • Crawley M.
      • Brown S.
      • Hails R.
      • Kohn D.
      • Rees M.
      Biotechnology: transgenic crops in natural habitats.
      ,
      • Peterson G.
      • Cunningham S.
      • Deutsch L.
      • Erickson J.
      • Quinlan A.
      • Raez-Luna E.
      • et al.
      The risks and benefits of genetically modified crops: a multidisciplinary perspective.
      ]. Despite the benefits, public concern regarding the potential impacts of GMO on human health and environment has increased over the years [
      • Bawa A.
      • Anilakumar K.
      Genetically modified foods: safety, risks and public concerns—a review.
      ,
      • Hails R.S.
      Genetically modified plants-the debate continues.
      ]. These concerns have fuelled the implementation of strict legislation on the planting, marketing, labelling and trade of GMOs. The collaboration between research and enforcement laboratories (e.g., the European Network of GMO Laboratories) has resulted in the harmonisation and standardisation of means and methods for GMOs analysis. The use of certified reference materials (CRMs) is mandatory for the certification and accreditation of laboratories [
      • Žel J.
      • Milavec M.
      • Morisset D.
      • Plan D.
      • Van den Eede G.
      • Gruden K.
      How to Reliably Test for GMOs.
      ]. The CRMs are available in the form of powder, extracted DNA or plasmids with transgenic DNA of a known GMO and are used for the validation of analytical procedures regarding the detection and quantification of GMOs [
      • Trapmann S.
      • Schimmel H.
      • Kramer G.N.
      • Eede Gvd Pauwels J.
      Production of certified reference materials for the detection of genetically modified organisms.
      ]. In contrast with the accessibility of CRMs, professionals working with GMOs face great difficulties when looking for reference DNA sequences of transgenic elements. Several countries have established a legal framework for risk assessment and authorization of GMOs that includes freely accessible databases [
      • Block A.
      • Debode F.
      • Grohmann L.
      • Hulin J.
      • Taverniers I.
      • Kluga L.
      • et al.
      The GMOseek matrix: a decision support tool for optimizing the detection of genetically modified plants.
      ,
      • Bonfini L.
      • Van den Bulcke M.H.
      • Mazzara M.
      • Ben E.
      • Patak A.
      GMOMETHODS: the European union database of reference methods for GMO analysis.
      ,
      • Dong W.
      • Yang L.
      • Shen K.
      • Kim B.
      • Kleter G.A.
      • Marvin H.J.
      • et al.
      GMDD: a database of GMO detection methods.
      ,
      • Petrillo M.
      • Angers-Loustau A.
      • Henriksson P.
      • Bonfini L.
      • Patak A.
      • Kreysa J.
      JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.
      ], however, there is no indication of which reference sequences should be used for indexing purposes. In fact, the transgenic sequences used in many GMOs are usually kept confidential by biotech companies in order to protect their technology against competitors. It is also difficult to find the most common transgenic sequences used in many commercialized GMOs by searching public databases (e.g., NCBI Entrez Nucleotide database). The problem often resides in the different names used for the same element (e.g., P35S, p35S, P-35s, CMV 35S, CaMV 35S), making it difficult to find the correct sequence. Moreover, the sequences used in GMOs (e.g., inserts or plasmids) are retrieved simultaneously with hundreds of sequences from population studies of the donor species, which makes it very difficult to choose an adequate sequence for reference purposes. In many cases, a search for a transgenic element only retrieves complete chromosome sequences or large sequence contigs, forcing the researcher to do additional sequence analyses with specialized software. Furthermore, there is no sequence information available for some elements in public databases, particularly for junction regions between elements, which are often the target of DNA-based detection methods. In this case, the researcher has to reconstruct the junction regions by the tedious process of downloading, aligning and editing sequences from different elements. The absence of reference sequences also hinders the description of methods and communication of results among laboratories. The simple process of describing polymorphisms on a transgenic element or sharing the location of PCR primers is problematic and highly prone to mistakes without a proper reference. By all these reasons, it is becoming evident that the existence of an organized list of reference sequences for the most common transgenic elements would facilitate the development, study and detection of GMOs. The use of reference sequences is a common practice on forensic investigations [
      • Linacre A.
      • Gusmao L.
      • Hecht W.
      • Hellmann A.
      • Mayr W.
      • Parson W.
      • et al.
      ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations.
      ,
      • Salas A.
      • Coble M.
      • Desmyter S.
      • Grzybowski T.
      • Gusmão L.
      • Hohoff C.
      • et al.
      A cautionary note on switching mitochondrial DNA reference sequences in forensic genetics.
      ]. Here we propose the standardization of transgenic reference sequences and provide a catalogue of curated sequences for the most common transgenic elements used in GM plants. The list is freely accessible via the web at http://portugene.com/GMOrefseq.html and a subset of the database is described in Table 1 as an example. The reference sequences are organized in a dynamic table with hyperlinks to the NCBI Sequence Viewer, Taxonomy and PubMed. The sequences can be visualized and downloaded with full annotations. Our reference dataset was also compared by blast with the GMO-related sequences available in the JRC GMO-Amplicons database [
      • Petrillo M.
      • Angers-Loustau A.
      • Henriksson P.
      • Bonfini L.
      • Patak A.
      • Kreysa J.
      JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.
      ].
      Table 1List of reference sequences for some common transgenic elements used in genetically modified organisms (GMOs). A full list of references can be found at http://portugene.com/GMOrefseq.html.
      Genetic element Reference sequence
      Abbreviation Genomic region Donor organism Accession number Source Reference
      P35S (P-35s) Cauliflower Mosaic Virus (CaMV) 35S promoter Cauliflower mosaic virus NC_001497.1 Cauliflower mosaic virus
      • Franck A.
      • Guilley H.
      • Jonard G.
      • Richards K.
      • Hirth L.
      Nucleotide sequence of cauliflower mosaic virus DNA.
      FMV35S (P-FMV) Figwort mosaic virus 35S promoter Figwort mosaic virus NC_003554.1 Figwort mosaic virus
      • Richins R.D.
      • Scholthof H.B.
      • Shepherd R.J.
      Sequence of figwort mosaic virus DNA (caulimovirus group).
      T-nos Nopaline Synthase Gene Terminator Agrobacterium tumefaciens EU880444.1 Oryza sativa Indica Group
      • Waiblinger H.-U.
      • Grohmann L.
      • Mankertz J.
      • Engelbert D.
      • Pietsch K.
      A practical approach to screen for authorised and unauthorised genetically modified plants.
      ,
      • Wu G.
      • Wu Y.
      • Nie S.
      • Zhang L.
      • Xiao L.
      • Cao Y.
      • et al.
      Real-time PCR method for detection of the transgenic rice event TT51-1.
      bar Glufosinate ammonium tolerance gene (codes for phosphinothricin acetyltransferase − PAT) Streptomyces hygroscopicus X05822.1 Streptomyces hygroscopicus
      • Thompson C.J.
      • Movva N.R.
      • Tizard R.
      • Crameri R.
      • Davies J.E.
      • Lauwereys M.
      • et al.
      Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus.
      Cry1Ab Cry1Ab delta-endotoxin gene Bacillus thuringiensis AX392802.1 Synthetic construct

      N. Carozzi, S. Rabe, P. Miles, G. Warren, P. De Haan, Novel insecticidal toxins derived from Bacillus thuringiensis crystal proteins, Patent WO0215701. 2002.

      cp4epsps 5-enolpyruvulshikimate-3-phosphate synthase gene (epsps) Agrobacterium tumefaciens strain CP4 AB209952.1 (298–1881) Synthetic construct (Glycine max) GenBank direct submission
      ctp2-cp4epsps Chloroplast transit peptide (ctp2) +

      5-enolpyruvulshikimate-3-phosphate synthase gene (epsps)
      Arabidopsis thaliana (ctp2) +

      Agrobacterium tumefaciens strain CP4 (epsps)
      FN550387.1 + JN400385.1 + AB209952.1 Concatenated sequence
      • Waiblinger H.-U.
      • Grohmann L.
      • Mankertz J.
      • Engelbert D.
      • Pietsch K.
      A practical approach to screen for authorised and unauthorised genetically modified plants.
      ,
      • Preuss S.B.
      • Meister R.
      • Xu Q.
      • Urwin C.P.
      • Tripodi F.A.
      • Screen S.E.
      • et al.
      Expression of the Arabidopsis thaliana BBX32 gene in soybean increases grain yield.
      P35S-bar CaMV P-35S promoter + Glufosinate ammonium tolerance gene Cauliflower Mosaic Virus + Streptomyces hygroscopicus NC_001497.1 (6101–7445) + X05822.1 Concatenated sequence (Cauliflower Mosaic Virus + Streptomyces hygroscopicus)
      • Franck A.
      • Guilley H.
      • Jonard G.
      • Richards K.
      • Hirth L.
      Nucleotide sequence of cauliflower mosaic virus DNA.
      ,
      • Thompson C.J.
      • Movva N.R.
      • Tizard R.
      • Crameri R.
      • Davies J.E.
      • Lauwereys M.
      • et al.
      Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus.
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