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New Breeding Techniques: Detection and Identification of the Techniques and Derived Products

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Abstract

Since the commercial releases of GMOs in the 90s, new genetic modification tools known as New breeding techniques have been developed for e.g. gene silencing or more precise genomic modifications such as Crispr-endonuclease based systems. As for GMOs several consumers view may prevail about the societal interest in agricultural production and food of such genetic modification. Ensuring the freedom of choice to consumers needs to develop detection tools which could infer the NBT nature of the modification technique used. This article reviews all the elements which could allow the identification and detection of such techniques and products.

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... These related techniques include (i) protoplastisation 1 GMO Risk Assessment Agency 2 Oligonucleotide directed Mutagenesis 3 Interfering RNA to obtain isolated cells,(ii) in vitro culture, (iii) delivery of reagents (DNA, RNA, or RNA-associated or non-RNA-associated proteins), (iv) the selection procedure of modifying cells, (v) the elimination of these same selection markers for example by the Cre-Lox recombinase system, (vi) the callus differentiation, and (vii) the regeneration of seedlings before (viii) the habituation to culture conditions. All these mutations and epimutations follow a dynamic that has not been studied in detail until now (Bertheau, 2019;Filipecki and Malepszy, 2006;Lee and Seo, 2018;Mendizabal et al., 2014). However, these old-chapped related techniques still constitute a bottleneck in large-scale technique applications that have not been mastered, as recently noted (Ledford, 2016). ...
... The delivery of reagents, whether DNA, RNA (messenger or not) or proteins (such as RNA-associated nucleases for CRISPR-Cas RNPs 5 contaminated by foreign DNA of the multiplication organism DNA despite purification steps), is a highly empirical technical phase that induces genomic and epigenomic scars (Bertheau, 2019;Khalil, 2020;Ledford, 2016). ...
... Transgenesis and genome and epigenome editing methods share these related techniques and their unintended effects. The scars left after the excision of selection markers from modified cells is only one example (Ates et al., 2020;Bertheau, 2019;Jansing et al., 2019;Manimaran et al., 2011;Marx, 2015;Miguel and Marum, 2011;Ruffoni and Savona, 2013;Scahill et al., 2008;Volkova et al., 2020;Yau and Stewart, 2013). Therefore, most of the authors consider it necessary for the NBT to avoid all these in vitro culture phases, but the suggested options have not proven themselves (Hamada et al., 2017;Maher et al., 2020). ...
... These related techniques include (i) protoplastisation 1 GMO Risk Assessment Agency 2 Oligonucleotide directed Mutagenesis 3 Interfering RNA to obtain isolated cells,(ii) in vitro culture, (iii) delivery of reagents (DNA, RNA, or RNA-associated or non-RNA-associated proteins), (iv) the selection procedure of modifying cells, (v) the elimination of these same selection markers for example by the Cre-Lox recombinase system, (vi) the callus differentiation, and (vii) the regeneration of seedlings before (viii) the habituation to culture conditions. All these mutations and epimutations follow a dynamic that has not been studied in detail until now (Bertheau, 2019;Filipecki and Malepszy, 2006;Lee and Seo, 2018;Mendizabal et al., 2014). However, these old-chapped related techniques still constitute a bottleneck in large-scale technique applications that have not been mastered, as recently noted (Ledford, 2016). ...
... The delivery of reagents, whether DNA, RNA (messenger or not) or proteins (such as RNA-associated nucleases for CRISPR-Cas RNPs 5 contaminated by foreign DNA of the multiplication organism DNA despite purification steps), is a highly empirical technical phase that induces genomic and epigenomic scars (Bertheau, 2019;Khalil, 2020;Ledford, 2016). ...
... Transgenesis and genome and epigenome editing methods share these related techniques and their unintended effects. The scars left after the excision of selection markers from modified cells is only one example (Ates et al., 2020;Bertheau, 2019;Jansing et al., 2019;Manimaran et al., 2011;Marx, 2015;Miguel and Marum, 2011;Ruffoni and Savona, 2013;Scahill et al., 2008;Volkova et al., 2020;Yau and Stewart, 2013). Therefore, most of the authors consider it necessary for the NBT to avoid all these in vitro culture phases, but the suggested options have not proven themselves (Hamada et al., 2017;Maher et al., 2020). ...
Chapter
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In 2018 the Court of Justice of the European Union recalled that organisms with genomes modified by artifactual techniques should be considered GMOs under European regulations. GMOs derived from cultures of cells isolated in vitro or from new genomic techniques must therefore be traceable. This chapter reviews the various technical steps and characteristics of those techniques causing genomic and epigenomic scars and signatures. These intentional and unintentional traces, some of which are already used for varietal identification, and are being standardized, can be used to identify these GMOs and differentiate them from natural mutants. The chapter suggests a routine procedure for operators and control laboratories to achieve this without additional costs.
... aflatoxin) or pathogens, GMOs are a highly political-technicalscientific issue. Thus, when they are discussed or explored in contemporary literature and media, the state-of-the-art techniques and scientific knowledge involved are only partially taken into account (Bertheau, 2019;Bertheau, 2007, 2009). Despite the passage of 30 years, the definition of GMO is still imprecise and contested. ...
... The use of different targets and signal amplification of variants, with or without hybridisation, makes available a range of qualitative and quantitative methods (QRT-PCR, LAMP, NASBA, LCR, SNPLex…) that can be chosen, depending on the target. These methodological approaches can be applied from the field to the laboratory, from point mutation to the junction sequence between rearranged elements and from single to multiple targets Bertheau, 2019;Chhalliyil et al., 2020). Associated with various decision support tools, they allow, via the matrix approach, the identification of any GMO. ...
Chapter
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Transgenic GMOs were welcomed in the 1990s due to the difficulties distinguishing genetic and epigenetic modifications from random mutagenesis and their ability to insert new nucleic sequences more rapidly but still randomly. Their marketing in Europe has been accompanied by health and environmental risk assessments, specific monitoring and traceability procedures to preserve the free choice of consumers and allow the coexistence of different supply chains. This chapter reviews the regulations, detection techniques, strategies and standards that have been put in place in the European Union since 1996 to ensure the analytical traceability of these GMOs. The capacity of the matrix approach, initially targeted at transgenic GMOs, to trace other types of GMOs is discussed in an accompanying chapter.
... Although the precision of genome editing and range of off-target and other unintended effects, especially in comparison with random mutagenesis, have been one focus of the discussion regarding regulatory status of genome-edited organisms [3,19,[22][23][24][25], a second, prominent focus has been feasibility of developing methods for detecting genome-edited organisms [3,7,10,[25][26][27][28][29]. One of the arguments put forward to justify regulating genome-edited crops differently from recombinant DNA-based GMOs is that Directive 2001/18/EC requires analysis-based surveillance of GMOs, while, it is claimed, there are many technical and regulatory challenges that make development of GMO regulation-compliant analytical identification and quantitation methods for genome-edited organisms difficult or even impossible [7,10,25,26,28,29]. ...
... One of the arguments put forward to justify regulating genome-edited crops differently from recombinant DNA-based GMOs is that Directive 2001/18/EC requires analysis-based surveillance of GMOs, while, it is claimed, there are many technical and regulatory challenges that make development of GMO regulation-compliant analytical identification and quantitation methods for genome-edited organisms difficult or even impossible [7,10,25,26,28,29]. However, these claims are controversial [3,27]. ...
Article
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Discussion regarding the regulatory status of genome-edited crops has focused on precision of editing and on doubts regarding the feasibility of analytical monitoring compliant with existing GMO regulations. Effective detection methods are important, both for regulatory enforcement and traceability in case of biosafety, environmental or socio-economic impacts. Here, we approach the analysis question for the first time in the laboratory and report the successful development of a quantitative PCR detection method for the first commercialized genome-edited crop, a canola with a single base pair edit conferring herbicide tolerance. The method is highly sensitive and specific (quantification limit, 0.05%), compatible with the standards of practice, equipment and expertise typical in GMO laboratories, and readily integrable into their analytical workflows, including use of the matrix approach. The method, validated by an independent laboratory, meets all legal requirements for GMO analytical methods in jurisdictions such as the EU, is consistent with ISO17025 accreditation standards and has been placed in the public domain. Having developed a qPCR method for the most challenging class of genome edits, single-nucleotide variants, this research suggests that qPCR-based method development may be applicable to virtually any genome-edited organism. This advance resolves doubts regarding the feasibility of extending the regulatory approach currently employed for recombinant DNA-based GMOs to genome-edited organisms.
... Zellkulturen sind in der Pflanzenzüchtung weit verbreitet, und Methoden zur Protoplasten-und Zellregeneration sind in der Pflanzenzüchtung oft unverzichtbar. Diese in vitro-Phasen können zu unerwünschten Effekten im Pflanzengenom führen, die als möglicher Ansatz für den Nachweis von NGT diskutiert wurden (Bertheau 2019). ...
Technical Report
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In the EU, genetically modified organisms (GMOs) are subject to the authorization require-ments of Directive 2001/18/EC or Regulation (EC) No. 1829/2003. Application for authoriza-tion of a genetically modified plant requires description of identification and detection meth-ods. Those methods are used by control laboratories of the Member States to detect and identify genetically modified plants and to quantify their occurrence in food and feed. In its ruling of July 25, 2018 (C-528/16), the European Court of Justice determined that plants pro-duced with directed mutagenesis (genome editing) are covered by the regulations under Di-rective 2001/18/EC on the release and placing on the market of GMOs. For control laborato-ries this poses specific challenges for detection, identification and quantification of genome-edited plants. Genome editing techniques allow targeted modifications at predefined sites in the genome of organisms. They belong to the "novel genomic techniques (NGT)", defined as “techniques capable to change the genetic material of an organism and that have emerged or have been developed since 2001”. The possible detection of genome-edited GMOs depends on the type of modification achieved and whether (transgenic) foreign DNA or genome-editing compo-nents may have been integrated into the plant genome. For detection, small mutations (point mutations) pose the greatest challenge. In this report, based on the currently used methods for the detection of classical GMOs, the existing possibilities and challenges for genome-edited plants are discussed. Detection of even very small sequence differences, such as point mutations, is possible with the current technical equipment of a control laboratory. Optimization steps (primer/probe de-sign, thermal profiles) may be required to increase sensitivity. To transfer detection methods into routine operation, the methods have to be validated. A characteristic-unique modification or the combined detection of multiple modifications in a tested genome may allow the identi-fication of genome-edited plants. Quantification of these analytical results by currently avail-able methods is technically possible using certain approaches. Accurate information on the modification made is the most important prerequisite for the de-velopment of methods for detection, identification and quantification of genome-edited GMOs. Such information could be gathered from publicly available sources. It is recom-mended combining information from different public documents, including scientific publica-tions, patents and regulatory documents from third countries. This information could be gath-ered in databases maintained by international organizations. Existing databases, such as the Biosafety Clearing House under the Cartagena Protocol on Biosafety or EUginius, could serve as a model for an international database for sharing information on globally marketed NGT products. It is recommended to establish such a database, maintained at the interna-tional level, to provide easy access for control laboratories to all relevant information. In order to be able to establish the developed methods in the laboratory, and for control pur-poses, reference material is necessary (biological material and derived material; includes the genome-edited plant and/or the parental line/starting material). Upon application for food, feed, or cultivation purposes, the applicant shall provide reference material. For GMO without application, the availability of reference material can be ensured by a central body (e.g., the Joint Research Centre, JRC) in cooperation with the developers. If biological material is not available, plasmids with the corresponding DNA sequences can also be synthesized based on sequence information, if available, and serve as reference material. Research is needed primarily in method development, characterization of genome editing applications (non-intended modifications, specificities, recognition sequences, etc.), and in the development of databases for pan-genomes to be used in control laboratories in the long term. It is recommended that this work be funded through European research funding pro-grammes and through mandates to JRC and EFSA.
... The use of different targets and signal amplification of variants, with or without hybridisation, makes available a range of qualitative and quantitative methods (QRT-PCR, LAMP, NASBA, LCR, SNPLex…) that can be chosen, depending on the target. These methodological approaches can be applied from the field to the laboratory, from point mutation to the junction sequence between rearranged elements and from single to multiple targets Bertheau, 2019;Chhalliyil et al., 2020). Associated with various decision support tools, they allow, via the matrix approach, the identification of any GMO. ...
... For others, the appearance of off-target effects such as tumors in humans (Haapaniemi et al., 2018) calls for caution in the use of these tools. Moreover, studies have traced changes caused by these technologies and thus attributed property rights to final products (Bertheau, 2019). ...
Article
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The evolution of varietal creation methods led in 2012 to the advent of the genome editing technique, CRISPR-CaS9. This technique would make it possible to create new varieties quickly and cheaply. Although some consider CRISPR-CaS9 to be revolutionary, others consider it a potential societal threat. To document the controversy, we explain the socioeconomic conditions under which this technique could be accepted for the creation of a rainfed rice variety in Madagascar. The methodological framework is based on 38 individual and semistructured interviews, a multistakeholder forum with organizations interviewed, and a survey of 148 rice producers. Results reveal that the acceptability of genome editing requires (i) strengthening the seed system through the operationalization of regulatory structures and the upgrading of stakeholders' knowledge of genetically modified organisms, (ii) assessing the effects of the edited variety on biodiversity and soil nitrogen dynamics, and (iii) strengthening the technical and human capacities of the biosafety body. Structural mechanisms for regulating the seed system are necessary to ensure safe experimentation of genome editing techniques. Organizational innovation also appears to be necessary. The study documents how collective learning between communities of scientists and nonscientists is a component of systemic processes of varietal innovation.
... If adequate sequence information is made available, distinct, unique genomic changes can be analytically detected [22]. These changes may either be (several) intended mutations or a combination of intended mutations with unintended changes that are simultaneously present in a genome-edited plant [23][24][25]. Plant genes with a low genetic distance may not be separable, a phenomenon frequently observed in classical breeding [26]. Similarly, mutations are expected to be genetically linked if located in the immediate vicinity. ...
Article
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It is difficult to trace and identify genome-edited food and feed products if relevant information is not made available to competent authorities. This results in major challenges, as genetically modified organism (GMO) regulatory frameworks for food and feed that apply to countries such as the member states of the European Union (EU) require enforcement based on detection. An international anticipatory detection and identification framework for voluntary collaboration and collation of disclosed information on genome-edited plants could be a valuable tool to address these challenges caused by data gaps. Scrutinizing different information sources and establishing a level of information that is sufficient to unambiguously conclude on the application of genome editing in the plant breeding process can support the identification of genome-edited products by complementing the results of analytical detection. International coordination to set up an appropriate state-of-the-art database is recommended to overcome the difficulty caused by the non-harmonized bio-safety regulation requirements of genome-edited food and feed products in various countries. This approach helps to avoid trade disruptions and to facilitate GMO/non-GMO labeling schemes. Implementation of the legal requirements for genome-edited food and feed products in the EU and elsewhere would substantially benefit from such an anticipatory framework.
... A key area of controversy is whether the product or the process is relevant for assessment and approval, because the products of NBTs are sometimes not distinguishable from those of conventional breeding methods [11]. The issue of detection remains controversial, and there have been calls for further research on this topic before NBTs are approved for commercial application [12]. Furthermore, while there was a legal obligation for developers and users of MON810 to limit 'contamination', i.e., the spread of transgenes, some products of NBTs incorporate 'gene drives', which are specifically designed to enable a particular genome to be propagated throughout a population [13]. ...
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Genomic evaluation has been successfully implemented in the United States, Canada, Great Britain, Ireland, New Zealand, Australia, France, the Netherlands, Germany, and the Scandinavian countries. Adoption of this technology in the major dairy producing countries has led to significant changes in the worldwide dairy industry. Gradual elimination of the progeny test system has led to a reduction in the number of sires with daughter records and fewer genetic ties between years. As genotyping costs decrease, the number of cows genotyped will continue to increase, and these records will become the basic data used to compute genomic evaluations, most likely via application of "single-step" methodologies. Although genomic selection has been successful in increasing rates of genetic gain, we still know very little about the genetic architecture of quantitative variation. Apparently, a very large number of genes affect nearly all economic traits, in accordance with the infinitesimal model for quantitative traits. Less emphasis in selection goals will be placed on milk production traits, and more on health, reproduction, and efficiency traits and on environmentally friendly production with reduced waste and gas emission. Genetic variance for economic traits is maintained by the increase in frequency of rare alleles, new mutations, and changes in selection goals and management. Thus, it is unlikely that a selection plateau will be reached in the near future.
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Sequence alignment is an active research area in the field of bioinformatics. It is also a crucial task as it guides many other tasks like phylogenetic analysis, function, and/or structure prediction of biological macromolecules like DNA, RNA, and Protein. Proteins are the building blocks of every living organism. Although protein alignment problem has been studied for several decades, unfortunately, every available method produces alignment results differently for a single alignment problem. Multiple sequence alignment is characterized as a very high computational complex problem. Many stochastic methods, therefore, are considered for improving the accuracy of alignment. Among them, many researchers frequently use Genetic Algorithm. In this study, we have shown different types of the method applied in alignment and the recent trends in the multiobjective genetic algorithm for solving multiple sequence alignment. Many recent studies have demonstrated considerable progress in finding the alignment accuracy.
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Because plants do not possess a proper germline, deleterious mutations that occur in the soma can be passed to gametes. It has generally been assumed that the large number of somatic cell divisions separating zygote from gamete formation in long-lived plants should lead to many mutations. However, a recent study showed that surprisingly few cell divisions separate apical stem cells from axillary stem cells in annual plants, challenging this view. To test this prediction, we generated and analysed the full genome sequence of two terminal branches of a 234-year-old oak tree and found very few fixed somatic single-nucleotide variants (SNVs), whose sequential appearance in the tree could reliably be traced back along nested sectors of younger branches. Our data indicate that the stem cells of shoot meristems in trees are robustly protected from accumulation of mutations, analogous to the germline in animals.
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