GMO Genetic Elements Thesaurus (GMO-GET): a controlled vocabulary for the consensus designation of introduced or modified genetic elements in genetically modified organisms.

BACKGROUND: Various databases on genetically modified organisms (GMOs) exist, all with their specific focus to facilitate access to information needed for, e. g., the assistance in risk assessment, the development of detection and identification strategies or inspection and control activities. Each database has its unique approach towards the subject. Often these databases use different terminology to describe the GMOs. For adequate GMO addressing and identification and exchange of GMO-related information it is necessary to use commonly agreed upon concepts and terminology. RESULT: A hierarchically structured controlled vocabulary describing the genetic elements inserted into conventional GMOs, and GMOs developed by the use of gen(om)e-editing is presented: the GMO genetic element thesaurus (GMO-GET). GMO-GET can be used for GMO-related documentation, including GMO-related databases. It has initially been developed on the basis of two GMO databases, i.e. the Biosafety Clearing-House and the EUginius database. CONCLUSION: The use of GMO-GET will enable consistent and compatible information (harmonisation), also allowing an accurate exchange of information between the different data systems and thereby facilitating their interoperability. GMO-GET can also be used to describe genetic elements that are altered in organisms obtained through current targeted genome-editing techniques.

Correction to: Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats.

In the original publication, the starting point in time for the three feeding trials.

Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats.

In 2012, a controversial study on the long-term toxicity of a Roundup herbicide and the glyphosate-tolerant genetically modified (GM) maize NK603 was published. The EC-funded G-TwYST research consortium tested the potential subchronic and chronic toxicity as well as the carcinogenicity of the glyphosate-resistant genetically modified maize NK603 by performing two 90-day feeding trials, one with GM maize inclusion rates of 11 and 33% and one with inclusion rates of up to 50%, as well as a 2-year feeding trial with inclusion rates of 11 and 33% in male and female Wistar Han RCC rats by taking into account OECD Guidelines for the testing of chemicals and EFSA recommendations on the safety testing of whole-food/feed in laboratory animals. In all three trials, the NK603 maize, untreated and treated once with Roundup during its cultivation, and the conventional counterpart were tested. Differences between each test group and the control group were evaluated. Equivalence was assessed by comparing the observed difference to differences between non-GM reference groups in previous studies. In case of significant differences, whether the effects were dose-related and/or accompanied by changes in related parameters including histopathological findings was evaluated. It is concluded that no adverse effects related to the feeding of the NK603 maize cultivated with or without Roundup for up to 2 years were observed. Based on the outcome of the subchronic and combined chronic toxicity/carcinogenicity studies, recommendations on the scientific justification and added value of long-term feeding trials in the GM plant risk assessment process are presented.

Semiautomated TaqMan PCR screening of GMO labelled samples for (unauthorised) GMOs.

In most countries, systems are in place to analyse food products for the potential presence of genetically modified organisms (GMOs), to enforce labelling requirements and to screen for the potential presence of unauthorised GMOs. With the growing number of GMOs on the world market, a larger diversity of methods is required for informative analyses. In this paper, the specificity of an extended screening set consisting of 32 screening methods to identify different crop species (endogenous genes) and GMO elements was verified against 59 different GMO reference materials. In addition, a cost- and time-efficient strategy for DNA isolation, screening and identification is presented. A module for semiautomated analysis of the screening results and planning of subsequent event-specific tests for identification has been developed. The Excel-based module contains information on the experimentally verified specificity of the element methods and of the EU authorisation status of the GMO events. If a detected GMO element cannot be explained by any of the events as identified in the same sample, this may indicate the presence of an unknown unauthorised GMO that may not yet have been assessed for its safety for humans, animals or the environment.

DNA enrichment approaches to identify unauthorized genetically modified organisms (GMOs).

With the increased global production of different genetically modified (GM) plant varieties, chances increase that unauthorized GM organisms (UGMOs) may enter the food chain. At the same time, the detection of UGMOs is a challenging task because of the limited sequence information that will generally be available. PCR-based methods are available to detect and quantify known UGMOs in specific cases. If this approach is not feasible, DNA enrichment of the unknown adjacent sequences of known GMO elements is one way to detect the presence of UGMOs in a food or feed product. These enrichment approaches are also known as chromosome walking or gene walking (GW). In recent years, enrichment approaches have been coupled with next generation sequencing (NGS) analysis and implemented in, amongst others, the medical and microbiological fields. The present review will provide an overview of these approaches and an evaluation of their applicability in the identification of UGMOs in complex food or feed samples.

One-year oral toxicity study on a genetically modified maize MON810 variety in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

The GRACE (GMO Risk Assessment and Communication of Evidence; www.grace-fp7.eu ) project was funded by the European Commission within the 7th Framework Programme. A key objective of GRACE was to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of a 1-year feeding trial with a GM maize MON810 variety, its near-isogenic non-GM comparator and an additional conventional maize variety are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 452. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after a chronic exposure.

Tuber proteome comparison of five potato varieties by principal component analysis.

BACKGROUND: Data analysis of omics data should be performed by multivariate analysis such as principal component analysis (PCA). The way data are clustered in PCA is of major importance to develop some classification systems based on multivariate analysis, such as soft independent modeling of class analogy (SIMCA). In a previous study a one-class classifier based on SIMCA was built using microarray data from a set of potatoes. The PCA grouped the transcriptomic data according to varieties. The present work aimed to use PCA to verify the clustering of the proteomic profiles for the same potato varieties. RESULTS: Proteomic profiles of five potato varieties (Biogold, Fontane, Innovator, Lady Rosetta and Maris Piper) were evaluated by two-dimensional gel electrophoresis (2-DE) performed on two immobilized pH gradient (IPG) strip lengths, 13 and 24 cm, both under pH range 4-7. For each strip length, two gels were prepared from each variety; in total there were ten gels per analysis. For 13 cm strips, 199-320 spots were detected per gel, and for 24 cm strips, 365-684 spots. CONCLUSION: All four PCAs performed with these datasets presented clear grouping of samples according to the varieties. The data presented here showed that PCA was applicable for proteomic analysis of potato and was able to separate the samples by varieties. © 2016 Society of Chemical Industry.

Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing.

The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.

A high-throughput method for GMO multi-detection using a microfluidic dynamic array.

The ever-increasing production of genetically modified crops generates a demand for high-throughput DNA-based methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis by DNA based methods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06 % were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection.

Ninety-day oral toxicity studies on two genetically modified maize MON810 varieties in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

The GMO Risk Assessment and Communication of Evidence (GRACE; www.grace-fp7.eu ) project is funded by the European Commission within the 7th Framework Programme. A key objective of GRACE is to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of two 90-day feeding trials with two different GM maize MON810 varieties, their near-isogenic non-GM varieties and four additional conventional maize varieties are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 408. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after subchronic exposure, independently of the two different genetic backgrounds of the event.

Safety assessment of plant varieties using transcriptomics profiling and a one-class classifier.

An important part of the current hazard identification of novel plant varieties is comparative targeted analysis of the novel and reference varieties. Comparative analysis will become much more informative with unbiased analytical approaches, e.g. omics profiling. Data analysis estimating the similarity of new varieties to a reference baseline class of known safe varieties would subsequently greatly facilitate hazard identification. Further biological and eventually toxicological analysis would then only be necessary for varieties that fall outside this reference class. For this purpose, a one-class classifier tool was explored to assess and classify transcriptome profiles of potato (Solanum tuberosum) varieties in a model study. Profiles of six different varieties, two locations of growth, two year of harvest and including biological and technical replication were used to build the model. Two scenarios were applied representing evaluation of a 'different' variety and a 'similar' variety. Within the model higher class distances resulted for the 'different' test set compared with the 'similar' test set. The present study may contribute to a more global hazard identification of novel plant varieties.

New EU legislation for risk assessment of GM food: no scientific justification for mandatory animal feeding trials.

This commentary focuses on the potential added value of and need for (sub)-chronic testing of whole genetically modified (GM) foods in rodents to assess their safety. Such routine testing should not be required since, due to apparent weaknesses in the approach, it does not add to current risk assessment of GM foods. Moreover, the demand for routine testing using animals is in conflict with the European Union (EU) Commission's efforts to reduce animal experimentation. Regulating agencies in the EU are invited to respect the sound scientific principles applied to the risk assessment of foods derived from GM plants and not to interfere in the risk assessment by introducing extra requirements based on pseudo-scientific or political considerations.

Comparison and transfer testing of multiplex ligation detection methods for GM plants.

BACKGROUND: With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study. RESULTS: Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands). CONCLUSIONS: From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible.

Development of a multiplex DNA-based traceability tool for crop plant materials.

The authenticity of food is of increasing importance for producers, retailers and consumers. All groups benefit from the correct labelling of the contents of food products. Producers and retailers want to guarantee the origin of their products and check for adulteration with cheaper or inferior ingredients. Consumers are also more demanding about the origin of their food for various socioeconomic reasons. In contrast to this increasing demand, correct labelling has become much more complex because of global transportation networks of raw materials and processed food products. Within the European integrated research project 'Tracing the origin of food' (TRACE), a DNA-based multiplex detection tool was developed-the padlock probe ligation and microarray detection (PPLMD) tool. In this paper, this method is extended to a 15-plex traceability tool with a focus on products of commercial importance such as the emmer wheat Farro della Garfagnana (FdG) and Basmati rice. The specificity of 14 plant-related padlock probes was determined and initially validated in mixtures comprising seven or nine plant species/varieties. One nucleotide difference in target sequence was sufficient for the distinction between the presence or absence of a specific target. At least 5% FdG or Basmati rice was detected in mixtures with cheaper bread wheat or non-fragrant rice, respectively. The results suggested that even lower levels of (un-)intentional adulteration could be detected. PPLMD has been shown to be a useful tool for the detection of fraudulent/intentional admixtures in premium foods and is ready for the monitoring of correct labelling of premium foods worldwide.

Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms.

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples.

Comparison of two GM maize varieties with a near-isogenic non-GM variety using transcriptomics, proteomics and metabolomics.

The aim of this study was to evaluate the use of four nontargeted analytical methodologies in the detection of unintended effects that could be derived during genetic manipulation of crops. Three profiling technologies were used to compare the transcriptome, proteome and metabolome of two transgenic maize lines with the respective control line. By comparing the profiles of the two transgenic lines grown in the same location over three growing seasons, we could determine the extent of environmental variation, while the comparison with the control maize line allowed the investigation of effects caused by a difference in genotype. The effect of growing conditions as an additional environmental effect was also evaluated by comparing the Bt-maize line with the control line from plants grown in three different locations in one growing season. The environment was shown to play an important effect in the protein, gene expression and metabolite levels of the maize samples tested where 5 proteins, 65 genes and 15 metabolites were found to be differentially expressed. A distinct separation between the three growing seasons was also found for all the samples grown in one location. Together, these environmental factors caused more variation in the different transcript/protein/metabolite profiles than the different genotypes.

Gene expression profiling for food safety assessment: examples in potato and maize.

Since the mid 1990s, microarray analysis has become one of the few tools that can analyze the entire contents of a cell regarding a specific information type. Especially since the development of whole genome microarrays the technique can be considered truly holistic. Most DNA based microarrays are used for the analysis of the total of messenger RNAs (transcriptome) and provide a snap-shot of what's going on in a cell population at the time of sampling. Within the last few years also full genome plant microarrays have become available for several crop species. With these it has been shown that several growing conditions can be separated based on their transcriptome pattern, such as location, year of harvest and agricultural input system, but also different cultivars of the same crop species, including genetically modified ones. A database comprising expression levels of the transcriptome in many different circumstances with a history of safe use would be a good comparator for evaluation of new agricultural practices or cultivars, genetically modified or otherwise obtained. New techniques as next generation sequencing may overcome issues on throughput time and cost, standard operation procedures and array design for individual crops.

Application of the Safe-By-Design Concept in Crop Breeding Innovation.

The present paper proposes the application of the safe-by-design concept to crop breeding innovation with the aim to accommodate safety considerations for new agricultural food and feed products. Safe-by-design can be implemented in all stages of the innovation cycle of agricultural products, from the early stages of research and development towards the post-market stage. Our proposed application of safe-by-design can be part of "responsible research and innovation" concepts, because they share features such as risk prevention strategies and a participatory approach. Early awareness of potential safety issues can guide the development of agricultural products towards safe options, both at the process and product level, and thus may help to reduce extensive pre-market assessment studies that might otherwise be needed further downstream for regulatory product approval. Here, it is discussed how the proposed safe-by-design approach can be introduced into the development of safe food crops using emerging technologies, such as gene editing and synthetic biology, and how this may help to safeguard the safety of our food and feed supply in the light of the ongoing global innovations in agricultural crop breeding.

Molecular Characterization and Event-Specific Real-Time PCR Detection of Two Dissimilar Groups of Genetically Modified Petunia (Petunia x hybrida) Sold on the Market.

Petunia plants with unusual orange flowers were noticed on the European market and confirmed to be genetically modified (GM) by the Finnish authorities in spring 2017. Later in 2017, inspections and controls performed by several official laboratories of national competent authorities in the European Union detected several GM petunia varieties with orange flowers, but also another group of unusually colored flowers. In the latter group, a so far undetected gene coding for a flavonoid 3'5' hydroxylase (F3'5'H) responsible for the purple color was identified by German and Dutch authorities, suggesting that the petunias found on the markets contain different genetic constructs. Here, a strategy is described for the identification of GM petunia varieties. It is based on an initial GMO screening for known elements using (real-time) PCR and subsequent identification of the insertion sites by a gene walking-like approach called ALF (amplification of linearly-enriched fragments) in combination with Sanger and MinION sequencing. The results indicate that the positively identified GM petunias can be traced back to two dissimilar GM events used for breeding of the different varieties. The test results also confirm that the transgenic petunia event RL01-17 used in the first German field trial in 1991 is not the origin of the GM petunias sold on the market. On basis of the obtained sequence data, event-specific real-time PCR confirmatory methods were developed and validated. These methods are applicable for the rapid detection and identification of GM petunias in routine analysis. In addition, a decision support system was developed for revealing the most likely origin of the GM petunia.