RESUMO
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.
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The objective of this study was to quantitatively assess potato omics profiles of new varieties for meaningful differences from analogous profiles of commercial varieties through the SIMCA one-class classification model. Analytical profiles of nine commercial potato varieties, eleven experimental potato varieties, one GM potato variety that had acquired Phytophtora resistance based on a single insert with potato-derived DNA sequences, and its non-GM commercial counterpart were generated. The ten conventional varieties were used to construct the one-class model. Omics profiles from experimental non-GM and GM varieties were assessed using the one-class SIMCA models. No potential unintended effects were identified in the case of the GM variety. The model showed that varieties that were genetically more distant from the commercial varieties were recognized as aberrant, highlighting its potential in determining whether additional evaluation is required for the risk assessment of materials produced from any breeding technique, including genetic modification.
Assuntos
Metaboloma , Plantas Geneticamente Modificadas/metabolismo , Solanum tuberosum/metabolismo , Transcriptoma , DNA de Plantas/química , DNA de Plantas/metabolismo , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Metabolômica , Plantas Geneticamente Modificadas/genética , Análise de Componente Principal , Medição de Risco , Análise de Sequência de RNA , Solanum tuberosum/genéticaRESUMO
Genetically modified (GM) maize and their non-modified counterparts were compared using MON810 varieties, the only GMO event cultivated in Europe. The differences in grain samples were analysed by omics profiles, including transcriptomics, proteomics and metabolomics. Other cultivated maize varieties were analysed as a reference for the variability that will exist between cultivated varieties. The observed differences between modified and non-modified maize varieties do not exceed typical differences between non-modified varieties. The use of these advanced analytical approaches to analyse novel plant materials as compared to the results from animal feeding trials with whole foods is assessed. No indications were observed for changes in the GM varieties that warrant further investigations. Furthermore, it was shown that such indications will be obtained if maize samples of inferior quality are analysed similarly. Omics data provide detailed analytical information of the plant material, which facilitates a risk assessment procedure of new (GM) plant varieties.
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Metabolômica , Plantas Geneticamente Modificadas/metabolismo , Proteômica , Zea mays/metabolismo , Ração Animal/análise , Animais , Cromatografia Líquida de Alta Pressão , Europa (Continente) , Genômica , Metaboloma , Plantas Geneticamente Modificadas/genética , Análise de Componente Principal , RNA de Plantas/química , RNA de Plantas/isolamento & purificação , RNA de Plantas/metabolismo , Espectrometria de Massas em Tandem , Zea mays/genéticaRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs). In parallel, the number of reports of unauthorised GMOs (UGMOs) is gradually increasing. There is a lack of specific detection methods for UGMOs, due to the absence of detailed sequence information and reference materials. In this research, an adapted genome walking approach was developed, called ALF: Amplification of Linearly-enriched Fragments. Coupling of ALF to NGS aims for simultaneous detection and identification of all GMOs, including UGMOs, in one sample, in a single analysis. The ALF approach was assessed on a mixture made of DNA extracts from four reference materials, in an uneven distribution, mimicking a real life situation. The complete insert and genomic flanking regions were known for three of the included GMO events, while for MON15985 only partial sequence information was available. Combined with a known organisation of elements, this GMO served as a model for a UGMO. We successfully identified sequences matching with this organisation of elements serving as proof of principle for ALF as new UGMO detection strategy. Additionally, this study provides a first outline of an automated, web-based analysis pipeline for identification of UGMOs containing known GM elements.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , Biologia Computacional/métodos , Alimentos Geneticamente Modificados , Gossypium/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Fluxo de Trabalho , Zea mays/genéticaRESUMO
DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.
Assuntos
Código de Barras de DNA Taxonômico , Espécies em Perigo de Extinção , Animais , Biologia Computacional , DNA de Plantas/genética , Plantas/classificação , Plantas/genética , Reprodutibilidade dos TestesRESUMO
This article contains data related to the research article entitled "A case study to determine the geographical origin of unknown GM papaya in routine food sample analysis, followed by identification of papaya events 16-0-1 and 18-2-4" (Prins et al., 2016) [1]. Quantitative real-time PCR (qPCR) with targets that are putatively present in genetically modified (GM) papaya was used as a first screening to narrow down the vast array of candidates. The combination of elements P-nos and nptII was further confirmed by amplification and subsequent sequencing of the P-nos/nptII construct. Next, presence of the candidate GM papayas 16-0-1 and 18-2-4 were investigated by amplification and sequencing of event-spanning regions on the left and right border. This data article reports the Cq values for GM elements, the nucleotide sequence of the P-nos/nptII construct and the presence of GM papaya events 18-2-4 and/or 16-0-1 in five samples that were randomly sampled to be analysed in the framework of the official Dutch GMO monitoring program for food.
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During routine monitoring for GMOs in food in the Netherlands, papaya-containing food supplements were found positive for the genetically modified (GM) elements P-35S and T-nos. The goal of this study was to identify the unknown and EU unauthorised GM papaya event(s). A screening strategy was applied using additional GM screening elements including a newly developed PRSV coat protein PCR. The detected PRSV coat protein PCR product was sequenced and the nucleotide sequence showed identity to PRSV YK strains indigenous to China and Taiwan. The GM events 16-0-1 and 18-2-4 could be identified by amplifying and sequencing events-specific sequences. Further analyses showed that both papaya event 16-0-1 and event 18-2-4 were transformed with the same construct. For use in routine analysis, derived TaqMan qPCR methods for events 16-0-1 and 18-2-4 were developed. Event 16-0-1 was detected in all samples tested whereas event 18-2-4 was detected in one sample. This study presents a strategy for combining information from different sources (literature, patent databases) and novel sequence data to identify unknown GM papaya events.
Assuntos
Proteínas do Capsídeo/análise , Carica/metabolismo , Análise de Alimentos/métodos , Plantas Geneticamente Modificadas/metabolismo , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Carica/genética , China , Geografia , TaiwanRESUMO
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.
Assuntos
Produtos Agrícolas/química , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , Proteínas de Vegetais Comestíveis/análise , Proteínas de Plantas/metabolismo , Tubérculos/química , Solanum tuberosum/química , Análise por Conglomerados , Produtos Agrícolas/metabolismo , Perfilação da Expressão Gênica , Países Baixos , Proteínas de Plantas/genética , Proteínas de Vegetais Comestíveis/biossíntese , Tubérculos/metabolismo , Análise de Componente Principal , Proteoma/biossíntese , Proteômica/métodos , Solanum tuberosum/metabolismo , Especificidade da Espécie , Eletroforese em Gel Diferencial BidimensionalRESUMO
Before commercial release, new potato (Solanum tuberosum) varieties must be evaluated for content of toxic compounds such as glycoalkaloids (GAs), which are potent poisons. GA biosynthesis proceeds via the cholesterol pathway to α-chaconine and α-solanine. The goal of this study was to evaluate the relationship between total glycoalkaloid (TGA) content and the expression of GAME, SGT1, and SGT3 genes in potato tubers. TGA content was measured by HPLC-MS, and reverse transcription quantitative polymerase chain reactions were performed to determine the relative expression of GAME, SGT1, and SGT3 genes. We searched for cis-elements of the transcription start site using the PlantPAN database. There was a relationship between TGA content and the relative expression of GAME, SGT1, and SGT3 genes in potato tubers. Putative promoter regions showed the presence of several cis-elements related to biotic and abiotic stresses and light. These findings provide an important step toward understanding TGA regulation and variation in potato tubers.
Assuntos
Alcaloides/biossíntese , Proteínas de Plantas/genética , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Transcrição Gênica , Alcaloides/toxicidade , Vias Biossintéticas , Proteínas de Plantas/metabolismo , Tubérculos/química , Tubérculos/genética , Tubérculos/metabolismo , Regiões Promotoras Genéticas , Solanina/análogos & derivados , Solanina/metabolismo , Solanina/toxicidadeRESUMO
Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples.
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Biologia Computacional/métodos , Perfilação da Expressão Gênica/normas , Genes de Plantas/genética , Tubérculos/genética , Solanum tuberosum/genética , Transcrição Gênica , Algoritmos , Variação Genética , Padrões de Referência , Análise de Sequência de RNARESUMO
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.
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Perfilação da Expressão Gênica , Modelos Teóricos , Plantas Geneticamente Modificadas/toxicidade , Solanum tuberosum/genética , TranscriptomaRESUMO
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.
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Agricultura/legislação & jurisprudência , Proteínas de Bactérias/genética , Gossypium/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Zea mays/genética , Contaminação de Alimentos/legislação & jurisprudênciaRESUMO
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.
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Beta vulgaris/genética , DNA de Plantas/isolamento & purificação , Glycine max/genética , Técnicas Analíticas Microfluídicas/instrumentação , Plantas Geneticamente Modificadas , Zea mays/genética , Calibragem , Primers do DNA , DNA de Plantas/genética , Ensaios de Triagem em Larga Escala , Humanos , Limite de Detecção , Reação em Cadeia da PolimeraseRESUMO
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.
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Tecnologia de Alimentos/métodos , Técnicas de Amplificação de Ácido Nucleico , Plantas Geneticamente Modificadas/genética , Sequência de Bases , DNA de Plantas/análise , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Sondas de Oligonucleotídeos/química , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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.
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Produtos Agrícolas/classificação , Produtos Agrícolas/genética , DNA de Plantas/análise , DNA de Plantas/genética , Análise de Alimentos/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Sondas de Oligonucleotídeos/genética , Oligonucleotídeos/genética , Oryza/genética , Controle de Qualidade , Triticum/genéticaRESUMO
BACKGROUND: To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. RESULTS: In this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation.In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. CONCLUSION: Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetected.