Analysis of the genetic stability of event NK603 in stacked corn varieties using high-resolution melting (HRM) analysis and Sanger sequencing

Castan, Magali; Ali, Sina-Elisabeth Ben; Hochegger, Rupert; Ruppitsch, Werner; Haslberger, Alexander G.; Brandes, Christian

doi:10.1007/s00217-016-2749-2

Cited by 10 publications

(7 citation statements)

References 35 publications

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“…While the first one contains the 5'-end of the rice actin sequence (ract1) promoter and the first intron upstream of the CTP sequence, the second CP4 EPSPS gene is under the control of the enhanced CaMV 35S promoter fused to the 0.8 kb intron sequence from the gene of maize heat shock 70 protein [ 89 ]. Castan et al [ 90 ] recently reported a study in which the NK603 construct was sequenced, with the aim of examining its genetic stability. The authors consistently detected two nucleotide insertions in all sequenced samples of NK603 × MON810 (varieties 631RR2/Bt and DKC 26–79 progeny), as well as the certified reference material, by comparing the sequenced construct NK603 to the published patent sequence [ 91 ].…”

Section: Resultsmentioning

confidence: 99%

Molecular responses of genetically modified maize to abiotic stresses as determined through proteomic and metabolomic analyses

et al. 2017

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Some genetically modified (GM) plants have transgenes that confer tolerance to abiotic stressors. Meanwhile, other transgenes may interact with abiotic stressors, causing pleiotropic effects that will affect the plant physiology. Thus, physiological alteration might have an impact on the product safety. However, routine risk assessment (RA) analyses do not evaluate the response of GM plants exposed to different environmental conditions. Therefore, we here present a proteome profile of herbicide-tolerant maize, including the levels of phytohormones and related compounds, compared to its near-isogenic non-GM variety under drought and herbicide stresses. Twenty differentially abundant proteins were detected between GM and non-GM hybrids under different water deficiency conditions and herbicide sprays. Pathway enrichment analysis showed that most of these proteins are assigned to energetic/carbohydrate metabolic processes. Among phytohormones and related compounds, different levels of ABA, CA, JA, MeJA and SA were detected in the maize varieties and stress conditions analysed. In pathway and proteome analyses, environment was found to be the major source of variation followed by the genetic transformation factor. Nonetheless, differences were detected in the levels of JA, MeJA and CA and in the abundance of 11 proteins when comparing the GM plant and its non-GM near-isogenic variety under the same environmental conditions. Thus, these findings do support molecular studies in GM plants Risk Assessment analyses.

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Section: Resultsmentioning

confidence: 99%

Molecular responses of genetically modified maize to abiotic stresses as determined through proteomic and metabolomic analyses

et al. 2017

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“…Due to the date of the publication, the publication by Castan et al (2017) was not included in the systematic literature search. This publication is discussed in Section 2.1…”

Section: 2mentioning

confidence: 99%

Assessment of genetically modified maize NK603 x MON810 for renewal of authorisation under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐RX‐007)

Naegeli¹,

Birch²,

Casacuberta³

et al. 2018

EFS2

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Following the submission of application EFSA-GMO-RX-007 under Regulation (EC) No 1829/2003 from Monsanto, the Panel on Genetically Modified Organisms of the European Food Safety Authority (GMO Panel) was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application of the herbicide-tolerant and insect-resistant genetically modified maize NK603 x MON810. The data received in the context of this renewal application contained post-market environmental monitoring reports, a systematic search and evaluation of literature, updated bioinformatic analyses, and additional documents or studies performed by or on behalf of the applicant. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. Under the assumption that the DNA sequence of the events in maize NK603 x MON810 considered for renewal is identical to the sequence of the originally assessed events, the GMO Panel concludes that there is no evidence in the renewal application EFSA-GMO-RX-007 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on maize NK603 x MON810.

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“…We have also performed a molecular analysis of NK603 maize and identified two insertions, which are not present in the NK603 patent sequence. These insertions were located in the transgenic promoter region; therefore, they may have an effect on the promoter activity and, consequently, also on transgene expression [14]. However, our analysis of the 5´-end of MON810 as a single event with Scorpion probes revealed no unintended effects or mutations [62].…”

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confidence: 71%

Analysis of transcriptomic differences between NK603 maize and near-isogenic varieties using RNA sequencing and RT-qPCR

et al. 2020

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Background The insertion of a transgene into a plant organism can, in addition to the intended effects, lead to unintended effects in the plants. To uncover such effects, we compared maize grains of two genetically modified varieties containing NK603 (AG8025RR2, AG9045RR2) to their non-transgenic counterparts (AG8025conv, AG9045conv) using high-throughput RNA sequencing. Moreover, in-depth analysis of these data was performed to reveal the biological meaning of detected differences. Results Uniquely mapped reads corresponded to 29,146 and 33,420 counts in the AG8025 and AG9045 varieties, respectively. An analysis using the R-Bioconductor package EdgeR revealed 3534 and 694 DEGs (significant differentially expressed genes) between the varieties AG8025RR2 and AG9045RR2, respectively, and their non-transgenic counterparts. Furthermore, a Deseq2 package revealed 2477 and 440 DEGs between AG8025RR2 and AG9045RR2, respectively, and their counterparts. We were able to confirm the RNA-seq results by the analysis of two randomly selected genes using RT-qPCR (reverse transcription quantitative PCR). PCA and heatmap analysis confirmed a robust data set that differentiates the genotypes even by transgenic event. A detailed analysis of the DEGs was performed by the functional annotation of GO (Gene Ontology), annotation/enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) ontologies and functional classification of resulting key genes using the DAVID Bioinformatics Package. Several biological processes and metabolic pathways were found to be significantly different in both variety pairs. Conclusion Overall, our data clearly demonstrate substantial differences between the analyzed transgenic varieties and their non-transgenic counterparts. These differences indicate that several unintended effects have occurred as a result of NK603 integration. Heatmap data imply that most of the transgenic insert effects are variety-dependent. However, identified key genes involved in affected pathways of both variety pairs show that transgenic independent effects cannot be excluded. Further research of different NK603 varieties is necessary to clarify the role of internal and external influences on gene expression. Nevertheless, our study suggests that RNA-seq analysis can be utilized as a tool to characterize unintended genetic effects in transgenic plants and may also be useful in the safety assessment and authorization of genetically modified (GM) plants.

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Analysis of the genetic stability of event NK603 in stacked corn varieties using high-resolution melting (HRM) analysis and Sanger sequencing

Cited by 10 publications

References 35 publications

Molecular responses of genetically modified maize to abiotic stresses as determined through proteomic and metabolomic analyses

Molecular responses of genetically modified maize to abiotic stresses as determined through proteomic and metabolomic analyses

Assessment of genetically modified maize NK603 x MON810 for renewal of authorisation under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐RX‐007)

Analysis of transcriptomic differences between NK603 maize and near-isogenic varieties using RNA sequencing and RT-qPCR

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