Identification of unknown genetically modified material admixed in conventional cotton seed and development of an event-specific detection method

Akritidis, Paschalis; Pasentsis, Konstantinos; Tsaftaris, Athanasios; Mylona, Photini V.; Polidoros, Alexios N.

doi:10.2225/vol11-issue2-fulltext-11

Cited by 12 publications

(6 citation statements)

References 9 publications

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“…Among these, TAIL-PCR and genome walking are commonly used approaches for isolating and cloning sequences flanking T-DNA (Daniela et al, 2013). Several junction sequences in transgenic soybean, maize, and cotton were successfully characterized using these methods (Windels et al, 2001; Yang et al, 2005; Akritidis et al, 2008; Wang et al, 2010; Fraiture et al, 2015b). However, these approaches are always laborious and expensive, and especially difficult to achieve high throughput.…”

Section: Introductionmentioning

confidence: 99%

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method

Guo

Hong

et al. 2016

Front. Plant Sci.

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Molecular characterization of sequence flanking exogenous fragment insertion is essential for safety assessment and labeling of genetically modified organism (GMO). In this study, the T-DNA insertion sites and flanking sequences were identified in two newly developed transgenic glyphosate-tolerant soybeans GE-J16 and ZH10-6 based on whole genome sequencing (WGS) method. More than 22.4 Gb sequence data (∼21 × coverage) for each line was generated on Illumina HiSeq 2500 platform. The junction reads mapped to boundaries of T-DNA and flanking sequences in these two events were identified by comparing all sequencing reads with soybean reference genome and sequence of transgenic vector. The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis. All these analyses supported that exogenous T-DNA fragments were integrated in positions of Chr19: 50543767–50543792 and Chr17: 7980527–7980541 in these two transgenic lines. Identification of genomic insertion sites of G2-EPSPS and GAT transgenes will facilitate the utilization of their glyphosate-tolerant traits in soybean breeding program. These results also demonstrated that WGS was a cost-effective and rapid method for identifying sites of T-DNA insertions and flanking sequences in soybean.

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Section: Introductionmentioning

confidence: 99%

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method

Guo

Hong

et al. 2016

Front. Plant Sci.

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“…Since DNA walking requires less prior knowledge about the sequence of interest than conventional PCR-based methods previously described, GMO with entirely or partially known sequences could be characterized. Therefore, in targeting key elements, such as p35S and tNOS that are highly frequent in GM crops, a broad range of GMO could be characterized [ 96 , 106 , 110 , 111 , 113 , 118 , 156 ]. In order to especially identify unauthorized GMO in European Union, a DNA walking approach using primers specific to the element t35S from the pCAMBIA vector, found in approximately 30% of transgenic plants, was developed [ 33 , 117 ].…”

Section: Gmo Detection Approachesmentioning

confidence: 99%

Current and New Approaches in GMO Detection: Challenges and Solutions

Fraiture

Herman

Taverniers³

et al. 2015

BioMed Research International

116

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In many countries, genetically modified organisms (GMO) legislations have been established in order to guarantee the traceability of food/feed products on the market and to protect the consumer freedom of choice. Therefore, several GMO detection strategies, mainly based on DNA, have been developed to implement these legislations. Due to its numerous advantages, the quantitative PCR (qPCR) is the method of choice for the enforcement laboratories in GMO routine analysis. However, given the increasing number and diversity of GMO developed and put on the market around the world, some technical hurdles could be encountered with the qPCR technology, mainly owing to its inherent properties. To address these challenges, alternative GMO detection methods have been developed, allowing faster detections of single GM target (e.g., loop-mediated isothermal amplification), simultaneous detections of multiple GM targets (e.g., PCR capillary gel electrophoresis, microarray, and Luminex), more accurate quantification of GM targets (e.g., digital PCR), or characterization of partially known (e.g., DNA walking and Next Generation Sequencing (NGS)) or unknown (e.g., NGS) GMO. The benefits and drawbacks of these methods are discussed in this review.

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“…Among these methods, thermal asymmetric interlaced PCR (TAIL-PCR) and genome walking are often used to isolate and clone T-DNA flanking sequences [9,10]. For example, by using the TAIL-PCR method by sequencing, several T-DNA flanking sequences were identified and characterized in transgenic maize [11], soybean [12], cotton [13], and alfalfa [14]. However, these PCR-based methods are labor-intensive and expensive.…”

Section: Introductionmentioning

confidence: 99%

Identification of genomic insertion and flanking sequences of the transgenic drought-tolerant maize line “SbSNAC1-382” using the single-molecule real-time (SMRT) sequencing method

Zeng

Zhang

et al. 2020

PLoS ONE

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Safety assessment of genetically modified (GM) crops is crucial at the product-development phase before GM crops are placed on the market. Determining characteristics of sequences flanking exogenous insertion sequences is essential for the safety assessment and marketing of transgenic crops. In this study, we used genome walking and whole-genome sequencing (WGS) to identify the flanking sequence characteristics of the SbSNAC1 transgenic drought-tolerant maize line "SbSNAC1-382", but both of the two methods failed. Then, we constructed a genomic fosmid library of the transgenic maize line, which contained 4.18×10 5 clones with an average insertion fragment of 35 kb, covering 5.85 times the maize genome. Subsequently, three positive clones were screened by pairs of specific primers, and one of the three positive clones was sequenced by using single-molecule real-time (SMRT) sequencing technology. More than 1.95 Gb sequence data (~10 5 × coverage) for the sequenced clone were generated. The junction reads mapped to the boundaries of T-DNA, and the flanking sequences in the transgenic line were identified by comparing all sequencing reads with the maize reference genome and the sequence of the transgenic vector. Furthermore, the putative insertion loci and flanking sequences were confirmed by PCR amplification and Sanger sequencing. The results indicated that two copies of the exogenous T-DNA fragments were inserted at the same genomic site, and the exogenous T-DNA fragments were integrated at the position of Chromosome 5 from 177155650 to 177155696 in the transgenic line 382. In this study, we demonstrated the successful application of the SMRT technology for the characterization of genomic insertion and flanking sequences.

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Identification of unknown genetically modified material admixed in conventional cotton seed and development of an event-specific detection method

Cited by 12 publications

References 9 publications

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method

Current and New Approaches in GMO Detection: Challenges and Solutions

Identification of genomic insertion and flanking sequences of the transgenic drought-tolerant maize line “SbSNAC1-382” using the single-molecule real-time (SMRT) sequencing method

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