Identification of T-DNA Insertion Site and Flanking Sequence of a Genetically Modified Maize Event IE09S034 Using Next-Generation Sequencing Technology

Siddique, Kiran; Wei, Jiaojun; Li, Rong; Zhang, Dabing; Shi, Jiannong

doi:10.1007/s12033-019-00196-0

Cited by 15 publications

(11 citation statements)

References 32 publications

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“…The next-generation Illumina-based sequencing technology is a powerful tool for identifying the insertion positions of GMOs (Polko et al, 2012 ; Lepage et al, 2013 ; Yang et al, 2013 ; Guo et al, 2016 ; and Siddique et al, 2019 ). In this study, the technology was utilized to identify the insertion of GM maize SK12-5.…”

Section: Resultsmentioning

confidence: 99%

“…Yet, these approaches always remain laborious and expensive, and it is especially difficult to achieve high throughput while using them. Instead, the next-generation Illumina sequencing technology has been used successfully to identify the insertion positions of the transgenes in GMOs, given its depth of sequencing capacity and low labor cost (Polko et al, 2012 ; Lepage et al, 2013 ; Yang et al, 2013 ; Guo et al, 2016 ; and Siddique et al, 2019 ). However, the next-generation Illumina sequencing-based method produces short reads; these pose a challenge for analyzing the data and resolving the insertion positions of the transgenes in many plant species having complex genomes primarily because of the issues related to genome rearrangements and copy-number variations, which may lead to inaccurately mapped locations (Li et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

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Using Combined Methods of Genetic Mapping and Nanopore-Based Sequencing Technology to Analyze the Insertion Positions of G10evo-EPSPS and Cry1Ab/Cry2Aj Transgenes in Maize

et al. 2021

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The insertion position of the exogenous fragment sequence in a genetically modified organism (GMO) is important for the safety assessment and labeling of GMOs. SK12-5 is a newly developed transgenic maize line transformed with two trait genes [i.e., G10evo-5-enolpyrul-shikimate-3-phosphate synthase (EPSPS) and Cry1Ab/Cry2Aj] that was recently approved for commercial use in China. In this study, we tried to determine the insertion position of the exogenous fragment for SK12-5. The transgene–host left border and right border integration junctions were obtained from SK12-5 genomic DNA by using the thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and next-generation Illumina sequencing technology. However, a Basic Local Alignment Search Tool (BLAST) analysis revealed that the flanking sequences in the maize genome are unspecific and that the insertion position is located in a repetitive sequence area in the maize genome. To locate the fine-scale insertion position in SK12-5, we combined the methods of genetic mapping and nanopore-based sequencing technology. From a classical bulked-segregant analysis (BSA), the insertion position in SK12-5 was mapped onto Bin9.03 of chromosome 9 between the simple sequence repeat (SSR) markers umc2337 and umc1743 (26,822,048–100,724,531 bp). The nanopore sequencing results uncovered 10 reads for which one end was mapped onto the vector and the other end was mapped onto the maize genome. These observations indicated that the exogenous T-DNA fragments were putatively integrated at the position from 82,329,568 to 82,379,296 bp of chromosome 9 in the transgenic maize SK12-5. This study is helpful for the safety assessment of the novel transgenic maize SK12-5 and shows that the combined method of genetic mapping and the nanopore-based sequencing technology will be a useful approach for identifying the insertion positions of transgenic sequences in other GM plants with relatively large and complex genomes.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Using Combined Methods of Genetic Mapping and Nanopore-Based Sequencing Technology to Analyze the Insertion Positions of G10evo-EPSPS and Cry1Ab/Cry2Aj Transgenes in Maize

et al. 2021

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“…Some groups have carried out a demanding whole genome sequencing, able to depict all possible kinds of variations caused by the process of transformation even at long distances from T-DNA integration site. There are examples in the model plant Arabidopsis as well as in important crops like rice, soybean, maize 38 – 41 . Other studies made use of a target enrichment step to focus the NGS analysis on the specific region of interest.…”

Section: Discussionmentioning

confidence: 99%

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

Costa

Piazza

Pompili

et al. 2020

Sci Rep

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Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

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“…Guo et al used the WGS technology to sequence and analyze the sequence information of two GM soybean events and successfully identified them from a single read analysis [6]. In the work of Siddique et al, the T-DNA insertion site and flanking sequence of the transgenic maize IE09S034 at the 3' end were identified by using NGS and PCR amplification [18]. Although several NGS-based methods have been developed to identify the molecular characteristics of genetically modified crops, some examples often failed to identify insertion sites and flanking sequences in GM crops.…”

Section: Discussionmentioning

confidence: 99%

“…With the emergence and rapid development of the next-generation sequencing (NGS) technology over the past several years, molecular characterization of insert locations, copy numbers, integrity, and stability of transgenic crops can be implemented in a relatively short time and at acceptable cost [15]. To date, a number of flanking sequences of exogenous genes in GM plants, such as Arabidopsis [16], rice [17], soybean [6], and maize [18], have been identified by NGS.…”

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 T-DNA Insertion Site and Flanking Sequence of a Genetically Modified Maize Event IE09S034 Using Next-Generation Sequencing Technology

Cited by 15 publications

References 32 publications

Using Combined Methods of Genetic Mapping and Nanopore-Based Sequencing Technology to Analyze the Insertion Positions of G10evo-EPSPS and Cry1Ab/Cry2Aj Transgenes in Maize

Using Combined Methods of Genetic Mapping and Nanopore-Based Sequencing Technology to Analyze the Insertion Positions of G10evo-EPSPS and Cry1Ab/Cry2Aj Transgenes in Maize

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

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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