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

Guo B, Guo Y, Hong H, Qiu LJ - Front Plant Sci (2016)

Bottom Line: Molecular characterization of sequence flanking exogenous fragment insertion is essential for safety assessment and labeling of genetically modified organism (GMO).The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis.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.

View Article: PubMed Central - PubMed

Affiliation: The National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI) and MOA Key Lab of Soybean Biology (Beijing), Institute of Crop Science, Chinese Academy of Agricultural Sciences Beijing, China.

ABSTRACT
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.

No MeSH data available.


Junction reads covering junctions of integrated T-DNA and host genomes in GE-J16 and ZH10-6. Junctions with transition on insert and chromosome 19 in GE-J16 (A) and transition on insert and chromosome 17 in ZH10-6 (B,C). The sequence of reference genome along the insertion site was underlined and the transition between soybean genome and T-DNA insertion was indicated by a gap. The part of each read belonging to the exogenous fragment was indicated in bold.
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Figure 2: Junction reads covering junctions of integrated T-DNA and host genomes in GE-J16 and ZH10-6. Junctions with transition on insert and chromosome 19 in GE-J16 (A) and transition on insert and chromosome 17 in ZH10-6 (B,C). The sequence of reference genome along the insertion site was underlined and the transition between soybean genome and T-DNA insertion was indicated by a gap. The part of each read belonging to the exogenous fragment was indicated in bold.

Mentions: In order to identify putative insertion sites of exogenous fragments, all clean reads were mapped to the sequence of pKT-rGE vector and soybean reference genome. The putative integration sites of transgenic events were characterized based on junction reads in which one end was mapped to the sequence of vector and the other end to the host genome. After detailed data analysis, six junction reads on chromosome 19 and 15 reads on chromosome 17 were identified from the sequence data of GE-J16 and ZH10-6 separately (Figure 2). According to physical positions of junction reads, the T-DNA is integrated at position around Chr19: 50,543,500-50,543,900 in GE-J16 and the insertion loci of ZH10-6 was located at position Chr17: 7,980,300-7,980,600. These results further confirmed a single insertion site of exogenous gene in the genome of these each transgenic line.


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

Guo B, Guo Y, Hong H, Qiu LJ - Front Plant Sci (2016)

Junction reads covering junctions of integrated T-DNA and host genomes in GE-J16 and ZH10-6. Junctions with transition on insert and chromosome 19 in GE-J16 (A) and transition on insert and chromosome 17 in ZH10-6 (B,C). The sequence of reference genome along the insertion site was underlined and the transition between soybean genome and T-DNA insertion was indicated by a gap. The part of each read belonging to the exogenous fragment was indicated in bold.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940375&req=5

Figure 2: Junction reads covering junctions of integrated T-DNA and host genomes in GE-J16 and ZH10-6. Junctions with transition on insert and chromosome 19 in GE-J16 (A) and transition on insert and chromosome 17 in ZH10-6 (B,C). The sequence of reference genome along the insertion site was underlined and the transition between soybean genome and T-DNA insertion was indicated by a gap. The part of each read belonging to the exogenous fragment was indicated in bold.
Mentions: In order to identify putative insertion sites of exogenous fragments, all clean reads were mapped to the sequence of pKT-rGE vector and soybean reference genome. The putative integration sites of transgenic events were characterized based on junction reads in which one end was mapped to the sequence of vector and the other end to the host genome. After detailed data analysis, six junction reads on chromosome 19 and 15 reads on chromosome 17 were identified from the sequence data of GE-J16 and ZH10-6 separately (Figure 2). According to physical positions of junction reads, the T-DNA is integrated at position around Chr19: 50,543,500-50,543,900 in GE-J16 and the insertion loci of ZH10-6 was located at position Chr17: 7,980,300-7,980,600. These results further confirmed a single insertion site of exogenous gene in the genome of these each transgenic line.

Bottom Line: Molecular characterization of sequence flanking exogenous fragment insertion is essential for safety assessment and labeling of genetically modified organism (GMO).The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis.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.

View Article: PubMed Central - PubMed

Affiliation: The National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI) and MOA Key Lab of Soybean Biology (Beijing), Institute of Crop Science, Chinese Academy of Agricultural Sciences Beijing, China.

ABSTRACT
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.

No MeSH data available.