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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.


Locations of primers and PCR validation of transgenic insertion sites. Schematic diagram of PCR validation primers were designed for GE-J16 (A) and ZH10-6 (B). Glyma.19G262700 and Glyma.17G101500 were genes located near the insertion sites of two transgenic events. PCR amplifications of junction sequences were carried out in GE-J16 (C,D) and ZH10-6 (E,F). M: 200 bp DNA Marker, 1: negative control of water; 2: negative control of non-transgenic soybean Jack (C,D) and ZH10 (E,F); 3, 4: transgenic plants of GE-J16 (C,D) and ZH10-6 (E,F).
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Figure 3: Locations of primers and PCR validation of transgenic insertion sites. Schematic diagram of PCR validation primers were designed for GE-J16 (A) and ZH10-6 (B). Glyma.19G262700 and Glyma.17G101500 were genes located near the insertion sites of two transgenic events. PCR amplifications of junction sequences were carried out in GE-J16 (C,D) and ZH10-6 (E,F). M: 200 bp DNA Marker, 1: negative control of water; 2: negative control of non-transgenic soybean Jack (C,D) and ZH10 (E,F); 3, 4: transgenic plants of GE-J16 (C,D) and ZH10-6 (E,F).

Mentions: In order to characterize exact positions of T-DNA insertions, PCR primers were designed based on speculated upstream and downstream flanking sequences and the T-DNA sequence (Figure 3). When using primer pairs with one primer annealing within putative flanking sequences (JackP-1, JackP-2, ZH10P-1, and ZH10P-2) and the other annealing to the exogenous genes (GAT-1, GAT-2, G2EP-1, and G2EP-2), gel electrophoresis revealed that PCR reactions of primer pairs JackP-1/GAT-1, G2EP-1/JackP-2, ZH10P-1/GAT-2, and G2EP-2/ZH10P-2 had generated products with single band in transgenic lines while no product could be detected from the non-transgenic control (Figure 3). Sanger sequencing of these junction fragments confirmed the putative insertion sites identified by WGS and exact positions of T-DNA insertions were also identified. The T-DNA of GE-J16 was integrated into physical position 50543767–50543792 on chromosome 19 while that of ZH10-6 was inserted into position 7980527–7980541 on chromosome 17 (Figure 4). Both exogenous fragments were all inserted in intergenic regions of the host genome and no functional gene was interrupted by T-DNA insertions. Accordingly, due to the transformation, 24-bp and 13-bp fragments of host genome sequences were replaced by insertions of T-DNA in GE-J16 and ZH10-6, respectively.


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)

Locations of primers and PCR validation of transgenic insertion sites. Schematic diagram of PCR validation primers were designed for GE-J16 (A) and ZH10-6 (B). Glyma.19G262700 and Glyma.17G101500 were genes located near the insertion sites of two transgenic events. PCR amplifications of junction sequences were carried out in GE-J16 (C,D) and ZH10-6 (E,F). M: 200 bp DNA Marker, 1: negative control of water; 2: negative control of non-transgenic soybean Jack (C,D) and ZH10 (E,F); 3, 4: transgenic plants of GE-J16 (C,D) and ZH10-6 (E,F).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Locations of primers and PCR validation of transgenic insertion sites. Schematic diagram of PCR validation primers were designed for GE-J16 (A) and ZH10-6 (B). Glyma.19G262700 and Glyma.17G101500 were genes located near the insertion sites of two transgenic events. PCR amplifications of junction sequences were carried out in GE-J16 (C,D) and ZH10-6 (E,F). M: 200 bp DNA Marker, 1: negative control of water; 2: negative control of non-transgenic soybean Jack (C,D) and ZH10 (E,F); 3, 4: transgenic plants of GE-J16 (C,D) and ZH10-6 (E,F).
Mentions: In order to characterize exact positions of T-DNA insertions, PCR primers were designed based on speculated upstream and downstream flanking sequences and the T-DNA sequence (Figure 3). When using primer pairs with one primer annealing within putative flanking sequences (JackP-1, JackP-2, ZH10P-1, and ZH10P-2) and the other annealing to the exogenous genes (GAT-1, GAT-2, G2EP-1, and G2EP-2), gel electrophoresis revealed that PCR reactions of primer pairs JackP-1/GAT-1, G2EP-1/JackP-2, ZH10P-1/GAT-2, and G2EP-2/ZH10P-2 had generated products with single band in transgenic lines while no product could be detected from the non-transgenic control (Figure 3). Sanger sequencing of these junction fragments confirmed the putative insertion sites identified by WGS and exact positions of T-DNA insertions were also identified. The T-DNA of GE-J16 was integrated into physical position 50543767–50543792 on chromosome 19 while that of ZH10-6 was inserted into position 7980527–7980541 on chromosome 17 (Figure 4). Both exogenous fragments were all inserted in intergenic regions of the host genome and no functional gene was interrupted by T-DNA insertions. Accordingly, due to the transformation, 24-bp and 13-bp fragments of host genome sequences were replaced by insertions of T-DNA in GE-J16 and ZH10-6, respectively.

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.