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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.


Schematic diagram of the work-flow for insertion site identification and validation.
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Figure 1: Schematic diagram of the work-flow for insertion site identification and validation.

Mentions: Data obtained from the sequencer was processed for quality control and raw reads were filtered by removal of adapter and low quality reads (Q < 20). Clean reads were individually aligned and mapped to Glycine max Wm82.a2.v1 reference genome from Phytozome and sequence of pKT-rGE vector using BWA with default parameters (Langmead and Salzberg, 2012). The pipeline for data analysis and validation was briefly described in Figure 1. After mapping of all reads against the reference genome and sequence of vector, they were classified into three groups: reads only mapped to the reference genome, reads only mapped to vector sequence and reads mapped to both sequences (junction reads). Physical positions of junction reads were indicated the integration sites and were used for further analysis.


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)

Schematic diagram of the work-flow for insertion site identification and validation.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Schematic diagram of the work-flow for insertion site identification and validation.
Mentions: Data obtained from the sequencer was processed for quality control and raw reads were filtered by removal of adapter and low quality reads (Q < 20). Clean reads were individually aligned and mapped to Glycine max Wm82.a2.v1 reference genome from Phytozome and sequence of pKT-rGE vector using BWA with default parameters (Langmead and Salzberg, 2012). The pipeline for data analysis and validation was briefly described in Figure 1. After mapping of all reads against the reference genome and sequence of vector, they were classified into three groups: reads only mapped to the reference genome, reads only mapped to vector sequence and reads mapped to both sequences (junction reads). Physical positions of junction reads were indicated the integration sites and were used for further analysis.

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.