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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.


Validation of the insertion sites in individuals of heterozygosis lines and segregation populations. (A) Validation of the insertion site in progeny of heterozygosis GE-J16. M: 200-bp DNA marker; 1, 3, 5, 7, 9, 12, 14, 15: glyphosate tolerant individuals; 2, 4, 6, 8, 10, 11, 13: glyphosate sensitive individuals; 16: negative control of water. (B) Validation of the insertion site in segregation populations derived from ZH10-6. M: 200-bp DNA marker; 1: negative control; 2,3: glyphosate sensitive individuals; 4–24: glyphosate tolerant individuals.
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Figure 5: Validation of the insertion sites in individuals of heterozygosis lines and segregation populations. (A) Validation of the insertion site in progeny of heterozygosis GE-J16. M: 200-bp DNA marker; 1, 3, 5, 7, 9, 12, 14, 15: glyphosate tolerant individuals; 2, 4, 6, 8, 10, 11, 13: glyphosate sensitive individuals; 16: negative control of water. (B) Validation of the insertion site in segregation populations derived from ZH10-6. M: 200-bp DNA marker; 1: negative control; 2,3: glyphosate sensitive individuals; 4–24: glyphosate tolerant individuals.

Mentions: In order to further validate the insertion sites, specific primer pairs for each event were applied to identify genotypes of individual plants from T2 and F2 populations. Genomic DNA isolated from random selected glyphosate tolerant and sensitive plants was used as template for PCR amplification. For primer pairs (JackP-1/GAT-1, G2EP-1/JackP-2 for GE-J16 and ZH10P-1/GAT-2, G2EP-2/ZH10P-2 for ZH10-6) amplifying upstream or downstream junction of the plant genome and T-DNA region, expected sizes of PCR products (1529-bp, 2203-bp for GE-J16 and 810-bp, 1626-bp for ZH10-6) were amplified in all glyphosate tolerant plants while no product was detected in all sensitive plants (Figure 5), indicating that glyphosate tolerant phenotype co-segregated with T-DNA insertion either in GE-J16 or ZH10-6. For primer pairs JackP-1/2 and ZH10P-1/2 used for amplifying flanking sequences of host genome, expected 1246-bp and 632-bp products were amplified in 13 progeny of heterozygous GE-J16 and 17 F2 individuals derived from ZH10-6, respectively. These 30 lines contain heterozygous lines if PCR amplification of upstream or downstream junction sequences could be detected and wild type if junction sequences could not be amplified. In addition, no PCR product of host genome could be detected from two and six glyphosate tolerant plants derived from GE-J16 and ZH10-6, respectively (Figure 5). These plants were regarded as homozygous lines since only junctions between T-DNA and host genome could be amplified. Further identification of phenotype in T3 generation and F2:3 populations also confirmed no segregation of glyphosate tolerant phenotype in these eight lines. This result suggested that the insertion of exogenous genes and glyphosate tolerance phenotype were co-segregated in these segregation populations.


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)

Validation of the insertion sites in individuals of heterozygosis lines and segregation populations. (A) Validation of the insertion site in progeny of heterozygosis GE-J16. M: 200-bp DNA marker; 1, 3, 5, 7, 9, 12, 14, 15: glyphosate tolerant individuals; 2, 4, 6, 8, 10, 11, 13: glyphosate sensitive individuals; 16: negative control of water. (B) Validation of the insertion site in segregation populations derived from ZH10-6. M: 200-bp DNA marker; 1: negative control; 2,3: glyphosate sensitive individuals; 4–24: glyphosate tolerant individuals.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Validation of the insertion sites in individuals of heterozygosis lines and segregation populations. (A) Validation of the insertion site in progeny of heterozygosis GE-J16. M: 200-bp DNA marker; 1, 3, 5, 7, 9, 12, 14, 15: glyphosate tolerant individuals; 2, 4, 6, 8, 10, 11, 13: glyphosate sensitive individuals; 16: negative control of water. (B) Validation of the insertion site in segregation populations derived from ZH10-6. M: 200-bp DNA marker; 1: negative control; 2,3: glyphosate sensitive individuals; 4–24: glyphosate tolerant individuals.
Mentions: In order to further validate the insertion sites, specific primer pairs for each event were applied to identify genotypes of individual plants from T2 and F2 populations. Genomic DNA isolated from random selected glyphosate tolerant and sensitive plants was used as template for PCR amplification. For primer pairs (JackP-1/GAT-1, G2EP-1/JackP-2 for GE-J16 and ZH10P-1/GAT-2, G2EP-2/ZH10P-2 for ZH10-6) amplifying upstream or downstream junction of the plant genome and T-DNA region, expected sizes of PCR products (1529-bp, 2203-bp for GE-J16 and 810-bp, 1626-bp for ZH10-6) were amplified in all glyphosate tolerant plants while no product was detected in all sensitive plants (Figure 5), indicating that glyphosate tolerant phenotype co-segregated with T-DNA insertion either in GE-J16 or ZH10-6. For primer pairs JackP-1/2 and ZH10P-1/2 used for amplifying flanking sequences of host genome, expected 1246-bp and 632-bp products were amplified in 13 progeny of heterozygous GE-J16 and 17 F2 individuals derived from ZH10-6, respectively. These 30 lines contain heterozygous lines if PCR amplification of upstream or downstream junction sequences could be detected and wild type if junction sequences could not be amplified. In addition, no PCR product of host genome could be detected from two and six glyphosate tolerant plants derived from GE-J16 and ZH10-6, respectively (Figure 5). These plants were regarded as homozygous lines since only junctions between T-DNA and host genome could be amplified. Further identification of phenotype in T3 generation and F2:3 populations also confirmed no segregation of glyphosate tolerant phenotype in these eight lines. This result suggested that the insertion of exogenous genes and glyphosate tolerance phenotype were co-segregated in these segregation populations.

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.