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

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.

Keywords: flanking sequence; insertion site; next generation sequencing; transgenic soybean; whole genome sequencing.

FIGURE 1

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

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.

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).

FIGURE 4

Schematic diagram of insertion loci and flanking sequences in GE-J16 (A) and ZH10-6 (B). The upper diagram of each chart indicated 20 chromosomes of soybean with chromosome numbers at the bottom. The numbers under the line of Chr 19 (A) and Chr 17 (B) indicates physical positions of these chromosomes.

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.