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.

Flanking Sequence and Copy-Number Analysis of Transformation Events by Integrating Next-Generation Sequencing Technology with Southern Blot Hybridization

“…A sequencing depth of 75× is often recommended for the molecular characterization of transformation events by NGS analysis (Kovalic et al 2012). Several studies have employed a 20×-30× sequencing depth to detect flanking sequences and inserts when studying transformation events (Yang et al 2013;Guo et al 2016). In this study, we considered that increasing sequencing depth provides access to relatively complete sequence information, which seems beneficial for revealing T-DNA integrations and numbers; however, increased sequencing depths involve higher costs.…”

“…The advent of next-generation sequencing (NGS) technologies opened a new era of genomics and molecular biology. In recent years, NGS-based molecular characterizations have been widely applied for studying many transgenic events (Daniela et al 2013;Lepage et al 2013;Park et al 2015;Guo et al 2016). In comparison with PCR, Sanger sequencing and Southern blot hybridization, NGS has been proven effective for detecting incomplete and multiple integration events (Zhang et al 2012).…”

Generation of Epichloë Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event

“…Traditionally, molecular techniques such as Southern blotting, PCR, and genome walking have been used for characterisation of T-DNA integration [21,57,68]. However, these approaches are not capable of identifying complex integration events [69]. Next-generation sequencing has been used more effectively for the analysis of T-DNA integration patterns.…”

Mapping of transgenic alleles in plants using a Nanopore-based sequencing strategy

“…Introduction 1 0 0 map transgenic alleles in plants due to its depth of sequencing capacity (Guo et al, 2016 Polko et al, 2012). However, because this method produces short reads, a high 1 0 2 degree of sequencing depth is needed, especially in crops that have large genomes that are rich in 1 0 3 repetitive sequence.…”