Abstract
Next-generation sequencing (NGS) approaches are attractive alternatives to the PCR-based characterisation of genetically modified plants for safety assessment and labelling since NGS is highly sensitive to the detection of T-DNA inserts as well as vector backbone sequences in transgenic plants. In this study, two independent transgenic male Populus tremula lines, T193-2 and T195-1, both carrying the FLOWERING LOCUS T gene from Arabidopsis thaliana under control of a heat-inducible promoter (pHSP::AtFT) and the non-transgenic control clone W52, were further characterised by NGS and third-generation sequencing. The results support previous findings that the T-DNA was hemizygously inserted in one genomic locus of each line. However, the T-DNA insertions consist of conglomerations of one or two T-DNA copies together with a small T-DNA fragment without AtFT parts. Based on NGS data, no additional T-DNA splinters or vector backbone sequences could be identified in the genome of the two transgenic lines. Seedlings derived from crosses between the pHSP::AtFT transgenic male parents and female wild type plants are therefore expected to be T-DNA splinter or vector backbone free. Thus, PCR analyses amplifying a partial T-DNA fragment with AtFT-specific primers are sufficient to determine whether the seedlings are transgenic or not. An analysis of 72 second generation-seedlings clearly showed that about 50% of them still reveal the presence of the T-DNA, confirming data already published. To prove if unanticipated genomic changes were induced by T-DNA integration, extended future studies using long-range sequencing technologies are required once a suitable chromosome-level P. tremula reference genome sequence is available.
Highlights
The European Union has established a number of legal frameworks regulating the practical use of genetically modified (GM) organisms, i.e. Directive 2001/18/EC on the deliberate release of GM organisms into the environment (European_Parliament_Council 2001).This strict legislation officially aims to ensure that the development of modern biotechnology, including GM organisms, takes place in safe conditions (Davison 2010)
These results indicate that both transgenic lines nearly completely include the T-DNA insert
We describe short and long read sequencing of two independent transgenic poplar lines (T195-1 and T193-2) carrying the heat-inducible FLOWERING LOCUS T gene from A. thaliana and of the non-transgenic control clone W52 in order to unravel the genomic integration site(s) and the possible existence of T-DNA splinters and/or vector backbone sequences
Summary
The European Union has established a number of legal frameworks regulating the practical use of genetically modified (GM) organisms, i.e. Directive 2001/18/EC on the deliberate release of GM organisms into the environment (European_Parliament_Council 2001). This strict legislation officially aims to ensure that the development of modern biotechnology, including GM organisms, takes place in safe conditions (Davison 2010). These EU regulations hamper, or even impede, the market introduction of GM plants, including trees (Custers et al 2016; Fladung et al 2012). In both cases, offspring is produced with theoretically 75% (selffertilization) or 50% (cross with non-GM) GM plants and 25% (self-fertilization) or 50%
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