MinION sequencing technology to characterize unauthorized GM petunia plants circulating on the European Union market

Abstract

In order to characterize unauthorized genetically modified petunia, an integrated strategy has been applied here on several suspected petunia samples from the European market. More precisely, DNA fragments of interest were produced by DNA walking anchored on key targets, earlier detected by real-time PCR screening analysis, to be subsequently sequenced using the MinION platform from Oxford Nanopore Technologies. This way, the presence of genetically modified petunia was demonstrated via the characterization of their transgene flanking regions as well as unnatural associations of elements from their transgenic cassette.

Event-specific qualitative polymerase chain reaction analysis for two T-DNA copies in genetically modified orange Petunia

“…The 1,078 bp sequence was deposited in the NCBI database (accession No. MT000723) and comprises identically a recently isolated junction sequence of 402 bp of GM petunia (Fraiture et al 2019 ). The entire sequence of the Pet_5′T-DNA2 fragment consists of 828 bp petunia DNA followed by 250 bp of a β -lactamase gene ( bla ) in antisense direction.…”

“…In 2017, it turned out that most of the orange petunia contain foreign DNA from maize and plant transformation vectors (Bashandy and Teeri 2017 ; Haselmair-Gosch et al 2018 ). Based on the current information, the transformation construct of the first petunia transformation experiments performed by Meyer et al ( 1987 ) can be assumed to be the source of those undeclared genetically modified (GM) orange petunia (Bashandy and Teeri 2017 ; Fraiture et al 2019 ; Haselmair-Gosch et al 2018 ). Subsequently, the cultivars, which were identified as GM, were removed from the market with some exceptions like e.g.…”

“…Recently, MinION sequencing technology was shown to be suitable for isolating a transgene flanking sequence part of GM petunia, which was identical in the 23 tested cultivars, suggesting a single origin of the GM plants (Fraiture et al 2019 ). However, for orange GM petunia, several transgenic lines with a different number of integrated copies of the transgenic sequence were reported from the initial transformation experiment (Linn et al 1990 ) but it is still unclear, which of these could be the source of the undeclared orange GM petunia.…”

Targeted MinION sequencing of transgenes

“…PCR-based DNA walking methods anchored on the detected transgenic elements, for instance p35S, tNOS, t35S pCAMBIA, or VIP3A elements, have been applied to GMOs including GMOs at trace levels, GMO mixtures and processed food matrices. The resulting amplicons were sequenced using NGS technology and the transgene sequence and its flanking regions could be characterized [17][18][19][20][21][22][23][24][25] . Although effective, this technique is difficult to implement and remains time consuming.…”

“…In the unauthorized GMO context, our aim was to develop a rapid and simple method to sequence the transgene and its flanking regions with, as prior knowledge, the presence of a detected screening sequence. The method was developed and assessed on available GM petunia plants, for which part of the transgene sequence was available on NCBI and of which the 5′ flanking region had recently been described 17 . In 1988, Ochman et al described a protocol of inverse PCR allowing the amplification of regions of unknown sequence flanking a specified segment of DNA 27 .…”