Molecular Characterization and Event-Specific Real-Time PCR Detection of Two Dissimilar Groups of Genetically Modified Petunia (Petunia x hybrida) Sold on the Market

Abstract

Petunia plants with unusual orange flowers were noticed on the European market and confirmed to be genetically modified (GM) by the Finnish authorities in spring 2017. Later in 2017, inspections and controls performed by several official laboratories of national competent authorities in the European Union detected several GM petunia varieties with orange flowers, but also another group of unusually colored flowers. In the latter group, a so far undetected gene coding for a flavonoid 3'5' hydroxylase (F3'5'H) responsible for the purple color was identified by German and Dutch authorities, suggesting that the petunias found on the markets contain different genetic constructs. Here, a strategy is described for the identification of GM petunia varieties. It is based on an initial GMO screening for known elements using (real-time) PCR and subsequent identification of the insertion sites by a gene walking-like approach called ALF (amplification of linearly-enriched fragments) in combination with Sanger and MinION sequencing. The results indicate that the positively identified GM petunias can be traced back to two dissimilar GM events used for breeding of the different varieties. The test results also confirm that the transgenic petunia event RL01-17 used in the first German field trial in 1991 is not the origin of the GM petunias sold on the market. On basis of the obtained sequence data, event-specific real-time PCR confirmatory methods were developed and validated. These methods are applicable for the rapid detection and identification of GM petunias in routine analysis. In addition, a decision support system was developed for revealing the most likely origin of the GM petunia.

Keywords: MinION sequencing; amplification of linearly-enriched fragments; event-specific detection; genetically modified (GM); petunia; real-time PCR; unauthorized GMO.

Figure 1

Photographs of individual petunia flowers representing G1 varieties (A, B) and G2 varieties (C, D).

Figure 2

Characterization of G1 (A) and G2 (B) petunias. A priori information is based on (real-time) PCR results and available literature, final information is based on Sanger and MinION sequencing. Light grey arrows indicate enrichment into known sequence; dark grey arrows enrichment into unknown sequence, dashed arrows indicate a putative orientation. Event specific primers and probe are indicated by black arrows and a horizontal dash, respectively; an asterisk indicates a generic GM sequence (e.g. plasmid, vector); the dagger represents a putative partial sequence.

Figure 3

Decision Support System (DSS) for stepwise assignment of GM petunias to the potential origin of the construct. References describing the respective construct developments for GM petunias are indicated. Decision points are based on PCR test results.

Figure 4

Sequence data of the junction region present in G1 petunia as determined by MinION sequencing and confirmed by Sanger sequencing (sequencing primers in bold letters). The junction site is indicated (*). The shaded sequence part in G1 shows 100% identity to the sequence recently reported by Fraiture et al. (2019) including their predicted junction site. Underlined are sequences with identity to Petunia ssp. sequences (Solgenomics database; alignments shown below sequence). Location of G1 primers and probe are bold italic.

Figure 5

Sequence data of the junction region present in G2 petunia as determined by MinION sequencing and confirmed by Sanger sequencing (sequencing primers in bold letters). Underlined are sequences with identity to Petunia ssp. sequences (Solgenomics database; alignments shown below sequence). Location of G2 primers and probe are bold italic.