Novel PCR-based assay for rapid identification of Red Fluorescent Proteins in GloFish and GloFish x wildtype zebrafish (Danio rerio) hybrids

Twenty-five zebrafish presenting three different colour variants were collected and analysed by molecular markers in order to define their species and to verify if the unusual colouration presented by two morphotypes was due to genetic engineering or were natural colour variants obtained by selective breeding or spontaneous mutations. The three morphotypes collected were (a) an unusual reddish-pink (suspected red GloFish®), 15 specimens confiscated by the operative ecological core of police officers; (b) the black/blue striped colouration typical of wildtype Danio rerio (five specimens) and (c) an hybrid morphotype with an atypical flashing red pigmentation of skeletal muscle and a blue-black striped pigmentation of skin (five specimens obtained by crossing suspected GM danios with wildtype line) obtained by crossing wild type danios with fish confiscated by police. Despite the three different colourations presented, all analysed samples were unequivocally Danio rerio, excluding the possibility of belonging to different species or natural variants, which should carry different skin colours. Except the five wild-type danios, all other samples analysed should belong to an engineered variant and thus forbidden by EU GMO legislation. Here we designed a new highly reliable and affordable PCR-based assay able to identify the presence of red fluorescent proteins not only in the 15 parental GM samples but also in the five F1 hybrids created ad hoc. The cross-breeding experiment clearly demonstrated that the exogenous construct made by red fluorescent protein under a fast-muscle promoter was inherited by F1. For this reason in our opinion also cross-hybrids obtained by GM danios and wildtype zebrafish should be included in the list of organisms regulated by GMO legislation. Finally, the obtained results suggest that the PCR-based assay here implemented could represent a cost-effective test to rapidly identify suspected GM fish, which carry RFP/dsRED isoforms