BIODIVERSITY ANALYSIS TROUGH DNA BARCODING Applications in agrifood and seafood products

The research activity, the results of which are the subject of doctoral dissertation, focused on the potentials of DNA barcoding, a genomic approach that exploits a short DNA sequence, a barcode, from a standardized region of the plastid genome, mitochondrial and chloroplast, as a universal and unique identification marker for animal and plant species. The main goal was to test a new accurate and automatable method for the genetic traceability of agri-food products, both of animal (fish, crustaceans and molluscs) and plant origin (bean and grapevine). First of all, we chose the specimens for the analysis: we selected pure lines of bean (Phaseolus vulgaris L.), clones of grapevine (Vitis vinifera L.) and samples of fish, crustaceans and molluscs purchased in famous GDO or local market in Chioggia e Sottomarina. Regarding the selection of seafood samples to analyze, we proceeded with the collection of the marine species most commonly involved in fraudulent substitutions. The experimental procedure adopted were the genomic DNA isolation from 37 specimens followed by the amplification of three target regions, cox1 (cytochrome oxydase subunit I), cob (apo-cytochrome b) and 16S-rDNA (ribosomal RNA small subunit) genes. Once obtained these data, we proceeded with a sequence similarity search using BOLD and GenBank as reference databases and each of the sequences as query. Overall, the phenetic approach proved to be an efficient tool to ensure the correct detection of seafood composition and thus to control the label information. In fact, for most of the samples it was possible to confirm the origin of the meat declared on the label, except in five situations where it was impossible to establish with no doubt the origin of the samples flagging them as likely falsification cases, voluntary or by accident. Cox1 gene proved to be a valid target for traceability aims, except in three genera, Thunnus, Macruronus and Gadus, where the identification was more problematic. Finally, even if GenBank database still remains the best web tool for forensic purposes, BOLD database proved to be enough rich to allow the correct recognition of almost all the specimens. Regarding plant DNA barcoding, the goal was to test DNA barcoding strategy as a tool to assess the distinctiveness of species and varieties of pure lines and clones. In the case of bean, we selected 54 pure lines of Phaseolus vulgaris species, 24 Italian pure lines, 18 Mesoamerican landraces and 12 Andean landraces, along with a few P. coccineus, P. lunatus and Vigna unguiculata accessions adopted as reference standards and out-types. These samples were characterized by means of the amplification of 7 chloroplast and two nuclear regions followed by the application of a phenetic approach. The procedure confirmed to be a powerful technique to correctly separate different species, whereas at the varietal level it revealed to be scarcely informative to discriminate gene pools and to identify varieties within P. vulgaris. Thus a second approach, the character-based system, was tested and it allowed to detect within P. vulgaris species a total of 16 haplotypes corresponding to as many subgroups, each one made up by Mesoamerican or Andean accessions along with Italian accessions that clustered with one or the other gene pool. Finally, a third case study is represented by V. vinifera and the potentials of DNA barcoding approach to distinguish grapevine cultivars used in the production of wines. We proceeded with the selection of 123 grapevine cultivars along with other 5 species of Vitis (V. rupestris, V. riparia, V. labrusca, V. cinerea e V. berlandieri) adopted as reference standards and out-types. After a preliminary analysis of the chloroplast DNA that resulted to be monomorphic, we decided to shift to the nuclear genome amplifying four ESTs and the GAI1 (gibberellins insensitive-like) gene. The analysis is still ongoing, but the preliminary results lead to think that a few haplotypes exist within V. vinifera and they could be use to resolve frequent cases of synonymies and homonymies in grapevine. Furthermore, an economically valuable application may be the exploitation of these haplotypes cultivar-specific for the genetic traceability of wines to avoid cases of falsification.