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Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been ‘eco-certified’ by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93–100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

Next-generation sequencing (NGS) technologies now offer the opportunity to move from population genetics to population genomics, even in non-model species. In the present study, two NGS platforms, the Roche 454 FLX and the Illumina Genome Analyzer were used to discover Single Nucleotide Polymorphisms (SNPs) in the muscle transcriptome of the European hake (Merluccius merluccius), which represents one of the most important demersal fisheries in Europe. Transcriptome sequencing and de novo assembly into unique contigs, in silico SNP detection, and validation by high-throughput genotyping yielded a set of 395 polymorphic SNP loci. Of these, 381 nuclear SNPs were used to scan genomic variation in 19 Atlantic and Mediterranean populations of M. merluccius. As all SNPs reside in the functional (transcribed) portion of the hake genome, it is expected that a subset of sequence variants might be located in genomic regions potentially involved in local adaptation, conveying greater resolving power for the detection of spatial structuring on a large and/or local scale. Combining genome scans and correlation analysis between SNP allele frequencies and environmental factors, several candidate loci that are potentially under divergent selection were identified, mainly genes involved in muscles structural organization/function and protein synthesis regulation, or implicated in energy metabolism. Such “outlier” loci showed high performance in resolving population structure, unveiling distinct genetic clusters at multiple spatial scales, which possibly correspond to locally adapted populations. Following a precautionary approach, these results suggest the need to revise the current management strategies of European hake stocks, particularly within basins.

The research presented in my PhD thesis is part of a wider European project, FishPopTrace, focused on traceability of fish populations and products. My work was aimed at developing and analyzing novel genetic tools for a widely distributed marine fish species, the European hake (Merluccius merluccius), in order to investigate population genetic structure and explore potential applications to traceability scenarios. A total of 395 SNPs (Single Nucleotide Polymorphisms) were discovered from a massive collection of Expressed Sequence Tags, obtained by high-throughput sequencing, and validated on 19 geographic samples from Atlantic and Mediterranean. Genome-scan approaches were applied to identify polymorphisms on genes potentially under divergent selection (outlier SNPs), showing higher genetic differentiation among populations respect to the average observed across loci. Comparative analysis on population structure were carried out on putative neutral and outlier loci at wide (Atlantic and Mediterranean samples) and regional (samples within each basin) spatial scales, to disentangle the effects of demographic and adaptive evolutionary forces on European hake populations genetic structure. Results demonstrated the potential of outlier loci to unveil fine scale genetic structure, possibly identifying locally adapted populations, despite the weak signal showed from putative neutral SNPs. The application of outlier SNPs within the framework of fishery resources management was also explored. A minimum panel of SNP markers showing maximum discriminatory power was selected and applied to a traceability scenario aiming at identifying the basin (and hence the stock) of origin, Atlantic or Mediterranean, of individual fish. This case study illustrates how molecular analytical technologies have operational potential in real-world contexts, and more specifically, potential to support fisheries control and enforcement and fish and fish product traceability.

In the Mediterranean, the common (Solea solea) and Egyptian (S. aegyptiaca) soles are two of the most valuable flatfish fishery resources. In the past, S. aegyptiaca was erroneously synonymised with Solea solea because of the great similarity in external morphology. Recently molecular markers have enabled their reliable identification, showing that the two species seem to co-occur in several areas of the Mediterranean, forming sympatric demes. The sympatric distribution and the close phylogenetic relationship allow a scenario for potential ecological and evolutionary interactions. Here, we aimed at providing new insights on the taxonomy, phylogeography and evolutionary interactions of the Mediterranean soles by i) developing PCR tests for the rapid screening of the two cryptic species, ii) analysing the species composition at several geographical locations, iii) assessing the phylogeographic pattern and genetic structure of populations and iv) the possible occurrence of interspecific hybridization in sympatric populations. In total 435 sole individuals were sequenced for a mitochondrial fragment (cytB gene) and 217 genotyped at two nuclear marker classes (ITS rDNA gene and 8 microsatellite loci). The analysis of species composition revealed a wider distribution of S. aegyptiaca in the Mediterranean than previously described. The observed frequent sympatry with S. solea could have lead to their frequent mis-identification. All nuclear markers however consistently revealed a clear genetic separation of the two species. Both mitochondrial and nuclear data showed the structuring of populations according to a longitudinal gradient, between the Western and Eastern basin, with different degree of differentiation in the two sole species. Higher genetic diversity was observed in S. solea for all markers. Further multidisciplinary data (combining data from morphology, reproductive biology and life history with genetic) are needed to unravel distribution and ecology of S. aegyptiaca in the Mediterranean and its ecological interactions with the cryptic species S. solea. This assessment will have important implications for sustainable management and conservation of fishery resources, because different species can differently respond to environmental pressures and changes.

Il nasello rappresenta una delle risorse ittiche demersali più importanti per la pesca dell'Atlantico Nord-Orientale e del bacino Mediterraneo. La limitata disponibilità di strumenti molecolari adeguati ha impedito di risolvere il differenziamento genetico tra popolazioni a differenti scale spaziali, lasciando irrisolte alcune questioni di interesse gestionale. Nell'ambito del progetto europeo FishPopTrace circa 400 SNPs (polimorfismi a singolo nucleotide) sono stati sviluppati su sequenze nucleari espresse ottenute da individui di nasello rappresentativi dell’intero areale di distribuzione. Un totale di 19 campioni geografici da Atlantico e Mediterraneo sono stati analizzati con il pannello di marcatori sviluppato. Un subset di 10 loci SNP potenzialmente sotto selezione sono risultati discriminanti per risolvere la tracciabilità tra le popolazioni atlantiche e mediterranee. La debole struttura di popolazione mostrata dal nasello entro bacino è riconducibile ad elevati livelli di connettività. Sono tuttavia evidenti segnali di differenziamento dovuti probabilmente ad adattamento locale. La disponibilità di numerosi marcatori legati a sequenze espresse rende possibile risolvere con maggiore affidabilità i pattern di struttura genetica delle popolazioni di nasello sia in termini di stima di connettività e flusso genico, sia di potenziale adattativo a condizioni ambientali, ottenendo informazioni fondamentali per una gestione sostenibile della risorsa.

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).