An innovative index to incorporate transcriptomic data into weight of evidence approaches for environmental risk assessment.

The sharp decrease in the cost of RNA-sequencing and the rapid improvement in computational analysis of eco-toxicogenomic data have brought new insights into the adverse effects of chemicals on aquatic organisms. Yet, transcriptomics is generally applied qualitatively in environmental risk assessments, hampering more effective exploitation of this evidence through multidisciplinary studies. In view of this limitation, a methodology is here presented to quantitatively elaborate transcriptional data in support to environmental risk assessment. The proposed methodology makes use of results from the application of Gene Set Enrichment Analysis to recent studies investigating the response of Mytilus galloprovincialis and Ruditapes philippinarum exposed to contaminants of emerging concern. The degree of changes in gene sets and the relevance of physiological reactions are integrated in the calculation of a hazard index. The outcome is then classified according to five hazard classes (from absent to severe), providing an evaluation of whole-transcriptome effects of chemical exposure. The application to experimental and simulated datasets proved that the method can effectively discriminate different levels of altered transcriptomic responses when compared to expert judgement (Spearman correlation coefficient of 0.96). A further application to data collected in two independent studies of Salmo trutta and Xenopus tropicalis exposed to contaminants confirmed the potential extension of the methodology to other aquatic species. This methodology can serve as a proof of concept for the integration of "genomic tools" in environmental risk assessment based on multidisciplinary investigations. To this end, the proposed transcriptomic hazard index can now be incorporated into quantitative Weight of Evidence approaches and weighed, with results from other types of analysis, to elucidate the role of chemicals in adverse ecological effects.

Helping decision making for reliable and cost-effective 2b-RAD sequencing and genotyping analyses in non-model species.

High-throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b-RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b-RAD protocols on non-model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b-RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade-off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective-base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b-RAD protocols on non-model organisms with different genome sizes, helping decision-making for a reliable and cost-effective genotyping.

RAD SNP markers as a tool for conservation of dolphinfish Coryphaena hippurus in the Mediterranean Sea: Identification of subtle genetic structure and assessment of populations sex-ratios.

Dolphinfish is an important fish species for both commercial and sport fishing, but so far limited information is available on genetic variability and pattern of differentiation of dolphinfish populations in the Mediterranean basin. Recently developed techniques allow genome-wide identification of genetic markers for better understanding of population structure in species with limited genome information. Using restriction-site associated DNA analysis we successfully genotyped 140 individuals of dolphinfish from eight locations in the Mediterranean Sea at 3324 SNP loci. We identified 311 sex-related loci that were used to assess sex-ratio in dolphinfish populations. In addition, we identified a weak signature of genetic differentiation of the population closer to Gibraltar Strait in comparison to other Mediterranean populations, which might be related to introgression of individuals from Atlantic. No further genetic differentiation could be detected in the other populations sampled, as expected considering the known highly mobility of the species. The results obtained improve our knowledge of the species and can help managing dolphinfish stock in the future.

Methodological assessment of 2b-RAD genotyping technique for population structure inferences in yellowfin tuna (Thunnus albacares).

Global population genetic structure of yellowfin tuna (Thunnus albacares) is still poorly understood despite its relevance for the tuna fishery industry. Low levels of genetic differentiation among oceans speak in favour of the existence of a single panmictic population worldwide of this highly migratory fish. However, recent studies indicated genetic structuring at a much smaller geographic scales than previously considered, pointing out that YFT population genetic structure has not been properly assessed so far. In this study, we demonstrated for the first time, the utility of 2b-RAD genotyping technique for investigating population genetic diversity and differentiation in high gene-flow species. Running de novo pipeline in Stacks, a total of 6772 high-quality genome-wide SNPs were identified across Atlantic, Indian and Pacific population samples representing all major distribution areas. Preliminary analyses showed shallow but significant population structure among oceans (FST=0.0273; P-value<0.01). Discriminant Analysis of Principal Components endorsed the presence of genetically discrete yellowfin tuna populations among three oceanic pools. Although such evidence needs to be corroborated by increasing sample size, these results showed the efficiency of this genotyping technique in assessing genetic divergence in a marine fish with high dispersal potential.