Sardines at a junction: Seascape genomics reveals ecological and oceanographic drivers of variation in the NW Mediterranean Sea.

By evaluating genetic variation across the entire genome, one can address existing questions in a novel way while raising new ones. The latter includes how different local environments influence adaptive and neutral genomic variation within and among populations, providing insights into local adaptation of natural populations and their responses to global change. Here, under a seascape genomic approach, ddRAD data of 4609 single nucleotide polymorphisms (SNPs) from 398 sardines (Sardina pilchardus) collected in 11 Mediterranean and one Atlantic site were generated. These were used along with oceanographic and ecological information to detect signals of adaptive divergence with gene flow across environmental gradients. The studied sardines constitute two clusters (FST  = 0.07), a pattern attributed to outlier loci, highlighting putative local adaptation. The trend in the number of days with sea surface temperature above 19°C, a critical threshold for successful sardine spawning, was crucial at all levels of population structuring with implications on the species' key biological processes. Outliers link candidate SNPs to the region's environmental heterogeneity. Our findings provide evidence for a dynamic equilibrium in which population structure is maintained by physical and ecological factors under the opposing influences of migration and selection. This dynamic in a natural system warrants continuous monitoring under a seascape genomic approach that might benefit from a temporal and more detailed spatial dimension. Our results may contribute to complementary studies aimed at providing deeper insights into the mechanistic processes underlying population structuring. Those are key to understanding and predicting future changes and responses of this highly exploited species in the face of climate change.

New molecular tools for the identification of 2 endangered smooth-hound sharks, Mustelus mustelus and Mustelus punctulatus.

The smooth-hounds represent a significant proportion of the elasmobranch catch in the Adriatic basin of the Mediterranean Sea, where the common (Mustelus mustelus) and blackspotted (Mustelus punctulatus) smooth-hounds co-occur. The 2 species share several morphological and morphometric characters that lead to frequent misidentification. In order to provide information useful for their species identification, we performed a morphological identification of several Mustelus specimens to select individuals unambiguously attributed to 1 of the 2 species, and assayed these with 3 new molecular tests. First, we developed and validated a mitochondrial DNA assay based on species-specific amplification of the cytochrome c oxidase subunit 1 (COI). Second, a fragment analysis of 15 microsatellites cross-amplified from several triakid species was performed to identify diagnostic loci. Finally, a length difference was identified in the internal transcribed spacer 2 (ITS2) region and a diagnostic test based on its amplification was established. All the samples classified morphologically as M. mustelus and M. punctulatus showed a species-specific profile using all the 3 molecular tests. In addition, cross-amplification of microsatellites allowed identification of 9 highly polymorphic loci that will be useful for the study of the mating system and population differentiation of the 2 species.

Outlier SNP markers reveal fine-scale genetic structuring across European hake populations (Merluccius merluccius).

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome-wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large- and fine-scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F(CT) = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F(CT) range 0.275-0.705) and fine-scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.

Spatio-temporal population structuring and genetic diversity retention in depleted Atlantic bluefin tuna of the Mediterranean Sea.

Fishery genetics have greatly changed our understanding of population dynamics and structuring in marine fish. In this study, we show that the Atlantic Bluefin tuna (ABFT, Thunnus thynnus), an oceanic predatory species exhibiting highly migratory behavior, large population size, and high potential for dispersal during early life stages, displays significant genetic differences over space and time, both at the fine and large scales of variation. We compared microsatellite variation of contemporary (n = 256) and historical (n = 99) biological samples of ABFTs of the central-western Mediterranean Sea, the latter dating back to the early 20th century. Measures of genetic differentiation and a general heterozygote deficit suggest that differences exist among population samples, both now and 96-80 years ago. Thus, ABFTs do not represent a single panmictic population in the Mediterranean Sea. Statistics designed to infer changes in population size, both from current and past genetic variation, suggest that some Mediterranean ABFT populations, although still not severely reduced in their genetic potential, might have suffered from demographic declines. The short-term estimates of effective population size are straddled on the minimum threshold (effective population size = 500) indicated to maintain genetic diversity and evolutionary potential across several generations in natural populations.

I Like This New Me: Unravelling Population Structure of Mediterranean Electric Rays and Taxonomic Uncertainties within Torpediniformes.

The present study focused on the three species of electric rays known to occur in the Mediterranean Sea: Torpedo torpedo, Torpedo marmorata and Tetronarce nobiliana. Correct identification of specimens is needed to properly assess the impact of fisheries on populations and species. Unfortunately, torpedoes share high morphological similarities, boosting episodes of field misidentification. In this context, genetic data was used (1) to identify specimens caught during fishing operations, (2) to measure the diversity among and within these species, and (3) to shed light on the possible occurrence of additional hidden species in the investigated area. New and already published sequences of COI and NADH2 mitochondrial genes were analyzed, both at a small scale along the Sardinian coasts (Western Mediterranean) and at a large scale in the whole Mediterranean Sea. High levels of genetic diversity were found in Sardinian populations, being significantly different from other areas of the Eastern Mediterranean Sea due to the biotic and abiotic factors here discussed. Sardinian torpedoes can hence be indicated as priority populations/areas to be protected within the Mediterranean Sea. Moreover, sequence data confirmed that only the three species occur in the investigated area. The application of several 'species-delimitation' methods found evidence of cryptic species in the three species outside the Mediterranean Sea, as well as in other genera/families, suggesting the urgent need for future studies and a comprehensive revision of the order Torpediniformes for its effective conservation.

To Be, or Not to Be: That Is the Hamletic Question of Cryptic Evolution in the Eastern Atlantic and Mediterranean Raja miraletus Species Complex.

Despite a high species diversity, skates (Rajiformes) exhibit remarkably conservative morphology and ecology. Limited trait variations occur within and between species, and cryptic species have been reported among sister and non-sister taxa, suggesting that species complexes may be subject to stabilising selection. Three sibling species are currently recognised in the Raja miraletus complex: (i) R. miraletus occurring along the Portuguese and Mediterranean coasts, (ii) R. parva in the Central-Eastern Atlantic off West Africa and (iii) R. ocellifera in the Western Indian Ocean off South Africa. In the present study, the genetic variation at mitochondrial and nuclear markers was estimated in the species complex by analysing 323 individuals sampled across most of its geographical distribution area to test the hypothesis that restricted gene flow and genetic divergence within species reflect known climate and bio-oceanographic discontinuities. Our results support previous morphological studies and confirm the known taxonomic boundaries of the three recognised species. In addition, we identified multiple weakly differentiated clades in the Northeastern Atlantic Ocean and Mediterranean, at least two additional cryptic taxa off Senegal and Angola, a pronounced differentiation of ancient South African clades. The hidden genetic structure presented here may represent a valuable support to species' conservation action plans.

Matching an Old Marine Paradigm: Limitless Connectivity in a Deep-Water Fish over a Large Distance.

Investigations of population structuring in wild species are fundamental to complete the bigger picture defining their ecological and biological roles in the marine realm, to estimate their recovery capacity triggered by human disturbance and implement more efficient management strategies for fishery resources. The Blackspot Seabream (Pagellus bogaraveo, Brünnich 1768) is a commercially valuable deep-water fish highly exploited over past decades. Considering its exploitation status, deepening the knowledge of intraspecific variability, genetic diversity, and differentiation using high-performing molecular markers is considered an important step for a more effective stock assessment and fishery management. With one of the largest efforts conceived of and completed by countries overlooking the Atlantic and Mediterranean coasts in recent years, a total of 320 individuals were collected from different fishing grounds in the Mediterranean Sea and Atlantic Ocean and analysed using 29 microsatellite loci. We applied multiple statistical approaches to investigate the species' connectivity and population structure across most of its described distribution area. Considering the incomplete knowledge regarding the migratory behaviour of adults, here we suggest the importance of egg and larval dispersal in sustaining the observed genetic connectivity on such a large geographical scale.

Molecular Taxonomy and Diversification of Atlantic Skates (Chondrichthyes, Rajiformes): Adding More Pieces to the Puzzle of Their Evolutionary History.

Conservation and long-term management plans of marine species need to be based upon the universally recognized key-feature of species identity. This important assignment is particularly challenging in skates (Rajiformes) in which the phenotypic similarity between some taxa and the individual variability in others, hampers accurate species identification. Here, 432 individual skate samples collected from four major ocean areas of the Atlantic were barcoded and taxonomically analysed. A BOLD project ELASMO ATL was implemented with the aim of establishing a new fully available and well curated barcode library containing both biological and molecular information. The evolutionary histories of the 38 skate taxa were estimated with two concatenated mitochondrial markers (COI and NADH2) through Maximum Likelihood and Bayesian inference. New evolutionary lineages within the genus Raja were discovered off Angola, where paleogeographic history coupled with oceanographic discontinuities could have contributed to the establishment of isolated refugia, playing a fundamental role among skates' speciation events. These data successfully resolved many taxonomic ambiguities, identified cryptic diversity within valid species and demonstrated a highly cohesive monophyletic clustering among the order, laying the background for further inference of evolutionary patterns suitable for addressing management and conservation issues.

Ancient DNA SNP-panel data suggests stability in bluefin tuna genetic diversity despite centuries of fluctuating catches in the eastern Atlantic and Mediterranean.

Atlantic bluefin tuna (Thunnus thynnus; BFT) abundance was depleted in the late 20th and early 21st century due to overfishing. Historical catch records further indicate that the abundance of BFT in the Mediterranean has been fluctuating since at least the 16th century. Here we build upon previous work on ancient DNA of BFT in the Mediterranean by comparing contemporary (2009-2012) specimens with archival (1911-1926) and archaeological (2nd century BCE-15th century CE) specimens that represent population states prior to these two major periods of exploitation, respectively. We successfully genotyped and analysed 259 contemporary and 123 historical (91 archival and 32 archaeological) specimens at 92 SNP loci that were selected for their ability to differentiate contemporary populations or their association with core biological functions. We found no evidence of genetic bottlenecks, inbreeding or population restructuring between temporal sample groups that might explain what has driven catch fluctuations since the 16th century. We also detected a putative adaptive response, involving the cytoskeletal protein synemin which may be related to muscle stress. However, these results require further investigation with more extensive genome-wide data to rule out demographic changes due to overfishing, and other natural and anthropogenic factors, in addition to elucidating the adaptive drivers related to these.

Canning Processes Reduce the DNA-Based Traceability of Commercial Tropical Tunas.

Canned tuna is one of the most widely traded seafood products internationally and is of growing demand. There is an increasing concern over the vulnerability of canned tuna supply chains to species mislabelling and fraud. Extensive processing conditions in canning operations can lead to the degradation and fragmentation of DNA, complicating product traceability. We here employed a forensically validated DNA barcoding tool (cytochrome b partial sequences) to assess the effects of canning processes on DNA degradation and the identification of four tropical tuna species (yellowfin, bigeye, skipjack and longtail tuna) collected on a global scale, along their commercial chains. Each species was studied under five different canning processes i.e., freezing, defrosting, cooking, and canning in oil and brine, in order to investigate how these affect DNA-based species identification and traceability. The highest percentage of nucleotide substitutions were observed after brine-canning operations and were greatest for yellowfin and skipjack tuna. Overall, we found that DNA degradation significantly increased along the tuna canning process for most specimens. Consequently, most of the specimens canned in oil or brine were misidentified due to the high rate of nucleotide substitution in diagnostic sequences.

Author Correction: Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The population genomics of yellowfin tuna (Thunnus albacares) at global geographic scale challenges current stock delineation.

Yellowfin tuna, Thunnus albacares, is one of the most important seafood commodities in the world. Despite its great biological and economic importance, conflicting evidence arises from classical genetic and tagging studies concerning the yellowfin tuna population structure at local and global oceanic scales. Access to more powerful and cost effective genetic tools would represent the first step towards resolving the population structure of yellowfin tuna across its distribution range. Using a panel of 939 neutral Single Nucleotide Polymorphisms (SNPs), and the most comprehensive data set of yellowfin samples available so far, we found genetic differentiation among the Atlantic, Indian and Pacific oceans. The genetic stock structure analysis carried out with 33 outlier SNPs, putatively under selection, identified discrete populations within the Pacific Ocean and, for the first time, also within the Atlantic Ocean. Stock assessment approaches that consider genetic differences at neutral and adaptive genomic loci should be routinely implemented to check the status of the yellowfin tuna, prevent illegal trade, and develop more sustainable management measures.

Natural history and molecular evolution of demersal Mediterranean sharks and skates inferred by comparative phylogeographic and demographic analyses.

BACKGROUND: The unique and complex paleoclimatic and paleogeographic events which affected the Mediterranean Sea since late Miocene deeply influenced the distribution and evolution of marine organisms and shaped their genetic structure. Following the Messinian salinity crisis and the sea-level fluctuations during the Pleistocene, several Mediterranean marine species developed deep genetic differentiation, and some underwent rapid radiation. Here, we consider two of the most prioritized groups for conservation in the light of their evolutionary history: sharks and rays (elasmobranchs). This paper deals with a comparative multispecies analysis of phylogeographic structure and historical demography in two pairs of sympatric, phylogenetically- and ecologically-related elasmobranchs, two scyliorhinid catsharks (Galeus melastomus, Scyliorhinus canicula) and two rajid skates (Raja clavata, Raja miraletus). Sampling and experimental analyses were designed to primarily test if the Sicilian Channel can be considered as effective eco-physiological barrier for Mediterranean demersal sympatric elasmobranchs. METHODS: The phylogeography and the historical demography of target species were inferred by analysing the nucleotide variation of three mitochondrial DNA markers (i.e., partial sequence of COI, NADH2 and CR) obtained from a total of 248 individuals sampled in the Western and Eastern Mediterranean Sea as well as in the adjacent northeastern Atlantic Ocean. Phylogeographic analysis was performed by haplotype networking and testing spatial genetic differentiation of samples (i.e., analysis of molecular variance and of principal components). Demographic history of Mediterranean populations was reconstructed using mismatch distribution and Bayesian Skyline Plot analyses. RESULTS: No spatial genetic differentiation was identified in either catshark species, while phylogeographic structure of lineages was identified in both skates, with R. miraletus more structured than R. clavata. However, such structuring of skate lineages was not consistent with the separation between Western and Eastern Mediterranean. Sudden demographic expansions occurred synchronously during the upper Pleistocene (40,000-60,000 years ago) in both skates and G. melastomus, likely related to optimal environmental conditions. In contrast, S. canicula experienced a slow and constant increase in population size over the last 350,000 years. DISCUSSION: The comparative analysis of phylogeographic and historical demographic patterns for the Mediterranean populations of these elasmobranchs reveals that historical phylogeographic breaks have not had a large impact on their microevolution. We hypothesize that interactions between environmental and ecological/physiological traits may have been the driving force in the microevolution of these demersal elasmobranch species in the Mediterranean rather than oceanographic barriers.

Spatial dynamics and mixing of bluefin tuna in the Atlantic Ocean and Mediterranean Sea revealed using next-generation sequencing.

The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated with shared fisheries management. In an effort to resolve the species' stock dynamics, a genomewide search for spatially informative single nucleotide polymorphisms (SNPs) was undertaken, by way of sequencing reduced representation libraries. An allele frequency approach to SNP discovery was used, combining the data of 555 larvae and young-of-the-year (LYOY) into pools representing major geographical areas and mapping against a newly assembled genomic reference. From a set of 184,895 candidate loci, 384 were selected for validation using 167 LYOY. A highly discriminatory genotyping panel of 95 SNPs was ultimately developed by selecting loci with the most pronounced differences between western Atlantic and Mediterranean Sea LYOY. The panel was evaluated by genotyping a different set of LYOY (n = 326), and from these, 77.8% and 82.1% were correctly assigned to western Atlantic and Mediterranean Sea origins, respectively. The panel revealed temporally persistent differentiation among LYOY from the western Atlantic and Mediterranean Sea (FST  = 0.008, p = .034). The composition of six mixed feeding aggregations in the Atlantic Ocean and Mediterranean Sea was characterized using genotypes from medium (n = 184) and large (n = 48) adults, applying population assignment and mixture analyses. The results provide evidence of persistent population structuring across broad geographic areas and extensive mixing in the Atlantic Ocean, particularly in the mid-Atlantic Bight and Gulf of St. Lawrence. The genomic reference and genotyping tools presented here constitute novel resources useful for future research and conservation efforts.

Genetic differentiation and phylogeography of Mediterranean-North Eastern Atlantic blue shark (Prionace glauca, L. 1758) using mitochondrial DNA: panmixia or complex stock structure?

BACKGROUND: The blue shark (Prionace glauca, Linnaeus 1758) is one of the most abundant epipelagic shark inhabiting all the oceans except the poles, including the Mediterranean Sea, but its genetic structure has not been confirmed at basin and interoceanic distances. Past tagging programs in the Atlantic Ocean failed to find evidence of migration of blue sharks between the Mediterranean and the adjacent Atlantic, despite the extreme vagility of the species. Although the high rate of by-catch in the Mediterranean basin, to date no genetic study on Mediterranean blue shark was carried out, which constitutes a significant knowledge gap, considering that this population is classified as "Critically Endangered", unlike its open-ocean counterpart. METHODS: Blue shark phylogeography and demography in the Mediterranean Sea and North-Eastern Atlantic Ocean were inferred using two mitochondrial genes (Cytb and control region) amplified from 207 and 170 individuals respectively, collected from six localities across the Mediterranean and two from the North-Eastern Atlantic. RESULTS: Although no obvious pattern of geographical differentiation was apparent from the haplotype network, Φst analyses indicated significant genetic structure among four geographical groups. Demographic analyses suggest that these populations have experienced a constant population expansion in the last 0.4-0.1 million of years. DISCUSSION: The weak, but significant, differences in Mediterranean and adjacent North-eastern Atlantic blue sharks revealed a complex phylogeographic structure, which appears to reject the assumption of panmixia across the study area, but also supports a certain degree of population connectivity across the Strait of Gibraltar, despite the lack of evidence of migratory movements observed by tagging data. Analyses of spatial genetic structure in relation to sex-ratio and size could indicate some level of sex/stage biased migratory behaviour.

Improving the Conservation of Mediterranean Chondrichthyans: The ELASMOMED DNA Barcode Reference Library.

Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental change because of their K-selected reproductive strategy. Accurate data from scientific surveys and landings are essential to assess conservation status and to develop robust protection and management plans. Currently available data are often incomplete or incorrect as a result of inaccurate species identifications, due to a high level of morphological stasis, especially among closely related taxa. Moreover, several diagnostic characters clearly visible in adult specimens are less evident in juveniles. Here we present results generated by the ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Mediterranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode reference library. This library will support and improve the molecular taxonomy of this group and the effectiveness of management and conservation measures. We successfully barcoded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera), including four endemic and several threatened ones. Morphological misidentifications were found across most orders, further confirming the need for a comprehensive DNA barcoding library as a valuable tool for the reliable identification of specimens in support of taxonomist who are reviewing current identification keys. Despite low intraspecific variation among their barcode sequences and reduced samples size, five species showed preliminary evidence of phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific features in otherwise taxonomically problematic groups for biodiversity management and conservation actions.

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.

Molecular Identification of Atlantic Bluefin Tuna (Thunnus thynnus, Scombridae) Larvae and Development of a DNA Character-Based Identification Key for Mediterranean Scombrids.

The Atlantic bluefin tuna, Thunnus thynnus, is a commercially important species that has been severely over-exploited in the recent past. Although the eastern Atlantic and Mediterranean stock is now showing signs of recovery, its current status remains very uncertain and as a consequence their recovery is dependent upon severe management informed by rigorous scientific research. Monitoring of early life history stages can inform decision makers about the health of the species based upon recruitment and survival rates. Misidentification of fish larvae and eggs can lead to inaccurate estimates of stock biomass and productivity which can trigger demands for increased quotas and unsound management conclusions. Herein we used a molecular approach employing mitochondrial and nuclear genes (CO1 and ITS1, respectively) to identify larvae (n = 188) collected from three spawning areas in the Mediterranean Sea by different institutions working with a regional fisheries management organization. Several techniques were used to analyze the genetic sequences (sequence alignments using search algorithms, neighbour joining trees, and a genetic character-based identification key) and an extensive comparison of the results is presented. During this process various inaccuracies in related publications and online databases were uncovered. Our results reveal important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods. While less than half of larvae provided were bluefin tuna, other dominant taxa were bullet tuna (Auxis rochei), albacore (Thunnus alalunga) and little tunny (Euthynnus alletteratus). We advocate an expansion of expertise for a new generation of morphology-based taxonomists, increased dialogue between morphology-based and molecular taxonomists and increased scrutiny of public sequence databases.

Detection and characterization of pathogenic vibrios in shellfish by a Ligation Detection Reaction-Universal Array approach.

Vibrios are a group of major foodborne pathogens widely distributed in marine environment. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the pathogenic species of Vibrio that pose the greatest threat to human health. However, other vibrios, e.g. Vibrio alginolyticus, Vibrio mimicus and Grimontia hollisae, apparently less relevant in the group of foodborne pathogens, have been sporadically found in outbreaks. For seafood safety and economic purposes, a rapid and powerful method for the specific identification of harmful Vibrio strains is needed. We developed a PCR-Ligase Detection Reaction-Universal Array (PCR-LDR-UA) assay for the simultaneous identification of pathogenic vibrios and detection of virulence coding genes. The entire procedure was validated on a total of 31 reference strains and isolates from clinical and environmental samples, as well as on bivalve tissue homogenates infected with different strains of target Vibrio species. Twenty-three shellfish samples directed to human consumption were successfully screened, thus demonstrating that the developed microarray-based platform could be a reliable and sensitive detection tool for the identification of harmful Vibrio strains in seafood.

SNP discovery using Next Generation Transcriptomic Sequencing in Atlantic herring (Clupea harengus).

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild.