Contents lists available at ScienceDirect Fisheries Research journal homepage: www.elsevier.com/locate/fishres Full length article SNP identification and validation on genomic DNA for studying genetic diversity in Thunnus albacares and Scomberomorus brasiliensis by combining RADseq and long read high throughput sequencing Zoila Raquel Siccha-Ramireza,b,⁎,1, Francesco Marosoc,d,1, Belén G. Pardoc, Carlos Fernándezc, Paulino Martínezc,⁎⁎, Claudio Oliveiraa a Departamento de Morfologia, Instituto de Biociências, UNESP, Botucatu, São Paulo, Brazil b Instituto del Mar del Perú, Laboratorio Costero de Tumbes, Zorritos, Peru c Departamento de Zooloxía, Xenética e Antropoloxía Física, Facultade de Veterinaria, Universidade de Santiago de Compostela, Campus de Lugo, Lugo, 27002, Spain d Dipartimento di Biomedicina Comparata e Alimentazione (BCA), Università degli Studi di Padova, Italy A R T I C L E I N F O Keywords: Scomberomorus brasiliensis Thunnus albacares ddRAD Long-read high throughput sequencing Genetic structure A B S T R A C T A combination of a RADseq method (ddRAD) with long read high throughput sequencing (Roche 454) was tuned up in order to identify and validate a set of SNPs useful for gene diversity analysis in two important South American commercial tuna (Thunnus albacares and Scomberomorus brasiliensis). A total of 11 and 21 individuals of T. albacares and S. brasiliensis, respectively, were used for SNP identification. DNA was individually digested with two restriction enzymes (SbfI and SphI) and fragments between 300 and 600 bp selected. Combinatorial barcoding was used to identify individuals by including short sequences (5–7 bp) in the adaptors of each re- striction site (P1 and P2). After adaptor ligation, samples were pooled and size-selected, amplified by PCR, and sequenced on a 454 GS-Junior sequencer. A total of 180,779 reads were produced with an average length and coverage of 287 bp and 26x, respectively. Sets of 60 and 79 SNPs were in silico selected for T. albacares and S. brasiliensis, respectively, and were tested and validated in 74 and 66 individuals, respectively, on a MassARRAY platform. A total of 36 and 47 SNPs were polymorphic and useful for population analysis. A preliminary study on two distant Brazilian populations of both species (∼3000 km) with these SNPs suggested the absence of sig- nificant structure among local populations of both species. Our results demonstrate the possibility of combining ddRAD with long read high throughput sequencing for marker development in species with scarce genomic resources. 1. Introduction Next Generation Sequencing (NGS) has revolutionized the field of genetics (Guo et al., 2016; Mardis, 2008; Metzker, 2010) allowing in- vestigation on non-model species with unprecedented genomic cov- erage. An increasingly used application is the search, validation and large-scale genotyping of genetic markers using different methodolo- gies (Helyar et al., 2012). SNPs are stable and usually bi-allelic poly- morphisms (Mullikin et al., 2000), found in coding and non-coding regions (Bruneaux et al., 2013; Stölting et al., 2013), and homogeneously distributed across the genome at high densities (Du et al., 2012); thus, SNPs are the most common genetic markers for genomic screening (Vera et al., 2013). In fish, SNPs are found every ∼100 bp (Pardo et al., 2008; Vera et al., 2013). These properties make SNPs ideal for comparative genomics (Bouza et al., 2012), evolutionary genomics (Du et al., 2012), fine mapping of genes associated with productive traits (Sánchez et al., 2009), genomic screening in popula- tions for conservation and management (Albaina et al., 2013; Glaubitz et al., 2003; Kuhner et al., 2000; Vilas et al., 2015), and hybridization and impact of biological invasions (Bers et al., 2010). http://dx.doi.org/10.1016/j.fishres.2017.09.002 Received 3 March 2017; Received in revised form 30 August 2017; Accepted 4 September 2017 ⁎ Corresponding author at: Departamento de Morfologia, Instituto de Biociências, UNESP, Botucatu, São Paulo, 18618-970, Brazil. ⁎⁎ Corresponding author. 1 These authors contributed equally to this work. E-mail addresses: rsicchar@gmail.com (Z.R. Siccha-Ramirez), paulino.martinez@usc.es (P. Martínez). Abbreviations: NGS, Next Generation Sequencing; SNP, Single Nucleotide Polymorphisms; RRL, Reduced Representation Library; RADseq, Restriction-site associated DNA sequencing; ddRAD-seq, double-digestion RADseq; RE, restriction enzyme; PCR, polymerase chain reaction; HE, expected heterozygosity; HO, observed heterozygosity; MAF, minimum allele fre- quency; H-W, Hardy-Weinberg; FIS, inbreeding coefficient; FST, coefficient of relative differentiation between populations; NCBI, National Center for Biotechnology Information; BLAST, Basic Local Alignment Search Tool; WGS, whole genome shotgun; nr, non redundant; ts, transition; tv, transversion Fisheries Research 198 (2018) 189–194 Available online 18 September 2017 0165-7836/ © 2017 Elsevier B.V. All rights reserved. MARK http://www.sciencedirect.com/science/journal/01657836 http://www.elsevier.com/locate/fishres http://dx.doi.org/10.1016/j.fishres.2017.09.002 http://dx.doi.org/10.1016/j.fishres.2017.09.002 mailto:rsicchar@gmail.com mailto:paulino.martinez@usc.es http://dx.doi.org/10.1016/j.fishres.2017.09.002 http://crossmark.crossref.org/dialog/?doi=10.1016/j.fishres.2017.09.002&domain=pdf Reduced Representation Library (RRL) sequencing provides a high potential for efficient SNP discovery at very low cost (Sánchez et al., 2009), because sequencing is targeted to a small and adjustable genome fraction flanking the chosen RE sites (Du et al., 2012; Robledo et al., 2017). Since restriction sites are common to most individuals of a species, high throughput sequencing using DNA barcodes to label in- dividuals, populations or species provides a set of reads at each re- striction site to be explored bioinformatically. Depending on the goals of the study, SNP identification and genotyping can be jointly per- formed as a single step, or alternatively a subset of SNPs can be selected for further validation and genotyping using specific platforms (SNaP- shot, Sequenom, Truseq, Ampliseq, etc.) (Davey et al., 2011; Robledo et al., 2017). This last option offers the possibility of developing a panel of adjustable SNP number to be routinely used at lower cost depending on the goals of the study. RADseq is a RRL strategy which has gained popularity in non-model organisms because it allows obtaining huge SNP genotyping data at very low cost (Baird et al., 2008; Gompert et al., 2010; Vandepitte et al., 2013). ddRAD-seq is a variant of the classical RADseq (Baird et al., 2008), which eliminates random shearing and explicitly uses size selection to recover a tunable number of regions according to the objectives of the study (Peterson et al., 2012). ddRAD not only allows high-throughput multiplexed sequencing amenable for genotyping, but also provides improved efficiency and robustness compared to classical RADseq (Zhou et al., 2014). However, the short length of reads of the commonly used platforms for RADseq hampers the subsequent development of SNP panels for genotyping in PCR-based platforms, if no reference genome is available. Consequently, com- bining ddRAD libraries with longer-read sequencing platforms could aid to solve this drawback making affordable the selection and vali- dation of a subset of SNPs. The yellowfin tuna (Thunnus albacares, Scombridae) is a migratory species with high commercial value found in tropical and subtropical waters all over the world (Guo et al., 2016). This species is currently overfished and appears on the Red List as near threatened species (Collette et al., 2011). A variety of studies have been done to assess the population structure of T. albacares using different approaches, in- cluding genetic markers (Aires-da-Silva and Maunder, 2012; Alvarado Bremer et al., 1997; Appleyard et al., 2001; Dammannagoda et al., 2008; Ely et al., 2005; Kunal et al., 2013; Ward et al., 1994; Wu et al., 2009), but its genetic structure is still controversial. The Spanish mackerel (Scomberomorus brasiliensis, Scombridae) (Collette et al., 1978) is a neritic tuna (Fonteles Filho, 1989) distributed from Belize to south Brazil (Carvalho-Filho, 1999) of high commercial interest in Trinidad and Tobago and Venezuela (Hodgkinson-Clarke, 1990), and in Brazil, especially in the state of Maranhão (Fonteles-Filho, 1988). Few studies have been conducted in this species (Batista and da Fabré, 2001; Maia et al., 2015), and only one aimed at evaluating its genetic struc- ture (Gold et al., 2010). This work describes how the identification and validation of a large number of SNPs is achievable combining ddRAD and long read high throughput sequencing (e.g. 454 pyrosequencing) in T. albacares and S. brasiliensis for their routine genotyping on a Se- quenom platform. Validated SNPs were further tested on a preliminary analysis of genetic diversity and structure in both species using two geographically separated Brazilian samples. 2. Material and methods 2.1. Biological material and DNA extraction Samples of both species were collected in Brazil from areas sepa- rated by ∼3000 km: T. albacares was collected in Natal (n = 50, 05°50′24.90″S, 34°59′24.34″W) and Santos (n = 24, 24°16′24.88″S, 45°49′51.79″W) and S. brasiliensis in Penha (n = 29, 26°47′24.14″S, 48°33′37.43″W) and Bragança (n = 37, 00°49′4.63″S, 46°30′0.40″W). A total of 11 T. albacares and 21 S. brasiliensis individuals including samples from both populations were used for in silico SNP discovery and subsequent validation on a Sequenom platform. Seventy-four in- dividuals of T. albacares and 66 of S. brasiliensis were genotyped using validated SNPs for a preliminary evaluation of genetic diversity and structure of species. Representative specimens and all tissues were de- posited in the fish collection of Laboratório de Biologia and Genética de Peixes of the Universidade Estadual Paulista (Botucatu, São Paulo, Brazil). Genomic DNA was obtained from ethanol-preserved tissues. Samples were lysed in 300 μl of SSTNE extraction buffer (Blanquer, 1990) plus SDS (0.1%) and 5 μl of proteinase K (20 mg/ml) for 3 h at 55 °C. After 20 min at 70 °C, samples were treated with7.5 μl of RNAse (10 mg/ml) for 1 h at 37 °C for RNA degradation. Total DNA was pur- ified after protein precipitation (5 M NaCl) with freezing absolute ethanol (1 ml). DNA quality (high molecular weight > 20 kb) was first evaluated on agarose gels and the DNA quantity was measured using the NanoDrop ® ND-1000 spectrophotometer (NanoDrop® Technologies Inc) and PicoGreen kit (Molecular Probes) according to the kit in- structions. Finally, DNA concentration was accurately measured on a Qubit fluorometer (Life Technologies). 2.2. Library construction A reduced fraction of the genome of the two species was sequenced using a modified ddRAD protocol (Peterson et al., 2012) in a single 454 GS Junior run (Fig. 1). Briefly, the same amount of DNA (78 ng) of each sample (11 T. albacares and 21 S. brasiliensis) was individually digested with SbfI and SphI restriction enzymes (RE). Subsequently adaptors for each RE were ligated to both ends of digested fragments. Adaptors in- cluded: i) complementary cohesive ends for the correspondent RE; ii) barcodes to identify individuals; and iii) primers for an intermediate PCR amplification. Barcoded samples were then pooled and run in agarose gels 1.1% for fragment selection (300–600 bp), followed by extraction using Qiagen MinElute Gel Extraction kit. After selection, the target DNA fragments were amplified by PCR to obtain the required concentration for sequencing in the 454 GS Junior platform: initial denaturation and enzyme activation at 98 °C for 30s; 14 cycles at 98 °C for 10 s (denaturation step), 65 °C for 30 s (annealing) and 72 °C for 30 s (extension); final extension at 72 °C for 5 min. The PCR products were purified using the Qiagen MinElute PCR Purification kit followed by a magnetic bead clean-up/size selection using an equal volume of Beckman Coulter AMPure XP beads. This protocol ensured that only those fragments including SbfI and SphI target sites were amplified and further sequenced. 2.3. Sequencing, assembly and SNP identification and selection The final library was sequenced in a single shotgun run on a 454 GS Junior sequencer (Roche Diagnostics) available at the Sequencing and Functional Genomics Platform of the University of Santiago de Compostela (USC, Campus Lugo, Spain) (Fig. 1). Sequencing reads were filtered using default parameters, classified per individual according to barcodes and assembled with Newbler software (specifically designed for 454 GS series data). Alignments were then parsed with Tablet (Milne et al., 2010) in order to identify the most consistent contigs and detect SNPs in the assembled sequences. Only contigs containing a se- quencing depth > 6 were retained for further analysis to reduce SNPs attributable to sequencing errors. SNPs were selected according to the presence of enough flanking regions for primer design (± 100 bp) and the absence of other DNA polymorphism (SNPs and indels) in those regions that could interfere with primer annealing. Additionally, only those SNPs with at least three sequences of the least common allele were selected. 2.4. SNP genotyping In silico selected SNPs were validated and genotyped using the Z.R. Siccha-Ramirez et al. Fisheries Research 198 (2018) 189–194 190 MassARRAY platform (Sequenom, San Diego, CA, USA) at the USC node of the Spanish National Centre of Genotyping (CeGen ISCIII) following the protocols and recommendations provided by the manufacturer (Fig. 1). Briefly, target regions were PCR amplified using primers de- signed on contig sequences obtained in the previous step, and target SNPs were genotyped by single-base extension using an oligonucleotide primer that anneals immediately upstream to the polymorphic site (SNP) of interest (Buetow et al., 2001; Oeth et al., 2009). MALDI-TOF mass spectrometry analysis in an Autoflex spectrometer was used for allele scoring. Two SNPs multiplexes for each species were designed in silico using Assay Design 3.1 program (Sequenom, San Diego, CA), which maximizes the number of SNP per multiplex and minimizes the number of multiplexes. Validated SNPs (markers with reliable geno- types) and “failed assays” (majority of genotypes not scored or difficult to cluster according to genotype) were classified by manual inspection. SNPs validated were genotyped on the 74 and 66 individuals of T. al- bacares and S. brasiliensis, respectively, for population analysis. 2.5. Gene diversity and annotation Genepop 1.2 (Raymond and Rousset, 1995; Rousset, 2008) was used to estimate genetic diversity (HE, HO and MAF); to check for deviations from H-W equilibrium and their sense (FIS), and from linkage dis- equilibrium; and to check for population differentiation (FST). The complete enumeration approach was used to check for H-W equilibrium (Louis and Dempster, 1987) and the Weir and Cockerham method (Weir and Cockerham, 1984) to check the sense and magnitude of the de- viation (FIS). Linkage (genotypic) disequilibrium was analyzed for each pair of loci using the log likelihood ratio statistic (G-test). Bonferroni correction was considered when multiple tests were performed. BLASTn was used to look for significant hits in whole genome se- quencing (WGS) databases of six fish species selected among those phylogenetically closer and with best characterized genomes: Pacific bluefin tuna (Thunnus orientalis), fugu (Takifugu rubripes), stickleback (Gasterosteus aculeatus), zebrafish (Danio rerio), medaka (Oryzias latipes) and tetraodon (Tetraodon nigroviridis). Threshold significance was set at 10e-5. In addition to BLASTn searching, SNP containing sequences in both species were blasted against nr protein database from NCBI, using BLASTx. 3. Results and discussion 3.1. SNP discovery and validation The Roche 454 GS Junior run rendered a total of 246,663 reads, 180,779 of them passing the quality filter (73.3%) (Fig. 1). Average read length was 287.0 bp and the average Phred quality per read was 30.2. High quality reads were separated per species using barcodes and then assembled. A total of 1715 contigs were obtained for T. albacares, with an average length of 374.9 bp and average coverage of 25.5 reads per contig, while for S. brasiliensis, the number of contigs was 2274, with an average length of 374.6 bp and an average coverage of 26.2 reads (Table 1). The coverage achieved in the 454 GS Junior platform was enough to detect a high amount of SNPs in the assembled contigs distributed across the genome, representing a reservoir of markers for future studies in these species depending on the goals pursued. An al- ternative approach to reduce the genome looking for SNPs is to target coding regions for sequencing. This has been carried out in different fish species like Atlantic herring (Clupea harengus, Helyar et al., 2012) and pacú (Piaractus mesopotamicus, Mastrochirico-Filho et al., 2016) starting from cDNA of specific tissues sequenced on a Roche 454 plat- form. However, this approach requires very fresh samples for RNA extraction and storage to construct cDNA libraries, and this is not al- ways achievable, especially in deep sea fishing species like many tuna. A total of 60 SNPs for T. albacares and 79 for S. brasiliensis were initially selected for subsequent validation according to the criteria outlined before. Contigs containing selected SNPs averaged 290.1 bp for T. albacares and 298.7 bp for S. brasiliensis. After manual inspection, 50 SNPs (83.3% of the 60 SNPs selected) for T. albacares and 55 (69.6% of the 79 SNPs selected) for S. brasiliensis were finally chosen for vali- dation (Fig. 1). The design of flanking primers for subsequent amplifi- cation and genotyping of selected loci was feasible thanks to the length Fig. 1. Workflow of the technical procedure and main results achieved. Z.R. Siccha-Ramirez et al. Fisheries Research 198 (2018) 189–194 191 of the contigs obtained relying on the long read sequencing platform used. Markers were combined in two multiplex reactions for each species including 30 and 20 SNPs each for T. albacares and 30 and 25 for S. brasiliensis, respectively (Supporting information Tables S1 and S2). A total of 36 and 47 SNPs T, were polymorphic in T. albacares and S. brasiliensis, respectively. This represents a validation success of 72.0% and 85.5%, respectively, in the range previously documented (Vera et al., 2013). The number of transitions (ts) and transversions (tv) for SNPs was calculated in both species. Out of 36 variable markers in T. albacares dataset, 30 (83.3%) were transitions and 6 (16.7%) trans- versions, while in S. brasiliensis 17 SNPs (36.2%) represented transitions out of 47 markers scored and the remaining 30 (63.8%), transversions (Fig. 2). Accordingly, ts/tv ratio was greatly different between both species (5.000 vs 0.567, respectively), and further, in the extremes of the range regarding previous ts:tv ratios in other fish species (∼1.5, Cenadelli et al., 2007; Vera et al., 2013; Zhu et al., 2012). We cannot discard some bias produced during the process of SNP selection and further data will be required to confirm the observed trend in both species. Raw sequencing reads for both species have been deposited in NCBI Sequence Read Archive (SRA) (accession number SRR5998416). The consensus sequences of the selected 105 SNP-containing contigs were used for searching in NCBI public databases to look for annota- tion. As expected, the best significant BLASTn hits were obtained against the available T. orientalis genome (Supporting information Table S3). Average E-values were much higher for T. albacares (8 E-63) than for S. brasiliensis (3.71 E-28; Supporting information Table S3), an expected outcome considering the closer phylogenetic relationship be- tween both congeneric species. Twelve and 14 sequences out of 105 showed a significant hit (E-value < 10−5) against NCBI’s nr protein database for T. albacares and for S. brasiliensis, respectively (Supporting information Table S1), but mostly showing a not consistent annotation (predicted, hypothetical or unnamed proteins). The proportion of sig- nificant hits to protein databases (13%) is in the range expected con- sidering the ∼800 Mb of the Pacific bluefin tuna genome (Thunnus orientalis; Nakamura et al., 2013), where exons represent around 10% of the genome. The low consistency of annotations could be in part due to the high diversification and annotation success of fish, but a bias in the amplicon distribution due to the ddRAD protocol cannot be discarded. Globally, our results are very similar to those obtained in a parallel study in stingrays from South America using the same methodology (Cruz et al., 2017), confirming the consistency of the method. A similar approach was used by Jansson et al. (2016) to detect and genotype SNPs in goldsinny wrasse (Ctenolabrus rupestris), but using Illumina MiSeq for sequencing fragments obtained from ddRAD methodololgy. The length of the reads in this study was smaller than in ours (160 bp vs 287 bp), thus limiting the SNPs useful for validation due to the smaller flanking regions for primer design. However, the higher coverage of Illumina vs Roche 454, compensated this drawback and a high amount of contigs with SNPs were available for validation, which was still higher than in our study (92.3% vs 85.5%–72%). The longer read technology recently released by Illumina MiSeq (∼600 bp using pair- end sequencing) should ensure longer contigs and high coverage, thus representing the best option for this approach, but still needs for vali- dation. 3.2. Genetic diversity and structure A preliminary evaluation of genetic diversity and structure was addressed with the validated SNPs in both species using two popula- tions separated by around 3000 km. The MAF for T. albacares ranged from 0.007 to 0.464, with an average of 0.177, and for S. brasiliensis from 0.008 to 0.485, averaging 0.209 (Supplementary Table 2). HE ranged from 0.024 to 0.495 (average 0.180) for T. albacares and from 0.020 to 0.500 (average 0.240) for S. brasiliensis. The average hetero- zygosity for S. brasiliensis (0.240) was similar to that detected in other tuna species (Thunnus alalunga: 0.278 and T. thynnus: 0.272, Albaina et al., 2013) and within the range described in other marine species like European sea bass (Dicentrarchus labrax; Souche et al., 2015), Atlantic herring (Clupea harengus), turbot (Scophthalmus maximus; Vera et al., 2013) and Asian sea bass (Lates calcarifer; Wang et al., 2015). In the case of T. albacares, genetic diversity was somewhat lower than the aforementioned figures, and further work will be needed to confirm if this is a genomic feature of the species, but it is possibly bias due to the number of markers used. Hardy-Weinberg exact tests only detected deviations due to heterozygote deficit (FIS > 0): at one locus in T. al- bacares (p < 0.05), but none after Bonferroni correction, and at eight loci in S. brasiliensis (p < 0.05), six of them after Bonferroni correction (Supporting information Table S2). The consistency of the deviations across populations and its magnitude strongly suggests the presence of null alleles at these loci in S. brasiliensis. However, we cannot discard some degree of population admixture in this species (e.g. Walhund ef- fect), considering the structuring suggested for this species (Gold et al., Table 1 Characteristics of 454 GS Junior run and genotyping SNPs. Sequencing results Roche 454 GS-Junior stats Number of HQ reads 180,779 Total megabases (Mb) 51,879,629 Average length of reads 287 N° individuals sequenced 32 Assembly results T. albacares S. brasiliensis N° individuals sequenced 11 21 Number of aligned reads 42,875 59,124 Total n° of contigs 1715 2274 Average contig length 374.9 374.6 Average coverage per contig 25.3 26.2 Genotyping T. albacares S. brasiliensis N° individuals 74 66 N° markers 50 55 N° variable markers 36 47 Fig. 2. Frequency of transitions and transversions in the SNPs validated for Thunnus albacares and Scomberomorus brasiliensis. Z.R. Siccha-Ramirez et al. Fisheries Research 198 (2018) 189–194 192 2010). No pair of loci showed significant deviation from linkage equi- librium after Bonferroni correction in both species (630 and 859 tests, respectively). Our analysis suggested the absence of significant genetic structure in both T. albacares and S. brasiliensis. For T. albacares, our results are in accordance with previous reports carried out with microsatellites and SNPs that could not find significant population structure within oceanic basins likely related to its high migration capacity (Ely et al., 2005; Pecoraro et al., 2016a). Grewe et al. (2015) suggested the existence of differentiation in this species within the Pacific Ocean using 215 SNPs potentially under selection, but no differentiation was found at the remaining 5054 presumably neutral loci. Pecoraro et al. (2016b) found discrete genetic structure only between different ocean basins em- ploying 6772 SNPs. Conversely, a previous study of the Spanish mackerel detected significant genetic differentiation between geo- graphic areas (Gold et al., 2010). In our study, despite the overall ab- sence of significant differentiation (FST), five loci showed significant FST values ranging between 6.2% and 10.3% (Supporting information Table S2). Furthermore, we cannot discard that the heterozygote deficit de- tected at several loci in this species could be related to population ad- mixture suggestive of underlying structuring, which suggests the need for further work on the species using a representative sample across its distribution area. 4. Conclusions A combination of RADseq (ddRAD) and long read high throughput sequencing (454 pyrosequencing) was successfully used to identify a high amount of SNPs in two non-model fish species lacking a reference genome. The longer sequences obtained with this sequencing approach facilitated primer design for validating and genotyping a subset of SNPs accommodated to the goals of the study in a MassARRAY platform. This method enabled repeatable and tunable recovery of thousands of sampled regions from T. albacares and S. brasiliensis genomes demon- strating its utility to identify hundreds of SNPs for population studies applicable to other non-model species. This approach is faster and cheaper than other high throughput SNP genotyping methods when the number of SNPs required is not too high (i.e. ∼50–100), which is though enough for estimating population parameters with reasonable accuracy. SNPs developed with this approach demonstrated to be useful for a preliminary evaluation of genetic diversity and structure on T. albacares and S. brasiliensis. Acknowledgements We are grateful to the following people, who helped during the sampling process: Leonardo Machado (UNESP), Jonathan Ready (UFPA), Ricardo Britzke (UNESP) and all fishery owners who kindly help us to get the samples from their boats. We thank Susana Sánchez, Lucía Insua and María Villar for technical support. We also thank CeGen for the genotyping of our samples. This work was supported by São Paulo Research Foundation (grant 2011/00881-1 and grant 2013/ 07560-1) and the investigation funding provided by Xunta de Galicia local Government (GRC2014/010). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fishres.2017.09.002. References Aires-da-Silva, A., Maunder, M.N., 2012. Status of bigeye tuna in the eastern Pacific Ocean in 2010 and outlook for the future. IATTC Stock Assess. Rep. 13. 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