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Reduced SNPs panels for assigning Atlantic albacore and Bay of Biscay anchovy individuals to their geographic origin: toward sustainable fisheries management Iratxe Montes, Urtzi Laconcha, Mikel Iriondo, Carmen Manzano, Haritz Arrizabalaga, and Andone Estonba J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017

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Journal of Agricultural and Food Chemistry

Reduced SNPs panels for assigning Atlantic albacore and Bay of Biscay anchovy individuals to their geographic origin: toward sustainable fisheries management Iratxe Montes1* CO, Urtzi Laconcha1,2 CO, Mikel Iriondo1, Carmen Manzano1, Haritz Arrizabalaga2, Andone Estonba1 1

Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain 2 AZTI, Marine Research Division, Pasaia, Spain

Keywords: traceability; illegal fishing; fraud; food safety; false eco-certification; origin protection; Bay of Biscay.

Author contribution: IM, UL and AE designed the study. IM and UL carried out the laboratory work and analyzed data. IM, UL and MI interpreted the data. IM wrote the paper. MI, CM, HA and AE gave technical support and conceptual advice, and edited the manuscript. CO

equal contribution

* Author for correspondence [email protected]

ACS Paragon Plus Environment


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ABSTRACT

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There is an increasing trend on adding detailed description of the origin of seafood

3

products driven by a general interest on the implementation of sustainable fishery

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management plans for the conservation of marine ecosystems. Atlantic albacore

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(“Bonito del Norte con Eusko Label”) and Bay of Biscay anchovy (“Anchoa del

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Cantábrico”) are two commercially important fish populations with high economical

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value and vulnerable to commercial frauds. This fact together with the overexploited

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situation of these two populations makes necessary to develop a tool to identify

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individual origin and to detect commercial fraud. In the present study, we have

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developed and validated a traceability tool consisting of reduced panels of gene

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associated SNPs suitable for assigning two species’ individuals to their origin with

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unprecedented accuracy levels. Only 48 SNPs are necessary to assign 81.1% albacore

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and 93.4% anchovy individuals with 100% accuracy to their geographic origin. The

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total accuracy of the results demonstrates how gene associated SNPs can revolutionize

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food traceability. Gene associated SNP panels are not of mere commercial interest, but

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they also can result in a positive impact on sustainability of marine ecosystems through

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conservation of fish populations through establishing a more effective and sustainable

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fishery management framework, and contributing to the prevention of falsified

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labelling.

1 ACS Paragon Plus Environment

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INTRODUCTION

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The concept of sustainable fisheries management is based on the idea that each

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population has a limited exploitable biomass, which must not be exceeded for the

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perpetuation of the population. In a sustainable fishery management system each stock,

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or management unit or fishery, corresponds to a unique natural population with defined

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recruitment and mortality patterns. If this is not met, fish populations could be

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overexploited and reduced1-4. If overexploitation persists in time, fish populations may

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suffer loss of genetic diversity, and might finally result in the collapse of the fishery or

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even in the extinction of the population5,6. Therefore, a key objective for sustainable

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fishery management is to describe the population structure of exploited fish species,

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incorporating it into management plans7,8. However, this is not applied to numerous

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worldwide commercially relevant fish populations that are nowadays overexploited.

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Atlantic albacore (Thunnus alalunga, Bonn.) is eco-labelled as “Bonito del Norte con

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Eusko Label” in the Basque Country, and Bay of Biscay anchovy (Engraulis

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encrasicolus, L.) is eco-labelled as “Anchoa del Cantábrico”. These are two fish

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populations with major socio-economic importance9,10, but nowadays overexploited11,12.

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Accounting for the albacore, Illegal, Unreported and Unregulated (IUU) fishing

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practices have led to economic losses of millions of Euros per year; and the situation is

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even more delicate for Bay of Biscay European anchovy, with a collapse episode that

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led to the closure of this fishery from 2005 to 201011,13. Thus, the overexploitation of

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both species is likely due to unsustainable fishery management plans, which do not

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consider these species’ population structures, and also to the high market value of their

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canned products that has led to illegal fishing practices. Additionally, this high market

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value has made Atlantic albacore and Bay of Biscay anchovy very vulnerable to fraud in

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terms of product labelling14. This reality makes necessary to implement protection



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measures and to increase the control of the origin of the fishing products for these

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species (both in the port and in commercial products). In fact, international rules

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establish that fresh and refrigerated seafood products consumed in the European Union

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(EU) must include catch certificates containing the species scientific name and

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geographic origin, among others15,16. According to these regulations, many local

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fisheries are requesting eco-labelling as a credential of sustainability17. Hence, a tool for

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determining the stock of origin for the products eco-labelled as “Bonito del Norte con

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Eusko Label” (Thunnus alalunga from the Atlantic Ocean, fished in the Cantabrian Sea)

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and “Anchoa del Cantábrico” (Engraulis encrasicolus from the Bay of Biscay, fished in

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the Cantabrian Sea) would be highly beneficial.

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Fish is often consumed as fillets and as processed or frozen fish products, and

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monitoring labelling regulation of these products, which are not directly identifiable,

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might be difficult. In the last decade, genetic markers have become powerful tools for

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traceability of fishing derived products, whether fresh or preserved8,18-29. In 2011, two

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methods based on SNPs (Single Nucleotide Polymorphisms) were developed and

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patented by the University of the Basque Country (UPV/EHU) and AZTI for identifying

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the stock of origin of Atlantic albacore30 and Bay of Biscay anchovy31. Both tools used

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2 SNPs for species identification, and a subset of 16 SNPs for enabling identification of

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the native origin of both species. However, their accuracy values, both considerably

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below 100% and, in the case of albacore also the low percentage of assigned

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individuals, are the soft underbelly of these tools. Both, assignment and accuracy

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values, should be further improved in order to avoid false positives (assigning an

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individual to an incorrect origin) which could be the major drawback of using

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traceability tools in court cases.


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The aim of the present study is to develop a system based on genomic tools with total

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accuracy for Atlantic albacore (“Bonito del Norte con Eusko Label”) and for Bay of

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Biscay anchovy (“Anchoa del Cantábrico”) origin assignment. These tools could be

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applied to strengthen the fight against commercial fraud of fish products and, finally, to

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the conservation of these species. For this purpose, we took advantage of the already

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developed gene associated SNPs for both species, and of outlier SNPs (potentially under

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diversifying selection). We chose this strategy because the study of gene associated

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SNPs, in coding regions, enables assessment of polymorphisms potentially affecting the

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phenotype32; thus making feasible to discover outlier SNPs, which have proven to

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increase the accuracy of individual assignment tests8,328,33. We tested two panels of 32

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and 48 gene associated SNPs to trace the origin of albacore and European anchovy

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individuals. Results are referred to 460 albacore and 690 anchovy individuals covering

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the most commercially relevant fisheries of both species.

82 83 84

MATERIALS AND METHODS

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Albacore and anchovy samples

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We analyzed 460 albacore (already studied by Laconcha et al.34) and 690 European

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anchovy individuals (studied by Montes et al.35) that covered both species’

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commercially relevant fisheries (Table 1). Sampled individuals were collected during

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scientific surveys, or as part of authorized routine fishing procedures; therefore, no

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special permission was required for sampling. Tissues were stored in ethanol or at -20ºC

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until further processing. No experimentation with live animals was performed. No other

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ethical issue was applied to the present research project.



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Genomic DNA from all samples was extracted from 50 to 75 mg of muscle tissue using

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NucleoSpin® 96 Tissue kit (Macherey–Nagel). The amount and quality of DNA was

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quantified in a NanoDrop ND-8000 spectrophotometer (Thermo Fisher Scientific). In

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all, 137 SNPs (9 mitochondrial and 128 nuclear gene associated SNPs) previously

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discovered in albacore34,36 were genotyped for albacore individuals. In the case of

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European anchovy, all individuals were genotyped for 477 SNPs (15 mitochondrial and

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462 nuclear SNP including 1 intergenic and 461 gene associated ones) previously

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described for the species37,38. All individuals were genotyped using TaqMan®

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OpenArrayTM Genotyping System (Life Technologies) at the Sequencing and

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Genotyping Service (SGIker) of the University of the Basque Country (UPV/EHU).

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For both species, population genetic structure was estimated with unbiased FST39, and

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globally corrected p-values for pairwise FST were obtained from Fstat (100,000

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permutations)40. In the case of the Bay of Biscay anchovy samples, according to the

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existence of two genetically distinct populations of anchovies coexisting in the Bay of

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Biscay35, before developing the traceability tool, individuals from this region were

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assigned to their population (offshore or coastal). This assignment was done based on

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the STRUCTURE v2.3.441,42 output from Montes et al.35; estimated membership

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coefficients for each individual in each cluster (Q) were considered, and values of Q

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above 0.8 were required for classifying an individual to its population (see Figure 2 in

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Montes et al. 35).

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According to population genetic structure, individuals from each population were

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divided into reference (80% of individuals) and test (20% of individuals) samples

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(Table 1). Reference samples were those used to estimate gene frequencies of each

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population and to select loci for traceability. Test samples were analyzed as blind

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samples for validating the traceability tool.


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Apart from reference and test samples, 159 additional individuals from mixed hauls in

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the Bay of Biscay35 (hauls composed of Biscay-coastal and -offshore individuals) were

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considered for an additional validation of the traceability tool regarding the Bay of

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Biscay anchovy. These individuals correspond to samples 2-3, 5-6, 8 and 11, according

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to their nomenclature in Montes et al.35, and were analyzed as blind samples for

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validating the traceability tool.

124 125

Reduced SNP panels

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The SNP panels consisted of 2 SNPs for species identification, also included in the

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previous traceability patents of albacore30 and anchovy31. These SNPs can distinguish

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Thunnus alalunga from other Thunnus species, and Engraulis encrasicolus from other

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Engraulis species.

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Then, a set of SNPs for individual assignment to its origin was designed for each

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species according to their population genetic structure. Selected populations were

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clustered in population pairs, and SNP selection was performed in two consecutive

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steps. In the First-step, SNPs showing the highest pairwise FST values between the most

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different population pairs from each species were selected; and in the Second-step SNPs

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with the highest pairwise FST values between the most similar population pairs were

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selected. Pairwise FST estimates between population pairs and FST values for each SNP

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were estimated using BAYESCAN V2.143 using 20 pilot runs of 5,000 iterations, and an

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additional burn-in of 50,000 iterations (sample size of 5,000 and thinning interval of

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10). Critical values for the test were adjusted with false discovery rate (FDR)

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procedure44 (q < 0.1), as recommended in BAYESCAN V2.1 protocol43. Neighbor-Joining

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trees were calculated based on estimates of Reynolds weighted genetic distance (Drw)

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using POPULATIONS v1.2.31 software45.



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The number of selected SNPs from each population pair was inversely proportional to

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their FST value (Figure 1). Two traceability panels for each species were considered so

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as to create the smallest SNP panel with the highest power for individual assignment.

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Therefore, for each population pair the most discriminatory SNPs were selected and

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combined in order to reach the final numbers of 32 or 48 SNPs, which correspond to the

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commercially available genotyping formats of 32 assays available in TaqMan®

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OpenArray® Genotyping Plates (Thermo Fisher, Waltham, MA, USA), and to the

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192.48 Genotyping IFC formats for genotyping 48 SNPs (Fluidigm, San Francisco, CA,

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USA).

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Validation of the traceability tool

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The two SNP panels for each species were validated by assigning blind test samples to a

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reference population (composed of reference samples, Table 1) based on their allelic

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frequencies using GENECLASS v2.0 software46, and then blind samples were de-

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codified. This analysis was repeated for assigning 159 anchovy individuals from Bay of

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Biscay mixed hauls to their population (Biscay-offshore or Biscay-coastal). From these

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analyses we obtained the assignment probability score and the threshold score for each

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individual. Assignment probability score of an individual is the likelihood of its

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membership to each of the reference populations. Threshold score was defined as the

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minimum value that the assignment probability score must reach so an individual to be

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assigned to a reference population. Threshold score of 85 was determined for albacore,

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and 99 for anchovy. Differences in threshold scores used are due to distinct genetic

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differentiation levels, since larger FST values are found for anchovy (FST = 0.013-0.270)

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than for albacore (FST = 0.007-0.051) populations. Finally, an individual was assigned


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to a reference population if its assignment probability score was higher than the

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threshold score, on the contrary, the individual was considered as not assigned.

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Final traceability tool (selected SNP panel) for each species was the one that better met

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two criteria: (1) the highest proportion of assigned individuals (assignment percentage),

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and (2) the highest proportion of correctly assigned individuals (accuracy).

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RESULTS

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Definition of populations and geographic origins

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The traceability tools were designed taking into account population genetic structure of

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each species, and validated according to its objective: assigning individuals to their

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origin. According to population genetic structure, for albacore three origins were

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considered: Indo-Pacific, Atlantic and Mediterranean; and for European anchovy, four

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origins were considered: E-Atlantic, NW-Mediterranean, Biscay-offshore and Biscay-

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coastal.

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Design of albacore and anchovy reduced SNP panels

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Albacore and European anchovy show two levels of population genetic structure, with

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some highly differentiated populations (albacore FST > 0.033; anchovy FST > 0.097), but

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others genetically close (albacore FST < 0.025; anchovy FST < 0.030) (Figure 1).

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For species identification, two mitochondrial SNPs for each species, already described

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in previous patents30,31, were included in present traceability tools (Figures 2A and 3A).

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Albacore origin traceability panels consisted of 32 and 48 SNPs. In these panels, almost

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all the SNPs markers were new with respect to the previous patent30. Only 2 SNPs in the

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32 SNPs panel (rs193631170 and rs193631188), or 3 SNPs in the 48 SNPs panel 8 ACS Paragon Plus Environment


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(rs193631161, rs193631170 and rs193631188) had been also used in the previous

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albacore traceability patent30 (File S1). The 30% of SNPs from each panel were

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powerful markers for differentiating the two genetically most distant groups:

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Mediterranean and Atlantic/Indo-Pacific (FST = 0.035) (Figures 1A and 2B). Then, 70%

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of SNPs were powerful markers to distinguish between Atlantic and Indo-Pacific

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origins (FST = 0.017) (Figures 1A and 2C).

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Anchovy SNP panels included 32 and 48 SNPs. The 20% of SNPs from each panel

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were powerful markers for distinguishing the two genetically most distant groups: E-

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Atlantic/Biscay-coastal and NW-Mediterranean/Biscay-offshore (FST = 0.096) (Figures

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1B and 2B). Then, 30% of SNPs were powerful markers to discriminate between E-

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Atlantic and Biscay-coastal populations (FST = 0.060) (Figures 1B and 3C). Finally,

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50% of SNPs had power enough to distinguish the genetically closest population pair of

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NW-Mediterranean and Biscay-offshore (FST = 0.026) (Figures 1B and 3C).

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The accession numbers of the SNPs included in each panel are included in File S1.

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Validation of the traceability tools

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The 32 SNPs panel for albacore traceability showed assignment percentages of 86.7%,

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55.2% and 74.2% for Mediterranean, Atlantic and Indo-Pacific origins respectively; all

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of them with total accuracy of the method. This panel was unable to assign the 27.8% of

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albacore individuals, but all individuals were correctly assigned. The 48 SNPs panel

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showed assignment percentages of 90%, 69% and 83.9% for Mediterranean, Atlantic

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and Indo-Pacific origins, respectively; all of them with total accuracy of 100%. In this

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case, only 18.9% of albacore individuals was not assigned, and again no incorrectly

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assigned individuals were found (Table 2).


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In the case of the European anchovy (Table 3), the 32 SNPs panel showed assignment

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percentages of 83.3% for the Biscay-offshore, 93.3% for the Biscay-coastal, 83.3% for

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the NW-Mediterranean, and 94.7% for the E-Atlantic origins. All the anchovy

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assignments reached total accuracy. For this panel, only 13.2% of the individuals were

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not assigned, and no incorrectly assigned individuals were found. The 48 SNPs panel

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did not change assignment percentages for NW-Mediterranean, neither for E-Atlantic,

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but higher assignment percentages were obtained for Biscay-offshore and Biscay-

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coastal origins, 92.4% and 100% respectively. Only 6.6% of individuals were not

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assigned, and no individual was incorrectly assigned.

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Traceability tool for the Bay of Biscay anchovy

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The anchovy traceability tool of 48 SNPs was tested for assigning 159 anchovy

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individuals from Bay of Biscay mixed hauls to their population (Biscay-offshore or

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Biscay-coastal). These hauls were overall composed of 85 offshore and 74 coastal

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individuals, based on hundreds of SNP markers that represent whole genome variability.

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The test resulted in an overall assignment percentage of 94.3% of the individuals. A

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total of 76 offshore (89.4%) and 73 coastal (98.6%) individuals were correctly assigned

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to their population, 10 individuals were not assigned (6.3%), and no individual was

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incorrectly assigned.

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DISCUSSION

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In this study we have developed and validated a control method (either for management

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or traceability) to be included in the management policies, or incorporated into the

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commercial sector, for conserving albacore and European anchovy species’ diversity.



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The two developed tools have been designed by selecting SNPs based on the population

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genetic structure of both species, which is essential for conservation purposes7. In

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addition, we prioritized reaching total accuracies in order to avoid false positives

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(individuals assigned to an incorrect origin). In terms of complexity, cost and time

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required to assign an individual to its origin, the tool is easy to use and cost-effective;

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with results in a 4-5-days time period by less than 10Euro per tissue sample.

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In the case of albacore, 4 genetically distinct populations have been defined:

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Mediterranean Sea, Atlantic, Indian, and Pacific Oceans34,36. The two latter populations

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are genetically so close (FST = 0.002-0.003) that differential assignment between them

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was not efficient (data not shown). However, the developed traceability tool aims to

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protect Atlantic albacore (“Bonito del Norte con Eusko Label”); therefore, Indian and

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Pacific populations were merged into a single reference population for the development

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of this tool. Consequently, three albacore reference origins were considered for

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traceability purposes: Mediterranean, Atlantic and Indo-Pacific; but the traceability tool

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developed here would strictly be applicable to the Atlantic and Mediterranean albacore

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populations.

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With regard to European anchovy samples, 4 genetically distinct populations have been

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defined:

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Interestingly, two genetically distinct anchovy populations coexist in the Bay of Biscay

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(Biscay-coastal and Biscay-offshore) and individuals from both populations are fished

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together35. However, for assessment and management purposes, the Bay of Biscay

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anchovy is considered as a single stock11,13, likely due to the ignorance or few

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knowledge about the coastal population, recently genetically described35. Therefore, this

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case is a clear example of a non sustainable fishery management, which is currently

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including two populations in a single stock47-49. For the main objective of the present

Biscay-offshore,

Biscay-coastal,

NW-Mediterranean


and

E-Atlantic.

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study, developing a traceability tool that would allow protecting Bay of Biscay anchovy

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by means of sustainable management plans, we have considered the two Bay of Biscay

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anchovy populations as different origins: coastal and offshore. The specific test for the

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Bay of Biscay anchovy indicated that the traceability tool developed in this study is

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suitable for helping to the sustainability of both populations by detecting mixed hauls50.

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The commercial fraud of “Anchoa del Cantábrico” arises from replacing individuals

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from the Bay of Biscay with individuals from the NW-Mediterranean and E-Atlantic

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populations51, which constitute big fisheries in the Iberian peninsula. According to all

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this information, four anchovy reference origins matching population genetic structure

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of the species were defined in this study: Biscay-offshore, Biscay-coastal, NW-

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Mediterranean and E-Atlantic.

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After a preliminary analysis of species identification, the selection of SNPs for

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designing the reduced panels was performed in two consecutive steps. The First-step

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discriminated the origin in a wide-scale perspective and the Second-step was a fine-

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scale origin assignment. Molecular genetic markers have performed good individual

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fish assignment in a single-step at the species level8,18-26,29. However, this is not true for

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population (or origin) assignment because of the low population genetic differentiation

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level of marine species, with significant FST values below 0.0152-55. Therefore, the

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strategy followed in the present study, where SNPs sets have been designed in a two-

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step approach, is optimum for the pursued objective.

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The present study has shown the appropriateness of the following statement: the smaller

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genetic distance between two groups, the higher number of neutral markers needed for

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discriminating them. The genetic differentiation values obtained in the present study

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point out to a lower genetic differentiation for albacore (FST = 0.007-0.051) than for

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anchovy (FST = 0.013-0.270), according to literature34-36,56. This is likely explained



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because of albacore’s higher gene flow due to its higher migratory capacity and larger

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effective population sizes (North Atlantic albacore Ne = 13,267 ± 6,049)34 (Bay of

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Biscay anchovy Ne = 6,342 ± 1,050)57. Consequently, to obtain similar results on

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geographic origin assignment, more neutral genetic markers are required for albacore

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than for anchovy: using the 48 SNPs traceability panels, 81.1% of the albacores were

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correctly assigned, whereas this percentage was 93.4% for European anchovy. In

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conclusion, the most likely reason of assignment percentage difference is the genetic

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differentiation level differences between both species.

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Assignment percentage and accuracy are the two parameters that determine the quality

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of any traceability tool. Based on our results, the sets consisting of 48 SNPs were

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selected as suitable traceability tools for albacore and anchovy; with one exception:

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NW-Mediterranean and E-Atlantic anchovy origins, which show the same results for

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the two SNPs panels tested. Therefore, for traceability purposes focused on these two

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origins authentication the 32 SNPs panel should be used.

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We aimed to produce SNP panels with high statistical power, but also sufficiently small

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in SNP number to be time and cost-effective tools. Reaching total accuracies in order to

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avoid false positives (incorrectly assigned individuals) was also a priority. This is an

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essential rule for performing a reduced SNP panel; however, obtaining high individual

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assignment percentages (with total accuracy) for fish populations with low genetic

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differentiation levels is a big challenge. For cod species, 8 SNPs are able to correctly

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assign 98% of individuals from two geographic origins (FST = 0.07-0.51)8. There are

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other examples such as hake (FST = 0.08-0.29), herring (FST = 0.01-0.19), or sole (FST =

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0.01-0.05) populations, for which 13, 32 and 50 SNPs, respectively, are necessary to

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correctly assign 93-100% of individuals to their geographic origins8. These assignment

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percentages are much higher than the one obtained in the present study for albacore


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(81.1% albacore individuals correctly assigned using 48 SNPs) but similar to the one

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obtained for anchovy species (93.4% anchovy individuals correctly assigned using 48

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SNPs). These differences are likely due to the lower genetic differentiation levels

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observed for albacore and some anchovy populations compared to those from cod, hake,

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herring and sole populations: FST = 0.02 between Indo-Pacific and Atlantic albacore

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populations, FST = 0.03 between Bay of Biscay offshore and NW-Mediterranean

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anchovy populations, and FST = 0.05 between E-Atlantic and Bay of Biscay coastal

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anchovy populations. In any case, it must be taken into account that for obtaining those

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results in cod, hake, herring and sole8, neither validation step was carried out nor were

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blind samples used.

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Results in the present study have overall improved the outcomes of the existing patents

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to assign albacore30 and anchovy31 individuals to their origin (Tables 2 and 3). First, the

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percentage of assigned individuals has been improved in all cases; however, the number

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of not assigned individuals is still notable (18.9% for albacore and 13.2% for anchovy).

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But this is a basic problem because improvements on accuracy are at the expense of

331

assignment percentage. In this study we have given priority to accuracy instead of

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assignment percentage, therefore, high threshold score have been considered. In fact, in

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this study we have reached accuracy of 100% for all assignments. Second, related to the

334

previous, while the existing patents reached accuracies of 91.9% for albacore and 96.5%

335

for anchovy, the present tools reached total accuracies (100%) in all cases. Finally, the

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anchovy traceability tool has incorporated the coastal population from the Bay of

337

Biscay, which was neglected in the existing patent. This addition has been essential for

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the successful results obtained and also for the properness of the design and future

339

application of the traceability tool. In sum, designed traceability tools provided

340

accuracies of 100% for all origin assignments and good assignment percentages in the



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validation step (most of them above 80%), significantly improving results from the

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already existing patents. The higher assignment power obtained in the present study

343

might be due to: (1) the higher threshold score applied in the case of anchovy (99

344

compared to 95 threshold score of the existing patent) that minimize type I errors or

345

false positives, (2) the higher number of reference samples, that define a more accurate

346

reference allele frequency spectrum, and more importantly, (3) the nature of the markers

347

used, which are gene associated; this fact makes easier to find adaptive markers among

348

them that have population specific or diagnostic alleles. Although Atlantic albacore

349

percentage of correctly assigned individuals was improved, this population was the sole

350

case for which assignment percentage was lower than 80%. Future studies should make

351

a special effort on discovering more powerful genetic markers to get a better

352

discrimination (increasing assignment percentages) of Atlantic albacores. In any case,

353

the results of this study are overall satisfactory, including this latter case because while

354

increasing assignment percentages is important, reaching total accuracies is mandatory

355

in order to avoid false positives, which are not allowed for detecting commercial fraud

356

(false eco-certification) in a court of law.

357 358 359

ACKNOWLEDGEMENT

360

Authors are grateful to the following institutes and people for sampling assistance:

361

Galway-Mayo Institute of Technology (GMIT, Ireland), Instituto Español de

362

Oceanografía (IEO, Spain), ICCAT (International Commission for the Conservation of

363

Atlantic Tunas), E. Jimenez, M.A. Pardo, M. Santos, K. Escuredo, I. Fraile, I. Arregi, J.

364

Lopez, J. Filmarter, J. Areso, D. Brophy, K.Schaefer, D. Fuller, and V. Allain. Finally,


Page 16 of 33

Page 17 of 33


365

the authors thank for technical and human support provided by Sequencing and

366

Genotyping SGIker unit of UPV/EHU and European funding (ERDF and ESF).

367 368 369

FUNDING SOURCES

370

This work was supported by the Ministry of Science and Research of the Government

371

of Spain (grant number MICINN CTM2009-13570-C02-02); the Basque Government

372

(grant numbers 351BI20090047, SPE10UN92 and IT558-10); the Foundation “Centros

373

Tecnológicos Iñaki Goenaga” and the University of the Basque Country, UPV/EHU

374

(grant number 3571/2008). The funders had no role in study design, data collection and

375

analysis, decision to publish, or preparation of the manuscript. The authors declare no

376

competing financial interest.

377 378 379 380

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Page 26 of 33

587

TABLES AND ARTWORK

588

Figure captions

589

Figure 1. Neighbor-Joining trees for albacore (A) and European anchovy populations

590

(B). In each branch, information of population pairs’ differentiation level (FST value),

591

and percentage of selected SNPs is included. Numbers of selected SNPs in the 32 and

592

48 SNP panels for are indicated between brackets and separated with a comma.

593

Figure 2. Descriptive diagram of the SNP selection and design of traceability panels for

594

albacore. Numbers above and below tree branches correspond to the number of SNPs

595

that constituted the 32 and 48 SNPs traceability panels, respectively.

596

Figure 3. Descriptive diagram of the SNP selection and design of traceability panels for

597

anchovy. Numbers above and below tree branches correspond to the number of SNPs

598

that constituted the 32 and 48 SNPs traceability panels, respectively.

599 600

Tables

601

Table 1. Sample number: numbers of the samples that have been included in the present

602

study according to their nomenclature in Laconcha et al.34 for albacore, and in Montes et

603

al.35 for anchovy. Number of albacore and European anchovy individuals included in

604

the reference (N reference) and test (N test) panels. Species

Albacore

European anchovy

N reference (1-2, 5)34 Mediterranean Sea 94 34 (10, 14, 18) Atlantic Ocean 143 (20)34 Indian Ocean 18 34 (24-26) Pacific Ocean 120 Total= 370 35 (1, 4, 7, 12-18, 20-21) Bay of Biscay (offshore) 263 35 (9-10, 19) Bay of Biscay (coastal) 62 35 (31) NW-Mediterranean 23 35 (27-30) E-Atlantic 77 Population (genetics)

Sample number


N test 24 32 4 27 90 66 15 6 19

Page 27 of 33


Total =

425

106

605 606

Table 2. Albacore traceability panels, including already designed patent, with

607

corresponding assignment percentages to stock of origin. Accuracy percentages below

608

100% are indicated (in brackets). Albacore traceability panel

Number of SNPs

Mediterranean

Atlantic

Indo-Pacific

Patent30

18

50.9% (95.8%)

40.0% (82.8%)

40.0% (94.9%)

32

86.7%

55.2%

74.2%

48

90.0%

69.0%

83.9%

Present study 609 610

Table 3. European anchovy traceability panels, including already designed patent, with

611

corresponding assignment percentages to stock of origin. Accuracy percentages below

612

100% are indicated (in brackets). Anchovy Number traceability of SNPs panel Patent31 Present study

Biscayoffshore

Biscaycoastal

NWMediterranean

E-Atlantic

18

53.0% (94.4%)

-

56.5% (96.7%)

81.5% (98.4%)

32

83.3%

93.3%

83.3%

94.7%

48

92.4%

100%

83.3%

94.7%

613 614

Figure graphics



615 616

617 618


Page 28 of 33

Page 29 of 33


619 620 621 622

SUPPORTING INFORMATION

623

File S1. List of NCBI ss accession numbers of the SNP markers included in albacore

624

and anchovy traceability panels.



Figure 1. Neighbor-Joining trees for albacore (A) and European anchovy populations (B). In each branch, information of population pairs’ differentiation level (FST value), and percentage of selected SNPs is included. Numbers of selected SNPs in the 32 and 48 SNP panels for are indicated between brackets and separated with a comma. 179x160mm (96 x 96 DPI)


Page 30 of 33

Page 31 of 33


Figure 2. Descriptive diagram of the SNP selection and design of traceability panels for albacore. Numbers above and below tree branches correspond to the number of SNPs that constituted the 32 and 48 SNPs traceability panels, respectively. 139x54mm (96 x 96 DPI)



Figure 3. Descriptive diagram of the SNP selection and design of traceability panels for anchovy. Numbers above and below tree branches correspond to the number of SNPs that constituted the 32 and 48 SNPs traceability panels, respectively. 139x54mm (96 x 96 DPI)


Page 32 of 33

Page 33 of 33


TOC graphic 254x209mm (96 x 96 DPI)


Reduced Single Nucleotide Polymorphism Panels ... - ACS Publications

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