Fish and seafood traceability based on AFLP markers: Elaboration of a species database

Aquaculture 261 (2006) 487 – 494 www.elsevier.com/locate/aqua-online

Fish and seafood traceability based on AFLP markers: Elaboration of a species database Milena Maldini a , Francesco Nonnis Marzano a,⁎, Gloria González Fortes a,b , Riccardo Papa a , Gilberto Gandolfi a a

Dipartimento di Biologia Evolutiva e Funzionale, University of Parma, Parco Area delle Scienze 11/A, 43100 Parma, Italy b Departamento de Genética, University of Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain Received 8 March 2006; received in revised form 10 July 2006; accepted 10 July 2006

Abstract Several sociological, health and conservation arguments request a correct labelling of seafood products. Nowadays, molecular genetics is a useful tool for food chain traceability, particularly in regards to species identification. Among the variety of PCRbased molecular markers, AFLPs (Amplified Fragment Length Polymorphisms) have recently been used to investigate genomes of different complexities. This paper assesses the potential use of the AFLP technology to determine fish and seafood species in processed commercial products and domestic stocks. In particular a species database of fish, molluscs and crustaceans has been created with the aim to identify species of origin of seafood products by previously defined AFLP patterns. Different EcoRI and TaqI primer combinations were selected from 20 screened combinations in relation to the total number of detected fragments and polymorphic ones. Most informative combinations were E32/T32, E32/T33, E33/T33, E33/T37, E33/T38, E40/T33, E40/T37, E42/T32, E42/T37. The comparison of informative markers between unknown frozen or fresh products and reference samples has enabled the accurate identification of 32 different species. The taxonomic characterization has been performed either at the species or at the population level depending on the number of available individuals. AFLP variation at the population level is particularly helpful for the stock traceability of domestic strains. Size homoplasy was also investigated in one species to assess the rate of nonhomologous comigrating fragments and to detect additional polymorphic markers to be used in stock identification. Results of Band Sharing Index (BSI) and percentage of polymorphic fragments are presented and are discussed in relation to the wide applicability of AFLPs both for fish and seafood safety and authenticity testing in such fields as food traceability and restocking management. The database, available upon request at [email protected], will be continuously updated. © 2006 Elsevier B.V. All rights reserved. Keywords: Molecular biotechnology; Fingerprinting; Food safety; Traceability; Authenticity testing; Homoplasy

1. Introduction Several sociological, health and conservation arguments request the correct labelling of seafood products. ⁎ Corresponding author. Tel.: +39 521 905643; fax:+39 521 905657. E-mail address: [email protected] (F. Nonnis Marzano). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.07.010

The frequent practice of mislabelling involves such questions as truth in advertising, species substitution, consumer protection and management of depleted stocks and their monitoring (Marko et al., 2004). It is crucial that products be identified and examined in each step from fishing area and fish farms to trade and selling (Asaro, 2004). The precautionary measures are

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necessary due to the fact species substitution of food fish occurs frequently, particularly for imported products which are not recognizable by sight and are indistinguishable on the morphological base after processing and freezing. A recent paper by Marko et al. (2004) showed that some three-quarters of the fish sold in the U.S. such as the red snapper Lutjanus campechanus, belong to another species. Nowadays, fishery chain traceability is the most important tool for fish food safety and for consumers protection (Borresen, 2004). Innovative and safe technologies are therefore necessary to assess species identification and authenticity testing. Among the variety of methods which are able to identify commercially imported fish and seafood species, molecular biotechnologies are becoming more widely utilized and are gaining increased attention (Lockely and Bardsley, 2000). In the past, authenticity testing of fish products has been frequently carried out using protein-based techniques (Lundstrom, 1980; Rehbein, 1990; Lockely and Bardsley, 2000; Sussi et al., 2002). However, biochemical methods frequently do not accomplish 100 percent accuracy and protein patterns have limited identification power. As a result, nucleic acid analytic methods have become popular for species identification because of their specificity and sensitivity (Allmann et al., 1993; Carrera et al., 1998; Sotelo et al., 1993). The extension of these methods to the traceability of domestic stocks has been suggested (Policansky and Magnuson, 1998). Microsatellites would potentially offer a high diagnostic power. However, they require the previous knowledge of the species dealing with. MtDNA is nearly ideal. While for mtDNA, there are universal primers and DNA sequence data which can discriminate between closely related species, mtDNA is probably not polymorphic enough to distinguish stocks. The choice of a specific molecular marker must corespond to the specific biological question (Parker et al., 1998). Among the vast array of Polymerase Chain Reaction (PCR)-based markers, AFLPs (Amplified Fragment Length Polymorphisms) are gaining increasing attention among animal geneticists (Voss et al., 2001; Watanabe et al., 2004; Papa et al., 2005). This technique is based on the PCR amplification of restricted fragments ligated to synthetic adapters and then amplified using primers which carry selective nucleotides at their 3′ ends (Vos et al., 1995). AFLP generates hundreds of informative genetic markers that increase the probability of detection of species-specific and population-specific polymorphisms (Gomez-Uchida et al., 2003; Mickett et al., 2003; Papa et al., 2003; Liu and Cordes, 2004). Additional benefits of AFLPs over other techniques are referred to their potential power for stock identification, and do not require any

upfront knowledge of the species dealing with. Due to their high rate of polymorphisms, AFLPs are helpful to differentiate inbred populations and domestic strains. Among vertebrates, Razzoli et al. (2003) demonstrated the power of discrimination and the suitability of AFLPs to detect genetic differentiation at strain level in a group of inbred domestic rodents. Unfortunately, the same approach and the application of these markers to fish and other aquatic taxa are still limited. For this reason, an innovative approach based on AFLP markers used to assess species identification and authenticity testing in fish and seafood samples, is presented. We discuss the suitability of the AFLP fingerprinting technique for classification at species level of commercial products which have been imported from various countries and are not classifiable on a morphological basis. The results have been organized with the aim of creating an AFLP database of reference species which is useful in order to identify unknown commercial fish and seafood products. The analytical approach is presented in conjunction with the effectiveness of Genographer elaboration software in order to define diagnostic AFLP markers among different species. The investigation has also been extended to the molecular characterization of domestic strains of salmonid, turbot and crustacean to define levels of AFLP variability to be used for the traceability of domestic stocks. In relation to this, size homoplasy was also investigated according to O'Hanlon and Peakall (2000) to enlarge the set of markers to be used within the same turbot brood. Size homoplasy refers to electromorphs that have identical size but are not identical in their sequences (Estoup et al., 2002; Vekemans et al., 2002). They therefore represent a source of hidden variability. This investigation is the first attempt in Europe to optimize innovative technologies based on AFLPs to be applied to food chain traceability and frauds detection of commercial fish and seafood from individuals to species. 2. Materials and methods 2.1. Samples Samples of different unknown fish and seafood species were collected from frozen processed panels imported by Italian fish-trading factories. Whole unprocessed fish, molluscs and crustaceans which were used as reference samples, were either delivered by the same trading companies in the case of foreign species or were collected in natural waters and aquaculture plants in the case of autochthonous Mediterranean ones. Reference fish were classified on the basis of morphological characters before genetic analyses. To assess stock traceability, samples of

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domestic origin were analyzed for such widely cultivated taxa as salmonids, turbot, mussels and crayfish. The entire set of samples was representative of different freshwater and seawater areas of different countries, private hatcheries or fish farms. In Table 1 a description of the samples, their origin and the number of analyzed individuals for each species is reported. A total number of 409 individuals belonging to 32 different species were analyzed (Table 1).

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case of cultured fish and live crayfish, fin tissues and fragments of pleiopods were respectively collected from live samples. The soft part of molluscs was collected far from possible sources of endogenous and exogenous contamination. DNA was extracted either according to the classical SDS-proteinase K and phenol–chloroform technique described by Moore (1999) or alternatively by means of the Aquapure genomic DNA kit (Biorad). DNA quality was assessed by means of 1% agarose gel in TAE buffer.

2.2. DNA extraction and purification 2.3. Production and scoring of fluorescent AFLP markers High molecular weight genomic DNAs were extracted and purified from ethanol-fixed muscle tissue or fin fragments. Muscle tissues were collected from frozen samples (−20 °C) and were placed in ethanol before melting. In the

Between 400 and 700 ng of genomic DNA were first digested 1.5 h at 65 °C in a 25 μl volume containing 5 U of TaqI endonuclease, 1X “one phor all buffer plus”

Table 1 List of investigated species with area of fishing and country of purchasing Common name

Scientific name

n

Origin

Area

Country

Arctic charr Arctic charr Arctic charr Brook trout Rainbow trout Brown trout Brown trout Sea trout Chum salmon Grey mullet Thinlip mullet Golden Grey mullet Flathead mullet Thicklip grey mullet Argentine hake Shallow water Cape hake European seabass Gilthead seabream European eel Smooth hound Turbot Swordfish Striped bass European squid Wellington flying squid Common octopus Common cuttlefish Green Lipped Mussel Common mussel Manila clam Oyster Prawn Tiger prawn Red crayfish Italian crayfish

Salvelinus alpinus Salvelinus alpinus Salvelinus alpinus Salvelinus fontinalis Oncorhynchus mykiss Salmo macrostigma Salmo trutta Salmo trutta Oncorhynchus keta Liza saliens Liza ramada Liza aurata Mugil chephalus Chelon labrosus Merluccius hubbsi Merluccius capensis Dicentrarchus labrax Sparus auratus Anguilla anguilla Mustelus punctulatus Scophthalmus maximus Xiphias gladius Morone sp. Loligo vulgaris Nototodarus sloanii Octopus vulgaris Sepia officinalis Perna canaliculis Mytilus galloprovincialis Tapes philippinarum Crassostrea gigas Palaemon elegans Penaeus monodon Procambarus clarki Austropotamobius pallipes

15 40 25 10 5 30 30 5 2 20 20 20 20 15 5 5 10 10 2 5 20 3 10 5 5 5 5 2 10 10 10 5 5 10 10

Domestic Domestic Domestic Domestic Domestic Domestic Domestic Natural Natural Extensive farming Extensive farming Extensive farming Extensive farming Extensive farming Natural Natural Extensive farming Extensive farming Extensive farming Natural Extensive farming Natural Domestic Natural Natural Natural Natural Natural Extensive farming Extensive farming Extensive farming Natural Extensive farming Natural Natural

Bodensee S. Wolfgangsee Morgex Hatchery Hatchery Hatchery Hatchery Tyrrhenian Sea Pacific Ocean Adriatic lagoons Adriatic lagoons Adriatic lagoons Adriatic lagoons Adriatic lagoons Atlantic Ocean Atlantic Ocean Tyrrhenian Sea Tyrrhenian Sea Tyrrhenian Sea Tyrrhenian Sea Atlantic Ocean Mediterranean Sea Hatchery Tyrrhenian Sea Pacific Ocean Mediterranean Sea Adriatic Sea Pacific Ocean Adriatic lagoons Adriatic lagoons Adriatic lagoons Adriatic lagoons Pacific Ocean Po river Po river

Germany Austria Italy Italy Italy Italy Denmark Italy USA Italy Italy Italy Italy Italy Argentine Argentine Italy Italy Italy Italy Spain Italy Italy Italy New Zealand Morocco Italy New Zealand Italy Italy Italy Italy Indonesia Italy Italy

n = number of analyzed samples.

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(Amersham Pharmacia Biotech), 50 ng/μl BSA (Bovine Serum Albumin) and 5 mM DTT (dithiothreitol). Thereafter, a solution containing EcoRI (5U) and equal proportions of DTT, BSA and buffer composition were added to attain a final volume of 40 μl and the restriction continued at 37 °C for 2 h. Ligation of synthetic adapters to restriction fragments was carried out for 16–18 h at 16 °C adding to the 40 μl restriction solution 10 μl of a solution containing 1 U of T4 DNA Ligase (USB Cleveland, Ohio), EcoRI adapters (5 pmol), TaqI adapters (50 pmol), 1 mM adenosine triphosphate (ATP), 5 mM DTT, 50 ng/μl BSA in “one phor all buffer plus”. Following restriction-ligation, the 50 μl reaction volume was diluted 4 fold in sterile apirogen water and processed further. Preamplification with primers carrying one selective nucleotide was carried out in 50 μl volumes containing 15 μl of diluted ligated DNA (about 25–50 ng of template DNA), equal quantities of EcoRI and TaqI primers (75 ng), 0.2 mM each of the four dNTPs, 1 U of Taq polymerase (Roche Molecular Biochemical, Mannheim Germany), 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl. The PCR reaction was carried out at the following amplification conditions: denaturation for 30 s at 94 °C, annealing for 1 min at 56 °C, extension for 1 min at 72 °C (30 cycles), followed by 7 min at 72 °C to complete partial amplifications. Pre-amplifications were diluted 30 fold with sterile apirogen water, and 5 μl of diluted product were used for selective amplification in 20 μl of PCR reaction mix containing EcoRI labelled (Cy5) primer (10 ng), unlabelled TaqI primer (30 ng), 1 U of Taq polymerase (Roche Molecular Biochemical, Mannheim Germany), 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.2 mM each of the four dNTPs. For TaqI primers with three selective bases the PCR amplification profile was the following: an initial 2 min at 94 °C denaturation; 13 cycles of touch down amplification consisting in 94 °C 30 s denaturation, 65–56 °C 30 s (0.7 °C decrease at each cycle) annealing, 72 °C 2 min extension; 30 additional cycles at 94 °C 30 s, 56 °C 30 s, 72 °C 2 min; a final extension of partial amplifications at 72 °C for 7 min. To avoid mismatch annealing described by Vos et al. (1995) when primers with four selective bases are employed, more stringent PCR conditions were used with annealing temperatures from 70 to 61 °C decreasing 1 °C each cycle (Krauss, 1999; O'Hanlon and Peakall, 2000). A 1.7 μl volume of PCR product and 0.3 μl of DNA internal size standards (CEQ DNA Size standard — 600 Beckman — Coulter, Fullerton, CA) were added to 40 μl of deionized formamide (J.T. Baker, Phillipsburg, NJ).

Samples were then loaded into the “CEQ™ 8000 DNA Analysis System” (Beckman Coulter). Running conditions for capillary electrophoresis, raw data elaboration and reproducibility are reported in Papa et al. (2005). Analysed data was exported to Genographer software (Vers.1.6.0, Benham J.J., Montana State University 2001), for single specimen comparison and scoring. Genographer allows the construction of a virtual gel (Fig. 1) with bands shaped on the base of peak height, resolution and mobility and permits a thorough analysis of single fragments. Genetic variation of each group was assessed on the basis of estimated proportion of polymorphic fragments (loci), that is the ratio between variable and total fragments (95% criterion). In addition, considering pairwise comparisons of all fragments among different groups to define the degree of their similarity, the Band Sharing Index (BSI) was calculated by means of the R-package (available at http://www.r-project.org): BSIxy ¼ 2nxy =nx þ ny Lynch (1990) where nx is the number of fragments in the sample x, ny is the number of fragments in the sample y, and nxy is the number of shared fragments between x and y. 3. Results A total of 20 primer pairs were randomly screened in five different species belonging to phylogenetically distant taxa: a bony fish, a cartilagineous fish, a cephalopod mollusc, a bivalve mollusc and a crayfish. Among tested combinations in 5 individuals for each species, primer pairs E32/T32, E32/T33, E33/T33, E33/T37, E33/T38, E33/T49, E40/T33, E40/T37, E42/T32, E42/T37 were chosen according to the bands resolution, their size range, the total number of fragments and polymorphisms. These ten primer combinations were then used to obtain species-specific AFLP profiles in 32 different species (Table 1). Fragments in the size range 70–600 bp were scored using the Genographer detection method (Papa et al., 2005). The total number of fragments varied between 14 and 90, and major differences were observed in different systematic groups. The total number of bands considering both monomorphic and polymorphic fragments was over 40 in each one of these combinations. The comparison between unknown samples and previously genotyped reference samples was performed in relation to band sharing within 2 different primer combinations. It must be noted that the possibility of using even monomorphic bands (66–100% per combination) allowed for comparisons based on a limited number of primer pairs. The Genographer analysis of AFLPs profiles

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Fig. 1. Comparison of AFLP \ Genographer profiles of reference samples (A, Merluccius hubbsi; B, Merluccius capensis; C, Oncorhynchus keta; D, Oncorhynchus mykiss; E, Salvelinus alpinus; F, Salvelinus fontinalis; G, Salmo trutta) and three unknown samples of each species.

among reference and unknown samples allowed the correct graphical species identification (Fig. 1) and all different species were correctly genotyped. Whenever the number of specimens from each group was consistent with fine systematic classification and population considerations, BSI and the percentage of polymorphic bands were calculated. From a taxonomy point of view, band sharing index (BSI) of the two primer

combinations analyzed in closely related samples (within species level) was in the range 97–100% with the only exception of Arctic charr (see below). BSI among different species was much lower and variable between 50 (congeneric species) and 20% (not congeneric). The BSI variation between populations which belong to the same species yet come from separate areas was determined by the AFLP profiles of a larger set of

Table 2 Levels of AFLP variability detected in fish and crayfish species with primer combinations E32/T32, E32/T33, E33/T33, E40/T33, E40/T37 Scientific name

n

Area

Total number of fragments

Polymorphic fragments

% Polymorphisms

Salvelinus alpinus Salvelinus alpinus Salvelinus alpinus Salvelinus fontinalis Oncorhynchus mykiss Salmo trutta Liza saliens Liza ramada Austropotamobius pallipes

15 40 25 10 5 30 20 20 10

Bodensee S. Wolfgangsee Morgex Hatchery Hatchery Hatchery Adriatic lagoons Adriatic lagoons Po river

220 223 223 251 182 240 205 220 212

15 27 19 12 5 21 64 82 58

6.82 12.11 8.51 4.78 2.75 8.75 31.19 37.27 27.36

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M

morphisms are reported in Table 2. The percentage of polymorphic fragments ranged from 2.75–37.27% and seemed quite affected by ecological characteristics of the species and stocking conditions. In fact, high values were determined for the two Liza species (31.19–37.27%) and the natural crayfish population (27.36%), while low variability emerged in salmonid species subject to continuous inbreeding (2.75–12.11%). Although detected on a limited number of specimens, the low value (2.75%) of the hatchery reared individuals of O. mykiss may presumably be put in relation to possible allelic loss and reduced molecular variability (consanguinity) due to the particular farming conditions (full biological cycle in captivity without any outbreeding). Using AFLP technology to evaluate genetic diversity could result in an underestimation of such variability because of the effect of size homoplasy (Vekemans et al., 2002). For this reason, the presence of non-homologous fragments among co-migrating AFLPs generated with primer combination E33/T49 was verified in a turbot (S. maximus) brood. Considering only the strictly monomorphic bands after critical evaluation of band intensity and resolution, size homoplasy was determined following the approach based on 4-nt selective primers proposed by O'Hanlon and Peakall (2000). Using an additional selective nucleotide in the T49 primer (TaqI-CAGA, TaqI-CAGC, TaqI-CAGG, TaqI-CAGT) the frequency of non-homologous co-migrating E33/T49 fragments was assessed in the size range 60–450 bp (Table 3). Ten out of 31 (32%) monomorphic fragments resulted variable after adding an additional selective nucleotide (either A, C, G and T) to the E33/T49 primer pair. Most of this variability (70%) was detected in the small fragments (equal or less than 300 bp) in agreement with Vekemans et al. (2002) who proposed that size homoplasy is more frequent in small fragments.

M M

4. Discussion

primers: the primer combinations E32/T32, E32/T33, E33/T33, E40/T33, E40/T37. The results determined in different hatcheries and natural stocks of S. alpinus were 0.62–0.81. The high degree of differentiation of BSI in Arctic charr populations or stocks is due to their geographical separation (land-locked populations). On the contrary, the results determined in the diadromous L. saliens were in the range 0.93–0.95. Population differentiation was performed only when a consistent number of individuals was available for each geographical group. To assess AFLP diversity in farmed fish and crayfish in terms of percentage of polymorphic fragments, different populations were analyzed using the above cited 5 primer combinations. In particular, 2 extensively aquacultured populations of L. ramada and L. saliens, a variety of both intensively and extensively farmed salmonids, a stocked turbot family and a natural population of the endangered Italian crayfish were considered. Results of AFLP polyTable 3 Frequency of possible homoplasic fragments in turbot determined for the selective primer combination (E33/T49) Fragment size

E33/T49

66 74 84 91 96 106 113 118 125 137 140 144 150 175 186 192 235 239 248 257 277 308 328 341 350 368 381 388 424 433 449

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

tCAGA

tCAGC

tCAGG

tCAGT

M M M M

M P

M P M M

P

M M

M M P M M P

M M

M

P M

M M

M M P M M M M

M M M P

P M M

M

M = Monomorphic; P = Polymorphic.

M M M M

M M P M

AFLPs appear to be extremely informative markers for species identification and authenticity testing of unknown fish and seafood species (Yu and Guo, 2004). With respect to previous investigations based on mitochondrial markers (Carrera et al., 1998; Lockely and Bardsley, 2000), AFLPs are more polymorphic and combine universal applicability with high power of discrimination. The consistent number of fragments obtained for each primer combination and the high reproducibility (Papa et al., 2005) of the fingerprinting pattern within single species, has enabled the identification of exact correspondence between unknown samples and reference specimens previously classified morphologically. Each

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single primer combination has allowed direct graphical determination among phylogenetically distant species on the basis of virtual AFLP bands reported on the Genographer software. Compared to traditional methods, the use of capillary systems and automated analysis increases data throughput and scoring reliability. In general, the most abundant fragments were detected for the combinations E32/T32, E32/T33, E33/T33, E40/ T33, E40/T37. The number of primer combinations has to be increased as the taxonomic distance reduces but the possibility of using also monomorphic fragments allows the correct diagnosis at species level with a limited number of primer pairs whenever proper reference samples are available. For this reason, AFLP profiles have been obtained in this work on a certain number of teleosts, molluscs and crustaceans. Thirty two species were genotyped with the aim of starting a proper AFLP reference database. This approach appears to be a powerful tool for species traceability of processed freshwater and marine food samples which are not classifiable by sight. From an analytical point of view, two critical points must be considered in the application of this methodology: first, the DNA quality is a limiting point in the application of AFLPs since it can be partially degraded particularly in frozen imported stocks from overseas, yet another critical factor is the availability of reference AFLP profiles to be compared to unknown material and their analytical reproducibility. The first limiting step can be managed following the improvements reported in a protocol elaborated in our previous paper (see Papa et al., 2005). For the second aspect, the elaboration of a species-specific database of AFLP-fingerprinting profiles based on most informative primer combinations represents the starting point to design an effective strategy to implement AFLP testing. Our preliminary database for commercially important species of the Mediterranean area actually exhibits 32 species: 20 among freshwater and seawater fish, 4 crustaceans and 8 molluscs. Most of the considered species have a relevant commercial value either as food sources or for natural restocking practices. The database will be continuously updated with AFLP profiles determined in additional species of important commercial value and additional data concerning not only species traceability but also genetic differentiation at population and lower level. The database and experimental support are available upon request at: [email protected]. Traceability in aquacultural practices should not be utilized only for food purposes but it should also be extended to the correct identification of strains and genetic lineages. Although mtDNA is considered almost ideal for species identification (Marko et al., 2004), it is probably not sensitive enough to distinguish stocks. One of the

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potential benefits of AFLPs over other techniques is their sensitivity for stock identification without any upfront knowledge of the species. Definition of specific AFLP diagnostic bands at strain or stock level is useful to discover hybridization processes or to assess the level of genetic diversity in a population. In the case of large animal stocks statistical support can also be obtained with the determination of Band Sharing Index (BSI) or percentage of polymorphic fragments to define population differentiation. This aspect should not be avoided in future aquacultural activities as it should be an additional value of restocking practices based on a correct ecological management, particularly of land-locked salmonid populations (Policansky and Magnuson, 1998). To work at stock or strain level, it is necessary to evaluate the possible underestimation of AFLP variability due to the effect of size homoplasy (Vekemans et al., 2002). The extension of primer combinations to 4-nt selective primers is a helpful device to increase diagnostic markers, as well as to correct evaluation of genetic diversity within species and populations. It is noteworthy to observe that homoplasies are rarely investigated or considered in AFLPs profiles (O'Hanlon and Peakall, 2000). In this investigation homologous co-migrating fragments were analyzed in a farmed turbot family by adding an additional selective nucleotide to one specific primer combination. In this way, at least one third of fragments previously detected as monomorphic were polymorphic in at least one of the four nt-selective primer combinations. These polymorphisms hidden by co-migration of non-homologous fragments with equal size represented an increment in the estimation of genetic diversity within a population and increased the number of diagnostic markers potentially useful to assess differences among different cultivated turbot breeds (work in progress). Acknowledgements The research was partly financed in the framework of an EU-Spinner joint program on innovative tools for food traceability. The authors are thankful to P and A Seafood (Grottamare, Italy) for the delivery of foreign frozen samples and Kristin Brabender of Columbia University for revision of the English language. The manuscript has benefited from the suggestions of Prof. O. McMillan of Duke University. References Allmann, M., Candrian, U., Höfelein, C., Lüthy, J., 1993. Polymerase chain reaction (PCR): a possible alternative immunochemical methods assuring safety and quality of food. Z. Lebensm.-Unters. Forsch. 196, 248–251.

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