Aquaculture Research, 2006, 1^6 doi:10.1111/j.1365-2109.2006.01524.x

Microsatellite variability in natural populations of the blackspot seabream Pagellus bogaraveo (Bru¨nnick, 1768): a database to access parentage assignment in aquaculture

Andreia Lemos1, Ana Isabel Freitas2, Ana Teresa Fernandes2, Rita Goncalves2,Jose¤Jesus1, Carlos Andrade3 & Anto¤nio Brehm2 1Department of Biology,University of , Funchal, 2Human Genetics Laboratory,University of Madeira, Funchal, Portugal 3Centro de Maricultura da Calheta, Calheta, Portugal

Correspondence: A Brehm, Human Genetics Laboratory, University of Madeira, Campus of Penteada, 9000-390 Funchal, Portugal. E-mail: [email protected]

Abstract ing of natural populations is not now su⁄cient to supply the global demand for most species. Thus, the Eight microsatellite loci previously reported were as- need for aquaculture production is in continuous de- sessed for their utility in parentage assignment in 96 mand, which implies the development of ¢sh farming individuals belonging to natural populations of the programmes and the establishment of successful blackspot seabream Pagellus bogaraveo (Brˇnnick, breeding strategies.The sustainability of these strate- 1768) from the Mediterranean and Northeast Atlantic gies requires a precise knowledge of the genetic regions. At the mtDNA level, no di¡erentiation was variability of wild populations from which indivi- found between these two regions but based on micro- duals are selected to start breeding programmes satellite data an overall discrete genetic di¡erentiation (Knibb, Gorshkova & Gorshkov 1998). Knowing indi- is perceivable between the two regions separated by vidual genetic pro¢les is therefore essential if trait se- the Strait of Gibraltar.The number of alleles per locus lection (e.g. growth rate, disease resistance, age at ranged from 8 to 30. A database constructed with al- maturity) is an objective of such breeding pro- lele frequency data from six populations was used grammes and also the only way a producer has to in a simulation parentage assignment test using the avoid e⁄ciently, high rates of endogamy (inbreeding software CERVUS. The test showed that the number of and reduced genetic variation), which will be impor- markers used is enough to perform parentage assign- tant for long-term sustainable production (Olesen, ments with real data. The polymorphic information Groen & Gjerde 2000; Olesen, Gjedrem, Bentsen, content for each locus was very high (mean value of Gjerde & Rye 2003). Forensic identi¢cation of speci- 0.849), with a total exclusionary power of 0.9995. In mens usually requires a comparison of a known sam- summary, seven of the eight microsatellites analysed ple with a sample of unknown origin in order to proved to be su⁄cient and powerful tools for parent- quantify the probability of the unknown sample to age assignment in hatcheries and the allele frequency have originated from the individual donating the data given here can be used to perform pedigree ana- known sample (Hammond, Jin, Zhong, Caskey & lysis against which real data may be tested. Chakraborty1994). Much work has focused on the utility of molecular Keywords: Pagellus bogaraveo, microsatellites, ge- pedigree analysis in aquaculture (Norris, Bradley & netic structure, parentage, database Cunningham 2000) or pedigree tracking in the selec- tion of broodstock management procedures (Estoup, Introduction Gharbi, Sancristobal, Chevalet, Ha¡ray & Guyomard The worldwide exploitation of ¢sh wild stocks has in- 1998; Evans,White & Elliot 2000;Wilson & Ferguson creased in the last decades to the extent that harvest- 2002). In those cases where it is not possible to

r 2006 The Authors Journal Compilation r 2006 Blackwell Publishing Ltd 1 Microsatellite variability in Pagellus bogaraveo A Lemos et al. Aquaculture Research, 2006, 1^6 exclude all but one parent (or both), the use of likeli- P. bogaraveo populations based on microsatellite al- hood-based methods is necessary for parentage as- lele frequencies and (4) to present an allele frequency signment, and simulation procedures must be per- database to evaluate routine parentage testing data. formed to determine the signi¢cance of results (San- Cristobal & Chevalet 1997; Marshall, Slate, Kruuk & Pemberton 1998; Gerber, Mariette, Strei¡, Bode¤ne' s& Materials and methods Kremer 2000). Likelihood-based methods consider Sample collection all potential parents as possible candidates, avoiding the need to identify a known parent a priori. Microsa- Ninety-six individuals of P. bogaraveo were collected tellites have been widely used to examine breeding from four sites of the Atlantic Ocean (Ma-Madeira Is- systems in natural populations of several ¢sh species land, n 5 24; Az- Islands, n 5 20; NI-North- (Olsen, Bentzen & 1996; Fontaine & Dodson west Iberian coast, n 510 and Al-Algarve/South of 1999; Zane, Nelson, Jones & Avise 1999; Dewoody, Portugal, n 5 20), and a Mediterranean sample Fletcher, Wilkins & Avise 2000; Gerber et al. 2000; (Me 5 22) composed of12 specimens from Malta and Iyengar, Piyapattanakorn, Heipel, Stone, Howell, 10 from the Ionian Sea. Child & Maclean 2000; Norris et al.2000). Microsatel- lites have also been used in parentage assignments DNA extraction and ampli¢cation (Hatchwell, Ross, Chaline, Fowlie & Burke 2002) but always need to be ¢rst validated in wild animals and For DNA extraction, muscle samples from the dorsal their polymorphic content estimated. These molecu- region were collected and stored in 70% ethanol. In lar markers are also useful to estimate levels of gene the case of populations from Madeira and Northwest £ow and thus population relatedness (Hansen, Iberian Coast, blood was collected by punction and Kenchington & Nielsen 2001), as well as to examine maintained at 20 1C. Tissue was homogenized in strain genetic structure (Liu, Chen & Li 2005). STE 100 bu¡er (0.1M NaCl, 0.05 M Tris, 0.1M EDTA; This paper focuses on the microsatellite genetic di- pH 8.0) and10% SDS. DNAwas puried by extractions versity of the blackspot sea bream Pagellus bogaraveo with phenol, phenol/chloroform (1:1), chloroform and (Brˇnnick,1768) from the Mediterranean Sea and the precipitated with isopropanol (Sambrook, Fritsch & Atlantic Northeast region around theAzores and Ma- Manitaris 1989). After cleaning with ethanol, DNA deira. The species is a protandrous hermaphrodite was suspended in water and kept at 20 1C. A par- much in demand today for aquaculture breeding pro- tial mitochondrial cytochrome b coding region was grammes. The possible role of the Strait of Gibraltar ampli¢ed in all individuals with universal primers in promoting a genetic discontinuity of populations from Kocher, Thomas, Meyer, Edwards, Paabo and of several sparid species including P. bogaraveo was Wilson (1989). The length of the coding-region addressed by Bargelloni, Alarcon, Alvarez, Penzo, polymerase chain reaction (PCR) products was ap- Magoulas, Reis and Patarnello (2003), who found a proximately 370 base pairs (bp). Polymerase chain re- lack of variation within this species but not in other action fragments were sequenced in an ABI 310 sparid species. Recently, a study focused on popula- sequencer; ampli¢cation and sequence protocols are tions from the Azores Islands also found an absence available from the authors. Sequences were aligned of genetic diversity using mitochondrial markers and using CLUSTAL X (Thompson, Higgins & Gibson 1994) microsatellite analysis (Stockley, Menezes, Pinho & with minor adjustments made by eye. The sequences Rogers 2005). obtained were compared with two others available The objectives of the present study are: (1) to char- from GenBank (accession numbers AJ319818- acterize eight microsatellite loci that have been previ- AJ276880). ously described (Stockley, Rogers, Iyengar, Menezes, Eight microsatellites (PbMS2, PbMS4, PbMS6, Santos & Long 2000) for basic genetic parameters PBMS15, PbMS16, PbMS17,PbMS19and PBS20) were such as number of alleles and heterozygosity, (2) to ampli¢ed using the primers described by Stockley assess whether these microsatellites show enough et al. (2000), which are based on a sample from the variability in P. bogaraveo to be useful in pedigree Azores. Polymerase chain reaction conditions were analysis or parentage assignment using forensic re-designed to adapt the annealing temperatures to genetic indicators such as the polymorphism infor- our populations following di⁄culties in implement- mation content (PIC) and power of exclusion (PE), ing those described by Stockley et al. (2000). Polymer- (3) to examine the distribution of variation among ase chain reaction products were separated by 6%

2 r 2006 The Authors Journal Compilation r 2006 Blackwell Publishing Ltd, Aquaculture Research, 1^6 Aquaculture Research, 2006, 1^6 Microsatellite variability in Pagellus bogaraveo A Lemos et al. polyacrylamide gel electrophoresis and visualized by Table 1 Characterization of eight Pagellus bogaraveo micro- silver staining. Fragment sizes were estimated in satellite loci based on 96 individuals runs using DNAs with known sequences. Moreover, all individuals homozygous for a given allele at each Number Size Homozygotes of range locus were sequenced to determine the exact DNA Locus sequenced Repeat sequence alleles (bp) sequence (both in structure as well as number of re- peats) of the microsatellites. A DNA ladder made of PbMS2 32 (CTT)n — à an assortment of these homozygous fragments was 1 (CA)nTA(CA)n PbMS4 2 (CA)nT(CA)nGG(CA)n 16 162–194 constructed and used in subsequent polyacrilamide 2 (CA)nTA(CA)nTA(CA)n gel runs. The development of an allelic ladder with PbMS6 2 (CA)n 30 101–161 DNA fragments of the same size as the alleles found PbMS15 2 (CA)nGAG(TC)n 24 157–203 makes it possible to compare directly the runs using PbMS16 2 (CA)n 22 147–193 either manual or automated DNA electrophoresis. PbMS17 5 (GA)n 8 187–201 PbMS19 23 (CAG)n 8 168–189 Microsatellite PbMS2 was not used for allele compar- PbMS20 10 (GTGC)n(GT)n 20 148–188 isons because it was found that although it had the à same motif reported by Stockley et al. (2000), the se- Size range varied among specimens due to the insertion of an quence incorporates an insertion of11bp (TTATTCC- 11bp segment (TTATTCCCATC) placed within the repeat region regardless of the number of repeats present. CATC) not reported before, placed inside the repeat region immediately after the ¢rst CTT repeat. This in- sertion is widespread in all populations surveyed in formative a given genetic marker is and is described fragments with eight to 16 repeats. Thus, ampli¢ed in Bolstein,White, Skolnik and Dawis (1980). The PE fragments with the same number of repeats appear (or probability of exclusion) corresponds to the power with di¡erent lengths, depending on whether they with which a locus excludes an erroneously assigned incorporate the insertion or not, rendering its use im- individual from being the parent of an o¡spring (Weir possible at a population scale. Microsatellite PbMS4 1996). Therefore, both PIC and PE are heterozygosity had a di¡erent repeat motif from that described by indicators. Two exclusion probabilities were calcu- Stockley et al. (2000); three di¡erent motifs were lated based on the allele frequency data: PE1assumes found, each with variable number of repeats (Table genotypes are known for the o¡spring and a putative 1). All sequenced alleles were submitted to GenBank parent and, PE2 assumes that genotypes are known and have accession numbers DQ520559^DQ520585. for the o¡spring, one con¢rmed parent and a putative parent. Both PE1 and PE2 are calculated according to Jamieson and Taylor (1997). An analysis of molecu- Statistical analyses lar variance (AMOVA) was performed based on Eucli- Basic genetic parameters such as number of alleles at dean distances between haplotypes according to each microsatellite locus, percentage of polymorphic Exco⁄er, Smouse and Quattro (1992) using Arlequin loci, proportion of individual heterozygous samples (Schneider, Roessli & Exco⁄er 2000).The total genet-

(direct count heterozygosity, Ho, as well as the un- ic variation was partitioned among two groups of biased estimate, He) and population di¡erentiation individuals: Atlantic Ocean versus Mediterranean were calculated using GENEPOP V3.1D (Raymond & populations. Variance components, F-statistics and

Rousset 1995). The overall genetic diversity (HT), corresponding P-values were calculated by perform- within-population diversity (HS) and between-popu- ing 10000 permutations of the original matrix data lation diversity (DST) were also calculated. Deviation and comparing the results with the original values. from Hardy^Weinberg equilibrium per population A Mantel test available in the ARLEQUIN package and locus was calculated according to the Weir and (Schneider et al. 2000) was used to test for isolation

Cockerham (1984) FIS estimator.The signi¢cance va- by distance by providing a correlation coe⁄cient of lue of FIS was evaluated through a bootstrap techni- FST and geographic distance matrices. Microsatellite que based on random permutation of the original frequency data from all individuals were merged to a dataset, as implemented in FSTAT v.2.9.3. (Goudet single database, which was subsequently used to per- 2001). This program uses a Bonferroni’correction for form a simulation of parentage analysis for this set of all signi¢cance levels (Rice 1989). The PIC and the loci using CERVUS v2.0 using the default parameters of PE for every locus were also calculated within each the program (10 candidate parents, typing error population and overall. PIC values describe how in- 0.01%, 80% relaxed con¢dence and 95% strict con¢-

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dence, Marshall et al.1998, Slate, Marshall & Pember- Table 2 Genetic diversity (HT)ofPagellus bogaraveo and its ton 2000). Basically,this software package simulates distribution within (HS) and between populations (DST) a parentage analysis estimating the resolving power of the set of loci and the critical values of the log-like- Absolute values Relative values lihood statistic D (Delta, LOD scores). An LOD score of Locus HT HS DST HT DST zero implies that a putative father is as likely to be the true parent as any other randomly chosen one. The PbMS4 0.894 0.886 0.008 0.896 0.010 PbMS6 0.958 0.960 0.002 0.957 0.003 program allows for potential scoring errors that may PbMS15 0.939 0.943 0.004 0.938 0.005 occur in the dataset. These values can subsequently PbMS16 0.940 0.938 0.003 0.941 0.003 be used to test real data (descendants and parents), in PbMS17 0.707 0.714 0.007 0.705 0.009 a parentage analysis by evaluating the LOD scores of PbMS19 0.721 0.667 0.054 0.732 0.065 the most likely candidate parents against the likeli- PbMS20 0.924 0.900 0.024 0.929 0.029 Mean 0.869 0.858 0.011 0.871 0.013 hood of other unrelated candidate parents. CERVUS also allows the calculation of PE values according to two scenarios: PE1, in which genotypes are known (HS) and between populations (DST) (Table 2). A total for the o¡spring and a putative parent but genotypes of128 di¡erent alleles were recovered over the seven are unavailable for the known parent (one parent loci for all populations (excluding PbMS2). The num- missing), and PE2, in which genotypes are known ber of alleles per locus ranged from 8 to 30 (Table 1). for the o¡spring, one con¢rmed parent and one puta- Allele frequencies per locus in each population of tive parent (both parents genotyped). P. bogaraveo and estimated genetic variability para- meters are presented as supplementary material Results and discussion (www.uma.pt/bioarticles). All populations studied are at Hardy^Weinberg equilibrium and no genoty- Cytochrome b variability pic disequilibrium was detected among pairs of loci. Ampli¢cation of the partial cytochrome b gene frag- The observed genotypic frequencies do not di¡er sta- ment yielded a single haplotype that is similar to tistically from the values expected under HWat each those existing in the Genbank. Given the fact that locus and for each population.The high genetic diver- individuals belonged to Mediterranean and Atlantic sity explains in part the small FIS values that were locations, this result shows a remarkabley lack of var- either non-signi¢cant or changed sign among popu- iation in this mitochondrial region. This is surprising lations (Table 3). A small or negative value of FIS indi- because all individuals sequenced were wild-caught cates that the population is not subjected to pre-excluding the use of inbred specimens from inbreeding and is not subdivided (Table 3). There aquaculture facilities. This is concordant with a pre- was no correlation between geographical distances vious survey by Bargelloni et al. (2003), which found and FST values among populations (Mantel test, only marginally signi¢cant di¡erences between r 5 0.37, P40.05). Estimated genetic distances be- Atlantic and Mediterranean P. bogaraveo at the tween populations were small (average DST 5 0.013, mtDNA level. Recently,Stockley et al. (2005) reported Table 2). The AMOVA analysis con¢rms the lack of a lack of diversity in three populations from the north structure prevailing in P. bogaraveo populations. The Atlantic region. Using D-Loop sequences, all haplo- clustering of populations according to their geo- types recovered were linked by a maximum of two graphic locations yields an overall low but signi¢cant mutational steps from the putative ancestral haplo- FST value of 0.016 (Po0.0001, after 10000 permuta- type and the topology was a typical‘star-like’network tions), indicating an insipient amount of population indicative of a recently demographic expansion of a di¡erentiation.The percentage of variation attributed population, most probably from a small number of in- to di¡erences among populations within groups was dividuals (Fig. 2 from Slatkin & Hudson 1991, Stock- only 1.2%; 0.49% was explained as variation among ley et al. 2005). groups and 98.3% was attributed to di¡erences with- in populations. This shows that the populations from the Mediterranean and Atlantic regions do not di¡er Microsatellite analysis signi¢cantly and most of the variability found is due All seven microsatellite loci were polymorphic in all to within-population variation. Also, the high with- the populations studied.Table 2 shows the genetic di- in-population variation is most probably the explana- versity (HT)ofP.bogaraveo and its distribution within tion for the high gene diversity values found (Ho).The

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Table 3 Within-population genetic variation of six Pagellus bogaraveo populations based on seven loci

Population MNA Ho He FIS PIC PE1 PE2

Ma 13.57 0.816 0.873 0.067 0.839 0.9991 0.9999 Az 10.86 0.814 0.835 0.026 0.788 0.9967 0.9998 NI 9.71 0.886 0.862 0.030 0.796 0.9966 0.9998 Me 13.14 0.838 0.866 0.033 0.828 0.9987 0.9999 Al 11.86 0.864 0.864 0.000 0.826 0.9985 0.9999 Mean 0.020 0.849 0.9995 0.9998 ( 0.013)

MNA, mean number of alleles; Ho and He are the population’s observed and expected heterozigosity. FIS values follow the formulae of Weir and Cockerham (1984); PIC is the mean value over all loci. PE1 and PE2 are the total exclusion power of the ¢rst parent and the second parent respectively.

highest value of FST was recorded between the Malta Acknowledgments and the Azorean populations (F 5 0.032, ST The authors are grateful to Dr Jon Slate for comments Po0.0001) and the lower were for the pairs Ma- on CERVUS and to all those who provided us with ¢sh deira/Algarve, Northwest Iberian/Azores and Ionian specimens, and to one anonymous referee for must Sea/Azores, with non-signi¢cant P values. In the si- useful comments in an earlier version of the manu- mulation of the parentage analysis, we used the de- script. This study was partially ¢nanced by project fault values of CERVUS (10000 replicates or simulated MARINOVA, contract MAC/4.2/M11 (INTERREG). o¡spring, 10 candidate males of being the true par- ent, all loci typed and a typing error of 0.01). Given both scenarios of PE1 and PE2, our database gives References an average LOD score in the ¢rst scenario of 4.41 Bargelloni L., Alarcon J., Alvarez M., Penzo E., Magoulas A., higher than the LOD score of the next most likely Reis C. & Patarnello T. (2003) Discord in the family male 99.6% of the time (SD 51.52). In the second sce- Sparidae (Teleostei): divergent phylogeographical pat- nario, this value is1.96 (SD 51.01) 95.12% of the time. terns across the Atlantic^Mediterranean divide. Journal For each of the seven loci, the PIC value was high en- of Molcular Evolution16,1149^1158. ough for this set of loci to be considered powerful for Bolstein D., White R., Skolnik M. & Dawis R. (1980) determining parentage when using real data (PIC Construction of a genetic linkage map using restriction mean value was 0.849) with a total exclusionary fragment length polymorphisms. American Journal of power (PE) of 0.9995. Moreover, the values of PE1 Human Genetics 32,314^331. and PE2 are all above 99%, which indicates that the DeWoody J., Fletcher E., Wilkins D. & Avise J. (2000) Parentage and nest guarding in the tessellated darter individuals analysed are part of one single popula- (Etheostoma olmstedi) assayed by microsatellite markers tion (corroborating mtDNA information) and that (Perciformes: Percidae). Copeia 3, 740^747. the number of specimens analysed was enough to re- Estoup A., Gharbi K., SanCristobal M., Chevalet C., Ha¡ray P. cover most of the allelic variation of the loci analysed. & Guyomard R. (1998) Parentage assignment using mi- crosatellites in turbot (Scophthalmus maximus) and rain- bow trout (Oncorhynchus mykiss) hatchery populations. Conclusion Candian Journal of Fisheries and Aquatic Sciences 55, The present study analyses microsatellite diversity 715^725. at seven loci in six Atlantic and Mediterranean Evans B.,White R. & Elliot N. (2000) The use of microsatellite populations of P. bogaraveo. The populations show markers for parentage analysis in Australian blacklip and high levels of genetic diversity albeit low genetic dif- hybrid abalone. Journal of Shell¢sh Research19,511. Exco⁄er L., Smouse P.E. & Quattro J.M. (1992) Analysis of ferentiations, suggesting that these populations are molecular variance inferred from metric distances not isolated. This core set of seven microsatellite al- among DNA haplotypes: application to human mitochon- lele frequencies represents a powerful and e⁄cient drial DNA restriction data. Genetics131,479^491. method to use in any pedigree analysis for local Fontaine P.& DodsonJ. (1999) Ananalysis of the distribution hatcheries to quantify the number and genetic pro¢le of juvenile Atlantic salmon (Salmo salar)innatureasa of founders and can therefore be applied for both par- function of relatedness using microsatellites. Molecular entage assignment and trait selection programmes. Ecology 8,189^198.

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