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 Gonc°alves2,Jose¤Jesus1, Carlos Andrade3 & Anto¤nio Brehm2 1Department of Biology,University of Madeira, Funchal, Portugal 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-Azores Islands, n 5 20; NI-North- (Olsen, Bentzen & Seeb 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 À 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 À 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 — Ã an assortment of these homozygous fragments was 1 (CA)nTA(CA)n PbMS4 2 (CA)nT(CA)nGG(CA)n 16 162–194 constructed