Animal Cells and Systems Vol. 16, No. 1, February 2012, 50Á56 Development and characterization of microsatellite markers for an endangered species, Epinephelus bruneus, to establish a conservation program Hye Suck An*, Jae Woo Kim, Jang Wook Lee, Shin Kwon Kim, Bae Ik Lee, Dae Jung Kim and Yi Cheong Kim New Strategy Research Center, National Fisheries Research and Development Institute, Busan, 619-705, Korea (Received 29 April 2011; received in revised form 5 July 2011; accepted 19 July 2011) Kelp grouper (Epinephelus bruneus Bloch 1793) is a commercially important fish in Korea. In recent years, the catch of kelp grouper in the coastal waters of Korea has significantly declined. Despite its importance, little is known about its genetic diversity and conservation efforts are hampered. In this study, we isolated and characterized 12 microsatellite loci using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. All loci were readily amplified and contained TG/CA denucleotide repeats. To characterize each locus, 30 individuals from a natural E. bruneus population in the coastal waters of Jeju Island, Korea, were genotyped. All loci except three, KEm118, KEm154, and KEm219, were polymorphic, with an average of 8.1 alleles per locus (range 2Á18). The mean observed and expected heterozygosities were 0.47 (range 0.19Á1.00) and 0.61 (range 0.29Á0.92), respectively. A significant deviation from Hardy-Weinberg equilibrium was observed at three loci (KEm134, KEm184, and KEm283). These findings will be useful for effective monitoring and management of genetic variation of kelp grouper as well as for the implementation of a fisheries conservation program. Keywords: kelp grouper; Epinephelus bruneus; heterozygosity; microsatellite; genetic marker Introduction is increasing evidence that marine species exhibit The kelp grouper (Epinephelus bruneus, Perciformes greater population differentiation than that expected Serranidae) is narrowly distributed in tropical and based on dispersal capabilities alone (Riginos and temperate oceans of Korea, Japan, China, and Taiwan. Nachman 2001; Banks et al. 2007; Hedgecock et al. It inhabits rocky reefs and mud bottoms. Adults can be 2007; Pampoulie et al. 2008). These research findings found at depths of 20 to 200 m, while juveniles occur in support a need for more available genetic information shallow (Heemstra and Randall 1993). E. bruneus has about E. bruneus. been enlisted as Endangered in Earth‘s Endangered Despite the urgent need for conservation, little is Creatures (EEC) (http://earthsendangered.com) and as known about the species genetic diversity, which is of Vulnerable in the International Union for the Con- importance for planning a conservation strategy servation of Nature and Natural Resources (IUCN) (Frankham et al. 2002). Furthermore, currently no Red List Category & Criteria (www.iucnredlist.org) molecular markers are available. Suitable DNA mar- due to the sharp decline in the catch data. In Korea, kers are needed to identify single individuals and to E. bruneus is an important and very expensive marine assess parentage and genetic relationships. To address fish species. In response to decreases in natural this issue, we have attempted to develop species-specific resources and increases in prices, the aquacultural genetic markers for population genetic studies of kelp production of kelp grouper has been attempted for a grouper. long time. Recently, complete culturing, including Among available genetic markers, microsatellites reproduction control, captive spawning, hatching, and are recognized as an essential tool in population studies larval and juvenile rearing, became possible on a small because of their useful properties, such as high levels of scale. polymorphism, codominant inheritance, and good To develop appropriate recovery and conservation reproducibility (Tauz 1989; Feral 2002; Liu and Cordes management plans, an understanding of the population 2004). Over the past decade, microsatellites have structure, gene flow, and genetic diversity within and produced promising results in studies of genetic varia- among populations of a species is crucial. Marine tion in many marine species (Castro et al. 2003; Cruz species populations usually have low levels of geogra- et al. 2004; Chistiakov et al. 2006; An et al. 2010). phical differentiation because of the relative lack of Recently, numerous microsatellite markers have been barriers to gene flow (Ward et al. 1994). However, there identified for endangered species that can serve as *Corresponding author. Email: [email protected] ISSN 1976-8354 print/ISSN 2151-2485 online # 2012 Korean Society for Integrative Biology http://dx.doi.org/10.1080/19768354.2011.611255 http://www.tandfonline.com Animal Cells and Systems 51 genetic tools to help us create a more accurate picture denatured, and biotin-labeled dinucleotide repeat se- of pattern and process in endangered species (DeSalle quences ((CA)12GCTTGA; Li et al. 2002) were and Amato 2004). Analysis of the current endangered hybridized to the PCR products. The hybridization population state of a population or species using complex was lifted with streptavidin-coated magnetic microsatellite markers is helpful in the management spheres (Promega, WI, USA). After washing, the of retrieval programs for endangered species. bound, enriched DNA was eluted from the magnetic In this paper, we report the development of primer spheres. PCR amplification was performed with an sets for the 12 microsatellite loci from an E. bruneus adaptor sequence primer, and the PCR products were DNA library enriched for (CA)n repeats, and the purified using a QIAquick PCR purification kit genetic variability at these loci in a wild population of (Qiagen). kelp grouper in Korean coastal waters. Cloning and sequencing of microsatellite loci Materials and methods The purified PCR products were digested with NheI DNA extraction from kelp grouper and ligated into XbaI-digested pUC18 vector (Phar- macia, Piscataway, NJ, USA). The construct was used For microsatellite isolation and genetic characteriza- to transform Escherichia coli DH5a competent cells. tion analysis, a non-destructive sampling method was A small portion of each white colony was screened for employed and only fin-clips of each sample were the presence of a repeat insert, using PCR with collected. For genomic DNA isolation for microsatel- universal M13 primers and the non-biotin-labeled lite-enriched partial genomic library construction, fin (CA)10 primer (Li et al. 2002). The PCR products clips were collected from a kelp grouper individual and were checked on 2% agarose gels, and the inserts for genotyping, from a total of 30 individuals. All kelp producing two or more bands were considered to grouper used in this study were sampled from kelp contain a microsatellite locus. Positive clones were groupers which were captured near the coastal waters cultured and used for purification of plasmid using a of Jeju Island, Korea, and have been kept for a QIAprep Spin Miniprep kit (Qiagen) and sequenced conservation program by the National Fisheries Re- using a BigDye Terminator Cycle Sequencing Ready search & Development Institute. All the samples were Reaction kit (ver. 3.1; Applied Biosystems, Foster City, placed in absolute ethanol and kept frozen at 208C CA, USA) and an automated sequencer (ABI Prism until DNA extraction. The TNES-urea buffer method 310 Genetic Analyzer; Applied Biosystems). Primer (Asahida et al. 1996) was used to isolate high-mole- designs were based on sequences flanking the micro- cular-weight DNA for microsatellite isolation. For satellite motifs, using OLIGO software (ver. 5.0; genotyping, total DNA was extracted using a MagEx- National Biosciences, Plymouth, MN). tractor-genomic DNA purification kit (Toyobo, Japan) for the automated MagExtractor MFXÁ2100 DNA extraction system (Toyobo, Japan). The extracted Assessment of polymorphisms in microsatellite loci genomic DNA was stored at 208C until being used We tested newly designed PCR primer pairs to for PCR. optimize a distinct amplification using a gradient PCR with a 50Á608C range of annealing temperatures. To characterize each amplified locus, primer pairs were Isolation of microsatellite-containing DNA fragment tested for polymorphisms using samples from 30 We constructed a partial genomic library enriched for randomly selected kelp groupers (body length, average CA repeats using a slightly modified enrichment 52.5 cm; body weight, average 3.67 kg). PCR amplifi- procedure with pre-hybridization PCR amplification cation was performed in a 10-ml reaction volume (Gardner et al. 1999; Hamilton et al. 1999). Extracted containing 0.25 U of Extaq DNA polymerase (TaKaRa DNA (20 mg) was digested with the restriction Biomedical Inc., Shiga, Japan), 1 PCR buffer, 0.2 enzymes AluI, RsaI, NheI, and HhaI (New England mM dNTP mix, 10 pmol of each primer, and 100 ng of Biolabs, Beverly, MA, USA). DNA fragments ranging template DNA, using a PTC 200 DNA engine (MJ from 300 to 800 bp were isolated and purified using a Research). The forward primer of each pair was 5? end- QIAquick Gel Extraction kit (Qiagen, Hilden, Ger- labeled with 6-FAM, NED, and HEX dyes (Applied many). The selected fragments were ligated to an Biosystems). PCR reactions were as follows: 11 min at adaptor (SNX/SNX rev linker sequences), and the 958C followed by 35 cycles of 1 min at 948C, 1 min ligated DNA was amplified by PCR using SNX as a at annealing temperature