Development of Fifteen Novel Microsatellite Markers from Rock Bream ( Oplegnathus fasciatus )

Rock bream (Oplegnathus fasciatus) is one of the most economically valuable fish in Korea. In recent years, artificial breeding techniques with molecular and microsatellite markers have been developed to enhance rock bream resources. Microsatellite loci to define genetic diversity were screened in rock bream (n = 30) from Jeju areas of Korea and fifteen polymorphic microsatellite loci were newly identified and analyzed. The number of alleles per locus ranged from 9 to 34 while observed and expected heterozygosity ranged from 0.600 to 1.000 and from 0.772 to 0.977, respectively. These markers will serve as a foundation for future population genetic studies and the selective breeding technology of rock bream farming.


Introduction
Rock bream (Oplegnathus fasciatus) is primarily an inhabitant of estuaries throughout Korea, Japan, Taiwan, and Hawaii [1] [2].Recently, it is becoming one of the most valuable species in the fisheries industry in Korea.Therefore, the government has developed artificial breeding techniques to enhance rock bream resources [1].Although it is an important commercial fish species, little is known about the genetic background of rock bream.Accordingly, screening for useful molecular markers is necessary for analyzing genetic information in rock bream [3].
In recent years, many rock bream farms in Korea have suffered substantial financial loss due to the occurrence of red seabream iridovirus disease (RSIVD) during summer months [15] [16].To reduce this economic loss, we carry out experiments to isolate fish with iridovirus resistance.For this purpose, the high resolution of genetic map is necessary to facilitate searching for disease resistance traits and identifying candidate genes.Ozaki et al. (2010) reported linkage analysis of resistance to Streptococcus iniae infection in Japanese flounder based on 159 microsatellite markers [17].Great links of microsatellite markers with resistant traits provide rise to quantitative trait loci (QTL) for resistance to a specific pathogen [18].We isolated a polymorphic dinucleotide (CA) repeat sequence from CA repeat enriched genomic library.In this study, we reported on an additional 15 microsatellite markers for the rock bream.

Materials and Methods
Rock breams for constructing CA repeat enriched library and genotyping were purchased from culture farms in Jeju Province of Korea.CA repeat enriched library was constructed with an enrichment technique suggested by Hamilton et al. [19] Genomic DNA was extracted from 1 g of muscle tissue of cultured rock bream in Jeju Province of Korea by using standard phenol-chloroform extraction and ethanol precipitation.Purified genomic DNA was digested with RsaI, HaeIII, AluI, and NheI restriction enzymes (New England Biolabs, Beverly, MA, USA) with proper conditions.The digested DNA fragments were ligated with specific linkers for amplification.The biotinylated repeat oligo (dCA 16 ) was hybridized with the digested DNA to purify DNA fragments containing a CA repeat motif.The microsatellite enriched elutant was amplified with an oligo adaptor primer.Amplified DNA was purified using Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany) and cloned with pBluescript II SK(-) vector (Stratagene, La Jolla, CA, USA).Cloned plasmids were extracted and sequenced with an ABI-3130xl sequencer.Mass sequences were analyzed with bioinformatical pipelines of Phred, Cross-match, RepeatMasker and SeqClean, and assembled with TGICL.Primer designable contigs were selected and primers were designed using Primer3Plus software.
Thirty rock bream individuals from the Jeju Province of Korea were used for genotyping.The genomic DNA for PCR template was extracted through standard phenol-chloroform extraction and ethanol precipitation.Polymerase chain reaction (PCR) amplification was carried out in a ABI 2720 Thermal Cycler (Applied Biosystem, Foster City, CA, USA) in 25 µL of reaction mixture including approximately 50 ng of template DNA, 0.2 mMdNTP, 0.5 µM of each primer, ddH 2 O, 1X PCR buffer (50 mM KCl, 2 mM MgCl 2 , 10 mM Tris-HCl), and 1 unit Taq DNA Polymerase.Initial denaturation at 95˚C for 5 min, was followed by 35 cycles at 95˚C for 1 min, 50˚C for 1 min, 72˚C for 1 min and a final extension at 72˚C for 5 min.PCR products were electrophoresed on an ABI 3730xl Genetic Analyzer (Applied Biosystems, USA) using a POP7 gel matrix with GeneScan™-500 LIZ® Size Standard (Applied Biosystems, USA).GenSeScan Analysis (V.3.7,Applied Biosystems, USA) software was used to score microsatellite alleles, and allele size was manually verified.
We calculate the number of alleles per locus (k), observed and expected heterozygosities (Ho and He), polymorphic information content (PIC) and estimated null allele frequency (F) using the Curvus Program [20].Linkage disequilibrium and the Hardy-Weinberg Equilibrium were determined using Genepop v4.0 [21].

Results and Discussion
We isolated 2039 unique repeat-containing sequences from enriched genomic DNA library and 621 primer pairs were designed from those sequences using Primer3Plus software.Fifteen of the primers were end-labeled at one primer of each pair with a fluorescent dye, 6-FAM, after the first screening step.The characteristics of the 15 microsatellite loci are shown in Table 1.A total of 312 different alleles were observed at the fifteen loci.The average number of alleles per locus was 21, ranging from 9 (RbJJCA07) to 34 (RbJJCA20).The size of alleles ranged from 116 (RbJJCA15) to 270 (RbJJCA25).Comparatively, a previous study by An et al. [3] reported rock bream to have a mean of 8 alleles in 9 loci with a total of 73 alleles observed.
Based on this classification, all loci from our study show highly informative values, indicating that all loci from our research could be useful markers for population genetics and quantitative trait locus (QTL) study of rock bream.In the case of rock bream individual RBJJCA20 the highest PIC value (0.960) was observed.This is a relatively high polymorphic value compared to other microsatellite markers from different fish species [23] [24].Observed and expected heterozygosities ranged from 0.600 to 1.000 and from 0.772 to 0.977, respectively.After a Bonferroni correction (P = 0.0033) four microsatellite loci (RbJJCA04, RbJJCA17, RbJJCA28, RbJJCA39) exhibited significant departure from the Hardy-Weinberg equilibrium, possibly due to the presence of null alleles.It is thought that these null alleles were caused by genetic instability within this region.

Table 1 .
Characteristics of fifteen polymorphic microsatellite loci for rock bream Oplegnathus fasciatus.