Microsatellite markers are important for gene mapping and for marker-assisted selection. Sixty-five polymorphic microsatellite markers were developed with an enriched partial genomic library from olive flounder Paralichthys olivaceus an important commercial fish species in Korea. The variability of these markers was tested in 30 individuals collected from the East Sea (Korea). The number of alleles for each locus ranged from 2 to 33 (mean, 17.1). Observed and expected heterozygosity as well as polymorphism information content varied from 0.313 to 1.000 (mean, 0.788), from 0.323 to 0.977 (mean, 0.820), and from 0.277 to 0.960 (mean, 0.787), respectively. Nine loci showed significant deviation from the Hardy-Weinberg equilibrium after sequential Bonferroni correction. Analysis with MICROCHECKER suggested the presence of null alleles at five of these loci with estimated null allele frequencies of 0.126-0.285. These new microsatellite markers from genomic libraries will be useful for constructing a P. olivaceus linkage map.
Olive flounder
Because microsatellite markers have high levels of polymorphism, co-dominant inheritance, genome-wide distribution, and high reproducibility, they are the most popular and powerful molecular markers in population genetics and can be used to construct genetic linkage maps (Liu and Cordes, 2004). In recent years, microsatellite markers have become one of the most commonly used molecular markers in population and evolutionary biology research, and are applied widely in studies of biological breeding, genetic linkage maps, genetic diversity, and phylogeny (Goldstein and Pollock, 1997). Seventy-nine microsatellite markers have been developed previously for
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Isolation of microsatellites
A partial genomic library enriched with GT repeats was constructed using a slight modification of the procedures described by Hamilton et al. (1999). Genomic DNA was extracted from
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Primer design and genotyping
Primers were designed from the unique sequences flanking microsatellite motifs using OLIGO 5.0 software (National Biosciences, Plumouth, MN, USA). PCR conditions were initially optimized using DNA samples originally used for microsatellite isolation to establish whether the desired size product was amplified by changing the annealing temperature, the primers, and MgCl2 concentrations as well as the amplification profiles. Suitable microsatellite loci were genotyped to test the level of genetic polymorphism using 30
The number of alleles per locus, polymorphism information content (PIC), and observed and expected heterozygosity at each locus were calculated using CERVUS 3.03 (Marshall et al., 1998). Deviations from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium were estimated using GENEPOP 4.0 (Raymond and Rousset, 1995), and adjusted
In total, 800 white colonies with inserts were randomly selected and screened for the repeat using PCR, which yielded 425 (53.1%) true positive clones. These were sequenced producing 330 (41.3%) sequences containing simple sequence repeats, of which 184 (23%) were eliminated because they possessed no flanking sequence. A total of 146 (18.3%) sequences containing microsatellites were obtained, and primers were designed to amplify microsatellite-containing regions of the genome. Only 98 of the 146 primer pairs successfully amplified the target region, and the remaining pairs either failed to amplify or produced nonspecific bands. Finally, we chose 70 primer sets because they produced clear and reliable bands at high temperatures, and we tested polymorphisms in 30
[Table 1.] Characterization of 65 polymorphic loci in Paralichthys olivaceus
Characterization of 65 polymorphic loci in Paralichthys olivaceus
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We constructed a microsatellite enrichment library for olive flounder using (GT)10 biotin-labeled probes, and 78% (330/425) of the positive clones contained microsatellite repeats. This efficiency is lower than that in tilapia (96%) (Carleton et al., 2002) but higher than that in cutlassfish (48%) (An et al., 2010).
The 65 new polymorphic microsatellite loci developed in
Nine loci (KOP108, KOP113, KOP121, KOP123, KOP125, KOP146, KOP148, KOP150, and KOP164) deviated from HWE in the tested population after sequential Bonferroni correction (