flounder Paralichthys olivaceus (Pleuronectiformes, Pleuronectoidei, Paralichthyidae)
M . S E K I N O * and M . H A R A †
*Tohoku National Fisheries Research Institute, Shinhama, Shiogama, Miyagi, 985 – 0001, Japan, †National Research Institute of Aquaculture, Nansei, Watarai, Mie, 516 – 0193, Japan
Keywords: DNA, Japanese flounder, microsatellites, Paralichthyidae, Paralichthys olivaceus
Received 15 August 2000; revision accepted 22 August 2000 Correspondence: M. Sekino. Fax: + 81 22-367-1250; E-mail:
Table 2 Summary statistics for five tri- and tetranucleotide repeat microsatellite markers used in the genetic analysis of Cynopterus sphinx (from localities <18° N latitude) and C. brachyotis in peninsular India. n, number of bats genotyped per locus; NA, number of alleles per locus; HO, observed heterozygosity; HE, expected heterozygosity. In both species, locus CSP-7 segregated multiple alleles with lengths that differed by 2 bp, even though the cloned allele was a (TATC)n repeat
Locus
Cynopterus sphinx (southern localities) Cynopterus brachyotis
Allele size range n NA* HO HE Allele size range n NA HO HE
CSP-1 191 – 224 189 12 0.79 0.86 176 – 227 111 12 0.61 0.69
CSP-2 119 – 134 189 6(7) 0.74 0.71 101 20 1 0 0
CSP-5 130 – 190 189 11(16) 0.82 0.81 110 –166 111 11 0.29 0.34
CSP-7 227 – 285 189 21 0.78 0.84 229 – 263 111 17 0.72 0.86
CSP-9 286 – 302 189 5(6) 0.55 0.60 270 – 282 111 4 0.45 0.49
*Numbers in parentheses refer to numbers of alleles observed in the complete sample of C. sphinx, from Pune and the southern localities (n = 620 bats).
Japanese flounder Paralichthys olivaceus is an important species consisting coastal fisheries resources in Japan, and is of high commercial value. Interest has been directed toward resource enhancement, and accordingly, millions of P. olivaceus are released into Japanese coastal fisheries grounds every year (Furusawa 1997), yet little is known about reproductive success of the stocked fish. To promote effective stocking management, it is necessary to monitor the fate of stocked fish and their relatedness apart from naturally reproduced fish. Microsatellite DNA loci are expected to provide an invaluable tool for this purpose because of the power and ability of microsatellite markers in regard to resolution for genetic relatedness among individuals (Blouin et al.
1996) and parentage determination (O’Reilly et al. 1998).
Here, we describe the characterization of microsatellites isolated from P. olivaceus that will be useful to address the stocking effects.
The method described by Sekino et al. (2000) was used for cloning P. olivaceus microsatellites. In brief, genomic DNA was fragmented by sonication. Sonicated fragments were blunted by mung bean nuclease (Takara, Shiga, Japan), and the fragments ranging from 300 – 500 bp were recovered.
The fragments were ligated into SrfI site of pCR-Script Amp SK(+) vector (Stratagene, La Jolla, CA, USA), and recom-binant plasmid vector was transformed into XL2-Blue MRF′ ultracompetent cells (Stratagene). Single-stranded DNA was prepared, and selective second-strand DNA synthesis was employed using (CA)12 oligonucleotide and cloned pfu DNA polymerase (Stratagene). The resultant double-strand DNA was transformed into XL2-Blue MRF′
cells again and these transformants were referred to a (CA)n -enriched library. From the library, 80 clones were randomly chosen, and plasmid DNAs were purified using GFX Micro Plasmid prep kit (Amersham Pharmacia Biotech, Uppsala, Sweden). The DNA sequences were determined in both directions using Thermo Sequenase™ cycle sequen-cing kit (Amersham Pharmacia Biotech) in combination with KS and T3 primers and subjected to an ALFexpress automated DNA sequencer (Amersham Pharmacia Biotech).
Of the 80 clones, 59 contained one or more repeat sequences. We designed 27 polymerase chain reaction (PCR) primer pairs using a Premier software package (Premier Biosoft International, Palo Alto, CA, USA). To examine microsatellite polymorphisms, PCR was employed.
PCR amplification was carried out in a 20 µL reaction volume, which included 20 pmols of each primer set (one primer in each pair was 5′ end-labelled with Cy5), 100 µm of each dNTP, 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, 0.001% gelatin, 0.5 U of Ampli Taq GoldTM (Perkin Elmer, Foster City, CA, USA), and approximately 50 ng of template DNA using PC-960G gradient thermal cycler (Corbett Research, Mortlake, NSW, Australia). PCR ampli-fication cycles were as follows: 12 min at 95 °C, 35 – 40 cycles of 30 s at 94 °C, 1 min at a primer-specific temperature, 1 min at 72 °C, and final elongation for 5 min at 72 °C. Analyses of PCR products were performed using ALFexpress sequencer in combination with an Allelelinks software package (Amer-sham Pharmacia Biotech).
All 27 microsatellite loci were successfully amplified,
out of which we finally chose 16 primer sets (the remain-ing 11 havremain-ing been rejected because their polymorphisms were low, and/or they produced unexpected PCR products in an initial sample of P. olivaceus) and assessed further microsatellite polymorphisms in a natural P. olivaceus population collected from the Japanese coast of the Japan Sea.
As shown in Table 1, the number of alleles ranged from 4 – 40, and the observed and expected heterozygosity ranged from 0.43 – 0.99 and 0.43 – 0.97, respectively. All but one of the 16 loci conformed to Hardy–Weinberg’s (HW) equilib -rium in the Markov-chain method (parameters used; 100 000 Markov-chain steps; 10 000 dememorization steps), using an Arlequin verion 1.1 software package (Schneider et al.
1997). At the Po31 locus, the observed genotype frequen-cies showed significant departure from HW expectations (P < 0.05) with a large discrepancy between the observed and expected heterozygosity (0.34 and 0.91, respect-ively). This may be explained by sampling errors due to limited sample size or substructuring of the samples, how-ever, this seems unlikely because the observed genotype frequencies in all other 15 loci were consistent with the expectations. We believe that the presence of null alleles (Pemberton et al. 1995) may be a valid explanation causing these results. Further investigation of this topic is necessary. Microsatellite DNA loci described in the present study possess hypervariability, suggesting that these loci will be useful for genetic monitoring of stocked P. olivaceus in furthering our understanding of stocking effects.
Acknowledgements
We express gratitude to Dr H. Takahashi, National Research Institute of Agro-biological Resources, for the technical advises and contributions.
References
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Furusawa T (1997) Key problems of sea-farming associated with its perspective. In: Biology and Stock Enhancement of Japanese Flounder (eds Minami T, Tanaka M), pp. 117–126. Koseisha-koseikaku, Tokyo. (in Japanese).
O’Reilly PT, Herbinger C, Wright JM (1998) Analysis of parentage determination in Atlantic salmon (Salmo salar) using micro-satellites. Animal Genetics, 29, 363–370.
Pemberton JM, Slate J, Bancroft DR, Barrett A (1995) Nonamplify-ing alleles at microsatellite loci: a caution for parentage and population studies. Molecular Ecology, 4, 249–252.
Schneider S, Kueffer JM, Roessli D, Excoffier L (1997) Arlequin version 1.1: A software for population genetic data analysis.
Genetics and Biometry Laboratory, University of Geneva, Switzerland.
Sekino M, Takagi N, Hara M, Takahashi H (2000) Microsatellites in rockfish Sebastes thompsoni (Scorpaenidae). Molecular Ecology, 9, 634 – 636.
2202PRIMER NOTES
© 2000 Blackwell Science Ltd, Molecular Ecology, 9, 2155–2234
Table 1 Core repeat and primer sequences, PCR amplification conditions, and results of variability of the 16 microsatellite loci in a Paralichthys olivaceus population. HO is observed and HE is expected heterozygosity
Locus Core repeat sequence (5′−3′) Primer sequence (5′−3′) Anneal. (°C) Sample size No. of alleles Size range† (bp) HO HE P‡
GenBank accession no.§
Po1 (TG)3T2(TG)8 F-GCCTTTTGTCAGCCATTAACAGAGC 55 67 20 160 – 216 0.68 0.71 0.84 AB046745 R-CTGAGGCCAGACATGACATTACCTT
Po13 (TG)3GA(CA)13 F-CGGCCTAAACCTGGACATCCTCTCTA 58 69 23 206 – 276 0.78 0.92 0.18 AB046746 R-CGGGACAACGGAGGTTTGACTGAC
Po20 (CACG)4(CA)4CG(CA)18 F-TGCTCCTTCACCTGCACGGCCTCAAA 58 69 40 239 – 379 0.99 0.97 1.00 AB046748 C(GT)3 R-TGCACCCTGACCTGTCACTGGGGATT
Po25A (GATG)2A
2CA(GATG)10 F-TGAGGAGTCAGGTTTCAGGCCACT 55 68 12 201– 253 0.76 0.76 0.26 AB046749 R-TCGCAGGAACACCCAGAGTACAGA
Po26 (CA)6CGCACGGA(CA)7 F-ACACTGGGCCCTCTGTTAAACAC 55 67 5 141–159 0.73 0.65 0.72 AB046750 R-AGAGGAGAAAGGGCACCGAGATA
Po31 (CA)4(GA)2(CA)11 F-AGGGTTAATTATAGAGGACGCAG 57 69 25 129 –193 0.43 0.91 0.00* AB046751 R-CTGAAACAACAACTCAGAAGACG
Po33 (TG)5T2(TG)10 F-GTTGGTTTAACTGATTCATCTGCAG 55 69 10 257 – 290 0.74 0.68 0.82 AB046752 R-TTACATATCCCACAATGCTTCACTC
Po35 (CA)7 F-TGGTTCTAGTGTTTGTCTGGTGA 54 69 15 283 – 333 0.81 0.78 1.00 AB046753
R-CCTACAGCACAGATATGACCTTT
Po42 (CA)5(TA)13(CA)3 F-CGAGCGCTGTTTCAACTACGGTCATT 55 69 23 164 – 224 0.88 0.91 0.67 AB046754 R-ATGATGATCTAACCGTCCGGCTCCAT
Po48 (CACG)4(CA)5 F-GCCTCCAGAAACATTTATGGGG 55 64 6 126 –142 0.44 0.43 0.69 AB046755
R-TGTCTTGCCTCTGGTCCTTCTT
Po52 (CA)2CG(CA)6GA(CA)5 F-TCAGACAGAGGAGCGGGGTTGTTGC 58 64 4 155 –163 0.46 0.50 0.62 AB046756 R-GCTGTACCCAGGGTTCCGCTGAAGA
Po56 (AC)20 F-TCGAGCGTAAACAAACCAGCTAACA 55 69 26 139 – 205 0.94 0.94 0.62 AB046757
R-GCTGAAAATCGCTTTAGCTTCCCAT
Po58 (CA)11(GA)2GC(GA)9 F-GCCCCTCACTGAGACTGTGACA 52 69 27 101–159 0.84 0.90 0.52 AB046758 R-CAAGGTATGTGCATGAGCAGGC
Po83 (CA)5AG(CG)2(TG)3 F-TGCGGTCATCATGTCTTTAAAATA 57 68 32 227 – 313 0.91 0.93 0.18 AB046759 (CG)2(CA)15 R-AGCAAATGTTTGCTTTTGGATACA
Po89 TA3(CA)7 F-ATCAGAAGTCATCCATGCACTGGCAC 60 69 20 252 – 327 0.86 0.90 0.44 AB046760
R-AGCTACTTATCCACAGGTGTCGACGG
Po91 (CA)18 F-AGGTTTCAAGGTGTTCATTGCGAGTC 55 69 34 146 – 246 0.96 0.94 0.97 AB046761
R-TAAAGGAAGTGCCTCACTGTGGAGAA
mean 20.1 — 0.76 0.80 —
†Size is indicated as number of the base pairs of PCR products.
‡P is the exact P-value estimated by a test anologous to Fisher’s exact test described by Schneider et al. (1997). Significant departure of the observed genotype frequencies from H-W expectations was determined by adding *P < 0.05.
§The nucleotide sequence data will appear in the DDJB/EMBL/GenBank nucleotide databases with the accession numbers.
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