Genetic Similarity-dissimilarity Among Korea Chum Salmons of Each Stream and Their Relationship with Japan salmons

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1 The Sea Journal of the Korean Society of Oceanography Vol. 12, No. 2, pp , May 2007 [Note] {{ pn Œ ~ ~no d {{ ~ q rq * mnp mrofsnn Genetic Similarity-dissimilarity Among Korea Chum Salmons of Each Stream and Their Relationship with Japan salmons GO-EUN KIM, CHOONG-GON KIM AND YOUN-HO LEE* Korea Ocean Research and Development Institute, Ansan P.O. Box 29, , Korea kp q rp ll lp ll(oncorhynchus keta)p v p o l l p v kk. ll lp p orq pn p e pp, pm (SNP)p pn v p r p pn p. ll llp } or r o pr p llm orr o l, Š kp COIII-ND3-ND4L vl 720 bp m lp m. p 3 vl( }, kk }, nv m })l }v 108~m pp 2 vl( pp e m )l }v 44~ l 29p haplotypep p m. orr k p } lll q l q k. Pairwise F STm AMOVA pn v, ll } orr p p lp p llm v kk(f ST<0.07)., haplotype op ~p orr llp p(k 25%) p llm orr (lineage) pp, } } lll m } ll sq p lt. Analysis of population structure of Oncorhynchus keta, the most abundant salmon in the East Sea of Korea, has not been much carried out despite its importance as a fishery resource in the North Pacific. Currently, molecular methods are being applied to stock identification and a method of using single nucleotide polymorphisms (SNPs) is getting more popular. In this study, we analyzed the 720 bp long sequence of the mtdna COIII-ND3-ND4L region in order to examine genetic similarity-dissimilarity among the Korea chum salmons of each stream and their relationship with the Japan chum salmons. A total of 152 individuals were analyzed, 108 from 3 locations of Korea and 44 from 2 locations of Japan, which resulted in as many as 29 different haplotypes. Pairwise F ST and AMOVA tests of the populations show that there is no significant population-level genetic difference among the chum salmons analyzed (F ST < 0.07). On the other hand, haplotype relationships among the individuals reveal that approximately 25% of the Korea salmons consist genetic lineages independent of Japan salmons and also that a genetic lineage exists in the Puk river and the Namdae river salmons independent of the Wangpi river salmons of Korea. Keywords: Chum Salmon, Oncorhynchus keta, COIII-ND3-ND4L, Genetic Diversity, Population ll l lsp ˆk kl. pp ovp }l l q lp e q ~ p o e ˆl }p km. vv k p t, p pf. *Corresponding author: ylee@kordi.re.kr ˆkl e 7sp ll l t p q p s p ll(onchorhynchus keta; chum salmon)p. p l, p, ekm p llp } dp p n l qop veˆ o lp p lp e e. np n kl o o kkl ll 1985 llp l eq l 2000 l 1300 m(, 2004). 2010v 1l pp l l ll kp lsp o p o p. n llqop 94

2 llp } orr o pr p llm orr 95 m l r ˆk loo (North Pacific Anadromous Fish Commission, NPAFC)l w o (,, ek, p, )p p, ll qo l rrp tq eq m. llqol tq kˆ lp prp q o o llp vp r v p p n p. n ll vp r vl l lp eq p rp pl r m. (1992)p llp ˆr vp q,, vp p, tp v l ll v p (kk }, } t t}; } m e}, nv m } m me})p l v m. (1998)p kk }l llp ~q, l s l mp, (1999)p o l llp r vp ll ll } llp qp pl s rp r m. Kang and Kim(2004)p pp kek ll ll q ~tp rp pp tl pl p ppp vr m. llp n ~p lp lll l p 3.4l. llp ol r (2001)p sl l rel ˆk r~ o km o r l, ˆk y lkp q km ˆk o re m., Seo et al.(2006)p p llp o re Urawa(2000)p p l ll l k p ~ m p llm p o p m. llp vp orrp e isozyme p eql. Okazaki(1983) MDH-B vp p ll vp kek p mp, (1992)p ll p d o re m., DNA p r isozymep DNA ml pp p kv p v s l DNA pn eq m., p Š k DNA r orq 5~10 v p pl vp ƒ on rkl (Brown et al., 1982). Sato et al.(2001)p Š kp control regionp pn l p llp } orr p e mp, p 2004l(Sato et al., 2004) ˆk r ll }v ll l orr vp p/ek/k p re m. p ll ll }l }v e lp pllm p l p ˆ., p ll ˆk l l k 2100 lp p 30 haplotypel l, Š k control regionp p o rr kp ƒp p pp r. r (2003)p p p DNA Ogo5(Olsen et al., 1998) m Š k orq t pp p p kv ND3(Jacobs et al., 1988; Sunnucks, 2000)m t vl COIII, ND4L p mlp l p llm l l p ppp. Kim et al.(2007)p COIII-ND3- ND4L p l ˆk llp vs -p (ek Primorye )/ek-kd - / p re m. vv lll ll } orr pl p p plvv kk. Kim et al.(2007)p ˆk ll l kk } nv m }l }v l l e p p. ll p m q }l ll }v l mp kk } ll }v l m. qƒ s lm p o orq pp p p kr l p pnl m Š k DNA COIII-ND3- ND4L vlp ˆ m. mlp l } haplotypep sq l p mp, llm p ll l pr op m. m ys v l n ll ll 108~, p ll 44 ~f 152~p. p t, ll o kkp } p }, nvp m }l }v l(fig. 1). }l 1999, 2000, 2001, 2005l 6, 14, 15, 30~j }vlp }l 2005l 30~, m }l 2000l 13~ }vl. p ll 2000 l pp e shokanbetsu l 22~, hakodatel 20~ }vl. ~l p o svp } l 100% lˆml r DNA l pn m. DNA }} lˆml r el k mgp ll 3 v l DNeasy Tissue Kit(Qiagen)p pn l tlv Fig. 1. Sampling locations of Korea and Japan chum salmons (O. keta). (KS1: Namdae River, KangwonDo, 38 o 44'20''N 128 o 37'17''E; KS2: Wangpi River, KyoungsangbukDo, 36 o 59'23''N 129 o 24'15''E, KS3 : Puk river, Kangwondo, 38 o 23'13''N 128 o 28'07''E, JS1: Mashike, Hokkaido, 43 o 46'59'N 141 o 31'59'E, JS2: Hakodate, Hokkaido, 41 o 46'59''N 140 o 45'E).

3 96 pë Ëpo p e l DNA m. DNA 1% kd rl rm l ˆ p mp, DNAp r spectrophotometry pn l A260/280 r p f m. lnmm q im(pcr) DNAl t lpp pn l COIII- ND3-ND4Lorq ˆrp v m. vl n p COIIIm ND4L orq p m ll qpl (Domanico and Phillips, 1995; forward primer, COIII: 5'-TTA CAA TCG CTG ACG GCG-3'; reverse primer, ND4L: 5'-GGT GCG GTG AAA CGC GAG TG -3'). PCR p nkp 10 PCR bufferm 2.5 mmp dntp (datp, dttp, dctp, dgtp), 2.5 unit p TaKaRa Ex Taq polymerase p nkl forward primerm reverse primer 20pmole ~ l r~ 50 µl l. PCR pp DNA Engine (MJ Research, )l denaturation (94 o C, 45), annealing (60 o C, 45) extension (72 o C, 2)p l pp 30 m. lp r predenaturationrp 94 Cl 5 k pe lp l o final extension rp 72 Cl 10 k o pe. p PCR t 5 µl 1% kd rl rm l v DNAp p mp, vp p p QIA Quick PCR Purification Kit(Qiagen) pn l rr m. ii o rr DNA r m spectrophotomerty pn l m p m. p t p p e COIIIm ND4L p pn l k l vr e ep ee l mlp r m. p ep n Topo TA cloning kit(invitrogen) pn l rr DNA m. rp r l re p v mp, DNA pcrii-topo vectorl qs E. Coli p r m. v COIII-ND3-ND4L DNA lp k p o Invitrogenl r M13 forwardm M13 reverse p pn l PCRp ee kv r l rm l v DNA p m. p, k k l Qiaquick mini preparation kit(qiagen)p n l v DNA, T7promoter p pn l mlp m. ii s mlp p rp DNAssist p pn l re p MEGA2 p ClustalW pn l sr p r m. vp p o ARLEQUIN program(schneider et al. 2000)p pn l haplotypek(h) nucleotidek(π), pairwise F ST value(slatkin 1995) r m p, Tamura and Nei model(1993)p rn l AMOVA test (AMOVA, Excoffier et al. 1992) ee m., ~ vl orr o TCS (version 1.21, 95% maximum connection steps)p n m(clement et al., 2000). ihm g DNA, COIII-ND3-ND4sim i i ll 108~m p ll 44~p svp DNAl Š k DNA COIII-ND3-ND4 vl p mlp l. Domanico and Philips(1995) pn COIIIm ND4L p n pf k 780 bp p DNA p vl. v DNA rr, vr e e rp -e e rp ~ p p r n k 720 bpp mlp rl. mll 204 v COIII orqp lp, v m lp trna(gly) orqp, v ND3o rq, v trna(arg) orq, 693 ND4L o rqp 5' y eq p(fig. 2). p mlp NCBIl chum salmonp ml(accession number, D84147) 99% p p p mv 4p o l p p p ˆ(67w TËC, w TAËGG, 691w AËG) COIII-ND3-ND4L vl DNApp p pl. 152~ ll e l pp e ml p v kk. ~p pm (single nucleotide polymorphism, SNP)p 29p o l lp, SNP l p l m lp 29p haplotypep Fig. 2. The major haplotype (H01) sequence of the COIII-ND3-ND4 region among the chum salmons (O. keta).

4 llp } orr o pr p llm orr 97 Table 1. Comparison of the nucleotide sequences among the haplotypes. The nucleotide positions only with polymorphisms are shown. Dots represent the same nucleotide sequence as in the haplotype H01. Nucleotide Position Haplotype Number of Samples H01 90 T T G T G A T T G C A T G G C C C T G T A A A C A T G A C H T T H A H G H T H G C H T H A H C H G H G H C.. T H C G H G. H G H16 4 C H17 1 C A G.... H A G.... H A G. A.. H C G H C G.. G.... H22 1 C.. C G.. G.... H C G G H G.. C... G.. G.... H C C G H C C.... T..... G H C C G... A... H C C G. T..... H29 1. G. C..... C G l(table 1). 29p haplotypet 23 ~l ~ pr p(singleton)pl, 6 haplotype(h01, H16, H18, H20, H21, H25)p ~ pl rp v pr p ˆ. q p ~l rp lv haplotypep H01p 90~p ll p(table 1). H01 m p p p haplotypep 11 H03~H05, H07~H11, H14~H16 p, ll ll 9~m p ll 5~ p. p t H16p np v pr p(8 w ml TËC p) ll 4~., ll 8~ H18p H01 p o l p(246w ml GËA, 591w ml AËG p) p. H18p p H17 H19l rp lv, ~ pr p(singleton)p H17 H19 H01 H18 p ml p p. H17p 8w m l TËCp v, H19 619w ml GËAp rp m p pl. H20 H01 l 57w m ll TËC pm 534w m ll AËG p pl p ml p p. p p H21~H29 rp pl pr (lineage) pr p p lv., H24 57w p e CËT l r p p p p. 10~ H21p H20 p p : 591w m AËG m., 14p ll ~ H25p H21 o l H20 p p : 307 o l AËC p tranversionp p. p p H26~H29l r p pr pp p ˆ(Table 1). COIII-ND3-ND4L vl mlp pl haplotype orr m p ll ~p vl TCS diagraml q (Fig. 3). p l haplotype p op m, orr o op l p e. q p p H01p

5 98 pë Ëpo Table 2. Haplotype frequency in each sampling site Haplotype Number of Sampling site Samples KS1 KS2 KS3 JS1 JS2 H H H H H H H H H H H H H H H H H H H H H H H H H H H H H Total p ll l ˆ. Haplotypep orr l p l r: H01~H19m H20~H29. ~w p e H01~H16 H17~H19p 2p p, w p H20~H24m H25~H29p 2p p l. H01p tep H01~16p p H16p rn H01l Ž ~pr p p ll. H17~H19 plv p mv llp pp, H18p ll }v vl l ˆ. H20~H24 p p ll p, H25~H29 p llp p., p p H01 pp p ~l H25 tep pl vl pr p p sq lt. s haplotype lnn h ll e 108~l ˆ 20p haplotype p }vv KS1p n 65~l 18p haplotypep ˆ, KS2p n 13~l 4 Table 3. Haplotype diversity (h ± standard deviation) and nucleotide diversity (π ± average over loci) within population Population N h ð (%) KS ± ± 0.18 KS ± ± 0.08 KS ± ± 0.16 JS ± ± 0.17 JS ± ± 0.08 haplotype, KS3p n 30~l 4p haplotypep m. p ll e 44~l 12p haplotypep mp, }vv JS1l 24~ 9p haplotype, JS2l 20~ 5p haplotypep m. ARLEQUIN programp }vv haplotype kp KS1l q (0.72±0.06), JS2l q p (0.37±0.13)p ˆl. Nucleotide k le KS1(0.2±0.1 %)l q ˆp, JS2l q p (0.08±0.08 %) p l(table 3), ll p lll rp p p m. ll vp haplotype p, kk } (KS1), } m }(KS2) }(KS3)p ll e t 50% p p H01p p ˆ(KS1:33/65, KS2:10/13, KS3:18/ 30). ll vl haplotypep 16p, p t 2 pp ~l rp p p 3f H16(4 ~), H18(8~), H25(12~) p. H16p H01 1p m l p l, H18p 2p ml p p, H25 p n H01 3p ml p p p p l. n H18 1 p 2p m p p H17 H19p haplotypep ˆ. H25 1jp m l p p singleton groupp p p 4p haplotype le ll ~p l p p p (Fig. 3). p ll v le s vlp ll e t 50% p (JS1 : 13/24, JS2 : 16/20)p H01l l. H01 p ml p haplotypel 5p ~ singletonp ˆp 2p ml p haplotypel 2 ~ l. H01 pp ml p p 4p haplotypel 8p ~ l. p t 2p haplotypel ll lp, v p haplotype p p ll ~j singletonp ˆ. }vv p p ll pp orr p o AMOVA(Analysis of molecular variance) test ee (Excoffier et al. 1992), p ll p p r~ orq pp 0.38% n ˆp, p }vv p le 2.62% ˆ. l l el ˆ pp 96.99% haplotypep p ˆ. p p ll p orr vp p op v kpp lt, }vvp ll rp o rr vp lpp e. p llp }v v pairwise F STp r }vv l 0.07 p p p p l }vv rp orr vp r lpp ˆ(Table 4). ll }vv p F ST p l p, p ll }vv p p

6 llp } orr o pr p llm orr 99 Fig. 3. Genetic relationship among the haplotypes in the Korea and Japan chum salmons (O. keta) constructed by the TCS program (ver 1.21; Clement et al., 2000). Circles and lines indicate each haplotype and one nucleotide difference, respectively. Haplotype names are in italics and individual numbers are within the circle. Table 4. Pairwise F STs among the chum salmon populations KS1 KS2 KS3 JS1 JS2 KS1 KS KS JS JS a P<0.01 ˆp, 0 op p pv kk. llp } orr pr p ol l k n m. p (isozyme)l l (, 1992;, 1996)m p 18SrDNAm Š kp 12SrDNAm 16SrDNA l(seong, 1999) plp }p or vp l plp m l lm ell, }l, v l ll l sp t m. COIII-ND3-ND4L vlp p sp l(choi, 2002; r, 2003) p, llp orr p trp l ll v p p s rp k. ll ll kk } }, nv m }l }v ll 108~ p Š k orq COIII-ND3-ND4vl k 720 bpp mlp l pf } llp orr p k., p pp p v l }v ll 44 ~p orq ml m pf llm pl lp orr o prp š pl., kk } y llp n 1999, 2000, 2001, 2005l ~ }v e n pf }vl o pl. Š k DNA COIII-ND3-ND4vlp ml l ll }l orr vp v kkp, r~ vp tl ll p llm v kk. p lll p haplotypep 50% p p ~ o pl( 56.6%, p 65%). }vv p p ll p p AMOVA (Excoffier et al. 1992)l pp 96.99% haplotype p ˆp, p ll p pm }vv p n ˆ., p llp }vv pairwise F ST le el 0.07 p p p p l p } l l rp orr v sq v k p, p llm p l p ˆ(Table 4). p llp orr op p po vp e p mv kk p r ep kkp p ll v pl v vrp ~p pp pl pp, l pp 100 pp ps p l kk }l pf porp orq p plp p p(r, 2003)., haplotypep orr op TCS p ll pl orrp (lineage)p ~p sq p ltl. ll 29p haplotypel vpr p o 8p haplotype (H17~H19; H25~H29)p llp lp, ll ep k 25% p haplotypel p ˆ (Fig. 3). p ll p haplotype p plp 1p ~p ~pr p(singleton)m. p llm p ll v tl v kv, ~p orr sp ~ (lineage) tl

7 100 pë Ëpo p p e. ll haplotypep }l m llvp p }l m llv on qƒ pn pp p. TCS p } ll v pl tp p pp ppp lt. ll pr haplotype p H17~H19l kk }(KS1), nv m }(KS2), }(KS3)p ll l pp, p H25~H29l kk }(KS1) }(KS3)p ll p nv m }(KS2)p ll v k(fig. 3; Table 2). p orq H25~H29l p ll kp ll }l v k yp n~ pp p lt. p (1992)p ˆr v lm p p. (1992)p q,, vp p, tp v llp ˆr vp lkp ll vp p l(kk }, } t t}; } me}, nv m } m me}). lp v tl p } l lp orr p lp, p ll pl orr p l p ˆp ~p (lineage) tl orr p ppp lt. tp orr p ~p vp q ƒ n pp p. kp l p }l e }v p e, tp v pr qƒ k pp p, n ˆk loo (NPAFC) l p t ll rp ~ n p ~rp r q pp p. m l k r ll( )p v op k lp, ll n ll e r te o l lll p p jp. y r, ~o,, llp ˆÎ r l. kl BSPE , 160pp llp r vor r l. o,, 125pp }l ll mm pln. Ž, Ž t, 140pp.,, p, pv kk }l llp ~q, ls. vl, 54: po n llp q, o p Žk. klo BSPG , 201pp. roe, po, k, v, orr vl p ˆk ll(chum salmon, Oncorhynchus keta)p. v, 36(6): roe, po, e ~,, k Microsatellite DNA pn ll(oncorhynchus keta, chum salmon)m ll(o. gorbuscha, pink salmon)p. qo v, 4(1): Allendorf, F.W. and L.W. Seeb Concordance of genetic divergence among sockeye salmon populations at allozyme, nuclear DNA, and mitochondrial DNA markers. Evolution., 54: Brown, W.M., E.M. Prage, A. Wang and A.C. Wilson Mitochondrial DNA sequences of primates: Tempo and mode of evolution. J. Mol. Evol., 18: Clement, M., D. Posada, and K.A. Crandall TCS: a computer program to estimate gene genealogies. Mol. Ecol., 9(10): Choi, Y.S Cloning and Nucleotide Sequence of Mitochondrial NADH dehydrogenase subunit 3 DNA for Population Analysis of Chum salmon, (Oncorhynchus keta). Korea. MS. Thesis, Kangnung National University. Dominico, M.J. and R.B. Phillips Phylogenetic analysis of Pacific salmon (genus Oncorhynchus) based on mitochondrial DNA sequence data. Mol. Phylo. Evol., 4: Excoffier, L., P.E. Smouse, and J.M. Quattro Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data. Genetics, 131: Hong K.P., J.G. Myuong, J.K. Son and C.W. Park A biochemical study for the development of genetic marker in salmonids in Korea. Bull. Korean Fish. Soc., 27: Jacobs, H.T., D.J. Elliott, V.B. Math and A. Farquharson Nucleotide sequence and gene organization of sea urchin mitochondrial DNA. J. Mol. Biol., 202: Jung, W., Y.-H. Lee, S. Kim, D.H. Jin, K.B. Seong Genetic identification of the North Pacific Chum Salmon (Oncorhynchus keta) Stocks. J. Kor. Fish. Soc., 36(6): Kang, S. and S. Kim, Comparison of biological characteristics of chum salmon, Oncorhynchus keta from the eastern and western North Pacific. J. Korean Soc. Fish. Res., 6: Kim, G.-E., Y.-H. Lee, G. Kang, C.-G. Kim, W. Jung, K.-B. Seong, J.E. Seeb, S. Kim, S. Kang Genetic Diversity and Population Structure of the Chum Salmon in the North Pacific. NPAFC bulletin #4 (in press). Lee, H.J., J.Y. Park, J.H. Lee, K.S. Min, I.G. Jeon, M.A. Yoo and W.H. Lee Phylogeny of the subfamily Salmonidae distributed in Korea based upon nucleotide sequences of mitochondrial ribosomal RNA genes. J. Korean Fish. Soc., 33: McKay, S.J., R.H. Devlin and M.J. Smith Phylogeny of Pacific salmon and trout based on growth hormone type-2 and mitochondrial NADH dehydrogenase subunit 3 DNA sequences. Can. J. Fish. Aquat. Sci., 53: Okazaki, T Genetic structure of chum salmon Oncorhynchus keta river population. Bull. Jap. Soc. Sci. Fish., 49: Olsen, J.B., P. Bentzen and J.E. Seeb Characterization of seven microsatellite loci derived from pink salmon. Mol. Ecol., 7: Sato, S., H. Kojima, J. Ando, H. Ando, R.L. Wilmot, L.W. Seeb, V. Efremov, L. LeClair, W. Buchholz, D. Jin, S. Urawa, M. Kae-

8 llp } orr o pr p llm orr 101 riyama, A. Urano, and S. Abe Genetic population structure of chum salmon in the Pacific Rim inferred from mitochondrial DNA sequence variation. Environ. Biol. Fish., 69: Sato. S., J. Ando, H. Ando, S. Urawa, A. Urano and S. Abe Genetic variation among Japanese populations of chum salmon inferred from the nucleotide sequences of the mitochondrial DNA control region. Zool. Sci., 18: Schineider, S., D. Roessli, and L. Excoffier Arlequin version 2.000: A software for population genetics data analysis. Genetics and Biometry Lab., Univ. of Geneva, Switzerland. Seo, H., S. Kim, K. Seong, and S. Kang Variability in scale growth rates of chum salmon (Oncorhynchus keta) in relation to climate changes in the late 1980s. Prog. in Oceanogr., 68: Slatkin, M A measure of population subdivision based on microsatellite allele frequencies. Genetics, 139: Sunnucks, P Efficient genetic markers for population biology. TREE., 15: Tamura, K. and M. Nei Estimation of the number of the nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol., 10: Urawa, S., Ocean migration route of Japnaese chum salmon with a reference to future salmon research. Nat. Salmon Resources Center Newsletter, 5: 3 9. (in Japanese) o 7p or o 18p r }ˆ voo: k

Genetic Diversity and Population Structure of. salmon in the North Pacific.

North Pacific Anadromous Fish Commission Bulletin No. 4: 203 209, 2007 Genetic Diversity and Population Structure of Chum Salmon in the North Pacific Go-Eun Kim 1, Youn-Ho Lee 1, Geoyoung Kang 1, Choong-gon