Atlantic salmon populations reveal adaptive divergence of immune related genes - a duplicated genome under selection

RESEARCH ARTICLE Open Access A lan ic salmon popula ions e eal adap i e di e gence o immune ela ed genes - a duplica ed genome unde selec ion E ik Kjæ ne -Semb 1,2* , Fe nando Ayllon 1 , Tomasz Fu manek 1 , Vida Wenne ik 1 , Gei Dahle 1 , Ee o Niemelä 3 , Mikhail Oze o 4 , Juha-Pekka Vähä 4,6 , Ke in A. Glo e 1,2 , Ca l J. Rubin 5 , Anna Wa gelius 1 and Rol B. Ed a dsen 1* Abs ac Backg ound: Popula ions o A lan ic salmon display highly signi ican gene ic di e ences wi h un esol ed molecula basis. These di e ences may esul om sepa a e pos glacial coloniza ion pa e ns, di e si ying na u al selec ion and adap a ion, o a combina ion. Adap a ion could be in luenced o e en acili a ed by he ecen whole genome duplica ion in he salmonid lineage which esul ed in a pa ly e aploid species wi h duplica ed genes and egions. Resul s: In o de o elucida e he genes and genomic egions unde lying he gene ic di e ences, we conduc ed a genome wide associa ion s udy using whole genome esequencing da a om eigh popula ions om No he n and Sou he n No way. F om a o al o ~4.5 million sequencing-de i ed SNPs, mo e han 10 % showed signi ican di e en ia ion be ween popula ions om hese wo egions and en selec i e sweeps on ch omosomes 5, 10, 11, 13–15, 21, 24 and 25 we e iden i ied. These comp ised 59 genes, o which 15 had one o mo e di e en ia ed missense mu a ion. Ou analysis showed ha mos sweeps ha e pa alogous egions in he pa ially e aploid genome, each lacking he high numbe o signi ican SNPs ound in he sweeps. The mos signi ican sweep was ound on Ch 25 and ca ied se e al missense mu a ions in he an i i al mx genes, sugges ing ha hese popula ions ha e expe ienced di e ing i al p essu es. In e es ingly he second mos signi ican sweep, ound on Ch 5, con ains wo genes in ol ed in he NF-KB pa hway (nkap and nk ), which is also a known pa hogen a ge ha con ols a la ge numbe o p ocesses in animals. Conclusion: Ou esul s show ha na u al selec ion ac ing on immune ela ed genes has con ibu ed o gene ic di e gence be ween salmon popula ions in No way. The di e ences be ween popula ions may ha e been acili a ed by he plas ici y o he salmon genome. The obse ed signa u es o selec ion in duplica ed genomic egions sugges ha he ecen ly duplica ed genome has p o ided aw ma e ial o e olu iona y adap a ion. Keywo ds: Whole genome duplica ion, Adap a ion, Aquacul u e, Immune sys em, GWAS, Resequencing, Selec i e sweep, SNPs, Salmo sala Backg ound In addi ion o being one o he mos highly p ized esh- wa e ish o ec ea ional ishing, he A lan ic salmon (Salmo sala L.) is one o he mos economically impo - an aquacul u e species wo ldwide. I s na u al dis ibu ion is h oughou he No h A lan ic, anging om Long Is- land Sound o Unga a Bay in he wes and om No he n Po ugal o he Ba en s Sea in he eas [1]. This dis ibu ion is he esul o pos glacial coloniza ion o ecosys ems ha became a ailable when he glacial ice e ea ed abou 10,000 yea s ago [2]. A lan ic salmon is cha ac e ised by highly signi ican , hie a chically s uc u ed popula ion gene ic di e gence, wi h he la ges di e ences obse ed be ween he Eu opean and No h Ame ican lineages [3–5]. This di e gence is also obse ed on a egional scale, p esumably as a consequence o he coloniza ion p ocess associa ed wi h he e ea o he glacie [6, 7]. Mo eo e , local scale di e en ia ion ex- is s, o example be ween neighbou ing i e s [8–10] and * Co espondence: [email p o ec ed];[email p o ec ed] 1 Ins i u e o Ma ine Resea ch, Be gen, No way Full lis o au ho in o ma ion is a ailable a he end o he a icle © 2016 The Au ho (s). Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Kjæ ne -Semb e al. BMC Genomics (2016) 17:610 DOI 10.1186/s12864-016-2867-z among ibu a ies wi hin he same i e which migh be explained by es ic ed gene low, gene ic d i and adap a ion [11–13]. A lan ic salmon exhibi a ela i ely complex li e his o y ha includes spawning and ju enile ea ing in eshwa e ollowed by ex ended ocean mig a ions o he eeding g ounds [14]. As a consequence, salmon go h ough se - e al dis inc ansi ions ha a e cha ac e ized by changes in beha iou and physiology [15]. They a e also able o adap o a ying local condi ions h oughou hei ange o en i onmen s [16], exempli ied by hei abili y o in- habi i e s wi h a wide ange o empe a u es, om Spain o he colde A c ic la i udes [17]. P e ious s udies ha e shown di e ences in empe a u e and clima e o be as- socia ed wi h gene ic di e ences be ween salmon popu- la ions [7, 18], and la i ude also seems o be co ela ed wi h allele equencies o ma ke s ele an o immune esponse in Ame ican and Eu opean A lan ic salmon popula ions, possibly due o empe a u e induced di e - ences in pa hogen-d i en selec ion o o he en i onmen al ac o s [19–21]. In he wild, A lan ic salmon a e cons an ly con on ed wi h a ange o pa hogens, and ha e consequen ly de el- oped nume ous inna e and adap i e immune mechanisms o o e come in ec ious challenges [22]. Recen s udies sugges ha he p e alence o pa asi es and in ec ious dis- eases is inc easing in wild popula ions pa ly due o global wa ming [23, 24]. Gi en he comme cial ele ance o A lan ic salmon and he ecen elease o a e e ence genome [25], pa icula e o should be made o iden- i y genes a ge ed by na u al selec ion in wild A lan ic salmon popula ions ha ul ima ely can lead o op i- mized aquacul u e p ac ices. The po en ial ele ance o hese indings o he A lan ic salmon a ming indus y is exempli ied by he iden i ica ion o In ec ious Pan- c ea ic Nec osis (IPN) Vi us esis ance [26] and age a ma u i y associa ed genes [27, 28]. A ela i ely ecen whole genome duplica ion occu ed in he salmonid lineage some 80 million yea s ago [29], esul ing in a pa ly e aploid genome unde going ediploidiza ion. Consequen ly he genome con ains many pa alogous egions ha could p o ide aw ma e ial o e olu ion as pa alogous genes and egions can di e si y and acqui e new unc ions [30]. Based upon he analysis o mic osa elli e and SNP ma ke s, se e al s udies ha e demons a ed ha he e a e highly signi ican gene ic di e ences be ween A lan ic salmon popula ions loca ed in he no h and sou h o No way [31–33]. Howe e , he genomic egions and genes behind he di e ences ha e no been in es iga ed in de ail, and consequen ly, he po en ial adap i e sig- ni icance o his gene ic di e gence emains elusi e. Recen ly, a genome wide associa ion s udy (GWAS) based upon whole genome esequencing da a e ealed a selec i e sweep in A lan ic salmon s ongly associa ed wi h age o ma u a ion [27]. Using a simila me hodo- logical app oach, he p esen s udy aimed o iden i y genes and genomic egions di e ging be ween A lan ic salmon popula ions in he no h and sou h o No way. In o de o achie e his objec i e, salmon popula ions inhabi ing he ou i e s Tanael a, Laksel , Al ael a and Reisael a om No he n No way and he ou i e s Gloppenel a, Eidsel a, Suldalslågen and Å dalsel a om Sou he n No way we e chosen o esequencing using DNA pools (n= 30 ish pe i e , Fig. 1). The majo inding in his s udy was he obse a ion ha di e si ying na u al se- lec ion has ac ed on immune ela ed genes causing adap- i e di e gence be ween popula ions in he no h and sou h o No way. Resul s and discussion Wholegenomesequenceda a om eigh selec ed i - e s along he No wegian coas (Fig. 1) was mapped o he mos ecen A lan ic salmon e e ence genome (AKGD00000000.4). This yielded a 26.7× a e age dep h o co e age o uniquely mapped eads pe i e . SNP calling e ealed 4,450,990 high quali y SNPs. To quan i y he gen- e ic di e ence be ween popula ions o he chosen i e s, Hudson’s es ima o o W ig h’s ixa ion index (F ST ) [34] was calcula ed (Addi ional ile 1: Table S1). A phylogene ic ee was made using his dis ance ma ix o illus a e and con i m he epo ed la ge gene ic di e ence be ween he no he n and sou he n popula ions o A lan ic salmon in No way (Fig. 1). S a is ical analysis using he Coch an- Man el-Haenszel es o di e en allele equencies be- ween no he n and sou he n A lan ic salmon popula ions e ealed 474,410 SNPs wi h signi ican ly di e en allele equencies (0.1 % FDR, Fig. 2a). Genomic egions sub- jec ed o ecen posi i e selec ion a e expec ed o ha e lowe he e ozygosi y han o he egions, and i he selec i e p essu e di e s be ween popula ions, highe F ST is ob- se ed [35]. An app oach calcula ing F ST and he e ozygosi y in 50 kb sliding windows has p e iously been used o iden- i y genomic egions unde selec ion (selec i e sweeps) [36]. Thisme hodwasused o indselec i esweepswhichdi e be ween no he n and sou he n salmon popula ions in No way (Addi ional ile 1: Figu e S1). The combined F ST / he e ozygosi y app oach sugges ed 10 selec i e sweeps ha di e ed be ween he wo geog aphical egions. The sweeps anged om 75,000 o 575,000 bp in size, and we e ound in ch omosomes 5, 10, 11, 13–15, 21, 24 and 25 (Table 1). These sweeps con ained in o al 59 genes in ol ed in a numbe o di e en biological p ocesses including cell di - ision, cy okinesis, angiogenesis, de elopmen , ansc ip- ional egula ion and immune esponse. Fo a de ailed lis o gene ID and sho desc ip ion o unc ion see Addi ional ile 1: Table S2. Kjæ ne -Semb e al. BMC Genomics (2016) 17:610 Page 2 o 12 The high numbe o SNPs and genes in he selec i e sweeps complica es he ask o pin-poin ing he mos impo an gene ic di e ences. The e o e, we ocused on missense mu a ions ha induce amino acid changes in p o eins, since hese a e mo e likely o con e a di e - ence in biological unc ion. Wi hin he iden i ied sweeps 20 signi ican ly di e en ia ed missense SNPs we e ound, comp ising 15 di e en genes dispe sed in 6 selec i e sweeps (Table 2). Th ee missense mu a ions we e ob- se ed in he sweep on Ch 10, all in a single gene, anln, encoding an ac in-binding p o ein equi ed o cy okin- esis. Th ee genes on Ch 13 ha bo missense mu a ions: pc2, in ol ed in chemosenso y ansduc ion, and in e - es ingly knockou mice display changes in hei sexual, agg essi e, and pa en ing beha io s [37]; m1,anenzyme essen ial o he p oduc ion o deoxy ibonucleo ides; b1, which p omo es G0-G1 ansi ion when phospho yla ed by CDK3/cyclin-C ac s as a ansc ip ional ep esso o E2F1 a ge genes. Also in he Ch 14 sweep he e a e h ee genes wi h missense mu a ions: adnp,ahomeo- domain con aining DNA binding ansc ip ion ac o ; cps 1, encoding a componen o he clea age and polya- denyla ion speci ici y ac o complex; pa p10, encoding a ADP- ibosyl ans e ase in ol ed in apop osis, NF-kB signaling, and DNA damage epai [38]. The sweep on Ch 21 con ains one gene, naseh2b, which is linked o a ch onic in lamma o y diso de in humans [39]. The second mos signi ican selec i e sweep was ound on Ch 5 (Fig. 2b) and included he s ess and immune esponse ansc ip ion ac o genes nk and nkap;zb b33 encoding a ansc ip ional egula o binding o me hyla ed CpG dinucleo ides, and a gene wi h unknown unc ion, sowahc. Bo h Nk and Nkap a e ansc ip ion ac o s which egula e he NF-kB pa hway in which Nkap ac i- a es many cell p ocesses including in lamma ion, im- muni y, di e en ia ion, cell g ow h and apop osis, while Fig. 1 Geog aphical o e iew o sampled salmon popula ions. Resequenced genomes o A lan ic salmon om ou popula ions in No he n No way and ou popula ions in Sou he n No way we e analyzed in his s udy and a e shown as black do s on a map. A phylogene ic ee based on pai wise calcula ions o ixa ion index (F ST ) illus a es he gene ic dis ances be ween he sequenced popula ions. 19 addi ional popula ions om i e s along he No wegian coas we e analyzed using geno yping assays and he geog aphical sampling loca ions o hese a e indica ed acco ding o numbe s in he map Kjæ ne -Semb e al. BMC Genomics (2016) 17:610 Page 3 o 12 Nk media es ansc ip ional ep ession o ce ain Nkap esponsi e genes. Since NF-kB signaling pa hways ac i a e heimmunesys emin hehos , hesep o eins a e key a ge s o p o eases exp essed by in ading pa hogens [40]. Func ional s udies o he Nkap p o ein ha e e ealed oles o his p o ein in T-cell ma u a ion [41] and mRNA splicing [42]. To ou knowledge, no p e ious s udies ha e iden i ied unc ionally signi ican SNPs associa ed wi h any o he ou genes loca ed wi hin his sweep, howe e one o he SNPs ound in nkap is loca ed in a highly conse ed egion necessa y o ansc ip ional ep ession. He e he aline is conse ed in o he species ep esen ing he ances al a ian while in No he n No way me hionine is mos common (Addi ional ile 1: Figu e S2). This inding may be e- la ed o di e ences in immune de ense be ween salmon om hese wo egions, a sugges ion suppo ed by he ac ha he NF-kB pa hway is di e en ly egula ed in IPN esis an salmon [43]. Fu he s udies will e eal how hese SNPs modula e he unc ion o NF-kB and i us esponse o i o he unc ional p ope ies a e as- socia ed wi h he selec i e sweep on Ch 5. The mos signi ican sweep was ound on Ch 25 and con ained a clus e o i e mx (myxo i us esis ance) genes known o be in ol ed in de ense agains i uses. Th ee o hese mx genes con ained missense mu a ions; mx1-1,mx1-2 and mx2-1 (Fig. 2c). These p o eins a e dynamin-like GTPases induced upon i us in ec ion h ough he inna e in e e on sys em. I has been shown ha hey can ac b oadly agains bo h DNA and RNA i- uses and speci ically agains ce ain i uses [44] and s udies in mouse, human and chicken ha e shown ha single missense mu a ions in Mx1 and Mx2 can con e such speci ic esponses [45–48]. I is possible ha he iden i ied missense SNPs in he mx genes e lec speci ic adjus men s o di e en i al disease p essu es be ween Fig. 2 Iden i ica ion o genomic egions unde selec ion in No he n and Sou he n No way. aManha an plo showing di e en ia ed SNPs be ween no he n and sou he n popula ions o A lan ic salmon in No way. The x-axis indica es ch omosomal posi ions; he y-axis p esen s he nega i e loga i hm o he P- alue o allele equencies being di e en be ween he wo geog aphical egions. SNPs in selec i e sweep egions iden i ied using FST/he e ozygosi y a e indica ed by ed do s. SNPs abo e he dashed ho izon al line (p< 1.0658e-4, 0.1 % FDR) ha e signi ican ly di e en allele equencies be ween he wo geog aphical egions. band cMagni ica ion showing 500 kb o selec i e sweeps on Ch 5 and Ch 25. SNPs a e indica ed as black do s and missense mu a ions a e ma ked wi h ed squa es. The ack labeled “HET”shows he he e ozygosi y o salmon om No he n (blue) and Sou he n No way (g een) in 3 kb windows. The ack labeled “FST”shows he F ST be ween popula ions in No he n and Sou he n No way in 3 kb windows. In he bo om, iden i ied genes a e shown, wi h genes con aining di e en ia ed missense mu a ions colo ed black. The x-axis shows he ch omosomal posi ions gi en in kb Kjæ ne -Semb e al. BMC Genomics (2016) 17:610 Page 4 o 12 no he n and sou he n popula ions o salmon. We iden- i ied missense SNPs in all egions o he p o ein includ- ing a SNP in he an i i al speci ici y domain in exon 13 (Addi ional ile 1: Figu e S3). This SNP ep esen s a s uc u ally ele an amino acid subs i u ion, whe e a ginine seems o be he ances al a ian and cys eine he de i ed a ian domina ing in he no he n popula- ion (Ch 25 posi ion: 47,120,121). Likewise, SNPs in his domain ha e been associa ed wi h speci ic i us esis - ance in chicken [49, 50] and pig [51]. SNPs in mx genes Table 1 Selec i e sweeps. F ST and he e ozygosi y es ima ions in sliding windows we e used o iden i y di e en ia ed loci unde going selec ion in ei he no he n o sou he n popula ions o A lan ic salmon in No way. Genes ca ying di e en ia ed missense mu a ions a e shown in bold Ch omosome Ch omosomal egion Sweep leng h (bp) Genes in selec i e sweeps 5 22,475,000 –22,800,000 325,000 nmd, el 1, nk , sep 6, sowahc, pl39, up 3b, nkap,zb b33, a p1b4, lamp2, cul4b, mc s1, c1gal 1c1, clic2 10 76,250,000 –76,450,000 200,000 sep 7, anln, p m 7, coq9, pol 2c, dga 1, nedd4, ada 1, spi e2 11 19,225,000 –19,300,000 75,000 numa1 13 78,325,000 –78,425,000 100,000 pc2, m1, slc6a7, b1, lpa 6 13 81,025,000 –81,150,000 125,000 eda2 , a , ophn1 14 64,700,000 –65,275,000 575,000 pa d6g, bloc1s4, nu 2, adnp2, xnl4a, pqlc1, kcng2, c dp1, adck5, cps 1,pa p10, s 3gal1, khd bs3 15 46,925,000 –47,200,000 275,000 mdga1 21 24,850,000 –25,075,000 225,000 im13, naseh2b, n 0b1, il1 apl1 24 34,225,000 –34,525,000 300,000 edil3 25 47,075,000 –47,225,000 150,000 s xbp5l, g 2e1, mx1-1,mx1-2,mx2-1, smcp-1a, mx1-3 Table 2 Missense mu a ions in selec i e sweeps. Se e al missense mu a ions we e disco e ed in he selec i e sweeps. The able lis s esequencing de i ed e e ence alleles equencies in he no he n (N) and sou he n (S) popula ions o salmon in No way. SNPs om Ch 5 and Ch 25 selec ed o geno yping a e shown in bold Ch Posi ion -log 10 P Re e ence allele equency (S/N) Re /al nucleo ide Re /al amino acid Gene Desc ip ion o gene 5 a 22,641,277 29.6 1.00/0.34 A (G) Phe (Se ) nk NF-kB ep essing ac o . 5 22,691,092 27.5 0.95/0.29 G (T) Ala (Se ) sowahc Unknown unc ion. 5 a 22,708,279 28.8 1.00/0.33 C (T) Val (Me ) nkap NF-kB ac i a ing p o ein. 5 a 22,719,453 19.4 0.79/0.27 T (A) Val (Glu) zb b33 T ansc ip ional egula o . 10 b 76,272,006 18.7 0.94/0.46 T (G) Asp (Glu) anln Ac in binding p o ein. 10 76,272,245 18.4 0.96/0.49 C (A) P o (Gln) anln Same as abo e. 10 76,278,414 14.5 0.32/0.76 G (A) Ala (Th ) anln Same as abo e. 13 78,341,469 24.1 0.97/0.43 G (A) Gly (A g) pc2 T ansien Recep o Ca ion Channel. 13 78,349,376 20.3 0.98/0.40 T (G) Glu (Ala) m1 Ribonucleoside-diphospha e educ ase. 13 b 78,413,379 20.7 0.98/0.48 A (T) Gln (Leu) b1 Regula o o en y in o cell di ision. 14 64,739,123 20.0 0.55/0.01 T (C) Asn (Ile) adnp2 T ansc ip ion ac o . 14 b 64,988,859 11.9 0.36/0.79 A (T) Asn (Lys) cps 1 P e-mRNAs p ocessing. 14 b 65,006,543 20.6 0.38/0.94 A (C) Asp (Glu) pa p10 ADP-Ribosyl ans e ase. 21 b 24,974,056 19.7 0.98/0.46 T (A) Me (Lys) naseh2b Non ca aly ic subuni o RNase H2. 25 a 47,105,598 26.0 0.84/0.03 A (G) Th (Ala) mx1-1 In e e on-induced an i i al. 25 47,108,912 27.2 0.91/0.00 G (A) Val (Ile) mx1-1 Same as abo e. 25 47,111,137 18.0 0.79/0.06 G (A) Val (Me ) mx1-1 Same as abo e. 25 a 47,120,121 33.1 0.83/0.06 C (T) A g (Cys) mx1-1 Same as abo e. 25 47,147,348 27.2 0.22/0.98 G (T) P o (His) mx1-2 Same as abo e. 25 47,181,001 25.4 0.85/0.00 T (C) His (A g) mx2-1 Same as abo e. a Allele equencies om geno yping a e illus a ed in Fig. 4 b Allele equencies om geno yping a e illus a ed in Addi ional ile 1: Figu e S5 Kjæ ne -Semb e al. BMC Genomics (2016) 17:610 Page 5 o 12 ha e also been in es iga ed in ano he ish species, he u bo [52], howe e , p ope ies ela ed o p o ec ion agains i uses we e no in es iga ed in his s udy. In ainbow ou (Onco hynchus mykiss) gene ic a ia ion in mx be ween s ains in exon 3–6, was co ela ed wi h suscep ibili y o in ec ious hema opoie ic nec osis i us (IHNV) [53]. This i us also in ec s A lan ic salmon and ou disco e y o a missense mu a ion in exon 6 sug- ges s ha salmon could ha e adap ed o he IHNV (Addi ional ile 1: Figu e S3). In addi ion, di e en s ains o ainbow ou display a iable suscep ibili y o his i us [54]. In his s udy we canno elucida e he unc ional signi icance o he acqui ed SNPs in mx in No he n No way, howe e , u he s udies will e eal whe he any o hese changes ha e been in ol ed in hos - i us adap a ion [55]. We also in es iga ed whe he he selec i e sweeps on Ch 5 and Ch 25 had pa alogous egions in he pa ially e aploid salmon genome [56]. In silico analysis showed ha bo h sweeps ha e pa alogous egions loca ed on o he ch omosomes. The Ch 5 sweep has a pa alogous egion on Ch 9 (Addi ional ile 1: Figu e S4, posi ion 51,349,279 o 51,849,279), which did no con ain any di - e en ia ed SNPs. The syn eny is conse ed in o he species, and he exis ence o only one copy in zeb a ish (Danio e io), combined wi h he obse a ion ha mis- sense mu a ions on Ch 5 a e no p esen in he pa alo- gous genes on Ch 9, indica e ha he mu a ions a ose a e he salmonid speci ic whole genome duplica ion (WGD). Based upon his obse a ion, i is possible o specula e ha he WGD p o ided pa alogous egions whe e one copy was ee o sub- o neo- unc ionalize, much like he heo y o duplica ed genes [57] which has been sugges ed o be impo an o e olu iona y adap- a ion and inno a ion in salmon [58], in eleos s [59] and in gene al [30]. A simila pic u e is seen o he sweep on Ch 25 whe e he pa alogous egion ha bo s aclus e o h eemx genes on Ch 12 (posi ion 66,552,602 o 67,052,602), bu ca ies no di e en ia ed SNPs o missense mu a ions. While he sweeps on Ch s 11, 15, 21 and 24 ha e no clea pa alogous egions, he sweeps on Ch 10, Ch 14 and he wo sweeps on Ch 13 also ha e pa alogous egions wi h e y ew signi ican ly di e en ia ed SNPs, on Ch 16, 27 and 4, espec i ely (Fig. 3). Simila ly, in ou ecen disco e y o he loci in Ch 25 con olling age a ma u i y [27] we in es iga ed he wo pa alogous egions in Ch 21, bo h o which we e wi hou SNPs associa ed wi h he ai . Toge he , hese indings indica e ha he pa ially e aploid s age may be bene icial o adap a ion, since one gene copy o gene clus e can keep he o iginal unc ion while he o he can adap o a new si ua ion such as no el disease p essu es. In his s udy, he ini ial esequencing was based only upon males. This is because i allowed eusing sequence da a om ou p e ious wo k [27]. The a ge ed SNP analysis, used o alida e he esul s om esequencing in a la ge independen se o i e s, was conduc ed using bo h males and emales (Figs. 1 and 4). Geno yping o mixed sex salmon om 19 i e s (n= 20 salmon/ i e ) along he No wegian coas (Fig. 1) o i e missense SNPs on Ch 5 and 25 con i med s ong gene ic di e en ia ion be ween salmon popula ions om he no h and sou h o No way (Fig. 4). Popula ions om no he n i e s (1–9) displayed allele equencies in he ange 0–0.7, while hose om sou he n i e s (11–19) we e close o ixa ion o one allele a hese wo loci. Salmon om i e 10, Målsel , shows in e media e equencies, which co esponds well wi h wha has been epo ed in he li e a u e [31, 32]. These esul s also con i m allele equency es ima ions om he pooled esequencing (Table 2). In addi ion, we designed Sequenom assays o i e o he missense SNPs in o he egions; one SNP each o sweeps on Ch s 10, 13 and 21, and wo SNPs in Ch 14. Geno yping was pe - o med o all 19 i e s (Addi ional ile 1: Figu e S5). The allele equencies showed he same clea di e ence be- ween he no he n and sou he n popula ions. Fo he SNPs on Ch 14 he e appea s o be an addi ional gene ic shi be ween he i e s 14, S jø dalsel a and i e s sou h o his. In addi ion o he da a p oduced wi hin he p esen s udy, esequencing da a om a ecen publica- ion was downloaded and compa ed o ou esul s [28]. The downloaded da a include h ee indi idually se- quenced salmon om 4 sou he n and 3 no he n salmon i e s in No way. These da a co obo a e ou esequen- cing and geno yping esul s (Addi ional ile 1: Table S3). Ou su eyed SNPs he e o e also ep esen obus and good gene ic ma ke s o dis inguishing no he n and sou he n popula ions o A lan ic salmon in No way. Fu- u e s udies on an ex ended se o popula ions may e eal i hese a e also obus ma ke s o de ec ing gene ic s uc u ing in o he pa s o he dis ibu ion ange o he species. A lan ic salmon aquacul u e in ol es ea ing domes i- ca ed ish ha o igina e om comme cial b eeding p o- g ams. Fo y wild popula ions om bo h he no h and sou h o No way we e sampled when es ablishing he na ional b eeding p og ams o salmon [60]. Howe e , analyses o gene ic ma ke s demons a e ha he e is a dominance o salmon om Sou he n No way in he do- mes ica ed lines cu en ly in p oduc ion [61]. Gene ic analyses o a med salmon escapees in No way ha e un- co e ed gene ic in og ession in o na i e salmon popula- ions in bo h No he n and Sou he n No way, bu he biological consequence emains unknown [32, 61, 62]. Consequen ly he esul s om he p esen s udy, whe e adap i e gene ic di e gence be ween wild salmon om popula ions loca ed in he no h and sou h o No way was e ealed, i is likely ha he po en ial nega i e Kjæ ne -Semb e al. BMC Genomics (2016) 17:610 Page 6 o 12 gene ic impac o domes ica ed salmon in og ession is g ea e in popula ions loca ed in no he n egions, since he a med ish o igina e mos ly om wild Sou he n No way popula ions. Conclusion In his s udy we pe o med a GWAS by genome ese- quencing wi h he aim o sc een he A lan ic salmon genome o gene ic di e en ia ion be ween he no he n and sou he n popula ions in No way. By in es iga ing eigh i e s we unco e ed en pa icula ly s iking sweeps including wo clus e s o immune ela ed genes ha bo ing missense mu a ions. A easible in e p e a ion is ha di e en popula ions o A lan ic salmon ha e his- o ically been exposed o di e en selec ion p essu es in he o m o pa hogens. Some o hese adap ed alleles could be ad an ageous o aquacul u e p oduc ion which is cu en ly hampe ed by a numbe o diseases, including i us in ec ions [63]. Fu u e s udies should include gene edi ing o immune genes ound in hese selec i e sweeps [64, 65] in combina ion wi h i al exposu e expe imen s. Wi hin hese expe imen s, i uses ele an o salmon aquacul u e should be he p ima y ocus since inding speci ic esis ance alleles can be o signi ican alue o he indus y and could also be used o p o ec ing wild ish agains high disease p essu es posed by open cage aquacul u e [66]. Upon inding he p o ec i e alleles, se- lec i e b eeding on indi iduals wi h bene icial haplo ypes could lead o inc eased wel a e o aquacul u e salmon, dec eased disease p essu e on wild popula ions and could also be economically a o able o he indus y. On he o he hand, u he s udies should in es iga e he impac o gene ic in og ession om e ile aquacul u e escapees on he adap i e gene ic p ope ies in wild pop- ula ions. To educe he isk o his unwan ed loss o local adap a ion and al e a ion o i ness- ela ed ai s, a Fig. 3 Compa ison o sweeps and pa alogous egions. Rep esen a ion o he selec i e sweeps ha ing pa alogous egions in he salmon genome, displaying a 500 kb o e iew o he SNPs in he selec i e sweeps (le side) and in he co esponding pa alogous egions ( igh side). The y-axis shows –log 10 P o he SNPs being di e en in popula ions be ween he no h and sou h o No way, and he x-axis ep esen s he posi ion in he gi en ch omosome. The dashed lines indica e he genome wide signi icance h eshold (0.1 % FDR) Kjæ ne -Semb e al. BMC Genomics (2016) 17:610 Page 7 o 12 sus ainable solu ion would be he use o s e ile ish in aquacul u e, especially in No he n No way. Fu u e s ud- ies should also in es iga e whe he pa alogous egions o selec i e sweeps ha e unde gone posi i e selec ion o no , as he la e scena io would sugges an e olu iona y mech- anism which p o ides highe adap i e possibili ies when a genome is pa ially e aploid. Me hods Samples and sampling Scales om 30 A lan ic salmon males pe i e we e selec ed om a sample se o 26,000 samples collec ed in coas al ishe iesinNo he nNo way.In he Kola c ic Salmon p o- jec (h p://p osjek . ylkesmannen.no/Kola c icsalmon), he mul ilocus geno ypes o all indi iduals we e compa ed o a gene ic baseline consis ing o o e 180 i e s om No he n Russia and No way and we e assigned o i e o o igin. Samples ha we e assigned wi h high p obabili y o ou i - e s in No he n No way; Al ael a, Reisael a, Laksel and Tanael a we e gene ously made a ailable o his s udy. 30 salmon males om each o ou di e en i e s in Sou he n No way, including Å dalsel a, Eidsel a, Gloppenel a and Suldalslågen we e sampled and esequenced in a ecen s udy [27]. In addi ion o hese, we also used male and emale salmon DNA om 19 i e s along he No wegian coas . These included 20 pa indi iduals om each o he i e s G ense Jakobsel , Neiden, Be gebyel a, Komagel a, Kongs jo del a, Lang jo del a, Bø sel a, S ab- bu sel a, Reppa jo dsel a, Målsel , Laukhelle, Al s åg ass- d age , Å gå ds assd age , S jø dalsel a, Jøls a, Lyseel a, Bje k eimsel a, S o el a and Enningdalsel a ( ep esen ed by numbe s in Fig. 1). Wi h he excep ion o Enningdalsel a whe e he sample was ob ained om scales collec ed by ec ea ional ishe ies, hese samples we e ob ained om ins collec ed by elec o ishing o ju enile salmon om muli ple loca ions in he i e s. DNA ex ac ion and sequencing DNA om he 19 i e s o geno yping was ex ac ed om scales o in samples using Qiagen DNeasy Blood and Tissue Ki (Qiagen, Hilden, Ge many) acco ding o manu ac u e ’s ecommenda ions. F om salmon belong- ing o he ou popula ions in No he n No way o al DNA was ex ac ed om scales using Qiagen DNeasy Blood and Tissue Ki . Equal amoun s o DNA om en indi iduals we e pooled o make h ee pools pe i e , o aling 30 indi iduals om each i e . Pai ed-end lib a ies we e cons uc ed using he Genomic DNA Sample P epa - a ion Ki (Illumina, CA, USA) acco ding o manu ac u e ’s ins uc ions and sequenced on he Illumina HiSeq2000 pla o m (Illumina, CA, USA) a he No wegian Sequen- cing cen e (h ps://www.sequencing.uio.no, Oslo, No way) wi h each pool sequenced in sepa a e lanes. Sequence mapping and SNP calling To ensu e high quali y sequences, sequenced eads we e inspec ed wi h Fas QC (h p://www.bioin o ma ics. bab aham.ac.uk/p ojec s/ as qc/). Adap e sequence e- mo al and quali y imming was done wi h Cu adap [67], esul ing in 1,077,839,448 (SD 40,407,492) pai ed eads on a e age pe i e . Sequenced eads we e mapped o he mos ecen elease o he salmon genome Fig. 4 Allele equencies o i e missense mu a ions in popula ions along he No wegian coas . Th ee missense mu a ions iden i ied in selec i e sweeps on Ch 5 and wo on Ch 25 we e used in a geno yping assay o A lan ic salmon popula ions along he No wegian coas . The g aphs show equencies o he e e ence alleles. Ri e numbe s a e explained in Fig. 1. SNPs on Ch 5 included missense mu a ions in he genes nk , nkap and zb b33 (g een lines) and missense mu a ions on Ch 25 we e loca ed in he mx1-1 gene (blue lines) Kjæ ne -Semb e al. BMC Genomics (2016) 17:610 Page 8 o 12 (AGKD0000000.4) using Bow ie2 ( .2.1.0) [68] wi hou so clipping (end- o-end mode). To inc ease he sensi i - i y o he mapping, seed leng h (−L pa ame e ) was se o 18 and he in e al be ween ex ac ed seeds (−ipa ame e ) was se o S,1,1.5 co esponding o he unc ion (L) = 1 + 1.5*sq (L), whe e L is he ead leng h. Addi ionally, he maximum numbe o misma ches pe seed (−N pa ame e ) was se o L,0,0.1, co esponding o he unc ion (L) = 0 + 0.1*L, whe e L is he ead leng h, and minimum alignmen sco e (−− sco e-min pa ame e ) was se o L,-0.6,-0.4, co e- sponding o he unc ion (L) = −0.6 + −0.4*L, whe e L is he ead leng h. To emo e ambiguously mapped eads he mapping quali y h eshold was se o 20. To ob ain highe sequence co e age, he h ee sequenced pools pe i e we e me ged o a single BAM ile using SAM ools me ge. SNPs we e called using SAM ools mpileup [69] and he ou pu was pa sed using he PoPoola ion2 package (mpileup2sync.ja ) [70] wi h a minimum base quali y h eshold o 20. Fo a SNP o be included in he inal se o high quali y SNPs, minimum co e age o 10 and maximum co e age o 50 (99 % pe cen ile) was e- qui ed o each i e . In addi ion, he o al numbe o obse ed mino alleles was equi ed o be a leas 8. Re- cen ly published whole genome esequencing da a om indi iduals [28] was downloaded and mapped o he e e ence genome. The da a included h ee salmon om each o he i e s Tanael a, Reppa jo del a, Al ael a, Namsenel a, Å gå ds assd age , Naus a and Jøls a, whe e he i s h ee ep esen popula ions in No h e n No way and he las ou ep esen Sou he n No way. Accession numbe s o he samples a e shown in he cap ion o Addi ional ile 1: Table S3. S a is ical analysis Pai wise ixa ion index (F ST ) be ween all eigh sequenced popula ions was calcula ed o all high quali y SNPs using Hudson’s es ima o o F ST [34]. F ST alues we e a e aged o e all SNPs in each popula ion o gene a e a dis ance ma ix using F ST as gene ic dis ance. This ma ix was con e ed o a newick ee using NEIGHBOR om hePhylippackage[71]andaphylogene ic eewas c ea ed wi h NJplo [72]. To ind SNPs wi h signi ican ly di e en allele equencies (0.1 % FDR) be ween popula- ions om No he n and Sou he n No way he Coch an- Man el-Haenszel es o epea ed es s o independence om he PoPoola ion2 package (cmh- es .pl) [70] was used. The FDR h eshold was de e mined using he me hod desc ibed in [73]. Allele coun s o each i e we e me ged o ge he o al allele coun pe SNP in No he n and Sou he n No way, co esponding o 120 indi iduals pe geog aphical egion. F om his, F ST alues be ween he no he n and sou he n popula ions we e es i- ma ed using he F ST calcula ion om he PoPoola ion2 package ( s -sliding.pl) o each SNP, wi h –pool-size pa ame e se o 120. Genomic egions wi h low alues o he e ozygosi y may indica e SNPs unde selec ion. The e- o e he e ozygosi y alues we e es ima ed o no h and sou h o No way, sepa a ely, o each SNPs as 2 * (majo allele equency * mino allele equency). Sliding windows o 50 kb wi h s eps o 25 kb was used o ind genomic e- gions wi h high F ST alues and wi h low he e ozygosi y alues in ei he No he n o Sou he n No way. This ap- p oach is simila o one used o disco e genomic egions unde selec ion in o he animals [36]. To iden i y pu a i e selec i e sweeps i was equi ed ha he a e age F ST alue o he window was a leas 0.17 (abo e 99.9 % pe cen ile) and ha a e age he e ozygosi y o he window in ei he No he n o Sou he n No way was a mos 0.15 (below 5 % pe cen ile) (Addi ional ile 1: Figu e S1). The h esh- olds we e chosen wi h ocus on cap u ing he ou lie s in he FST and he e ozygosi y dis ibu ions. Pu a i e sweeps we e ex ended o he sides o as long as he neighbo ing windows had ei he a e age F ST o a leas 0.17 o he e o- zygosi y o a mos 0.15 in ei he No he n o Sou he n No way. I iden i ied sweeps we e less han 50 kb apa hese we e joined o a oid agmen a ion o he pu a i e selec i e sweeps. Genomic windows con aining mo e han 10 % ambiguous bases (Ns) in he e e ence assembly we e disca ded o exclude egions wi h high le els o unce ain y. SNP anno a ion Genes in he sweep egions we e ob ained om he o i- cial genome anno a ion (NCBI Salmo sala Anno a ion Release 100). Missense mu a ions in selec i e sweep e- gions we e iden i ied by manual inspec ion o he coding sequences. Amino acid sequences o i e mx genes ound in a selec i e sweep on Ch 25 we e aligned o he ho- mologs Mx1 and Mx2 om human and MxD and MxG om Zeb a ish using BLASTP (de aul pa ame e s). Func ional domains in he Mx p o eins we e assigned using domain in o ma ion o human Mx1 om Uni- P o . Amino acid sequences om ou genes con aining missense mu a ions in a selec i e sweep on Ch 5 (nk , sowahc,nkap and zb b33) we e aligned o homologous zeb a ish and No he n Pike genes using BLASTP wi h de aul pa ame e s. Syn eny be ween genes in he sweep on Ch 5 and o he animals was ound using he UCSC genome b owse (h ps://genome.ucsc.edu) o inspec he syn enic egions o zeb a ish, human and mouse. Pa alogous egions o he sweeps we e iden i ied using TBLASTN (de aul pa ame e s) wi h he genes in he sweeps agains he salmon genome. Geno yping Twen y salmon om 19 i e s along he No wegian coas line (n= 380) we e geno yped using en o he mos signi ican missensemu a ionsonaSequenom Kjæ ne -Semb e al. BMC Genomics (2016) 17:610 Page 9 o 12