Microsatellite-based genetic diversity and population structure of domestic sheep in northern Eurasia

RESEARCH ARTICLE Open Access Mic osa elli e-based gene ic di e si y and popula ion s uc u e o domes ic sheep in no he n Eu asia Miika Tapio 1 , Mikhail Oze o 1 , Ilma Tapio 1 , Miguel A To o 2 , Nu biy Ma zano 3 , Mi jana Ćinkulo 4 , Galina Goncha enko 5 , Ta yana Kiselyo a 6 , Maziek Mu awski 7 , Juha Kan anen 1* Abs ac Backg ound: Iden i ica ion o global li es ock di e si y ho spo s and hei impo ance in di e si y main enance is essen ial o making global conse a ion e o s. We sc eened 52 sheep b eeds om he Eu asian subcon inen wi h 20 mic osa elli e ma ke s. By es ima ing and weigh ing di e en ly wi hin- and be ween-b eed gene ic a ia ion ou aims we e o iden i y gene ic di e si y ho spo s and p io i ize he impo ance o each b eed o conse a ion, espec i ely. In addi ion we es ima ed how impo an wi hin-species di e si y ho spo s a e in li es ock conse a ion. Resul s: Bayesian clus e ing analysis e ealed h ee gene ic clus e s, e med No dic, Composi e and Fa - ailed. Sou he n b eeds om close o he egion o sheep domes ica ion we e mo e a iable, bu less gene ically di e en ia ed compa ed wi h mo e no he n popula ions. Dec easing weigh o wi hin-b eed di e si y componen led o e y high ep esen a ion o gene ic clus e s o egions con aining mo e di e ged b eeds, bu did no inc ease pheno ypic di e si y among he high anked b eeds. Sampling popula ions h oughou 14 egional g oups was sugges ed o maximized o al gene ic di e si y. Conclusions: Du ing ini ial s eps o es ablishing a li es ock conse a ion p og am popula ions om he di e si y ho -spo a ea a e he mos impo an ones, bu o he ull design ou esul s sugges ed ha app oxima ely equal popula ion p esen a ion ac oss en i onmen s should be conside ed. E en in his case, highe pe popula ion emphasis in a eas o high di e si y is app op ia e. The analysis was based on neu al da a, bu we ha e no eason o hink he gene al end is limi ed o his ype o da a. Howe e , a comp ehensi e alua ion o popula ions should balance p oduc ion sys ems, pheno ypic ai s and a ailable gene ic in o ma ion, and include conside a ion o p obabili y o success. Backg ound The domes ic sheep (O is a ies) has been an economic- ally and cul u ally impo an a m animal species since i s domes ica ion in he Nea Eas app oxima ely 9,000 yea s B.P. [1]. A no he n Eu asian sheep s ock o med some 6,000 yea s ago as sheep we e b ough o he B i - ish Isles, no he n Eu ope and Russia a e he expan- sion o he Eu opean con inen ia Danubian and Medi e anean ou es [2], and a possible ou e h ough Russia [3]. Sheep dispe sed ac oss Eu ope in empo ally sepa a e mig a o y episodes: he mos o iginal and a mo e p imi i e ype o domes ic sheep was la e eplaced by a mo e de eloped wool ype o sheep. Ances y om he i s immig an wa e seems o ha e su i ed only in no h-wes e n and no he n pe iphe ies o Eu ope [4]. A simila eplacemen p ocess is occu ing in mode n days. Global s anda diza ion o p oduc ion en i onmen s and b eed compe i ion ha e led o he disappea ance o many na i e b eeds. Food and Ag icul u e O ganiza ion o he Uni ed Na ions (FAO) has es ima ed ha 36% o he sheep b eeds o known census size a e ei he ex inc o endange ed [5]. Fu he mo e, he use o a ew high- quali y males o in ense ma ing has esul ed in he * Co espondence: [email p o ec ed] 1 Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, 31600 Jokioinen, Finland Full lis o au ho in o ma ion is a ailable a he end o he a icle Tapio e al.BMC Gene ics 2010, 11:76 h p://www.biomedcen al.com/1471-2156/11/76 © 2010 Tapio e al; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. educ ion o e ec i e popula ion size (N e ) o e ime and educed gene ic di e si y wi hin b eeds [6]. These p o- cesses will lead o he dec ease o e ec i e popula ion sizeo heen i especies.Thiscould es ic b eeding op ions and gene ic gain o b eeding p og ams o he ex en ha unp edic able u u e equi emen s migh no be me [6-8]. B eed conse a ion aims o main ain hese op ions, bu limi ed esou ces, e.g. inancial limi a ion, migh no allow conse a ion o all he b eeds. One can a gue ha he b eeds o igina ing om o close o he domes ica ion cen e s, such as he Nea Eas e n egion, should be pa icula ly p io i ized in con- se a ion p og ams. Mic osa elli e s udies in ca le (Bos au us) [9-11], goa (Cap a hi cus) [12] and sheep (O is a ies) [13] sugges ed ha he b eeds loca ed close o he pu a i e domes ica ion cen e s a e he mos a iable. These b eeds migh possess allelic a ia ions e ained om he wild ances o s ha ne e eached a eas u he om he cen e o o igin. Al hough one canno easily di e en ia e hese p ima y di e si y ho spo s om he seconda y ho spo s c ea ed by a mo e ecen c oss- b eeding, con inen -wide mapping o he egions o excep ional li es ock di e si y (gene ic di e si y ho - spo s) has been sugges ed as a means o a ge ing con- se a ion e o s o li es ock species [10,14]. DNA ma ke da a can be used o calcula e molecula coances- ies wi hin and be ween b eeds and de e mine con i- bu ions o each b eed o a pool o animals ha would maximize gene ic di e si y o he pool, i.e. minimize a e age molecula coances y [15]. These calcula ions can p o ide c i ical in o ma ion when he p io i iza ion o b eeds needs o be done o conse a ion o di e si y o domes ic animal species. Using his conse a ion app oach, i would be possible o maximize N e o he subdi ided species and hus minimize he deple ing e ec o gene ic d i on gene ic a ia ion. The e ha e been a ew qui e comp ehensi e gene di e si y s udies in sheep [13,16-18]. Howe e , none o hese ocused on b eed p io i iza ion o desc ibe gene al ends in he conse a ion o gene ic di e si y in sheep. Though genome-wide Single Nucleo ide Polymo phisms (SNP) da a a e becoming he s anda d o li es ock gene ics [18], hey can ha e a p oblem o asce ainmen bias o igina ing om SNP disco e y p o ocols [19]. Though he p oblem can be alle ia ed h ough using haplo ypic measu es [20] o h ough bias co ec ions [21], he es ablished baseline end using low bias ma - ke s such as mic osa elli es emains an impo an benchma k. We used a ep esen a i e se o sheep ypes ac oss he No he n Eu asia o explo e he di e si y pa - e ns and in e ed conse a ion p io i ies based on mic osa elli es. Fo b eed anking we applied he me hod based on he minimiza ion o molecula coan- ces y in a subdi ided popula ion by Caballe o and To o [15]. We es ed he e ec by weigh ing di e en ly he wo componen s o maximum gene ic di e si y, wi hin- b eed and be ween-b eed a ia ion, when doing p io i y se ings o b eeds. Based on he common s a emen ha popula ions om di e si y ho spo egions a e mo e impo an [9,14,10], we expec ed la ge numbe o b eeds om a ho spo egion o be highly p io i ized. Resul s Gene ic di e si y In o al, 342 alleles we e de ec ed a he 20 mic osa el- li e loci analyzed (Addi ional ile 1: Table S1). A sum- ma y o he gene ic di e si y pa ame e s compu ed o 16 egional g oups is p esen ed in Table 1 and he b eed-wise alues based, on an a e age, on 32 sheep pe b eed a e gi en in Addi ional ile 2: Table S2. The o al gene ic di e si y (H T ) a ied om 0.651 o 0.807 in he Danish and he Uk ainian egional g oups, espec i ely. The a ea ha ing egional g oups wi h H T alues abo e 0.8 (Uk aine, sou heas Eu ope, Kazakhs an and eas o he Caspian Sea, Bu ya ia and he sou he n Caucasus), was e med a di e si y ho spo . Among b eeds, he unbiased expec ed he e ozygosi y (H S ) anged om 0.613 ( he No wegian Che io ) o 0.806 ( he Russian Ka akul), wi h an a e age alue o 0.759. Allelic ichness a ied in he simila pa e n as o he wi hin-popula ion di e si y measu es (e.g. H T and H S )ac oss heb eeds (Table 1). The o e all es ima e o [22] was 0.011. The b eed-wise es ima es we e signi ican ly (P<0.05) g ea e han ze o only o he No wegian Rygja Sheep and he Swedish Rya Sheep sugges ing ha mos b eeds a e qui e uni o m (Addi ional ile 2: Table S2). Gene ic clus e analysis A model-based clus e ing was applied o esol e he popula ion gene ic s uc u e. A K=3,oneclus e was cons i u ed by he b eeds descending mainly om he no he nmos edge o he s udied dis ibu ion ( e med he ‘No dic clus e ’), while he a - ailed b eeds, o igina - ing mainly om he Caucasus and Caspian basin a eas, geog aphically close o he Nea Eas e n domes ica ion cen e , o med he second clus e ( e med he ‘Fa - ailed clus e ’). A hi d clus e mainly con ained he composi e sheep b eeds om cen al Eu asia ( e med he ‘Compo- si e clus e ’) (Figu e 1). The mean simila i y coe icien (SC) ac oss 10 uns was 0.984 a K=3.A K= 4, a spli wi hin he No dic clus e was obse ed, bu he d op o SC o 0.534 indica ed a iable assignmen s o b eeds ac oss uns and lack o addi ional s ong high-le el sub- s uc u e among he popula ions. The e o e, sepa a ing he en i e da ase in o h ee clus e s was chosen as he inal global con igu a ion. To dissec he gene ic s uc u e wi hin he h ee clus- e s, STRUCTURE analysis was u he applied o each Tapio e al.BMC Gene ics 2010, 11:76 h p://www.biomedcen al.com/1471-2156/11/76 Page 2 o 11 o hem sepa a ely. In he No dic clus e , he mos con- sis en g ouping o 11 no h Eu opean sheep b eeds was achie ed a K= 7 (SC = 0.641), wi h he mean SC an- ging om 0.250 o 0.314 a Ko he han se en. B eeds o igina ing om he same coun y (e.g. Finnsheep and Finnish G ey Land ace) o om he neighbo ing egions (e.g. he Icelandic Sheep and he Fae oe Island Sheep) ended o clus e oge he (Figu e 1). The Fa - ailed sheep clus e was composed mainly o he coa se-wool na i e b eeds om he Caucasus and s eppes o he Caspian basin and Kazakhs an. Su p isingly, he no he n sho - ailed coa se-wool Romano Sheep was Table 1 Gene ic di e si y wi hin 16 egional g oups Geog aphical egion Regional g oup N H T R Caucasus Sou h Caucasus* 6 0.802 0.020 6.57 No h Caucasus 5 0.795 0.017 6.24 S a opol 3 0.792 -0.005 6.04 Caspian dep ession 3 0.795 0.023 6.19 Asia Kazakhs an and eas o Caspian Sea* 6 0.804 0.037 6.45 Al ai 2 0.795 0.004 6.41 Bu ya ia* 2 0.802 -0.008 6.42 Eas e n inge o Eu ope Volga egion 2 0.779 0.015 5.88 Wes Russia 3 0.794 -0.041 5.45 Uk aine* 2 0.807 -0.004 6.43 Sou heas Eu ope* 4 0.806 -0.006 6.14 Poland 3 0.759 -0.015 6.24 Finland 2 0.758 0.007 5.35 Scandina ia 6 0.774 0.030 4.59 Denma k 1 0.651 0.028 4.41 Iceland and Fae oe Islands 2 0.746 -0.001 4.95 Numbe o b eeds (N), o al gene di e si y (H T ), depa u es om Ha dy-Weinbe g equilib ium ( ), and mean allelic ichness (R). * Regional g oups iden i ied as di e si y ho spo s. Figu e 1 Clus e ing o 52 sheep b eeds. Indi iduals a e p esen ed as e ical lines di ided in o Kcolo s, ep esen ing cons uc ed popula ions. The lowes ow ep esen s u he clus e ing o 3 g oups, iden i ied a K= 3, sepa a ely. The No dic g oup is di ided in o 7 subclus e s, while he Composi e (in he middle) and he Fa - ailed g oups each spli in o 3 subclus e s. Tapio e al.BMC Gene ics 2010, 11:76 h p://www.biomedcen al.com/1471-2156/11/76 Page 3 o 11 also assigned in o his clus e . The b eed’s es ima ed ac- ion o he Fa - ailed clus e was 0.59. Howe e , he mos consis en subclus e ing o he Fa - ailed clus e was ob ained a K= 3 (SC = 0.865), wi h he Romano sheep o ming a dis inc subclus e (Figu e 1). The Andi and he Ka akul ype sheep b eeds ancho ed he emaining wo subclus e s. Eigh ou o 14 b eeds showed pa ial and a ying membe ships o he wo subclus e s, indica ing hei admixed o igin (Figu e 1). The Composi e clus e hos ed he emaining 26 syn- he ic semi- and ine-wool sheep b eeds ha we e spli in o h ee gene ic subclus e s (SC = 0.932). The h ee subclus e s iden i ied ollowed a pa e n o geog aphical sepa a ion: long-wool Ma sh and Texel ype b eeds om he no h g ouped in o subclus e I (ligh blue); ine-wool b eeds om he Caucasus, Kazakhs an and Bu ya ia o med subclus e II (ligh g een) and he sou he n Eu - opean Zackel ype b eeds g ouped in o subclus e III (pink, Figu e 1). O he nine Eu opean, Caucasian and Asian sheep b eeds had pa ial membe ship o mul iple clus e s, which ep esen s mo e di e se ances ies in he p ocess o b eed de elopmen (Figu e 1). The PCoA esul s we e qui e in acco dance wi h he STRUCTURE esul s. The b eeds om he abo e men- ioned No dic clus e we e sepa a ed om he o he b eeds on Axis I, which explained 48% o he dis ance ma ix (Figu e 2). On Axis II, b eeds om he Fa - ailed clus e we e sepa a ed om he Composi e clus e b eeds, which explained an addi ional 7%. A no able excep ion was he Romano Sheep whose yellow ci cle in Figu e 2 (a -0.085,-0.004) sugges ed he b eed’sclus- e ing wi h he No dic a he han he Fa - ailed b eeds (Figu e 2). This ma ched ou p io expec a ions on he basis o pheno ypic cha ac e s be e han he STUC- TURE esul . The p opo ions o No dic, Fa - ailed and Composi e gene ic ances ies wi hin each o he egional g oups s udied a e p esen ed in Figu e 3. The highes p opo - ion o Fa - ailed ances y was eco ded a he sou he n pe iphe y o he s udied dis ibu ion, which g adually dec eased no hwa ds and was he smalles in he no h- e n egional g oups. The p opo ion o No dic ype ances y mi o ed his pa e n and was he la ges in he no he n egional g oups and dec eased sou hwa ds. The 16 egional g oups had simila p opo ions o Com- posi e ances y, wi h he excep ion o S a opol and Caspian dep ession egional g oups, whe e he p opo - ion o Composi e ances y was highes , and he no h- e nmos and sou he nmos egional g oups, whe e he Composi e ances y p opo ion was leas . Geog aphical pa e ns in gene ic di e si y To educe he e ec o possible ecen b eed-speci ic ac o s on he o e all geog aphical dis ibu ion o gene ic di e si y, a syn he ic map o gene ic di e si y was based on he o al gene di e si y (H T ) o iple so neighbo ing b eeds (Figu e 4). The highes di e si y was ound in he sou he n egion o he s udied a ea: Bu ya- ia (sou h Sibe ia), Caspian Sea and Black Sea basins. I dec eased g adually in Cen al and no he n Eu ope and he lowes H T alues we e eco ded o sou he n Scandi- na ia (Figu e 4). The end can be obse ed also based on wi hin b eed es ima es (Addi ional ile 3: Figu e S1). A signi ican bu weak posi i e co ela ion ( =0.382,P < 0.05) was calcula ed be ween he expec ed he e ozyg- osi y and he le el o admix u e based on global STRUCTURE esul s (K= 3) o he 52 sheep b eeds s udied, sugges ing ha admix u e does no explain he p esence o di e si y ho spo s, hough i can con ibu e o i in some a eas. Analysis o molecula a iance (AMOVA) We es ed he ex en o popula ion di e en ia ion using AMOVA in he whole da ase , as well as g ouping Figu e 2 P incipal coo dina e plo o b eeds based on Cho d dis ance. Axis I explains 48% o he a ia ion, axis II explains 7% o he a ia ion. B eeds om No dic clus e (based on STRUCTURE) a e ma ked wi h ed, b eeds om Fa - ailed clus e a e ma ke wi h yellow and b eeds om he Composi e clus e a e ma ked wi h black ci cles. Tapio e al.BMC Gene ics 2010, 11:76 h p://www.biomedcen al.com/1471-2156/11/76 Page 4 o 11 b eeds acco ding o geog aphical egions ( he Caucasus, Asia, o he eas e n inge o Eu ope), and acco ding o he 15 egional g oups (excluding he Danish g oup ep esen ed by a single b eed) (Table 2). As expec ed, mos gene ic a ia ion (> 86%) was e ained wi hin he b eeds, whe eas only 0.41% o 0.95% (P<0.001)o he a ia ion could be explained by geog aphical pa i ioning (Table 2). The be ween-b eed a ia ion wi hin each o he h ee gene ic subclus e s was signi ican (P<0.001), anging om 2.48% (Fa - ailed subclus e ) o 13.71% (No dic subclus e ) (Table 2). In ou da a, using gene ic clus e ing in AMOVA gi es highe be ween g oups a - iance han using geog aphical ca ego iza ions. Co e-se analysis O he 52 sheep b eeds 24 had con ibu ions o he co e-se when he 4 weigh ings (l=0,0.2,0.5,1)o wi hin-b eed di e si y we e conside ed. The 24 b eeds ep esen ed all he 16 egional g oups, excep hose o he Al ai and Bu ya ia egions. The dis ibu ion o b eeds was ela i ely e en, wi h 1 o 2 sheep b eeds pe egion, he excep ion being he Scandina ian egional g oup, which con ibu ed 5 sheep b eeds o his accu- mula ed co e-se (Addi ional ile 4: Table S3). Looking a he ou co e-se s sepa a ely, he numbe o con i- bu ing b eeds inc eased om 8 o 17 when weigh o wi hin-b eed a iabili y inc eased om 0 o 1 (Table 3). Resul s o analysis based on gene ic clus e ing a e com- pa able o hose based on geog aphic egions (Addi- ional ile 5: Table S4). E e y es ed scena io wi h educed weigh o wi hin- b eed a ia ion (l< 1) ga e a signi ican ly highe num- be o b eeds wi h non-ze o con ibu ion om he a eas ou side he ho spo egions (all he wo- ailed P alues < 0.02 using Fishe ’s exac es o independence; Table 3, Addi ional ile 4: Table S3). Looking a he con ibu ion o he co e-se , hese non-ho spo egion popula ions comp ised o > 90% o he se . Howe e , op imizing o global di e si y (l= 1), he e is no signi ican di e ence ( he wo ailed P= 0.77) in he p opo ion o b eeds included be ween ho spo and non-ho spo egions. Ve y dis inc i ely, now he co e-se consis s o 65% o he b eeds om he ho spo egions because he included ho spo egion b eeds make a signi ican ly la - ge mean con ibu ion (each ~11%) han he included b eeds om he non-ho spo egions (each ~3%) (Welch wo sample - es P= 0.009). Thus he di e si y ho spo a eas we e impo an o conse ing o al gene ic Figu e 3 Dis ibu ion o h ee in e ed gene ic clus e s in he s udy egions. Slices in he pie diag ams ep esen Fa - ailed (yellow), Composi e (black) and No dic ( ed) clus e s. The Caucasus a ea is ep esen ed by ou egions: Sou h Caucasus (1), No h Caucasus (2), S a opol (3) and he Caspian dep ession (4). The Asian egion is ep esen ed by h ee egions: Kazakhs an and eas o he Caspian Sea egion (5), he Al ai egion (6) and he Bu ya ia egion (7). The emaining g oups belong o eas e n inge o Eu ope: he Volga egion (8), Wes Russia (9), Uk aine (10), Sou heas Eu ope (11) Poland (12), Finland (13), Scandina ia (14), Denma k (15) and Iceland and he Fae oe Islands (16). Figu e 4 Con ou syn he ic map o o al gene ic di e si y (H T ) calcula ed o iple s o neighbo ing b eeds. Da ke shading indica es highe le els o di e si y. Tapio e al.BMC Gene ics 2010, 11:76 h p://www.biomedcen al.com/1471-2156/11/76 Page 5 o 11 di e si y in e ms o he e o pe conse ed popula ion a he han p opo ion o numbe o b eeds o be conse ed. Conse a ion p og ams migh be ini ia ed wi h limi ed in o ma ion. In cases whe e esou ces allow keeping only a small numbe o b eeds and when he e is no aim o di e en ia e be ween hei con ibu ions o he co e-se (assuming equal con ibu ions), he maximum amoun o gene ic di e si y would be main ained by gi - ing p io i y o b eeds om he di e si y ho spo egions. In he scena io o 5 b eeds, ou o hem a e om he ho spo a eas (Addi ional ile 6: Table S5). The p opo - ion o ho spo b eeds was educed om 80% o 45% when assuming esou ces o keep 20 b eeds. This la e se is simila o 17 b eeds iden i ied as con ibu o s o he co e-se when l= 1 (Addi ional ile 4: Table S3), bu includes also h ee a - ailed popula ions om he Caucasus (Bozakh, Tushin and Lezian). These esul s ag ee wi h he idea o ha ing he ini ial conse a ion ocus on ho spo egions. Discussion We p esen he e a comp ehensi e gene ic analysis o sheep popula ions o igina ing om a b oad geog aphical a ea o he Eu asian subcon inen . Ou esul s de ec ed he p esence o a sheep gene ic di e si y ho spo loca ed close o he Nea Eas , he assumed sheep domes ica ion cen e , and highligh he impo ance o such an a ea in conse a ion planning. The esul s co espond well wi h he geog aphical pa e n o gene ic di e si y dis ibu ion epo ed o ca le (B. au us) [23] and goa (Cap a hi - cus) [12] as well as a p e ious s udy o Eu opean sheep Table 2 Analysis o molecula a iance Sample Numbe o b eeds Numbe o b eed g oups Pe cen age o a iance and signi icance (P) Wi hin b eeds Among b eeds wi hin g oups Among g oups Whole da a 52 1 93.56 (< 0.001) 6.44 (< 0.001) Th ee geog aphical egions* 52 3 93.43 (< 0.05) 6.17 (< 0.001) 0.41 (< 0.001) 15 egional g oups* 51 15 93.68 (< 0.001) 5.38 (< 0.001) 0.95 (< 0.001) Th ee s uc u e clus e s: 52 3 93.13 (< 0.001) 5.63 (< 0.001) 1.24 (< 0.001) No dic subclus e 11 1 86.29 (< 0.001) 13.71 (< 0.001) Composi e subclus e 26 1 95.45 (< 0.001) 4.55 (< 0.001) Fa - ailed subclus e 15 1 97.52 (< 0.001) 2.48 (< 0.001) * See Table 1 and suppo ing in o ma ion o de ails. Table 3 Dis ibu ion o co e-se con ibu ions Geog aphical Regional g oup l=0 l= 0.2 l= 0.5 l=1 egion B eeds Con B eeds Con B eeds Con B eeds Con Caucasus Sou h Caucasus* 00000020.17 No h Caucasus 000010.04 1 0.01 S a opol 00000020.04 Caspian dep ession 00000010 Asia Kazakhs an and* eas o Caspian Sea 00000010.14 Al ai 00000000 Bu ya ia* 00000000 Eas e n inge Volga egion 00000010.06 o Eu ope Wes Russia 1 0.04 2 0.11 2 0.20 1 0.03 Uk aine* 00000020.24 Sou heas Eu ope* 000010.04 1 0.10 Poland 1 0.11 1 0.09 1 0.05 1 0.02 Finland 00000010.03 Scandina ia 4 0.56 4 0.51 5 0.42 2 0.10 Denma k 1 0.19 1 0.17 1 0.13 0 0 Iceland and Fae oe Islands 1 0.10 1 0.11 2 0.13 1 0.06 Sum 8191131171 SD 1.03 0.14 1.09 0.13 1.33 0.11 0.68 0.07 Numbe o b eeds (B eeds) and he sum o hei op imal con ibu ions (Con ) o he co e se o each egional g oup using ou di e en weigh ings (l) o he wi hin-b eed a ia ion. * Regional g oups in di e si y ho spo . Tapio e al.BMC Gene ics 2010, 11:76 h p://www.biomedcen al.com/1471-2156/11/76 Page 6 o 11 [13] which ocused on mo e sou he n b eeds. The con- g uence ac oss s udies sugges s he pa e n o be genu- ine, hough la ge numbe o ma ke s could be desi able. Based on obse ed allele numbe , we can expec he eliabili y o be app oxima ely simila as in a s udy o 300-400 unbiased bi-allelic SNPs [24]. Howe e , since s udies o humans do no sugges g ea disc epan- cies ac oss nuclea ma ke ypes as long as asce ain- men bias can be a oided [19,20,25], we expec he p esen ed gene al di e si y pa e ns o be obus . Since in ou analysis conse a ion op imiza ion was based on hesameda aused ode ine hedi e si yho spo ,ou gene al ecommenda ion o conside ing ho spo egions ough o be sound. Li es ock gene ic di e si y ho spo s ha e been sug- ges ed o be e y impo an o conse a ion because he domes ic animal s ocks associa ed wi h hem migh possess allelic a ia ion om wild ances o s, which, due o a sequence o ounding e en s, was los du ing he dispe sion o animals owa ds he no he n pa s o he con inen [14]. Howe e , o he bes o ou knowledge his ques ion has no been di ec ly add essed p e iously. Ou esul s p o ide addi ional e idence o he impo - ance o hese egions, while indica ing an impo an e inemen o he conse a ion goal. Ou esul s do no sugges ha a la ge p opo ion o popula ions om hese a eas needs o be conse ed, bu a he sugges mo e emphasis be placed on each conse ed di e si y ho spo popula ion. This dis inc ion, howe e , is highly ele an o domes ic species, whe e managemen uni s a e in mos cases clea ly de inable as b eeds. Fu he he esul s suppo o di ec ing he i s conse a ion esou ces o wo k on ho spo egions. O he h ee iden i ied No he n Eu asian gene ic clus- e s, he No dic clus e was ep esen ed by na i e and old comme cial sheep b eeds adap ed o li e unde cold and we no he n Eu opean clima ic condi ions. This g oup includes b eeds such as Gu e, Icelandic Sheep and Finnsheep, which descended om he sheep s ock in he i s dispe sion e en o Eu ope [4]. S ic b eed bounda ies o e a long pe iod and geog aphical isola- ion, pa icula ly o insula b eeds ( he Icelandic Sheep and he Fae oe Island Sheep), a e cha ac e is ic o he g oup and ha e esul ed in a unique and gene ically highly he e ogeneous pool o No dic sheep popula ions (Table 2). The la ge Composi e clus e wi h pa ial ances y om imp o ed wes e n b eeds con ains gene ically a iable ine- and semi- ine-wool sheep b eeds o admixed o igin wi h mode a e di e en ia ion be ween he b eeds. The p esence o subs uc u e wi hin he clus e e lec s he di e ences in he b eeding ends wi hin he o me So ie Union ha ook place in he middle o he las cen u y. The sheep in he wes e n pa o Russia and Volga egions ha e Ma sh-Texel ype composi e ances- y esul ing om c ossing local popula ions wi h B i ish ype long-wool sheep (Figu e 1). The second subclus e wi hin he Composi e g oup includes he b eeds p e a- len in he Caucasus, he S a opol egion and he Cas- pian basin, ano he geog aphical cen e o pu pose ul c ossb eeding, wi h a signi ican gene ic componen o he Me ino ype sheep. The hi d subclus e wi hin he Composi e clus e is ancho ed by wo Zackel ype moun ain sheep popula ions, P amenka and Kuchugu , and e lec s a common ances y o he majo i y o b eeds wi hin he subclus e . The g ouping o Tsigai in he same subclus e con i ms he assump ion ha his b eed was s ongly in luenced by Zackel (e.g. see [17]). Mos o he popula ions o he Composi e clus e also ep esen gene ic di e si y o local o igins as he upg ad- ing was pe o med on he basis o local sheep popula- ions, mixing hem wi h a numbe o imp o ed b eeds o o eign ances y o combine desi ed p oduc ion and obus ness cha ac e is ics. The Fa - ailed clus e hos ed e y a iable na i e coa se-wool popula ions, li ing unde a a ie y o cli- ma ic condi ions, anging om semi-dese and s eppe egions a ound he Caspian Sea and Cen al Asia o Caucasian moun ain e ains. The di e en ia ion o a - ailed sheep om he o he s indica es es ic ed gene low be ween s eppe o moun ain en i onmen s in cen- al Eu asia and coole and mois e no he n a eas o he con inen . The gene pool o he a - ailed sheep di ided in o he moun ain ype sheep (e.g. Andi and Lezgian) and s eppe-dese ypes (e.g. Gala and Ka akul). Howe e , he majo i y o a - ailed b eeds ha e hei ances ies in bo h o hese subclus e s (Figu e 1), which oge he wi h low di e en ia ion es ima es indica es sub- s an ial gene low be ween hem. This ag ees wi h he adi ional sheep b eeding p ac ices in he Caucasus, which p omo e gene low h ough he long-dis ance nomadic pas u ing o animals. G ouping o Romano sheep wi hin he Fa - ailed clus e (Figu e 1) should be ega ded cau iously. Decisions on adap a ion conse a ion should la gely be based on eliable pheno ypic e alua ions. In humans, gene ic and pheno ypic di e si y ag ee [26], bu selec ion migh a ec pheno ypes educing co ela ion be ween pheno ypic di e gence and gene al genomic ela edness [27,28]. This is pa icula ly ue o li es ock which would imply need o es ing (ecological) exchangeabili y (as in [29]). Un o una ely his is e y di icul . A la ge p opo ion o he necessa y pheno ypic in o ma ion exis s only as in o mal knowledge o local b eede s. E en he mo e igidly collec ed da a is a ely compa - able be ween en i onmen s. Molecula da a can ha e a ole in poin ing ou po en- ial conse a ion gaps when pheno ypic knowledge is Tapio e al.BMC Gene ics 2010, 11:76 h p://www.biomedcen al.com/1471-2156/11/76 Page 7 o 11 limi ed. The usabili y o app oaches based on molecula ma ke da a in se ing conse a ion p io i ies can be g ea ly imp o ed by genome-wide su eys o molecula a ia ion [30]. Fo example, scanning ens o housands o SNP ma ke s has he po en ial o iden i y selec ed loci [31] and allow compa ison o he conse a ion alues o se e al popula ions, bo h in he neu al and non-neu al con ex [30]. Howe e , e en wi h ull gen- ome sequences, alua ion o popula ions can p o e o be di icul due o incomple e unde s anding o he biol- ogy o he o ganisms and poo ly de inable conse a ion goals. We used neu al molecula da a o a speci ic se o popula ions and applied he me hod o Caballe o and To o [15] o calcula e op imal con ibu ions o Eu asian sheep b eeds o he co e se , which would minimize he mean kinship in he se and maximize N e and gene ic di e si y o he species. While gi ing mo e emphasis o di e gence has heo e ical appeal, i did no inc ease ecological o pheno ypic he e ogenei y in he p e e ed se o b eeds compa ed wi h he maximiza ion o global di e si y (and N e ). Maximiza ion o global di e si y p io i ized a mo e di e se se o b eeds o igina ing om a ange o biogeog aphic en i onmen s and ha ing di - e en gene ic his o ies. Though he se looks easonable, we acknowledge ha i is based on incomple e da a and we a e hesi an o conclude ha his pa icula design is op imal. Conclusions Neu al a ia ion sugges ed a gene al ule o humb o a ou b eeds om he di e si y ho spo egions in he i s phase o in si u and ex si u conse a ion ac ions. In he inal design, howe e , app oxima ely equal popula- ion p esen a ion ac oss en i onmen s is ecommended, bu s ill highe pe popula ion emphasis in a eas o high di e si y is sugges ed. A comp ehensi e alua ion o b eeds, pa icula ly wi hin each physical en i onmen , should conside p oduc ion sys ems, impo an biologi- cal cha ac e is ics and a ailable gene ic in o ma ion, as well as conside a ion o he p obabili y o success and he ex inc ion isk o b eeds. Me hods Biological samples In o al, 1675 animals ep esen ing 52 sheep b eeds we e s udied (Addi ional ile 2: Table S2). Sheep we e sampled om h ee geog aphical egions: The Caucasus, Asia, and he eas e n inge o Eu ope, including cen al and wes e n Russia. Each geog aphical egion was u he subdi ided in o egional g oups. The Caucasian a ea was composed o he sou he n Caucasus ( he ol- lowing b eeds we e sampled: Aze baijan Moun ain Me - ino, Bozakh, Gala, Ka abakh, Mazekh, Tushin), no he n Caucasus (Andi, Dages an local, Dages an Moun ain Me ino, Ka achai, Lezgian), S a opol (Caucasian, No h Caucasian Mu on-Wool, S a opol), and he Caspian dep ession (Aksa aisk ype o So ie Mu on-Wool, G ozny, Volgog ad). The Asian a ea was subdi ided in o he Kazakhs an and eas o he Caspian Sea g oup (Dege es Mu on-Wool, Kazakh A kha -Me ino, Kazakh Edilbai, Kazakh Finewool, Russian Edilbai, Russian Ka a- kul), Al ay (Go no-Al ay local, Kulunda), and he Bu ya- ia g oup (Baida ak, T ansbaikal Finewool). The emaining nine g oups co e ed he eas e n inge o Eu ope: he Volga egion (Kuibyshe , Opa in), wes e n Russia (Kuchugu , Romano , Russian Romney Ma sh), Uk aine (Ca pa hian Moun ain, Sokolsk), sou heas Eu - ope (Molda ial Ka akul, Molda ial Tsigai, P amenka, Russian Tsigai), Poland (Olkuska, Swinia ka, W zo- sowka), Finland (Finnsheep, Finnish G ey Land ace), Scandina ia (Swedish Rya Sheep, Swedish Go land Sheep, Swedish Gu e Sheep, No wegian Rygja Sheep, No wegian Che io , No wegian Fe al Sheep), Denma k (Danish Texel), and Iceland and he Fae oe Islands (Ice- landic Sheep, Fae oe Island Sheep). Un ela ed animals we e sampled based on pedig ee eco ds ( wo p e ious gene a ions) o a me s’knowledge. Genomic DNA was ex ac ed om blood as desc ibed in [32], o om skin samples using DNeasy Tissue Ki (Qiagen, C awley, Wes Sussex, UK). P io o DNA ex ac ion, skin samples s o ed in e hanol we e washed wice wi h phospha e bu e ed saline o emo e ixa i es. Gene ic loci The polyme ase chain eac ions (PCR) o 20 mic osa- elli es (Addi ional ile 1: Table S1) we e pe o med as desc ibed in [33] and geno yped using he MegaBACE™ 500 DNA Sequence (Ame sham Biosciences). F agmen sizing was pe o med using he MegaBaceTM Gene ic P o ile 2.2 o F agmen P o ile 1.2 (Ame sham Bios- ciences). Geno ypes o 20 mic osa elli es we e a ailable in he ea lie s udies o he Romano sheep [34] and o he 11 b eeds om Finland, Scandina ia, Denma k, Iceland and he Fae oe Islands [16]. S a is ical analysis The mic osa elli e loci we e cha ac e ized by he o al numbe o alleles, expec ed he e ozygosi y o o al gene di e si y [35], sample-size-co ec ed allelic ichness [36] co esponding he e expec ed allele numbe in a sample o nine diploid indi iduals, and F-s a is ics using FSTAT 2.93 [37]. F-s a is ics we e es ima ed using Wei and Cocke ham [22] me hod whe e and θco espond o W igh ’s coe icien s F IS and F ST , espec i ely. The gene ic ela ionships among b eeds we e analyzed using p incipal coo dina e analysis (PCoA) as implemen ed in PAST 1.73 [38] using he Cho d dis ance [39]. Tapio e al.BMC Gene ics 2010, 11:76 h p://www.biomedcen al.com/1471-2156/11/76 Page 8 o 11 A model-based Bayesian clus e ing analysis was used o in e popula ion s uc u e and he le el o admix u e in he sheep b eeds implemen ed in STRUCTURE 2.2 [40]. The STRUCTURE algo i hm assumes Kpopula- ions, each o which is in Ha dy-Weinbe g and linkage equilib ium and cha ac e ized by a se o allele equen- cies a each locus. Analysis was pe o med wi h a bu n- in leng h o 20,000 ollowed by 100,000 Ma ko chain Mon e Ca lo i e a ions o each o K= 1 o 10, wi h en eplica e uns o each Kusing independen allele e- quencies and an admix u e model. Resul s ac oss en uns a each Kwe e compa ed based on simila i y coe - icien s (SC) as p e iously desc ibed in [41]. The b eeds we e assigned o wide clus e s based on majo ances y and submi ed o a second ound o STRUCTURE ana- lysis pe o med wi hin each wide clus e . A linea eg ession analysis was pe o med o s udy he in luence o b eed ances y di e si y (admix u e) on he le el o gene ic di e si y. Ances y di e si y o each b eed was calcula ed as 1-Σ(q k ) 2 ,whe eq k is an a e age ac ion o he b eed’s gene ic ances y om he ksepa a e gene ic clus e s a he op imal K,iden i- ied in STRUCTURE analysis. To examine he signi i- cance o mixed ances ies as sou ces o wi hin-b eed di e si y, he ob ained ances y di e si y alues we e compa ed wi h he unbiased expec ed he e ozygozi y es ima es. Fo he geog aphical plo ing o gene ic di e si y pa a- me e s, la i ude and longi ude alues o each b eed we e ob ained om he cen e o he sample dis ibu- ion. The A cView GIS 9.1 (En i onmen al Sys ems Resea ch Ins i u e, ESRI, Redlands, CA, USA) was used o map he allelic ichness andexpec edhe e ozygosi y o each b eed and he su ace was ex apola ed o a ull ec angle. This was based on he In e se Dis ance Weigh ed in e pola ion me hod [42], which assumes each inpu poin o ha e a local in luence ha diminishes wi h dis ance. A syn he ic map o he dis i- bu ion o local o al gene di e si y (H T ) and θcalcula ed o he geog aphically neighbo ing iple s o popula- ions was done simila ly. Popula ion iple s we e o med using Delaunay iangula ion me hod implemen- ed in he p og am T iangle [43]. Componen s o wi hin- and be ween-b eed gene ic di e si y we e calcula ed based on he molecula coan- ces y o popula ions ollowing he me hod desc ibed by Caballe o and To o [15]. The molecula coances y be ween wo indi iduals is he p obabili y ha wo alleles a he locus aken a andom om each indi i- dual a e alike in s a e. In a s uc u ed popula ion wi h n b eeds he molecula coances y be ween b eeds iand j ( ij ) is he a e age ac oss loci and ac oss indi iduals. De ining he wi hin-b eed a e age coances y as n ij ij =∑, 2, he o al popula ion coances y as n ij ij =∑, 2,Nei’s minimum dis ance as D ij ii jj ij =+− 2and he a e age Nei’s minimum dis- ance as D D n ij ij =∑, 2, hen he o al gene di e si y o expec ed he e ozygosi y ()GD T=−1is pa i ioned in o componen s wi hin b eeds ()GD WS =−1and ano he be ween b eeds ()GD D BS =−= . The impo ance o di e en b eeds has been calcula ed based on he con ibu ion o each b eed o a pool o ani- mals o a co e se ha would maximize i s gene ic di e - si y (e.g. [15,44]). In he p esen s udy, he co e se e e s o he smalles se o sheep b eeds ha s ill encompasses he neu al gene ic di e si y in he species using he co- ances y measu e de ailed abo e. These op imal con i- bu ions can also be applied wi h a weigh ed (l) combina- ion o wi hin- and be ween-b eed componen s o gene di e si y  ()1−+  D . Maximizing global di e si y is achie ed by gi ing equal weigh s o wi hin- and be ween- b eed di e si y (l= 1), while maximizing be ween-b eed a ia ion is achie ed by igno ing wi hin-b eed di e si y (l= 0). Two in e media e l alues we e ecommended in ea lie s udies. Piyasa ian and Kingho n [45] sugges ed gi ing i e imes weigh o he be ween b eed a ia ion as o he wi hin-b eed a ia ion (l= 0.2), e lec ing he speed by which gene ic change can be made ac oss popu- la ions compa ed wi h selec ion wi hin one la ge mixed popula ion. Bennewi z and Meuwissen [46] p oposed a weigh ing based on maximizing he o al gene ic a iance o a hypo he ical quan i a i e ai , which is equi alen by using a weigh ing ac o o l=0.5.These ou l alues we e applied in es ima ing he op imal con ibu ions using a simula ed annealing algo i hm [47]. Addi ional ma e ial Addi ional ile 1: Table S1 - Ma ke di e si y pa ame e s. PDF ile wi h lis o mic osa elli es and hei ch omosomal loca ion, o al numbe o alleles, expec ed unbiased he e ozygosi y, and es ima es o wi hin- popula ion ( ) and among-popula ion (θ) ixa ion indices. Addi ional ile 2: Table S2 - Table o he name o sheep b eeds, hei o igin, demog aphic s a us and di e si y pa ame e s. PDF ile wi h da a on pe popula ion sample size, expec ed he e ozygosi y, wi hin-b eed ixa ion index ( ), allelic ichness, and numbe o p i a e alleles. Addi ional ile 3: Figu e S1 - Addi ional syn he ic maps. PDF ile syn he ic maps o wi hin-b eed di e si y and b eed di e en ia ion. Addi ional ile 4: Table S3 - B eed-wise op imal con ibu ions o a co e-se o di e en weigh ings o he wi hin-b eed a ia ion.PDF ile wi h de ailed da a summa ized in Table 3. Tapio e al.BMC Gene ics 2010, 11:76 h p://www.biomedcen al.com/1471-2156/11/76 Page 9 o 11