Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools

Gene . Sel. E ol. 35 (2003) 533–557 533 © INRA, EDP Sciences, 2003 DOI: 10.1051/gse:2003038 O iginal a icle Biodi e si y o 52 chicken popula ions assessed by mic osa elli e yping o DNA pools Jossi H ILLEL a∗, Ma ien A.M. G ROENEN b, Michèle T IXIER -B OICHARD c, Ab aham B. K OROL d, Lio D AVID a, Vale y M. K IRZHNER d, Te y B URKE e, Asili B ARRE -D IRIE , Richa d P.M.A. C ROOIJMANS b, Ka i E LO g, Ma cus W. F ELDMAN h, Paul J. F REIDLIN a, Asko M ÄKI -T ANILA g, Ma ian O ORTWIJN b, Pippa T HOMSON e, Alain V IGNAL i, Klaus W IMMERS j, S e en W EIGEND aDepa men o Gene ics, The Heb ew Uni e si y o Je usalem, Facul y o Ag icul u e, Food and En i onmen al quali y sciences, Reho o 76100, Is ael bIns i u e o Animal Sciences, Wageningen Ag icul u al Uni e si y, Wageningen, The Ne he lands cIns i u na ional de la eche che ag onomique, Cen e de Jouy-en-Josas, F ance dIns i u e o E olu ion, Uni e si y o Hai a, Is ael eDepa men o Animal and Plan Sciences, She ield Uni e si y, S10 2TN, UK Ins i u e o Animal Science, Fede al Ag icul u al Resea ch Cen e, Ma iensee, 31535 Neus ad , Ge many gAg icul u al Resea ch Cen e, Ins i u e o Animal P oduc ion, 31600 Jokioinen, Finland hDepa men o Biological Sciences, S an o d Uni e si y, S an o d, CA 94305, USA iIns i u na ional de la eche che ag onomique, Cen e de Toulouse, F ance jIns i u e o Animal B eeding Science, Rheinische F ied ich-Wilhelms-Uni e si a Bonn, Ge many (Recei ed 2 Sep embe 2002; accep ed 13 Ma ch 2003) ∗Co espondence and ep in s E-mail: [email p o ec ed] 534 J. Hillel e al. Abs ac – In a p ojec on he biodi e si y o chickens unded by he Eu opean Commission (EC), eigh labo a o ies collabo a ed o assess he gene ic a ia ion wi hin and be ween 52 pop- ula ions om a wide ange o chicken ypes. Twen y- wo di-nucleo ide mic osa elli e ma ke s we e used o geno ype DNA pools o 50 bi ds om each popula ion. The polymo phism meas- u es o he a e age, he leas polymo phic popula ion (inb ed C line) and he mos polymo phic popula ion (Gallus gallus spadiceus) we e, espec i ely, as ollows: numbe o alleles pe locus, pe popula ion: 3.5, 1.3 and 5.2; a e age gene di e si y ac oss ma ke s: 0.47, 0.05 and 0.64; and p opo ion o polymo phic ma ke s: 0.91, 0.25 and 1.0. These we e in good ag eemen wi h he b eeding his o y o he popula ions. Fo ins ance, unselec ed popula ions we e ound o be mo e polymo phic han selec ed b eeds such as laye s. Thus DNA pools a e e ec i e in he p elimina y assessmen o gene ic a ia ion o popula ions and ma ke s. Mean gene ic dis ance indica es he ex en o which a gi en popula ion sha es i s gene ic di e si y wi h ha o he whole es ed gene pool and is a use ul c i e ion o conse a ion o di e si y. The dis ibu ion o popula ion- speci ic (p i a e) alleles and he amoun o gene ic a ia ion sha ed among popula ions suppo s he hypo hesis ha he ed jungle owl is he main p ogeni o o he domes ica ed chicken. gene ic dis ance / polymo phism / ed jungle owl / DNA ma ke s / domes ica ed chicken 1. INTRODUCTION I is widely accep ed ha all popula ions o domes ica ed chickens descend om a single ances o , he Red Jungle Fowl (RJF) (Gallus gallus), o igina ing in Sou heas Asia. Al hough i has been claimed ha o he wild species o Gallus migh ha e con ibu ed o he domes ica ed chicken [5], he mo e widely accep ed iew is ha Gallus gallus gallus alone is su icien o accoun o he ma e nal ances y o he domes ica ed chicken [14,15]. A he p esen ime, he imp o ed Medi e anean ype popula ions a e he mos closely ela ed o he RJF, which we e he i s chickens b ough in o Eu ope [33]. Much la e , wi h he massi e use o selec ion and c ossb eeding, local b eeds and lines in di e en pa s o Eu ope we e de eloped, and Asian b eeds o he Chinese and Malay ypes we e in oduced. All o hese sou ces con ibu ed o he mode n biodi e si y o chicken popula ions. In e -c ossing, howe e , may ha e pa ly ex inguished di e ences among g oups o b eeds, wi h he esul ha gene ic ela ionships be ween chicken popula ions a e no always de ini i e. Fu he mo e, only some o hese sou ces we e used o de elop he popula ions which cu en ly domina e he wo ld’s poul y indus y [6,27]. Since he s a o comme cial poul y b eeding in he middle o he 20 h cen u y, chicken gene ic di e si y has become pa i ioned among ela i ely ew highly specialized lines. As a consequence, many dual-pu pose b eeds, esul ing om cen u ies o domes ica ion and b eeding, a e now a he isk o being los . These b eeds may, howe e , ep esen a esou ce o genes o u u e b eeding and esea ch pu poses. The e o e, i is necessa y o assess he di e si y a he molecula le el in a wide ange o chicken popula ions, including comme cial lines, adi ional b eeds, expe imen al lines and he ed jungle owl, in o de o p o ide ecommenda ions ega ding u u e managemen o conse a ion o Chicken biodi e si y assessed by DNA pools 535 hese popula ions. Al hough decisions on conse a ion o gene ic esou ce popula ions should ely upon se e al sou ces o in o ma ion, including speci ic ai s o in e es , molecula ma ke s may se e as an impo an ini ial guide [1]. In he p ocess o de eloping s a egies o conse e gene ic di e si y in domes ic chickens, i is impo an o assess quan i a i ely he gene ic uniqueness o a gi en popula ion [43], which may be deduced om gene ic dis ances. Recen ad ances in molecula echnology ha e p o ided new oppo uni ies o assess gene ic a iabili y a he DNA le el. Cu en ly, mic osa elli es a e widely used since hey a e nume ous, andomly dis ibu ed in he genome, highly polymo phic, and show co-dominan inhe i ance [20,30]. Many mic osa elli es ha e ecen ly become a ailable in chickens, and ha e been mapped in e e ence popula ions [4,8,9,22]. These ma ke s p o ide a powe ul ool o QTL esea ch, and ha e also been success ully used o s udy he gene ic ela ionship be ween and wi hin chicken popula ions [36,40,41,45,48]. Reliable in o m- a ion on allele equencies was ob ained om chicken blood o DNA pools using minisa elli e ma ke s [12,13,24,25], as well as mic osa elli es [10,31]. This a icle epo s on he esul s o he AVIANDIV EC- unded esea ch p ojec [26,44,47]. The aim o he s udy was o e alua e he gene-pool o 52 chicken popula ions om a wide ange o o igins. The esul s may p o e o be aluable o he u u e conse a ion o gene ic esou ces o chickens. 2. MATERIAL AND METHODS Whe e possible and ele an , h oughou his epo , chicken lines, popula- ions, and b eeds will be e e ed o as popula ions. Gene al in o ma ion on hese popula ions is p esen ed in Table I and de ails we e epo ed by Tixie - Boicha d e al. [44]. In o ma ion on he ma ke s is gi en in Table II and a b ie desc ip ion o he popula ions, ma ke s and geno yping is gi en below. 2.1. Popula ions and animals Fi y- wo popula ions we e chosen o ep esen as many Eu opean coun ies as possible and o co e a wide ange o popula ions di e ing by selec ion his o y and cu en managemen . We aimed o sample he same numbe o males and emales om as many amilies as possible, o a o al o 50 chickens pe popula ion. Sampled popula ions we e classi ied a p io i in o six ypes (Tab. I). Type 1 includes wo subspecies o he Red Jungle Fowl, Gallus gallus gallus and G. g. spadiceus which we e ecen ly caugh in he wild. Type 2 ep esen s i e domes ica ed bu unselec ed chicken popula ions wi h subs an ial mo phological a ia ion. Type 3 ep esen s 23 s anda dized b eeds, whe e selec ion has been done, ei he ecen ly o in he pas , on mo phological ai s acco ding o a pheno ypic s anda d. Type 4 encompasses 13 expe imen al o comme cial, whi e- o b own-egg laye lines. Type 5 includes eigh lines 536 J. Hillel e al. Table I. In o ma ion on popula ions. (con inued on he nex page) Popula ion Type ∗ O igin Founde ∗∗ C ea ion ∗∗∗ Size ( ange) Name No. Gallus gallus spadiceus 101 1 Thailand b 1997 100 Gallus gallus gallus 102 1 Thailand b 1997 165 Fayoumi 04 3 Egyp b 1978 50–300 Bedouin 05 2 Is ael a 1995 30–50 Wes aeliche To lege 06 3 Ge many b 1904 900 Sundheime 07 3 Ge many c 1886 100–500 Ligh B own Legho n 08 3 Ge many b 1870 (la ge) Owl-bea ded (Uilenbaa de ) 09 3 The Ne he lands a 1650 200 F iesian owl 10 3 The Ne he lands a (unknown) 50 B esse noi e 11 4.2 F ance b 1995 400–2500 Houdan 12 3 F ance c 1994 50–200 Ma ans 13 3 F ance c 1988 200–350 Do king 14 3 G ea -B i ain b 1986 85 Cochin 15 3 China b 1946 130 Icelandic land ace 16 2 Iceland a 900 2000–4000 Finnish Land ace 17 2 Finland c 1900 600–1000 Old Scand. Re . Pop. 18 3 Denma k c 1969 200–700 Jae hoens 19 3 No way a 1916 300–400 Sicilienne Bu e cup 20 3 I aly- Sicilia b 1990 150 Pado ana 21 3 I aly b 1986 35–350 Black Cas ellana 22 3 Spain a 1975 200–300 Red Villa anquina 23 3 Spain a 1980 200–300 Czech Golden Pencilled 24 3 Czech Republic c 1995 500–1000 O a ka hen 25 3 Slo akia c 1994 50–100 T anssyl . Naked Neck 26 3 Hunga y a 1990 70–220 G een-legged Pa idge 27 3 Poland a 1950 1600 O lo 28 3 Russia c 1960 2000 Yu lo c owe , in Russia 2901 3 Russia c 1976 10 000 Yu lo c owe , in Uk ainia 2902 2 Uk aine b 1870 140–700 Uk ainian bea ded 30 2 Uk aine b 1850 74–105 Pol a a clay 31 3 Uk aine b 1870 2000–6000 C line 32 6 Czech Republic a 1932 180–600 Gödöllö Nhx, 33 4.15 Hunga y c 1996 600–8000 Line Sa coma-Resis an 3401 4.10 USA, now in Ge many b 1965 200 Line Sa coma-Suscep ible 3402 4.10 USA, now in Ge many b 1965 200 Whi e-egg laye A 37 4.3 comme cial b 1959 6000 B own-egg laye A 38 4.4 comme cial b 1979 5000 B oile dam line A 39 5.1 comme cial c 1980 10 000 B oile si e line A 40 5.2 comme cial c 1980 10 000 B oile dam line B 41 5.1 comme cial a 1960 5000–30 000 B oile si e line B 42 5.2 comme cial b 1970 10 000–70 000 B own-egg laye B 43 4.4 comme cial c 1960 5000 B own-egg laye line C 44 4.4 comme cial b 1960 5000 B own-egg laye line D 45 4.4 comme cial b 1962 1000 Chicken biodi e si y assessed by DNA pools 537 Table I. Con inued. Popula ion Type ∗ O igin Founde ∗∗ C ea ion ∗∗∗ Size ( ange) Name No. B own-egg laye line E 46 4.4 comme cial b 1955 600 B oile si e line C 47 5.2 comme cial a 1974 con iden iel B oile dam line C 48 5.1 comme cial a 1974 con iden iel B oile si e line D 49 5.2 comme cial a 1992 8000 B oile dam line D 50 5.1 comme cial b 1970 5000–20 000 Ab line, high 51 4.15 The Ne he lands c 1980 75–300 Ab line, low 52 4.15 The Ne he lands c 1980 75–300 Ab line, con ol 53 4.15 The Ne he lands c 1980 110–250 ∗ ype: 1 =wild popula ion; 2 =domes ica ed unselec ed b eed; 3 =s anda dized b eed selec ed on mo phology; 4 =Laye s, selec ed on quan i a i e ai s; 5 =B oile s, selec ed on quan i a i e ai s; 6 =inb ed line. De ailed in o ma ion o ype 4: 4.10 =expe imen al Whi e Legho n; 4.15 =expe imen al b own egg laye ; 4.2 =comme cial whi e egg laye no Whi e Legho n; 4.3 =comme cial Whi e Legho n; 4.4 =comme cial b own-egg laye . De ailed in o ma ion o ype 5: 5.1 =comme cial b oile dam line; 5.2 =comme cial b oile si e line. ∗∗ ounde : a =small lock; b =one b eed; c =a c oss be ween se e al b eeds. ∗∗∗ es ima e o he C ea ion Time o he sampled locks. o mea ype chickens: ou b oile dam-lines and ou b oile si e-lines. Fo lines o ypes 4 and 5, selec ion has been applied on a quan i a i e ai o on an index. Expe imen al lines included wo se s o di e gen ly selec ed lines. Type 6 is an inb ed line. 2.2. Blood and DNA samples Blood samples o 2–4 mL we e collec ed om he wing ein wi h Sa s ed Sy inges con aining EDTA as an an i-coagula ing agen . In 20 popula ions, blood samples we e collec ed on he same day and blood cells om indi idual bi ds we e ob ained om esh blood by cen i uga ion and we e e-suspended in an equal olume o PBS/Suc ose (1 /1 ). DNA pools om each popula ion we e used o educe he amoun o geno yping. An aliquo o 50 µL o blood cells in PBS/Suc ose om each bi d in a popula ion was aken o p epa e i s blood pool. Indi idual blood samples and blood pools we e ozen a −25 ◦C. High molecula weigh DNA was ex ac ed om 80 µL o he blood pool, ollowing se e al s eps o haemolysis, p o einase K incuba ion, p ecipi a ion in dime hyl- o mamide/ace one (95 /5 ), e-suspension in TE, e hanol p ecip- i a ion and inal e-suspension in 2 mL TE. Fo he o he 32 popula ions, a blood pool could no be made p ope ly, ei he because he indi idual blood samples had been ozen, o because hei quali y was no good enough. Consequen ly, DNA was ex ac ed om indi idual blood samples and he pooled DNA sample 538 J. Hillel e al. Table II. Polymo phism measu es o he 22 mic osa elli es; ma ke names, loca ion: ch omosome and posi ion, allele size ange, numbe o alleles ac oss popula ions and pe popula ion, a e age, minimum and maximum alues o gene di e si y and equency o polymo phic popula ions. Loci a e so ed by dec easing a e age alues o gene di e si y. Ma ke Loca ion Allele size* Numbe o alleles Gene di e si y F eq. polym. popula ions (P) Ch omosome Posi ion A e . S.D. Range (bp) Ac oss popula ions Pe popula ion A e . Min. Max. MCW34 2 230 230 5.9 30 16 6.5 0.68 0.19 0.88 0.98 ADL268 1 288 112 3.3 20 10 4.5 0.63 0.13 0.78 0.98 MCW69 E60C04W23 23 161 4.1 23 12 5.4 0.61 0.18 0.88 1.00 LEI192 6 114 271 14.7 74 23 5.4 0.59 0.28 0.80 0.96 MCW183 7 79 307 8.2 36 16 5.1 0.59 0.02 0.81 0.92 ADL112 10 0 132 2.5 12 7 3.8 0.55 0.15 0.75 1.00 MCW295 4 75 96 3.6 16 9 3.4 0.51 0.15 0.80 0.94 MCW206 2 104 233 4.1 31 14 4.0 0.51 0.07 0.77 0.98 MCW111 1 118 104 2.3 20 9 3.6 0.51 0.13 0.75 1.00 MCW14 6 96 184 5.7 26 13 4.2 0.50 0.11 0.78 1.00 MCW330 17 41 280 8.9 39 10 3.1 0.48 0.10 0.81 0.86 ADL278 8 87 118 4.2 15 5 2.8 0.47 0.32 0.71 0.92 MCW78 8 87 143 2.5 13 7 3.4 0.44 0.16 0.73 0.90 MCW37 3 317 157 1.5 8 5 2.4 0.43 0.03 0.67 0.87 MCW67 10 61 181 2.8 10 6 2.5 0.41 0.06 0.74 0.92 MCW81 5 123 122 8.7 23 12 4.2 0.41 0.02 0.79 0.88 MCW222 3 86 223 2.1 10 6 2.4 0.39 0.02 0.71 0.85 MCW216 13 28 146 1.4 14 6 2.2 0.38 0.06 0.67 0.86 MCW284 4 167 245 4.8 46 10 2.0 0.33 0.13 0.81 0.84 MCW103 3 210 273 2.0 12 7 2.5 0.33 0.10 0.66 0.86 MCW248 1 20 218 2.7 10 5 2.1 0.31 0.04 0.66 0.71 MCW98 4 217 264 1.0 6 4 1.8 0.29 0.06 0.55 0.69 A e age 191 4.4 22.4 9.6 3.5 0.47 0.11 0.75 0.91 ∗allele size =size o he PCR p oduc which includes he mic osa elli e epea s. Chicken biodi e si y assessed by DNA pools 539 was p epa ed by aliquo ing equal amoun s o indi idual DNA, as measu ed by spec opho ome y. P io o geno yping, he concen a ion o he DNA pools was s anda dized o 100 ng ·µL−1in TE solu ion. The geno yping o he 52 popula ions was done o 22 mic osa elli e loci. 2.3. Geno yping a mic osa elli e loci A se o 22 (CA)n di-nucleo ide mic osa elli e ma ke s, which a e as uni- o mly dis ibu ed as possible h oughou he chicken genome, was es ed o hei use in DNA pools. The ma ke s and hei genomic posi ion a e lis ed in Table II. Peak sco ing on an ABI sequence was used o es ima e mic osa elli e allele equencies as de ailed in C ooijmans e al. [10]. PCR p oduc s o di e en ma ke s we e pooled in such a way ha each ma ke signal on he ABI sequence s did no exceed a peak heigh o abou 1000 o 1500. F agmen sizes we e de e mined ela i e o he GENESCAN-350 TAMRA wi h he GENESCAN agmen analysis so wa e (Pe kin Elme , Applied Biosys ems Di ision). Subsequen ly GENOTYPER so wa e (Pe kin Elme , Applied Biosys ems Di ision) was used o au oma ed agmen calling and he gene a ion o an ou pu able con aining he agmen s o he di e en loci and he peak a eas o each o hese agmen s. Finally, hese peak a eas we e used o calcula e he ela i e agmen equencies o all peaks o each locus. These equencies we e used in his s udy as he allele equencies. Howe e , alleles sco ed om DNA pools using an ABI sequence , migh be a e ac s and s u e bands a he han eal alleles. We assumed ha his di icul y would be ele an equally o all popula ions and mos ma ke s. 2.4. Gene di e si y es ima e Using he calcula ed allele equencies Pmi o popula ion mand allele i, gene di e si y (Hm), namely he expec ed he e ozygosi y unde Ha dy-Weinbe g assump ions, is: Hm=1−X i P2 mi.(1) An a e age Hwas calcula ed o each locus ac oss popula ions and o each popula ion ac oss loci. 2.5. Gene ic dis ance es ima es Th ee gene ic dis ances based on allele equencies we e used: he Nei gene ic dis ance [34], Ca alli-S o za cho d measu e [3] and Reynolds gene ic dis ance [38]. Addi ionally, he del a-mu-squa ed dis ance [18,19], based on allele size, was applied. Pai wise dis ances be ween each pai o he 52 popula ions (1326 es ima es) we e calcula ed o each measu e. 540 J. Hillel e al. 2.6. S a is ical analyses Co ela ion coe icien s and ank co ela ions we e es ima ed using JMP 4.0 so wa e [28]. 2.7. Calcula ion o he mu a ion a e We used he in o ma ion on he his o y o he wo Sa coma lines 3401 and 3402 (see Tab. I) and hei mic osa elli es da a o es ima e he mu a ion a e o mic osa elli es in chickens. These lines we e di e gen ly selec ed o esis ance o suscep ibili y o Rous Sa coma Vi uses (RSV) A and B o 2- o-3 wk old chickens [23]. The wo sub-lines ha e been kep sepa a ely o 25 gene a ions. The wo lines had 74 di e en alleles o which only 46 (62%) we e sha ed. A each o he 25 gene a ions, en si es we e selec ed om each line and ma ed o app oxima ely 100 dams. In his p ocess, 220 game es we e in ol ed a each gene a ion and o each line, o p oduce he nex gene a ion. We sco ed he numbe o mic osa elli e alleles speci ic o each o he wo lines in compa ison o he o he one. 3. RESULTS Raw da a and basic esul s such as gene ic dis ances be ween popula ion pai s o he cu en s udy, can be ob ained om he Poul y Biodi e si y da abase a : h p://w3. z . al.de/a iandi /index.h ml. A o al o 3760 allele equencies we e ob ained. Amongs he 1144 possible ypings (22 ma ke s ×52 popula ions), 77 (6.7%) we e missing due o echnical di icul ies, mainly o h ee loci: ADL278,MCW14, and MCW330, wi h missing da a on 27, 15, and 15 popula ions, espec i ely. Fo he emaining 19 ma ke s, only 20 (2.0%) geno yping da a poin s we e missing. 3.1. Polymo phism o ma ke s As shown in Table II, all 22 ma ke s we e polymo phic in a leas 69% o he popula ions, and 91% o he popula ions we e polymo phic. The mean numbe o alleles was 9.6 ac oss popula ions and 3.5 wi hin popula ions, and a e age gene di e si y was 0.47. Among he 22 es ed ma ke s, he mos polymo phic was MCW34 wi h 16 alleles ac oss popula ions and, on a e age, 6.5 alleles pe popula ion. The gene di e si y o MCW34 was 0.68 and 98% o he popula ions we e polymo phic o his ma ke . A he o he ex eme, ma ke MCW98 was he leas polymo phic, wi h ou alleles ac oss popula ions, 1.8 alleles pe popula ion, and a gene di e si y o 0.29; in addi ion i was polymo phic in 69% o he popula ions. Chicken biodi e si y assessed by DNA pools 541 Table III. Co ela ion coe icien s be ween polymo phism measu es o ma ke s and hei s a is ical signi icance (p). Polymo phism measu e Alleles ac oss popula ions Alleles pe popula ion Expec ed he e ozygosi y Size di e ence 0.87 0.51 0.41 (0.00001) (0.01) (n.s.†) Alleles ac oss popula ions 0.81 0.65 (<0.00001) (0.0011) Alleles pe popula ion 0.91 (<0.00001) †n.s. =non signi ican . The associa ions be ween he a ious measu emen s o ma ke polymo ph- ism a e p esen ed in Table III. The numbe o alleles ac oss he popula ions was highly co ela ed wi h he di e ence be ween he smalles and he la ges allele a a locus ( =0.87) and as expec ed, wi h he numbe o alleles pe popula ion ( =0.81). Gene di e si y was highly co ela ed wi h he numbe o alleles pe popula ion ( =0.91) bu o a lesse ex en wi h he numbe o alleles ac oss popula ions ( =0.65). Based on hese associa ions, i u ned ou ha he ma ke s LEI192 and MCW284 had mo e alleles ac oss popula ions han expec ed om hei a e age numbe o alleles pe popula ion and ma ke MCW34 had less alleles ac oss popula ions han expec ed om i s a e age pe popula ion. 3.2. Di e si y o popula ions Table IV p esen s he di e si y measu es o he 52 popula ions. A e age gene di e si y (H) wi hin he 52 popula ions ac oss all 22 loci was 0.47 and he a e age numbe o alleles was 3.5. The leas polymo phic popula ion was he inb ed “C line”, wi h a gene di e si y (H) o 0.05 and 1.3 alleles pe locus ac oss all ma ke s. The nex o lowes was Pado ana, a ancy b eed wi h a na ow base in No he n I aly, wi h H=0.17, and 1.8 alleles. The mos polymo phic popula ion was he Gallus gallus spadiceus, wi h H=0.64 and an a e age o 5.2 alleles, ollowed by he popula ion o Yu lo C owe in Russia, wi h H=0.62 and 4.8 alleles. Wi hin hese ex eme popula ions, he e was a ia ion ac oss ma ke loci. In he inb ed C line, he polymo phism anged om H=0.39 and wo alleles o ma ke ADL268 o H=0 a 15 o he emaining loci. Simila ly, in Gallus gallus spadiceus, polymo phism a ied be ween H=0.88 and 11 alleles (MCW69), o H=0.20 and 2 alleles (MCW222). 548 J. Hillel e al. 4. DISCUSSION 4.1. Da a eliabili y In gene al, he esul s epo ed he e we e in good ag eemen wi h wha is known o he his o y o he popula ions and wi h p e ious scien i ic epo s. Fo ins ance, he e al popula ions and he domes ica ed unselec ed popula ions we e he mos polymo phic, while he inb ed and he Whi e Legho n lines we e a less polymo phic. This sugges s ha use o DNA pools, he chosen ma ke s and he biome ical ools desc ibed in his epo , p o ide eliable es ima es o he popula ion’s biodi e si y (see also [10,12,13,24,25,31]). Fu he mo e, polymo phism es ima es ob ained om hese DNA pools we e ound o be in good ag eemen wi h hose ob ained om indi idually yping a subse o 30 bi ds om 20 popula ions (unpublished da a). Fo ins ance, he co ela ion coe icien be ween H alues o he cu en s udy and obse ed he e ozygo e equency is =0.85 (p=0.002)and be ween he numbe o alleles pe locus is =0.91 (p=0.0002). Da a eliabili y was also suppo ed by he same o de o popula ion H alues, based on 22 subse s, wi h single ma ke s sys ema ically excluded. 4.2. Di e si y o mic osa elli es and i s mu a ion a e The wide ange o biodi e si y in he sampled popula ions and he high le el o polymo phism o mic osa elli es we e e lec ed by he inding ha e en he leas polymo phic locus, MCW98 was ound o be polymo phic in 69% o he popula ions while ou o he 22 ma ke s we e polymo phic in all 52 popula ions (Tab. II). The co ela ion coe icien s be ween some o he polymo phism measu es a e high and e y signi ican ( =0.81 −0.91, see he diagonal o Tab. III). On he con a y, allele-size ange had lowe co ela ion coe icien s wi h he numbe o alleles pe popula ion (na)( =0.51, p<0.01) and wi h he expec ed he e ozygo e equency H( =0.41, p>0.05). On close exam- ina ion, o h ee loci, namely LEI92,MCW284 and MCW330, size anges a e la ge han expec ed, based on hei naand H alues. Fo LEI192, six o he six een alleles ac oss popula ions we e p esen in only a single popula ion and wo alleles in wo popula ions. Excluding hese eigh alleles, educed he ange om 74 bp o 46 bp. Simila ea men o he loci MCW284 and MCW330 educed he numbe o alleles ac oss popula ions om ele en o wo and om en o ou , espec i ely. Simila ly, allele size anges educed om 46 bp o 8 bp o MCW284 and om 39 bp o 30 o MCW330. These can be in e p e ed ei he as popula ion-speci ic (p i a e) alleles o as signal peaks in he sequence machine ha a e no alleles. Disc imina ion be ween hese is Chicken biodi e si y assessed by DNA pools 549 possible only by analyzing sibships, which is ou o he scope o he p esen epo . In s udies o human gene ic a ia ion, a he con inen al le el he e is good ag eemen in he o de ing by egion o gene di e si y measu es among di e en kinds o ma ke s, once asce ainmen bias is emo ed [29,39]. The same is ue o he o de ing o gene ic dis ances among popula ions assessed o di e en ma ke s [29]. I is easonable he e o e o belie e, ha he o de ing among chicken b eeds o di e si y and dis ances seen he e o mic osa elli es in DNA pools would no be e y di e en o o he gene ic ma ke sys ems. The mic osa elli e loci used he e we e selec ed o be polymo phic o use in gene mapping. Howe e , Rosenbe g e al. [42] ob ained di e si y and gene ic dis ance pa e ns o humans ha la gely ag eed wi h hose o Bowcock e al. [2] e en hough he o me s udy used mic osa elli es om a mapping se and he la e used ma ke s ha we e no selec ed. Asce ainmen bias is, he e o e, no expec ed o ha e had a majo e ec on he o de ing o s a is ics o ou chicken da a. The es ima ion o he mu a ion a e using he di e gen ly selec ed (RSV) lines is based on he assump ion ha he 22 mic osa elli e ma ke s a e no linked o genes ha a e a ec ed by he selec ion c i e ia. Webe and Wong ound a mu a ion a e o 5.6×10−4 o di-nucleo ide loci in humans by pedig ee analysis [46]. Based on 22 di-nucleo ide loci, ou es ima e was abou i e imes lowe , bu may unde es ima e he ac ual alue since i was assumed ha no e e se o ecu en mu a ions occu ed. Since his es ima e is om a single se o di e gen ly selec ed lines, we canno es ima e con idence limi s o his a e. Fu he analysis o chickens is needed o p o ide a eliable es ima e o mu a ion a e. 4.3. Gene ic di e si y o popula ions A gene di e si y o 0.48 was ob ained om DNA pools in a subse o 20 popula ions. Based on indi idual yping o he same subse , he equency o he e ozygo es was 0.47 (unpublished da a). These es ima es a e simila o he a e age Hin all 52 popula ions indica ing he good eliabili y o ou es ima es. Di e si y es ima es in his s udy a e lowe han he obse ed equencies o he e ozygo es epo ed in o he species using mic osa elli e ma ke s. Fo ins ance, in human popula ions he a e age he e ozygo e equency anges be ween 0.7 and 0.8 [2], in ca le- 0.6 [11], in pigs- 0.68 [21] and in ish- 0.86 [16]. Al hough such compa isons a e di icul o in e p e , he lowe a iabili y in chickens calls a en ion o he impo ance o conse ing he chicken gene pool. I is wo h men ioning, howe e , ha SNP equency in he chicken genome was ound o be qui e high and signi ican ly highe han ha ound in he human genome (unpublished da a). 550 J. Hillel e al. 4.4. Di e si y be ween popula ions wi hin ypes Ca ego iza ion o popula ions in his s udy was done acco ding o he pop- ula ions’ his o y and hei b eeding pu pose, which ha e a ec ed he gene ic di e si y. Indigenous s ocks a e domes ica ed bu unselec ed local b eeds ( ype 2) ha ha e pe sis ed in he ag icul u al socie ies o decades o e en cen u ies. Thei long his o y in ce ain egions migh ha e led o speci ic adap a ion o local en i onmen al condi ions and gene ic changes due o na u al selec ion and o some deg ee due o gene ic d i as well. In p ac ice, ou ou o he i e popula ions o ype 2 had ela i ely high polymo phism, which may e lec hei a he la ge e ec i e popula ion size and he ac ha hese s ocks we e no subjec ed o in ense selec ion o p oduc ion. The g oup o s anda dized b eeds ( ype 3) selec ed o mo phological ai s co e s a wide ange o a ious b eeds kep by ancie s. This ype has la ge a iance in polymo phism le els (Tab. IV) as also e lec ed in he sca e ed pa e n o i s popula ions in Figu e 1. Based on gene ically dis inc local popu- la ions, pu e b eeds we e de eloped ha di e ed in many pheno ypic ai s such as plumage colo , plumage pa e n, and comb ype. Gene ic changes in ancy b eeds may occu a he apidly in hese ela i ely small popula ions because o in ense selec ion o exhibi ion ai s, inb eeding, c ossb eeding gene ic d i , bo leneck and ounde e ec s [17,35,36]. This complexi y o his o ies and b eeding p ac ices may explain he he e ogenei y o polymo phism alues ha cha ac e izes ype 3. Types 4 (laye s) and 5 (b oile s) o chicken lines, which a e selec ed o quan i a i e ai s, encompass he majo comme cial poul y indus y. Es im- a es o b oile s (H=0.57) we e in ag eemen wi h a p e ious epo , in which H=0.53 was ob ained om pooled blood samples [10]. The Whi e Legho n laye line in ou s udy (sub- ype 4.3) was ound o ha e low gene di e si y (0.33), sligh ly highe han he alue o 0.27 in he p e ious epo [10]. Among he laye s his line had he lowes polymo phism. In he 1940s, poul y b eeding began o de elop as a business. Pu e b ed lines we e used o de elop specialized comme cial b eeding s ocks o able egg and mea p oduc ion by applying highly e icien and in ense b eeding p og ams. Indus ial egg and mea s ocks a e b ed by la ge mul ina ional b eeding companies, and he “end p oduc s” a e hyb ids based mos ly on ou highly selec ed g andpa en al lines. In pa icula du ing he las decades e o s ha e been made o limi inb eeding in hese g andpa en al pu e b ed lines [37]. La ge lock sizes and limi ing inb eeding may be e lec ed by he deg ee o polymo phism o hese ypes (4 and 5) which is compa able e en o hose o popula ions ha we e no subjec ed o in ensi e b eeding. I should be no ed he e ha he comme cial lines in ou s udy a e all pu e b ed lines. The e o e, hei le el o polymo phism can be conside ed as a eliable es ima e o he polymo phism o he pu e-b ed g andpa en al lines. Di e ences wi hin Chicken biodi e si y assessed by DNA pools 551 ypes 4 and 5 migh be explained by he di e en o igin o whi e egg laye s, b own egg laye s and b oile s. Whi e egg laye s a e chie ly ep esen ed by one b eed, he Single Comb Whi e Legho n b eed, while he gene ic basis o b own egg laye s has been somewha b oade , mainly coming om he Rhode Island Red, New Hampshi e, Plymou h Rock and Aus alo p b eeds. A simila pic u e is cha ac e is ic o he poul y mea p oduc ion sec o . The pa e nal g andpa en lines a e mainly de i ed om he Whi e Co nish b eed, while ma e nal g andpa en s a e hea ily based on Whi e Plymou h Rocks. The Co nish was de eloped in England om Asia ic igh ing s ocks, and he Whi e Plymou h Rock was de i ed om an Ame ican pa en b eed [6]. The low deg ee o gene di e si y in he inb ed C line esul ed di ec ly om he b o he -sis e ma ing scheme used o de elop his line and possibly om gene ic d i due o he limi ed e ec i e popula ion size. 4.5. Gene ic dis ance measu es The h ee gene ic dis ance measu es which a e based on gene equencies we e in good ag eemen wi h he gene ic di e si y o he examined b eeds, indica ing ha hese app oaches i he his o y o he domes ica ed chickens well. Howe e , he (δµ)2app oach ha is based on he allele size which is ypical o mic osa elli es, was di e en and poo ly co ela ed wi h o he gene ic dis ances as well as measu es o di e si y. P obably he his o y o he domes ica ed chicken does no mee he assump ions behind his app oach especially he cons an popula ion size. 4.6. Concep ual aspec s o MGD A domes ica ed popula ion wi h a la ge numbe o alleles pe locus is expec ed o ep esen a la ge po ion o he s udied gene pool and he e o e o be gene ically close o all o he popula ions. Popula ions o ypes 1 and 2 a e polymo phic and ep esen he gene pool well and indeed ha e low alues o MGD. On he con a y, popula ions ha a e ela i ely monomo phic canno e lec he whole s udied gene pool and he e o e will ha e high MGD alues, as e idenced by he inb ed C line and a ew popula ions om ypes 3 and 4.3. Figu e 1 shows a nega i e and clea associa ion be ween he a e age numbe o alleles pe locus and he Ca alli-S o za MGD alues. A simila and igh e associa ion is appa en when gene di e si y alues a e examined. The clea associa ion discussed abo e be ween polymo phism and MGD alues sugges s ha he app oach o cons uc ing e olu iona y ees in domes ica ed popula ions is likely o gi e a misleading pic u e o he his o y. This is pa icula ly ue o oo ed ees, which assume a unidi ec ional e olu iona y p ocess. I seems ha he p ocesses o domes ica ion and b eeding ha e in ol ed mo e o less con inuous and mul idi ec ional gene low. 552 J. Hillel e al. Ca e ul examina ion o Figu e 1 e eals ha popula ions wi h a simila numbe o alleles may ha e di e en MGD alues. The join dis ibu ion o he numbe o alleles and MGD alues can be iewed wi h a eg ession line ha will be e med a “ end line”, as in Figu e 1. The unselec ed popula ions ( ype 2) a e highly polymo phic and loca ed nex o o below he end line, implying ha many o hei alleles a e common o he s udied gene pool. The wo Gallus gallus subspecies ha e a la ge numbe o alleles pe locus bu a e bo h loca ed abo e he end line, which implies some p opo ion o p i a e alleles. Indeed, i e p i a e alleles ha a e absen in he domes ica ed chicken we e ound o be p esen in G. g. gallus and six such alleles in G. g. spadiceus. 4.7. The chicken ances o and he need o conse a ion I is assumed ha all b eeds o he domes ica ed chicken descend om a single ances o , he RJF, which o igina ed in Sou heas Asia [5,14,15]. Since popula ions a he cen e o o igin should con ain he highes di e si y (as has been shown o humans and o he species [2]), RJF should p esen a high le el o polymo phism and also hold low alues o MGD. The wo sub-species o Gallus gallus (RJF) which a e ep esen ed in his s udy ha e high di e si y alues and low MGD alues, bu no he lowes (Tab. IV and Fig. 1). These esul s sugges he hypo hesis ha he Red Jungle Fowl is a majo con ibu o o he gene pool o he domes ica ed chicken. The p esence o p i a e alleles bo h in he RJF and in he domes ica ed gene pool does no necessa ily con adic his hypo hesis. These p i a e alleles could esul om gene ic d i and sepa a e e olu ion since he onse o domes ica ion. Addi ionally, p i a e alleles o he domes ica ed b eeds may indica e a con ibu ion o o he wild species as deba ed in C aw o d [5]. In s udies on he mi ochond ial DNA in chickens [14, 15], he au ho s sugges ha Gallus gallus gallus could ha e gi en ise o he di e se b eeds o he domes ica ed chicken, e en hough common pa e ns in domes ica ed b eeds and in G. g. spadiceus and G. g. banki a a e also obse ed. Wi h ega ds o he le el o gene ic polymo phism and he mean gene ic dis ance, he Red Jungle Fowl (Gallus gallus) appea s o be an impo an ese oi o he chicken polymo phism. Ne e heless, du ing domes ica ion, ex ensi e gene ic di e si y has accumula ed in he chicken, and his di e si y is displayed by he many b eeds and s ains di e ing in hei pheno ypes which a e he p oduc s o many gene ic, en i onmen al and managemen egimes. A comple e desc ip ion o each b eed o popula ion would en ail asce aining all genes ha con ibu e o any pheno ypic ai . I is e y unlikely ha such comple e knowledge can be achie ed in he nea u u e. The e o e, a mo e p ac ical al e na i e is needed. We belie e ha in o ma ion om DNA ma ke s oge he wi h pheno ypic pe o mance and popula ion his o y may p o ide eliable guidelines in choosing popula ions o p ac ical and o conse a ion pu poses. Howe e , se ing conse a ion p io i ies based exclusi ely on he Chicken biodi e si y assessed by DNA pools 553 di e si y o molecula ma ke s migh lead o he loss o locally adap ed popula ions [32]. In ecen yea s, b eede s o comme cial whi e-egg laye s ha e been con- ce ned abou educed gene ic a iabili y and u u e esponse o selec ion. The esul s epo ed in he p esen a icle suppo his conce n, pa icula ly o Whi e Legho n b eeds. The massi e me ging o b eeding companies in ecen yea s should call o a en ion o he need o conse a ion o gene ic a ia ion among b eeds and lines. App op ia e s a egies o conse a ion o popula ions is ou o he scope o he p esen epo bu is an impo an and con o e sial issue [7]. Ou esul s emphasize he need o conse e polymo phic popula ions such as Yu lo C owe , he O lo o he Finnish Land ace o he sake o global a iabili y, and o p ese e small popula ions o hei unique gene ic ea u es. MGD alues can assis in choosing popula ions mos aluable o he p ese a ion o gene ic a ia ion. ACKNOWLEDGEMENTS We wish o hank Jean-Luc Co ille and Camilo Vilela-Lamego, In a, Jouy- en-Josas, F ance, who we e in ol ed in he DNA p epa a ion and i s dis i- bu ion, and o he ollowing sub-con ac o s who p o ided he blood samples and b eed desc ip ions; wi hou hei help his s udy could no ha e been pe o med ( hey a e lis ed alphabe ically): D . G. Baldane, Pado a, I aly; D . J. Baumga ne , Resea ch Ins i u e, I anka p i Dunaji, Slo akia; D . Y. Bond- a enko, Resea ch Ins i u e; Bo ky, Kha ki , Alaska; D . J.L. Campo, INIA, Mad id, Spain; D. Cassu o, ANAK, Is ael; D . K. Cywa-Benko, Resea ch Ins i u e, Balice n C acow, Poland; D . E. Ey ho sdo i , RALA, Is ael; Y. Jego, Hubba d-ISA, F ance; D . J. Ka hle, NLH, No way; D . I. Moiseye a, Resea ch Ins i u e, Gubkin, Moscow, Russia; D . G. Pidone, Cala a imi, I aly; P o . R. P eisinge Lohmann, Ge many; M. P o ais, Hubba d-ISA, F ance; P. Raul , SYSAAF, Tou s, F ance; M s. V. Robe s, UK; D . P. So ensen, DIAS, Denma k; D . I. 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