Aurochs genetics, a cornerstone of European biodiversity

Aurochsgenetics,

acornerstoneofEuropeanbiodiversity

Picture:ManoloUno(c)StaffanWidstrand

Authors:

• drs.RonaldGoderie(TaurusFoundation);• dr.JohannesA.Lenstra(UtrechtUniversity,FacultyofVeterinaryMedicine);• MaulikUpadhyay(pHDWageningenUniversity);• dr.RichardCrooijmans(AnimalBreedingandGenomicsCentre,Wageningen

University);• ir.LeoLinnartz(ArkNature)

Summaryof:AurochsGenetics,acornerstoneofbiodiversity

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PrefaceIn2015areportiswrittenonAurochsgenetics,madepossiblebyagrantfromtheDutchLibertyWildlifefund.ThisfundprovidedtheTaurusfoundationwithagrantofEUR20.000toconductgeneticresearchonaurochsanditsrelationwithnowadaysso-called‘primitive’breeds.Thisisthesummaryofthatreport.Thissummaryshortlydescribesthecurrentstateofaffairs,whatwedoknowearly2015abouttheaurochs,aboutdomesticcattleandtherelationshipofaurochsandtheprimitivebreedsusedintheTaurosProgramme.

Nijmegen,December2015.


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TableofcontentsPreface2Tableofcontents.........................................................................................................3Summary.....................................................................................................................41 Introduction..........................................................................................................62 Aurochs:ashortdescription..................................................................................83 Thedomesticationofcattle.................................................................................124 Results.................................................................................................................155 ThemeaningofgeneticresearchfortheTaurosProgramme...............................19Glossary.....................................................................................................................21Literature...................................................................................................................22Annex1:LONGLISTOF‘ORIGINAL’BREEDS.........................................................................24Annex2:SetwithcriteriafortheTaurosProgramme.................................................25Annex3:Briefsummaryofthebreedsinvolved.........................................................27Annex4:Overviewofconductedresearch.................................................................29Annex5:Theresultsoftheinheritancecheck............................................................30


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SummaryTheTaurosProgrammeisaninitiativeoftheDutchTaurusfoundation,since2011inclosecooperationwithRewildingEuropeandARKNature.Theprogrammeaimsatrewildingcattleandwantstodevelopacrediblereplacementfortheextinctaurochs.ThisreplacementiscalledtheTauros.TheTauroswillbebasedonthebestavailablescientificknowledge,involvingarcheo-zoology,history,ecology,ethologyandgenetics.

In2013afundingofEUR20.000fromtheDutchLibertyWildlifeFund,providedbyRewildingEurope,wasanewstimulustotheresearch.WealreadyhadbeenabletomakeacomparisonbetweenAurochsandalistof30EuropeanprimitivebreedsbasedonthemitochondrialDNA(thisisasmallDNA-particleoutsideofthecell-nucleus).ButwiththefundingfromtheLibertyWildlifeFundwenow–forthefirsttime-wereabletomakethecomparisonbetweenAurochsandour30‘primitive’breedsbasedontheDNAasitisfoundinthecellnucleus(autosomal1DNA).ThistypeofDNAinsidethecell-nucleuscodesforthephysicalproperties.ThisuniqueresultcouldonlybeachievedbecauseofthecooperationwithaDublin-basedresearchconsortiumwhoveryrecently(2015)wasabletosequencethecompletegenomeof(oneindividualof)Aurochs.

Ashortsummaryoftheresults–atthismoment–wouldbe:

§ Wealreadyknewthat-basedontheearlieranalysisofmitochondrialDNA(mtDNA)whichspecificallyinheritsfrommothertochild-certainso-calledPodolianbreeds(breedsfromItalyandtheBalkancountries)showacloserelationshipwithAurochs.Theseresultsarebasedontheanalysesofagreaternumberofaurochsindividuals(sinceit’sfareasiertoreconstructthesesmallfragmentsofDNA,thantoreconstructthecompletegenome);

§ TheinformationwenowgotfromthecomparisonofAurochswithour30primitivebreedsbasedontheautosomalDNA,showsthatIberianbreedsingeneralshowacloserrelationshiptoAurochs.SincetheinformationbasedonnuclearDNAtellsusfarmoreaboutinheritance,atthismomentweconsidertheanalysesoftheautosomalDNAasthemostvaluable;

§ ‘Rankingthestars’:Oneanalyses(NEIgeneticdistance)calculatedthedistancebetweentheanalysedbreedsincomparisontotheoneanalysedAurochs.ItwasfoundthatfourofthebreedsthatdoplayamajorroleintheTaurosbreedingprogramme(threeIberianandonePodolianbreed)areinthetop10andsixofourbreedsareinthetop15.

Theseresultsleadtothefollowingconclusionsandrecommendationsforthebreedingprogrammeandfollow-upresearch:

Conclusions

§ Theleadingpractiseinthebreedingprogrammesofar–combiningIberianandpodolianbreeds/individualswithAurochs-likephysicalproperties–isbeingconfirmed(bythecombinedresultsofautosomalandmtDNAandtheNEI-analyses).HoweveroneshouldtakeinconsiderationthatatthistimeonlyonespecimenofAurochshasbeenfullysequenced(autosomalDNA),sonewadditionalinformation

1Geneticjargonismarkedorangewhenfirstused.Ashortexplanationcanbefoundintheglossary.


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couldleadtoadjustmentsofthisconclusion.Itisthereforethatthebreedingprogrammestillusesaratherwidevarietyofcrossings(withtheselectedbreeds);

§ Inthe‘top10’anumberofbreedsisapparentthatarenot(yet)usedinthebreedingProgramme.Thereasonforthiswasthatonfenotypicalfeatures(thewaytheanimalslook)betterbreedsareavailable.Howeverinfuturestudiesweneedtolookbetteratsomeofthesebreeds(suchasthehighrankingBusha(notusedintheprogrammebecauseofitslimitedsize)

§ AsthePodolicawasrankedlow,weshouldreconsidertheuseofthisbreed,whichalreadywasusedinaverylimitednumber.

Recommendationsare:

FutureresearchneedstodivedeeperintothegeneticfeaturesofAurochstobeabletoidentifyspecificgenesfordomestication(andthusgettingmore‘grip’ongenesthatcouldbecrucialfortheoppositedirection:therewildingofthespecies).

MorefullysequencedspecimensofAurochsthatcoverasubstantialpartoftheEuropeancontinentareneeded,tobeabletogetanideaofthegeneticdiversitywithinAurochs.

Theresearchneedstoresultinpracticalguidelines–ontheshortrun–asthebaseforfutureselection.


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1 Introduction

TheTaurosProgrammeisaninitiativeoftheDutchTaurusfoundation,since2011incooperationwithRewildingEuropeandARKNature.Theprogrammeaimsatrewildingcattleandwantstodevelopacrediblereplacementfortheextinctaurochs.ThisreplacementiscalledtheTauros.InthenearandmoredistantfutureherdsofTauroswillliveaswildcattleandwillcontributetothesustainableconservationofEurope’s,openlandscapesandbiodiversity.Atthismoment,breedinggroupsofTaurosarealreadygrazingareasintheNetherlands,Spain,Portugal,Croatia,CzechRepublicandRomania.

TheTauroswillbebasedonthebestavailablescientificknowledge,involvingarcheo-zoology,history,ecology,ethologyandgenetics.Duringmillionsofyearsaurochsanditspredecessorsbecamegeneticallyadaptedtotheirenvironment.Therefore,theaurochsgeneswilltellushowtobreedaneffectivereplacementonthebasisofthegeneticvariationoftheprimitivedomesticcattleusedforbreedingtheaurochs.

In2013afundingofEUR20.000fromtheDutchLibertyWildlifeFund,providedthroughRewildingEurope,wasanewstimulustotheresearch.Thisallowedacomparisonoftheautosomal2DNA’sfrom30EuropeanprimitivebreedswiththeDNAoftheaurochs.Inadditionotherbreeds–suchastheCroatianBoskarinandMalteseCattle–wereaddedtothedatabase.ThebudgetoftheLibertyWildlifeFundwasalsousedforanalysingtheoffspringoftheTauroscrossbreds,avitalpartofthetotalresearch,sincewithoutfundingofthispartoftheresearchitwouldbeimpossibletojudgewhetherornotthebreedingisintherightdirection.ThefundingoftheDutchLibertyWildlifeFundwasalsousedtowritethisreportandbydoingsoprovidingbroaderaccesstotheTaurosresearch.

On-goinggeneticresearch

Indifferentpartsoftheworldresearchgroupsareworkingonthegeneticstructureofaurochs.ThisstartedwiththesequencingofmitochondrialDNA(mtDNA),whichindicatesthematernallineage(i.e.,itistransmittedfrommothertocalve)andisbecauseofitsabundanceinacellrelativelyeasytoanalyse.Onemilestonehasbeenreachedrecently:theresearchgroupofDavidMacHugh(UniversityCollegeofDublin)hasbeenabletosequencethecompletegenomeofaBritishaurochsfrom6700BPTheseresultshavebeenpublishedrecently3.Sograduallythecontoursofaurochsgenesareemerging.However,takenintoaccountthatoncetheremusthavebeenmillionsoftheseanimalsroamingforseveralhundredthousandsyearsaroundEurope,AsiaandNorthernAfrica,theremusthavebeenasubstantialgeneticdiversitywithinthepopulations.Withthegenomeofonlyoneindividualbeingavailableatthismoment,itmustbeclearthatwe’restillmissingalotofinformation.

Theaurochsgenomicsequencecanbeinterpretedonthebasisofourgrowingunderstandingoftheroleofgenesandgenevariantsofcattleindetermining

2Geneticjargonismarkedorangewhenfirstused.Ashortexplanationcanbefoundintheglossary.3Parketal.GenomeBiology(2015)16:234DOI10.1186/s13059-015-0790-2


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phenotypesassize,appearance(e.g.,coatcolour,shapeofhornsincattle)andadaptation.

SurveyoftheTaurosResearchProgramme

IntheTaurosProgammeanumberofthingshasbeentriedtodelvedeeperintoAurochsgenetics.In2009/2010partsofaurochsskeletonsfrommuseumsalloverEuropewerecollected,togetherwithgeneticmaterialofaround30differentEuropeancattlebreedswith‘primitive’features.DNAwasextractedandthegeneticvariationwasmeasuredat770.000sites(SNPs,seebelow)inthegenome.Thisyieldedmostvaluablenewinformationonthegeneticstructureofca.30primitiveEuropeanbreeds.WefoundthatthemtDNAfromaurochsandtaurinecattlearedifferent,andwecouldconstructafirstgenealogicaltree,sketchingthegeneticrelationbetweenthesebreedsandaurochs.Thereweredisappointmentstoo.DNAextractedfromaurochsbonesprovedtobeunsuitableforthegeneticanalysis,precludingforthemomentadirectcomparisonoftheaurochsandcattleautosomalDNA.ThisearlypartoftheresearchwasfundedandcarriedoutbyWageningenUniversity,aspartoftheirbroaderinterestinthegeneticstructureofproductivecattle-breeds.

In2012anotherattempttosequenceAurochsgeneticmaterialwasmade.PartoftheskinofamedievalspecimenofaPolishaurochswasanalysedbytheresearchgroupofBethShapirooftheUniversityofCalifornia,oneoftheforemostresearchersinancientDNA.Howeverthisleadtonoresult:theDNAprovedtobetoodeteriorated.Fortunately,in2012-2013,DavidMacHughagreedtogiveWageningenUniversityaccesstothe–atthatmomentstillunpublished-sequenceoftheaurochsgenomes,creatingnewopportunities.

InadditiontothegrantoftheLibertyWildlifeFundin2013,in2014theTaurusfoundationandWageningenUniversityweregrantedaEuropeanresearchbudgetofEUR240.000aspartoftheEGS-ABGprogramme.Sointheautumnof2014,aPhDstudenthasstartedonafour-yearprogrammeatWageningenUniversityincooperationwiththeUniversityofUppsala.Thefirstresultsarebeingpublishedearly2015.

AnextcontributiontoourdatasetwillbetheDNAsequenceofsixtotencompletegenomesoftheimportantparent-animalsofthebreedingoftheTaurosProgram.ThiswillbefundedbygrantofEUR10.000providedbytheDutchProvinceofGelderlandaspartoftheir‘Foodvalley’-programmeandanadditionalEUR10.000providedbyREaspartofthebudgetforthetransportofSayaguesaanimalsfromSpaintoCroatia.

Thecomingyearstheknowledgeontheaurochsandonprimitivecattlewillbebuiltupfurther.AdditionalspecimensofaurochswillbeanalysedbytheUniversityCollegeofDublinandDublinUniversity-andourunderstandingofthehistoryofdomesticationofcattleisgrowing(partofaresearchprojectoftheUniversityofLyon).

Becauseimportantgeneticinformationisstillmissing,wemodifiedourinitialchoices.Insteadoffocussingonjusttwoorthreebreedstostartwith,wechoseforawidervarietyofbreeds.Thisgivesflexibility,forinstanceifon-goingandfuturegeneticresearchleadstonewinsights.AdirectandmostusefulapplicationofthegeneticanalysiscarriedoutwiththefundingofLibertyWildlifeFundisthatwewillbeabletodetermineaccuratelythebreedcompositionoftheTaurosoffspringandweareabletocomparetheoffspringwithAurochs.


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2 Aurochs:ashortdescription

Archaeology,archaeozoology,writtenhistoricalsources,cavepaintings,knowledgeaboutcattlegenetics,cattlebreedingandcattlebehaviourhaveallcontributedtoourunderstandingoftheaurochsinthelastdecades.Weknowwhattheanimallookedlike,weknowitsecology,

Thelooks

Thesizeoftheaurochsbullsvariedbetween155cmand180cm,dependingonthelocalcircumstances.Thesizeofcowswasbetween135cmand155cm.Bullsweresignificantlylargerthancows.Thebodyshapeofanaurochswasdifferentfrommanydevelopedcattlebreedstoday.Ithadanathleticbodyshapeandlonglegs,builtforlong-distancewalking.Itwasaswiftandveryagileanimal.Theskull,carryingthelargehorns,waselongatedandbigcomparedtotherestofthebody.Bull’shornscouldreachupto107cminlengthandbetween10and18cmindiameterattheirbase.Thecowshadhornsprobablyreachingupto70cminlength.Thebull’shornswerelarger,withthecurvaturemorestronglypronouncedincows.Bothsexeshadadistinctlight-coloured,oftenwhite,muzzleandadistincteelstripe(especiallywithbulls).

Range,habitat,ecologyandbehaviour

Attheendofthelastglacialperiod(12,000BP)aurochswasendemicovermostofAsia,Europe,NorthAfricaandtheatthetimegreenSahara(figure2.1,fromFeliuset.al.,2014)

Figure2.1:ThepresumedmaximumareawhereAurochshistoricallyroamedaround(mapbyMarleenFelius,Feliuset.al.,2014)

AurochsoriginatedinIndiasome2millionyearsago.DuringthePleistoceneepoch(2,5millionto12,000yearsago)thespeciesspreadacrossotherpartsofAsia,alsointonorthernAfricaandEurope.TheearliestremainsofaurochsfoundinGermanydate


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fromabout400,000yearsago,butitisestimatedtheanimalreachedEuropelongbeforethat,probablyaround700,000yearsago.

Aurochshabitat:forestdwellerorkingofthesavannah?

Untilrecently,theprevailingtheorywasthataurochsmainlylivedinclosedcanopyforestsandforestedriverinehabitatsorswamps.Thisviewhasbeenadjustedbyecologicalevidenceandthestudiesonaurochsteeth,andskeletons,suchastherecentstudyofBakkeret.al.,20154.Althoughthere’sstillsomescientificdebate,theviewpointofRewildingEuropeandTaurusfoundationisthatAurochswasagrass-eatingplainanimal.Duringthe‘end-game’theanimalwasdriventoasuboptimalhabitat,themoreorlessclosedforests,becausemenoccupiedthefertileplains.Bytheirgrazingactivitiesaurochsopenedupthevegetationandcreatedbiodiversemosaicsofvegetation.

Aurochsbehaviour

Cattledon’trunawayfromthreats,butgrouptogetheranddefendthemselvesifneeded.Therefore,cattlearenotevenlydistributedoverthelandscape,butformfamilygroupsandsometimesroamaroundinlargergroups.Thisresultsinatemporaryandlocallyhighgrazingpressure,dependingontheseasonalfoodavailability.Thelargerthearea,thegreatertheireffectisondifferentiatingthelandscape.

Figure2.1:Preferredhabitatofaurochs

Undernaturalconditions,aurochsprobablywithdrewinsummertothecoolmountains,wheregrasswasmoreavailablethaninthehotlowlands,whilesnowandcoldforcedthemdownagainduringthewinter.

4www.pnas.org/cgi/doi/10.1073/pnas.1502545112


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Aurochsgenetics

Alotofinformationisavailableonthegeneticsofcattleandthevariabilitywithinandacrossbreeds.ThecompletegenomeofaHerefordcowisavailablesince20095,Onthegenomeabout22,000genescodingforproteinshavebeenidentified.Theroleofmanygenesarenowbeingdiscovered,withanemphasisontheeconomicallyimportantgenesformilkinggift,meatqualitye.g.

ThefirststudiesofancientAurochsDNAfocusedonmtDNA.In2010thecompletesequenceofAurochsmtDNAwasdescribed.BasedonarecentmtDNAstudyofourmoderncattlebreedsithasbeenestimatedaround80femalesfromtheFertileCrescentstandatthebasisofallournowadayscattle.RecentlythecompletegenomeofoneBritishAurochshasbeenanalysedpublished(Parket.al.,2015).

In2010forthefirsttimethecompletemtDNAsequenceofanaurochs(wasestablished(Edwardsetal2010).ThissamplebelongedtotheaurochshaplogroupP(derivedfrom‘primigenius’).InItalianAurochsanotherhaplogroup‘T’wasfound.Figure2.1showsthetreeofthecommoncattleTmtDNA(hereindicatedasBRS),theaurochsPmtDNAandthesporadicQandRmtDNA.

Figure2.3:AtreeofBovinehaplogroupsofmtDNA6

AncientDNAanalyses

Amongothermethods,suchasSNPgenotypingandwholegenomeanalyses,thecurrentstudyusesancientDNA-analysesasasourceofinformation.Box1givesanoverviewofhowthisisdone.

5Elsik,C.G.etal,(BovineGenomeSequencingandAnalysisConsortium)(2009)."Thegenomesequenceoftaurinecattle:awindowtoruminantbiologyandevolution".Science324(5926):522–528.doi:10.1126/science.1169588.PMC2943200.PMID19390049.6source:http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015760


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Box1AncientDNA(fromMageeet.al.,2014)

ThestudyofancientDNAhasthepotentialtoanswerquestionsaboutthepastdirectly,thusprovidingatemporalaspecttomoderngeneticstudies.AncientstudieshaveshownthatDNAcansurviveintactformorethan500,000years,enablingmanyevolutionaryquestionstobeaddressedoverthistimeperiod,includingbiogeography,phylogeny,taxonomyandselectiveprocessesatthepopulationlevel,aswellasdomesticationorigins.AncientDNAcanbeextractedfromavarietyofanimalremains,ranginginconditionandage,themostpopularofwhicharebones,teeth,andhair.ThecytoplasmiclocationandhighcopynumberofmitochondrialchromosomeswithineacheukaryoticcellhasmademtDNAtheprimarygeneticmarkerforancientDNAstudies.However,recentadvancesingenomictechnologieshaveseenanincreaseinthenumberofancientDNAstudiesthatinvolvepartialorwholenucleargenomesequencesfromarchaeologicalsamples(ShapiroandHofreiter,2014).AnalyzingancientDNAisaveryspecializedtechnologyandrequiresdedicatedfacilitiestopreventcontaminationwithmodernDNA.


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3 Thedomesticationofcattle

OneofthemaingoalsoftheTaurosProgrammeisthede-domesticationofcattle.Thereforeitisimportanttosummarizewhatweknowonthedomesticationitself.

Earlydomestication:the‘covenantofthewild’

Untilrecentlymuchofourknowledgeondomesticationwasbasedonarchaeologicalexcavations,osteologicalanalysesandradiocarbondating.Nowadaysourknowledgeofearlydomesticationofcattlehasmadebigleapsbecauseoftheuseofmoderngenetics.

Wethinkthatafteralongperiodofco-evolutionofman,aurochs(andwolf)(co-)domesticationwasthenextstepinthisprocess.Domesticationledtoselectiveadvantagesforbothpartners:animalswereprovidedwithfoodandshelterhumanswithmilk,meatandworksupply.Domesticationhasbeendescribedasa“covenantofthewild”(Budiansky,1994inMageeet.al.,2014).Cattlewereprobablyoneofthemoredifficultspeciesfordomesticationbecauseoftheirsize,butmoreeasybecauseoftheirtendencytoholdgroundinsteadoffleeing(suchashorses).Theywereamongstthefirstwildgrazerstobedomesticated.

10,000BP:thestartofitall

Around10,750-10,250yearsBPthedomesticationofTaurinecattlestartedintheFertileCrescent(Mesopotamia).MolecularresearchhasindicatedthatthenumberofaurochsfirstdomesticatedintheMiddleEastwasverysmall,possiblyonly80individuals.Alongthedomesticationrouteswild-matingswithwildaurochsbullshavetakenplaceinAfricaandEurope.Thefirstselectiononcattle-traitswasdirectedonmanageablesizeanddocility.Asaresultofdomestication,cattlebegantodecreaseinsizethroughoutthemillenniathatfollowed.Cattleweremultipurposeandbredformeat,milksupplyandworkingpower.Bullswerecastrated.

Routesofdomestication

Domesticationprovedtobeaverysuccessfulinnovation.Thefirstfarmerssoonneedednewareasandtooktheiranimalsalongwiththem.ThisiswherethemythofthefoundingofEurope-ZeusdisguisedasabullwithEuropeonhisbackswimmingfromtheLibanesecoasttoCrete-meetsreality.Around10,000BPandfrom8,500BP,startingintheFertileCrescent,awestwardexpansionofagriculturalsocietiesbroughtthefirstdomestictaurinecattletocentralAnatoliaintoEurope7.AnotherroutewasalongtheNorth-AfricanMediterraneancoastandthenalongtheWest-AfricanAtlanticcoast.ThetypicalAfricanT1mtDNAhaplotypeisalsofoundinIberiancattle,anindicationofimmigrationofAfricancattleinEurope(Spain,Italy,Malta)acrosstheseastraits.ThemigrationfurtherintoEuropefollowedprobablytworoutes:viatheMediterraneancoastsandalongtheDanuberiver.Approximately7,500BPdomesticcattlereachedCentralEuropeviatheDanubianroute.NorthernEuropefollowedca.1,000yearslater.

7MainsourceFeliusetal2014


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DuringfurtherdomesticationinEuropeintrogressionofaurochs(bulls)mayverywellhavetakenplace(andinfacthastakenplaceasisproveninParket.al.,2015).Groupsofcattlewereherdedinremoteareasandwerenotprotectedbybarbedwire.

AccordingtoDeckeretal(2014),indicine(fromBosIndicus)influenceisfoundinthreePodolianbreeds(Chianina,RomagnolaandMarchigiana),whichprobablydescendfromHungariansteppebullsimportedduringthe14thtothe18thcentury.Lenstraet.al.(2014)findsindicineinfluenceinallPodolianbreeds.

Figure3.1:DomesticationroutesintoAfricaandEurope(fromFeliuset.al.,2014)

Bovinebreeds

Themigrationofcattletoallinhabitedcontinents,localadaptationandartificialselectionhasresultedintovarietiesofcattlebreedsweobservetoday.Sincethelast100-200years,manycattlebreedshavebeenintentionallymodifiedtoimproveandproducethetraitsbeneficialforhuman,resultingintohighoutputcattlebreedsspecializedforspecificpurposesTheprofitablespecializedcattlebreedshavereplacedseverallocalandoftenextremelywell-adaptedtraditionalbreeds.Asaresult,verymanycattlebreedsareatrisk,mostofthembeingtheprimitivecattlebreeds.Themechanizationofagriculturehasunderminedthemaintenanceofauthenticdraughtcattlewithprimitivegenevariants,leadingtoafurtherdriftfromthewell-adaptednaturalgrazer,theaurochs.AmajorconcernisthecrossbreedingoflocallyadaptedbullswithhighlyproductivebreedssuchastheLimousin.

ClassificationofEuropeanbreeds

Deckeretal.,(2014)givesaclassificationoffivemajorgroupsofbreedsandwithineachgroupseveralclusters.


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1. North-Europeancattle,comprisingeightbreedclusters.2. CentralEuropeancattle,withmanydualpurpose(beef-workordairy-work)and

triple-purposebreeds,comprisingthreefivebreedclusters:3. Iberiancattle,withalargevarietyofcoatcoloursandhornmorphologyandmainly

usedasbeef,workandfightingcattle;4. GreyPodoliancattle,themostlylong-hornedand,primarilydevelopedasbeefand

workanimals;5. BalkanandAnatolianbreed,agroupofgeneticallydiversebreedsstillrepresenting

theundevelopedtaurinecattle.

ThelonglistofbreedsfortheTaurosProgramme

IntheTaurosProgrammeafirstselectionwasmadefromthehundredsofbreedsstillpreservedinEurope,thelonglist.ThisselectionwasperformedbythescientificcommitteeoftheTaurosProgrammein2009andbaseduponspecificfeatures,history,purposeandmanagement-system.Basedonthesecriteria38cattle-breedswereselectedandalsousedinthegeneticanalyses,includingthebreeds((suchastheBoskarinandMalteseCattle)thatwereaddedlatertothislist.Annex1givesthetotallist.

Basedonmoredetailedknowledgeofthebreedsasub-selectionofthislonglistwasusedintheinitialstageoftheTaurosProgramme,theshortlist.ThesebreedsandthereasonsfortheirinitialselectionfortheTaurosProgrammearelistedinAnnex3.AbreedastheItalianChianinaforexamplehasbeenleftoutsincethePodolianMaremmanaprimitivawithlargerhorns,lessmeattypical,bettercolorationandamoreferalwayoflivingisthebetteralternative.


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4 Results

Researchquestions

IntheendtheTaurosProgrammeneedsanswersonthefollowingquestions:

§ Whatwasthegeneticconstitutionofandvariationwithinaurochs?§ Whichgenesincattlehavebeenchangedinresponsetodomestication?§ Whichgenesencodetheessentialaurochs-likefeatures?§ Whatisthegeneticconstitutionofthe(primitive)cattle-breedsbeingusedandhow

dotheyrelatetoeachotherandtotheaurochs?§ HowcanweusethisgeneticinformationintheactualTaurosbreedingprogramme?§ DoestheTaurosProgrammegeneticallymoveintherightdirection:doesthe

offspringapproachthegeneticallyaurochs?§ WilltheresultingTaurospopulationshaveenoughgeneticvariation?

Toprovidetheseanswers(atleastthebeginningoftheanswers)WageningenUniversitycarriesoutthegeneticresearch,inclosecooperationwithotherscientificinstitutes.Annex4givesanoverviewofthedifferentresearch-stepstakensofar.Theamountofresearchhasbeenlimitedbytheavailablebudget.Intheperiod2010-2015chronologicallythefollowingstepshavebeentakenaspartofthegeneticresearchspecificallyorientedtowardtheTaurosProgramme.TheresearchspecificallyfundedbytheLibertyWildlifefundwasstartedin2013andconcludedin2015:

§ Analysesof100crossbredsfromtheTaurosprogramme(inheritancecheck);§ 770kSNPanalysesofadditionalbreeds(24individuals);

ThisresearchismostrelevanttotheTaurosProgramme,sincewithouttheinheritancecheckandthefurtheranalysisoftheoffspringgenerations,wewillnotbeabletocheckiftheprogrammemovesintherightdirection.

Methodsfollowed

Anumberofdifferentmethodsofanalysishavebeencarriedout:ingeneralquitespecialistgenetictoolsandmethods,hardlyunderstandablefornon-geneticists.Thereforethemethodsarenotdescribedinthesummary.

Results

Theresultsofthedifferentanalysesarerepresentedinthreedifferentways.InMauliketal(2015)8aditionalanalyseshavebeencarriedoutanditsresultspresented,howeverthatdoesnotchangethegeneralconclusions.

1. CalculationoftheNeigeneticdistance;2. PopulationstructureanalysisbyModelbasedclustering;3. Parentagecheck.

NEIGeneticdistance:Rankingthestars

8Mauliket.al.,2015.Geneticorigin,admixtureandpopulationhistoryofprimitiveEuropeancattleandAurochs


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NeiGeneticdistanceestimatesthedifferencebetweenbreeds,akindof‘rankingthestars’.NeiGeneticdistanceshowshowclosedifferentbreedsaretothe(oneBritish)aurochs.Table4.1givestheresults.

TABLE4.1:GENETICDISTANCETOAUROCHS

Ranking NEIDistance Code Breed Country 4.1.1 0 4.1.2 AU4.1.3 Aurochs 4.1.4 Britain1 0,1245 PA Pajuna Spain2 0,1249 BU Busha Balkanregion3 0,1307 CA Cardena Spain4 0,1312 SA Sayaguesa Spain5 0,1365 FL Fleckvieh Switzerland6 0,1374 BN Berrendaennegro Spain7 0,1377 LM Limia Spain8 0,1385 BC BerrendaenColorado Spain9 0,1386 LI Lidia Spain

4.1.5 10 4.1.6 0,13904.1.7 MA4.1.8 Maremmana 4.1.9 Italy11 0,1391 AR Arouquesa Portugal12 0,1412 ME Maronesa Portugal13 0,1443 BS Brownswiss Switzerland14 0,1457 HL Highlander Scotland15 0,1465 JE Jersey JerseyIsland16 0,1465 KC Kerrycattle Ireland17 0,1481 GA Galloway Britain18 0,1486 HE Heck -19 0,1515 CH Chianina Italy20 0,1520 RO Romeniangrey Romania21 0,1533 WP Whitepark Britain22 0,1536 BK Boskarin Croatia23 0,1548 MN Marondesa Spain24 0,1553 CC Cachea Portugal25 0,1563 AL Alentejana Portugal26 0,1582 TU Tudanca Spain27 0,1587 DB Dutchbelted TheNetherlands28 0,1600 MT Maltese Malta29 0,1628 EL EnglishLonghorn Britain30 0,1638 MI Mirandesa Portugal31 0,1923 AN Angler Germany32 0,1925 CL Caldela Portugal34 0,1978 PO Podolica Italy35 0,2239 NE Nelore India

Table4.1showsthatfromthe34primitivebreedsanalysedsofarthefourmostfrequentlyusedbreeds(marked)intheTaurosProgrammeareinthetop10.Thisisaremarkableresult,sincebefore2015thechoiceofbreedswasmadeonthebasisofthephenotypeandthehistoryofabreed.Especiallythenr1rankingofthePajunabreedisencouraging.Withregardtotheotherbreedsinthetopten:thereweresoundphenotypicalreasonsnotwantingtousethemintheinitialphase,presentedherebelow.Howeverthisdoesnotmeanthatthosebreedswillnotbeusedatall,because


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apparentlytheypossessgenesthatratethemhighintherankingcomparedtotheonespecimenofAurochs.

Modelbasedclustering

Modelbasedclusteringgivesinsightintheancestry.Figure4.1showstheresultofthecalculations,assumingthreedifferentclusters:zebu(blue)northEuropeancattle(green)andsouth/Europeancattle.Theaurochsspecimen(therightmostcolumnoffigure4.1)containselementsfromallthreeclusters.ThepatternsuggestszebuinfluenceintheEast-European(Podolian,Busha)butnotintheIberianclusters.Fromthedomesticationhistorythismakessense,sincetheEast-EuropeancattlehasbeenincontactwithAnatoliancattle,whichunderwentalreadyzebuinfluencesince5000BP.

Parentagecheck

SinceTauros-herdsoftenhavemorematingbulls,it’simportanttohaveagoodparentagecheck.Theparentageofthefirst100offspringoftheTaurosProgrammehasbeencheckedwitha40kSNParray.The40kanalysesinawayareanoverkillfortheparentagecheck,butthesamedatawillbeusefultocheckthesegregationofspecifictraits,onceweknowforwhichspecificgenesandtraitswehavetolook.

TheresultsofthehereditycheckarepresentedinAnnex5.From126outofthe130sampledindividualsitwaspossibletopositivelydeterminetheidentityofatleastoneoftheparents.Inmostcasesthemotherwasalreadyknownfromobservationsinthefield,butinothercasesmaterialofthemotherwasnotavailableanymore.Mostfatherswereknownasdonorsofartificialinseminations,butseveralfathersofimportantanimalshavenotbeensampledandremain(asyet)unknown.OncewewillhavesampledmoreindividualswewillbeabletopositivelyidentifytheparentageofallTaurosoffspring.

TheparentagetestrevealedthatintheherdofKeentnearlyallmatingswerebytheMaremannaxPajunabull(ManoloUno,nrNL896492389)andthatasecondbullinline–nrNL896492390,oneyearyounger–didnotperform.IntheotherlargeareaintheNetherlands(Kempen-Broek)thediversityofreproducingbullsislarger,becausetheareaissplitintoseveralsmallerareaseachwithadifferentbullandifavailablemorebullsinonegroup.


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Figure4.1:AncestryassignmentofalltheindividualswithK=39

9ProbablytheWhiteParkCattleandRomanianGreysampleshavebeenswitchedinthisfigure.ThiswillbecorrectedinthefinalpaperMauliket.al.,2015.


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5 ThemeaningofgeneticresearchfortheTaurosProgramme

ThegeneticresearchisoneofthepillarsoftheTaurosProgramme.Ourinitialideaswereprettystraightforward:sampleafairnumberofAurochsindividualsandcomparethesewiththebreedsyouwanttouseandalreadyareusing,andchoosethemostaurochs-likeanimals.Ofcoursetheroadappearedbumpierthanweexpectedwithamajorset-backourfailureofanalysingaurochsmaterial.Thisapproachinitselfwasnotunrealistichowever.

Meaningoftheresults

Forthefirsttimeagenealogicaltableofaurochsandprimitivecattlebreeds,basedon770KSNPautosomalDNA,hasbeenconstructed(table4.1).Althoughstillpreliminary,itisencouragingthattheoutcomeoftheresearchconfirmsourbreedingprogrammesofar.Itroughlyconfirmsthatfourofthebreedsthatdoplayamajorroleinourbreedingprogrammeareinthetop10andsixofourbreedsareinthetop15.Weexpecttobeabletodrawmoredefiniteconclusionswhenmorespecimensofaurochswillhavebeenanalysed.

ThereareotherprimitivebreedswedidnotconsideryetsuchasUkranianGrey,HungarianSteppecattle,ortheoffspringofelderIberiancattlebreedsinSouthAmerica(Criollo).Especiallythelattercouldbeverypromisingsince,someoftheseherdsstemdirectlyfromoldIberianbreedswithouttoomuchexternalinfluence.

Thespeedofourresearchwasn’tashighaswehopedforinitially.ButtheinitialslowprogressmeantthatwewereabletoincludenewinsightssuchasFeliuset.al.(2014)andLenstraet.al.(2015).Theyprovidedthebigpictureofcattledomesticationandthespreadofgenes.Thisdetailedpicturewasearliernotavailable.Wecannowconcludethatourresultsareinlinewiththosetwopublications.

ThestrategyofcombiningIberianandPodolianprimitivebreedsisinlinewiththeoutcomeofthegeneticresearch.Bythisstrategytworoutesofcattle-domesticationandtwodifferentresourcesof‘primitive’genesarecombined.TheIberianbreedshaveretainedmuchgeneticdiversity,whichwasincreasedbytheinfluenceofAfricancattlewiththeAfricanaurochsancestry.Incontrast,PodolianbreedshaveadifferentoriginintheBalkansandmayverywellhavepickedupgenevariantsofEuropeanaurochs.

Nextsteps

Obviously,wewillhavetocontinueoutinvestigationsandalsocontinueourinternationalcollaborations.Ourplansarethefollowing:

§ Attheshortterm,aphenotypicaldescriptionofindividualsaswellasmorepracticalguidelinesforselection(basedonthegross-listofbreedingcriteria).Nowthatthefirst100offspringhavebeenanalysed,weneedtodescribethemostessentialfeatureoftheanimalsaswell.Wewillnowworkoutapracticalprogrammetomakemeasurementsatindividualanimals.Thiswillenableustopredictthedevelopmentofanimalsinanearlystageandtospeeduptheselectionprocess;


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§ Wewillcarryoutwholegenomesequencingofapproximately10Taurosfounderanimals;

§ Mostessentialwillbegenotypesofotheraurochsindividualstoassessthegeneticvariationwithinaurochs.ThisisbeingworkedonbytheresearchgroupsofMacHughandBradleyandalsoontheUniversityofLyon;

§ Onthelongtermweneedtohavemoreinsightinidentityofthegenesforrelevanttraits,sothatwewillbeabletopredicttoacertainextentwhatthedevelopmentofayoungindividualwillbe.ThisanalysiswillbeperformedbythepHDinhisnextyears.

SincetheTaurusFoundationhasnottherequiredresources,additionalbudgetswillbenecessarytocarryoutthisresearchandtosetpriorities.


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Glossary• Alleles:AlternativeformsofaDNAsequence• Autosomalchromosomes:AllchromosomesexcepttheXandYsexchromosomes.• CNV:Copynumbervariation.AcertainDNAsegmentcancontainmorecopiesofa

gene,eachcopynotnecessarilybeingaperfectcopy.Thenumberofcopiescandependontheindividualandthusisapolymorphism:theCNV.

• Chromosome:Alongdouble-strandedandfoldedDNAmoleculethatcontainsgeneticinformationandispassedtonextgeneration

• Diploid:Anorganismwithtwocopiesofeachchromosome,onefromeachparent.• DNA:Deoxyribonucleicacid;themoleculethatcarriesgeneticinformation• Dominant:Aphenotypeoritscorrespondingallelethatinaheterozygotemasksthe

presenceofthealternative,recessivephenotypeofthesamelocusintheheterozygote;

• F1generationFirst-generationoffspringofacross;• F2generationSecond-generationoffspringofacross;• Gene:Thefundamentalunitofheredity;aspecificandfunctionalsectionofDNA

withinachromosomethatcodesforaproteinorRNAmolecule• Geneticdistance:Astatistictoolusedtomeasurethedifferencesinallele

frequenciesbetweenspeciesorbreeds.Thesmallerthedistancethemoretwobreedsorspeciesarealike.AstandardtoolingeneticsisNEIgeneticsdistance.

• Genotype:Thegeneticmakeupofanindividual;theallelecombinationatagivenlocus

• Haplotype:Thecombinationofallelesinachromosome,geneormtDNAmoleculeiscalledahaplotype

• Haplogroup:Anumberofsimilarhaplotypesiscalledahaplogroup.Haplotypesandhaplogroupswithinspeciesoftenhaveageographicdistributionindicatinghistoriccolonization-routes

• Heterozygote:Anindividualwithtwodifferentallelesatagivengeneorlocus• Homozygote:Anindividualwithtwoidenticalallelesofagivengeneorlocus• Locus:Aspecificlocationonachromosome• mtDNA:MitochondrialDNAisoutsidethenucleus.Itdoesnottakepartinthesexual

reproduction,butistransmittedfrommothertooffspring.• mRNA:MessengerRNA(mRNA)directsthesynthesisofproteins• Phenotype:Thephysicalcharacteristicsofanindividual• Recessive:Aphenotypeexhibitedonlywhenhomozygousortheallelecorresponding

tothisphenotype.• Sexchromosomes:Chromosomescontrollingthegender.InmammalsthesearetheX

andYchromosomeswithtwoXchromosomes(XX)infemalesandoneXandoneY(XY)inmales.

• Sexlinked:phenotypecontrolledbyageneononeofthesexchromosomes


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Literature• BollonginoR.,J.Elsner,J.D.VigneandJ.Burger,2008:Y-SNPsdonotindicate

hybridisationbetweenEuropeanaurochsanddomesticcattle.PLoSONE2008,3(10).

• E.Wright,S.Viner-Daniels,2015.GeographicalvariationinthesizeandshapeoftheEuropeanaurochs(Bosprimigenius).JournalofArchaeologicalScience54(2015).

• EdwardsC.J.,D.A.Magee,S.D.E.Park,P.A.McGettigan,A.J.Lohan,A.Murphy,E.K.Finlay,B.Shapiro,A.T.Chamberlain,M.B.Richards,D.G.Bradley,B.J.Loftus,D.E.MacHugh,2011.ACompleteMitochondrialGenomeSequencefromaMesolithicWildAurochs(Bosprimigenius).PLoSONE5(2):e9255.

• Edwards,C.J,D.A.Magee.,S.D.E.Park,P.A.McGettigan,A.J.Lohan,2010.ACompleteMitochondrialGenomeSequencefromaMesolithicWildAurochs(Bosprimigenius).PLoSONE5(2):e9255.

• FeliusM.,M.L.Beerling,D.S.Buchanan,B.Theunissen,P.A.Koolmees,J.A.Lenstra5,2014.OntheHistoryofCattleGeneticResources.Diversity2014,6,705-750;

• Felius.M.,CattleBreeds:AnEncyclopedia,Doetinchem,1995.• GoderieR.,W.Helmer,H.Kerkdijk-Otten,S.Widstrand,2013.Borntobewild.

ThecomebackofaEuropeanicon.RoodbontPublishers,Zutphen.• LariM.,E.Rizzi,S.Mona,G.Corti,G.Catalano,K.Chen,C.Vernesi,G.Larson,P.

Boscato,G.DeBellis,A.Cooper,D.Caramelli,andG.Bertorelle,2011.TheCompleteMitochondrialGenomeofan11,450-year-oldAurochsen(Bosprimigenius)fromCentralItaly.BMCEvolutionaryBiology2011,11:32

• LenstraJ.A.,P.Ajmone-Marsan,A.Beja-Pereira,R.Bollongino,D.G.Bradley,L.Colli,A.deGaetano,C.J.Edwards,M.Felius,L.Ferretti,C.Ginja,P.Hristov,J.Kantanen,J.PedroLirón,D.A.Magee,R.NegriniandG.A.Radoslavov,2014.Meta-AnalysisofMitochondrialDNARevealsSeveralPopulationBottlenecksduringWorldwideMigrationsofCattle.Diversity2014,6,178-187.

• Lynch,A.H.,Hamilton,J.&Hedges,R.E.M.(2008).Wherethewildthingsare:aurochsandcattleinEngland.Antiquity82(318):1025-1039

• MageeD.A.,D.E.MacHughandCeiridwenJ.Edwards,2014.Interrogationofmodernandancientgenomesrevealsthecomplexdomestichistoryofcattle.AnimalFrontiers,Volume4,Issue3Pages7-22.

• Mauliket.al.,2015(inprep).• McInerney,Jeremy,(2010).‘TheCattleoftheSun.CowsandCultureinthe

WorldoftheAncientGreeks’.PrincetonUniversityPress.• Perez-PardalL.,L.J.Royo,A.Beja-Pereira,S.Chen,R.J.C.Cantet,A.Traore,I.

Curik,J.Sölkner,R.Bozzi,I.Fernandez,I.Alvarez,J.P.Gutierrez,E.Gomez,F.A.PoncedeLeonandF.Goyache,2009.MultiplepaternaloriginsofdomesticcattlerevealedbyY-specificinterspersedmultilocusmicrosatellites.Heredity(2010)105,511–519.

• Shapiro,B.,andM.Hofreiter.2014.Apaleogenomicperspectiveonevolutionandgenefunction:NewinsightsfromancientDNA.Science343:1236573.

• StefanoMona,G.Catalano,M.Lari,G.Larson,P.Boscato,A.Casoli,L.Sineo,C.DiPatti,E.Pecchioli,D.Caramelli,G.Bertorelle,2010.Populationdynamicofthe


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extinctEuropeanaurochs:geneticevidenceofanorth-southdifferentiationpatternandnoevidenceofpost-glacialexpansion.BMCEvolutionaryBiology10:83,2010.

• Vera,F.W.M.,1997.‘MetaphorsfortheWilderness:Oak,Hazel,CattleandHorse.’Ph.D.diss.,AgriculturalUniversity,Wageningen,TheNetherlands.

• Vuure,C.van,2005.RetracingtheAurochs:History,MorphologyandEcologyofanExtinctWildOx.PensoftPublishers.Sofia-Moscow,2005.

• Wright,E.(2013).ThehistoryoftheEuropeanaurochs(Bosprimigenius)fromtheMiddlePleistocenetoitsextinction:anarchaeologicalinvestigationofitsevolution,morphologicalvariabilityandresponsetohumanexploitation.Thesis.DepartmentofArchaeology,UniversityofSheffield.


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Annex1:LONGLISTOF‘ORIGINAL’BREEDS

Breed Code Country RegionAlentejana AL Portugal Iberian

Angler AN Germany Dutch

Arouquesa AR Portugal Iberian

Aurochs AU Britain BosprimigeniusBerrendaenColorado BC Spain IberianBerrendaennegro BN Spain Iberian

Boskarin BK Croatia BalkanandItalyBrownSwiss BS Switzerland Alps

Busha BU Balkanregion BalkanandItalyCachea CC Portugal Iberian

Caldela CL Portugal Iberian

Cardena CA Spain Iberian

Chianina CH Italy BalkanandItaly

EnglishLonghorn EL Britain English

Fleckvieh FL Switzerland AlpsGalloway GA Britain English

Heck HE Germany Heck

Jersey JE JerseyIsland Jersey

Kerrycattle KC Ireland English

Lidia LI Spain Iberian

Limia LM Spain Iberian

Maremmana MA Italy BalkanandItalyMarondesa MN Spain Iberian

Mirandesa MI Portugal Iberian

MT Maltese Malta BalkanandItalyNelore NE India BosIndicus

Pajuna PA Spain Iberian

Podolica PO Ukraine BalkanandItaly

Romeniangrey RO Romania BalkanandItaly

Sayaguesa SA Spain Iberian

ScottishHighlander HL Scoland English

Tudanca TU Spain Iberian

Whitepark WP Britain English


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Annex2:SetwithcriteriafortheTaurosProgrammeCharacteristics Bull Cow

Shoulderheight 160to185cm 145to150cm

Overallbuild • Narrow/slender• Forehand:dorsalprotrusionsofthevertebrae,attachedtothebackoftheskull.Thisisneededforthemusclestocarryalargeheadwithhorns

• S-shapedback/spinecurvatureasaresultofdorsalprotrusionsoftheshouldervertebraetoconnectneckmuscles

• Squarebuildregardingtrunklengthandleglength.

• Narrow/slender• • • • • S-shapedback/spinecurvature• • • Squarebuildregardingtrunklengthandleg

lengthHindquarters • Slender

• Straightlegs• Hipjoint:sloping

• Slender• Straightlegs• Hipjoint:sloping(tofacilitatenaturalcalvingease)

Muscling • Nodoublemuscling• Musclescanbesomewhatpronounced,butfunctional

• Neckismuscled• Notypicalmeatcattleconformation

• Nodoublemuscling• Musclescanbesomewhatpronounced,butfunctional

Coloration • Calvesarered• Colorationchangestoblack/darkbrownafterafewtosixmonths

• Selectionagainsttoolightlycoloredindividuals

• Lightersaddleontheback(shadingingeneral)ispermitted

• Lighteelstripe• Whiteorlightmuzzle(cangetlessdistinctwhenanimalisolderthan2years)

• Darknose,nopinknosecolorpermitted

• Calvesarered• Redtobrownishwhenmature• Selectionagainsttoodarkortoolightlycoloredindividuals;Cowsshouldbelightercoloredthanbulls

• Lightersaddleontheback(shadingingeneral)ispermitted

• Canhavealighteelstripe,butthisisnotmandatory

• Whiteorlightmuzzle• Darknose,nopinknosecolorpermitted

Legs • Comparativelylonglegs.• Squarebuildregardingtrunklengthandleglength.

• Comparativelylonglegs.• Squarebuildregardingtrunklengthandleglength.

Head • Longandslender.• Bigincomparisonwiththerestofthebody.

• Longandslender.• Bigincomparisonwiththerestofthebody.

Horns • Length:60to107cm• Circumferenceonthebase:10to18cm• Form:variable,butonaverageasfollows:inthebeginninggrowingsomewhatbackwardsandupwardsinanangleof0to30degrees,thenincurvingforwardandinwardsinanangleof60degreesinrelationtotheskullandfinallyupwardsandoutwards.

• Length:40to70cm• Circumferenceonthebase:7to10cm• Form:variable,butonaverageasfollows:inthebeginninggrowingsomewhatbackwardsandupwardsinanangleof0to30degrees,thenincurvingforwardandinwardsinanangleof60degreesinrelationtotheskullandfinallyupwardsandoutwards.


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Characteristics Bull Cow

Coat • Singleshortcoatinsummer• Doublelongercoatinwinter• Fastsheddingofwintercoatinspring

• Singleshortcoatinsummer• Doublelongercoatinwinter• Fastsheddingofwintercoatinspring

Dewlap NotoverlypresentlikeinZebu NotoverlypresentlikeinZebu

Hump(neck) NohumplikeinZebu,exceptforfunctionaldorsalprotrusionsofthevertebraetoconnectneckmuscles.

NotpresentlikeinZebu

Udder Small,hairyandhardlyvisible


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Annex3:BriefsummaryofthebreedsinvolvedMaremmana‘Primitiva’(centralItaly)

TheMaremmanabreedcomesfromMaremma,aprairieandcoastalduneregioninwesterncentralItaly.Maremmanacattleareadaptedtowetandmarshyareas,butdowellinmountainsaswell.TheMaremmanaprimitivahasstrongaurochsfeatureslikethesize,coloursetting,thickhorns,cleardifferenceincolourbetweenmalesandfemalesandanaturalherdstructure.Long-legged,largecattle,withbullsreachingupto170cmwitherheight,swung-backlineinbulls,causedbythestronglyexpressedshoulderarea,extremelyhardy.Maremmanaprimitivaareextremelyhardycattleandcansurvivewellonpoorforage.InItaly,theMaremmanaliveinnaturalsemi-feralherds.

Pajuna(southernSpain)

ThePajunaisararecattlebreedfromAndalucíainSouthernSpain,withalong-leggedathleticbody,allowingthemtomovearoundtheterrainwithgreatease.Pajunabullscanreachupto165cmshoulderheight.Thehorns,especiallyofthebulls,arefacingforwardsandhavealightcolourwithadarktip.Bullsaredarkbrowntoblackwithalighteelstripe,whilethecowsarereddish-brownwithalongface.Theyareabletosurviveunderharshconditionsandonpoorforage.HighintheSpanishSierraNevadatheylivemostlyfromthornyshrubs.Andinthelowlandvalleystheyareknowntosurviveonsemi-desert-likevegetation,ifneeded.Thebreedadaptseasilytochangingcircumstances,whichisalsoanancienttrait.InAndalucíatheyarefrequentlykeptinlargerareas,withanaturalherdstructure.

Limia(north-westernSpain)

TheLimiacattlearefromGalícia,innorthwesternSpainandarealarge-sizedbreedaswell.Limiacattlealsohavecleardifferenceincoatcolourandshoulderheightbetweenmalesandfemales.Withtheirslenderbodiestheymovewithgreatease,andtheyareabletosurviveonpoorforage.Galíciaisrelativelycoldandwet,sotheanimalsareusedtolivinginawetcountry,withcoldandsnowywinters,likeinthemorenorthernpartsofEurope.

Sayaguesa(north-centralSpain)

TheSayaguesaorZamoranaisanoldcattlebreedfromSayagointheprovinceofZamorawestofSalamancainSpain.Traditionally,Sayaguesaswereusedforpullingploughs,cartsandwagons.It’salarge-sizedbreed,withbullsweighingonaveragebetween1000and1100kgandcowsbetween650and700kg.Thebullsareblackandsometimesstillhavealighteelstripe.Thecowsoftenarejustslightlylighterthanthebulls,sincefarmersselectonblackcolour,butreddish-brownSayaguesacowsstillcanbefound.Theyalsohavethewhitecolouredmuzzleoftheaurochs.Thehornsareaurochs-likeandpointforward,especiallyinthebulls.Sayaguesaisalong-leggedbreedwithacurvedbacklineandalongface.Thebreedisknowntobehardyandrobustand


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doesn’tneedanyshelterorsupplementaryfeedingintheSpanishdehesa-countryside.Theirbehaviouriswatchfulandshy,andthecowswillstronglydefendtheircalves.

Maronesa(northernPortugal)

MaronesaisahardybreedfromtheTrás-os-MontesregioninnorthernPortugalamongwolves.Somelineagesareveryaurochs-like.Thebullsaremuchlargerthanthecows,withadarkbrowntoblackcoatcolourandalighteelstripe,alightlycolouredmuzzleandverylargehorns.Maronesacowsarereddish-brown.Theheadsofbullsarelargeandtheyarewellmuscledwithacurvedbacklinefromthestronglypronouncedshoulderpart.Thehornsarethickandpointforward.Maronesasareusedtoharshconditionswithsnowywintersandhotsummersandabletolivewithoutanyhumanmanagement,withcalvesgrowingupsuccessfullyalsowithwolvesaround.


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Annex4:OverviewofconductedresearchTheamountofresearchhasbeenlimitedbytheavailablebudget.Intheperiod2010-2015chronologicallythefollowingstepshavebeentakenaspartofthegeneticresearchspecificallyorientedtowardtheTaurosProgramme.

1. In2010,aspartofandfundedbyWageningenUniversity,departmentAnimalBreedingandGenetics(ABG)thefollowingresearchhasbeendone:§ Thecollectionandgeneticanalysisof30originalbreeds(togetherwith

commerciallyinterestingbreedssuchasHolstein-FrisianandMRY)withthe770KSNParraytechnology

§ Thecollectionandanalysiswiththe770KSNParrayofaround25differentmuseumspecimensofaurochs;

2. In2012wetriedviaCaliforniaUniversitytoobtainacompletegenomesequencefromamedievalPolishaurochs(fundedbyTaurusFoundation);

3. In2013-2015additionalresearchcarriedoutwiththefundingoftheLibertywildlifefundhasbeendone:§ Analysesof100crossbredsfromtheTaurosprogramme(inheritancecheck);§ 770kSNPanalysesofadditionalbreeds(24individuals);Althoughthispartoftheresearchisfinanciallynotthemostsubstantial,itismostrelevanttotheTaurosProgramme,sincewithouttheinheritancecheckandthefurtheranalysisoftheoffspringgenerations,wewillnotbeabletocheckiftheProgrammemovesintherightdirection;

4. In2014-2015thecompletegenomesofanumberofimportantanimalsatthebaseoftheTaurosProgrammewillbesequenced.ThispartoftheresearchhasbeenmadepossiblebythefundingbytheFood-ValleyprogrammeoftheProvinceofGelderlandwith50%co-financingoftheTaurusfoundation;

Overtheperiod2014-2018furtherresearchandanalyseswilltakeplace,whichwasmadepossiblebyEuropeanfundingoftheEGS-ABGprogramme(PhDstudent)andwillultimatelyleadtopracticalrecommendationsforthebreedingprogramme;


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Annex5:TheresultsoftheinheritancecheckResultsoftheinheritancecheck.Insomecasesnotenoughgeneticmaterialwasavailable,inthatcasethecellinthistableisablank.Thiswillbesolvedinlateranalyses.

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Documents

Aurochs genetics, a cornerstone of European biodiversity