Microsatellitemarkersfortwostifftailducks:the
white-headedduck,Oxyura leucocephala,andtheruddy duck,O.jamaicensis
VIOLETAMUÑOZ-FUENTES,*NICLASGYLLENSTRAND,†JUAN J.NEGRO,*ANDY J.GREEN*
andCARLESVILÀ‡
*EstaciónBiológicadeDoñana(C.S.I.C.),Avda.MaríaLuisas/n,41013Sevilla,Spain,†DepartmentofConservationBiologyand Genetics,UppsalaUniversity,Norbyvägen18D,SE-75236Uppsala,Sweden,‡DepartmentofEvolutionaryBiology,Uppsala University,Norbyvägen18D,SE-75236Uppsala,Sweden
Abstract
Hybridizationwithacloserelative,theNorthAmericanruddyduck(Oxyura jamaicensis), isamajor problemfor theconservationoftheendangeredwhite-headedduck(Oxyuraleu- cocephala). Wereportthedevelopment of11microsatellitemarkersthatcanfacilitate the identificationofhybrids aswell asthestudy ofthepopulationstructureofbothspecies acrosstheirdistributions.These markerswere tested in63white-headedducks and50 ruddyducksandshowalargerdiversityinthelatterspecies.
Keywords:hybridization,microsatelliteprimers,ruddyduck, white-headedduck
Thewhite-headedduck, Oxyuraleucocephala,hasafrag- menteddistributioninthewesternPalaearcticandisclassified asendangered bytheWorld ConservationUnion (IUCN) (Green Hughes2001).In Spain, the populationwas reducedtoafewdozenindividualsinthe1970s.Population recoverysincethenhasbeenmarredbytheintroduction ofthe congenericNorthAmericanruddy duck, Oxyura jamaicensis.Hybridizationand genetic introgressionwith this species is considereda major threatto the white- headedduck(GreenHughes2001).Theruddyduckwas introducedinGreatBritaininthe1950s,andlaterspreadto other Europeancountries.ItwasfirstrecordedinSpainin
1983. We developed nuclear microsatellitemarkers to assess the genetic structureand variabilityofthe white- headedduckandthatoftheruddyduckinbothitsoriginal andintroducedranges,andalsotoidentifyhybridsbetween thetwospecies.
Wedevelopedseparatemicrosatellitelibraries foreach species. DNA forlibrary constructionwasextractedfrom muscle tissue ofone female white-headedduck and one female ruddy duck using DNeasyTissue Kit(QIAGEN). Approximately3µgofextractedDNAwasdigestedusing MboI(Fermentas)andenrichedforCAandCATCrepeats
Correspondence:VioletaMuñoz-Fuentes,Fax:+349546211 25; E-mail:
followingtheprotocolofFleischerLoew(1995)withmodi- fications (availableuponrequest).Modificationsincluded biotinylatingthe3′endoftheoligonucleotides(Koblízková etal.1998)andaddingspacers(Kandpaletal.1994).Positive colonieswereamplifiedthroughpolymerasechainreaction (PCR)using themodifiedUNIprimer (5′-CGACGTTG- TAAAACGAGGCCAGT-3′)and the OMNI primer(5′- ACAGGAAACAGCTATGACCATGAT-3′). Amplified productsweresequencedonaMegaBACEcapillarysequ- encer(Amersham) using DYEnamicETTerminator Cycle SequencingKit(Amersham).
Sequenceswere visualizedusing autoassembler2.1
(AppliedBiosystems)andPCRprimersweredesignedusing primer3 (Rozen Skaletsky2000).Toavoid labelling individualprimers,weaddedanM13ReverseorCAGtag tothe5′end oftheforwardprimer,and added alabelled M13Reverseor CAG tag in the amplificationreactions (HauswaldtGlenn2003).WeaddedatailGTT,GTTT or GTTTCTtothe5′endofthereverseprimertopromoteadeny- lation and thereforedecrease stutteringand background noise(Brownsteinetal.1996).Modifiedprimerswereevalu- atedusing netprimer(PREMIERBiosoftInternational).
Primersweredesignedfor16loci,ninecorrespondingto
white-headedduckclonesandseventoruddyduckclones. PCRwascarriedoutin10-µLreactionscontaining1×Gold Buffer (15mmTris-HCl, pH8.0,50mmKCl;Applied
Table1Characterizationof11white-headedduck (Oxyuraleucocephala,leu)and ruddyduck (Oxyurajamaicensis,jam)microsatelliteloci.Speciesindicatesthespecies from which the microsatellitewasisolated
O.leucocephala
(n=63)
O.jamaicensis
(n=50)Totalno.
Locus / Species / Repeat motif / Primersecquence(5′–3′) / Third primer / Ta
(°C) / Size
range (bp) / Na / HO / HE / Na / HO / HE / different alleles
Oxy3 / leu / (CA)15 / F:CAGTCGGGCGTCATCACTGCTGGAGGGTAAC
R:GTTTAACAAATGGCCCAGCAC / CAGtag / 55 / 181–193 / 3 / 0.27 / 0.28 / 2 / 0.12 / 0.11 / 5
Oxy4 / leu / (TG)10 / F:GGAAACAGCTATGACCATCCCGTCTTACAGGAGA
R:GTTAGGCATTTGCACCCTATCAG / M13 / 57 / 236–250 / 3 / 0.49 / 0.45 / 8 / 0.29 / 0.69 / 10
Oxy6 / leu / (CA)10 / F:CAGTCGGGCGTCATCAAGATTCTGGGATTCAAA
R:GTTAAAAATGGGCTCTTGGAAGG / CAGtag / 57 / 245–249 / 2 / 0.53 / 0.43 / 2 / 0.45 / 0.48 / 3
Oxy10 / jam / (CA)13 / F:GGAAACAGCTATGACCATCACCAAGGGGAAGAGTCA
R:GTTTGTCTGAGGCATTTGCAC / M13 / 57 / 158–172 / 3 / 0.49 / 0.46 / 11 / 0.72 / 0.79 / 11
Oxy11 / jam / (CA)11 / F:CAGTCGGGCGTCATCATGCAGTGAAGTCTGG
R:GTTTAGCTCTGCATGGAATGGTG / CAGtag / 57 / 188–200 / 3 / 0.41 / 0.45 / 5 / 0.24 / 0.25 / 7
Oxy13 / jam / (ATGG)11 / F:CAGTCGGGCGTCATCAGGAATCAATGAGATTAG
R:GTTTATGGGGTGCTGCTTCTGAG / CAGtag / 57 / 193–228 / 1 / 0.00 / 0.00 / 17 / 0.86 / 0.87 / 18
Oxy15 / leu / (AC)12 / F:CAGTCGGGCGTCATCACAGAGGTCTCCTTGGTCC
R:GTTCAAGCCAGACCAGACGATTTC / CAGtag / 55 / 227–235 / 1 / 0.00 / 0.00 / 4 / 0.20 / 0.19 / 5
Oxy17 / jam / (CA)12 / F:CAGTCGGGCGTCATCAATTTAAGGCCATCCTC
R:GTTGGACTGAAAACAGCCACTTC / CAGtag / 57 / 209–221 / 1 / 0.00 / 0.00 / 6 / 0.72 / 0.72 / 6
Oxy19 / jam / (GT)10 / F:GGAAACAGCTATGACCATACGGTGTAGTTCCCTTC
R:GTTGATCCCATGGGCTAGTGAAC / M13 / 55 / 218–222 / 1 / 0.00 / 0.00 / 3 / 0.51 / 0.52 / 3
Oxy1 / leu / (TGGA)5
TAGA / F:CAGTCGGGCGTCATCAGTGGGTTAGATGGATG
R:GTTTCCTGCCACATCCCCTCAT / CAGtag / 55 / 134–154 / 2 / 0.00 / 0.03 / 3 / 0.28 / 0.25 / 4
Oxy14 / leu / (TGGA)3
(TG)15TT / F:GGAAACAGCTATGACCATCCACTACATGGGCATC / M13 / 55 / 131–147 / 2 / 0.15 / 0.14 / 8 / 0.50 / 0.71 / 9
(TG)6 / R:GTTATGGCTCATGGGGAAAAAC
Ta,annealingtemperature;bp,basepairs;Na,numberofalleles;HO,observed heterozygosity,HE,expectedheterozygosity;n,totalnumberofindividualstyped. AthirdprimerfluorescentlylabelledandcomplementarytothebeginningoftheforwardprimerwasincludedinthePCRamplification(seetext).
GenBank Accession nos.:AY827620–AY827632.
Biosystems),2.5mmofMgCl2,1mmofdNTPs(0.25mmeach),
0.5 µmofreverseprimer,0.45 µmoffluorescentlylabelled
primer,0.05µmoftag-labelledprimer,25–100ngofwhite-
headedduck orruddy duck genomicDNA and 0.35Uof AmpliTaqGold(AppliedBiosystems).PCRswereperformed inaPTC-225Tetrad ThermalCycler(MJResearch)usingthe followingconditions:94°Cfor6min;35cyclesof94°Cfor
40s,57°Cor55°Cdependingonprimers(seeTable1)for
20s,and72°Cfor30s;andafinalextensionat 72°Cfor10min. PCRproductswerescoredforamplificationinagarosegelsand thenelectrophoresedona MegaBACEsequencer(Amersham). Fragmentsizesweredeterminedusing geneticprofiler version2.0(Amersham)bycomparisontoasizestandard. These primerswere testedforamplificationand poly- morphismin12white-headed ducksand 11ruddyducks fromwidespreadlocalitiesacrosstheirranges.Elevenloci were polymorphicforatleast one species (Table1),one wasmonomorphic,itssizebeingthesameforbothspecies (Oxy2),onelocusamplifiedonly inruddyduck and was monomorphic(Oxy20),andthreefailedtoamplifyinboth species. Redesigningthe latter primersfailed toamplify theseloci.Sixofthe11polymorphiclocihadbeenisolated fromwhite-headedduckDNAclonesandfivefromruddy duckDNAclones.The11polymorphiclociwerethenused toscreena totalof57Spanishwhite-headedducks,sixGreek white-headedducksand50North Americanruddyducks. Wecalculatedobservedand expectedheterozygosities (Table1)and performedHardy–Weinbergand linkage disequilibriumtestsusing microsatellitetoolkit(Park
2001)andgenepopontheweb(RaymondRousset1995).
Table1summarizesthecharacteristicsofthesemarkers.The meannumberof allelesperlocuswas1.6forSpanishwhite- headedducks,1.8forGreekwhite-headedducks,and6.3for ruddyducks. Whenalllociwereconsidered,theobserved heterozygosity(±SD)was0.216±0.017forSpanishwhite- headedducks,0.161±0.046forGreekwhite-headedducks and 0.445 ± 0.022forruddyducks. Allthese measures consistentlysuggestthatthegenetic diversityislarger for ruddyducksthan forwhite-headed ducks. ForlociOxy4, Oxy10andOxy13inruddyducks,weidentifiedsomealle- lesdiffering byjust1bpfromeachother, which indicates additionalvariabilitybesidesthenumberoftandemrepeats inthemicrosatellite.After applyingBonferroni’ssequen- tialcorrection,Oxy1inthecaseofwhite-headedducksand Oxy4and Oxy14inthecaseofruddyducksdid notcon- formtoHardy–Weinbergexpectations.However, because thesamplesmayincludeseveral populations, wecannot evaluateifthesedeviationsimply presenceofnullalleles, matingbiases orjustpopulationfragmentation.Evidence forlinkage disequilibriumbetween Oxy4and Oxy10was foundin both white-headedducksand ruddyducks. However,thislinkage could alsoderive fromthepooling of individualsfrom differentpopulations and needs furtherinvestigation.No scoring errors associatedwith
large alleledropout orstutteringwere detectedusing the programmicro-checker(VanOosterhoutetal.2004).
These microsatellitemarkerscan be used for genetic populationstudiesand paternityanalysesparticularlyin ruddyducks. Because most ofthe alleles are uniquefor eachspecies (Table1),theuseofthese microsatelliteshas thepotentialtounambiguouslydistinguishhybrids from pure individualsandtoassesstowhatdegreenaturalpopu- lations havebeenaffected byhybridization.
Acknowledgements
WethankBarbara Gautschiand Kristina Sefcforhelpduringthe microsatelliteisolationprocess.ThanksalsogotoGunillaAnders- son,KarinBerggrenand Frank Hailer. Thelabwork wascarried outattheDepartment ofEvolutionaryBiology,Uppsala Univer- sity,Sweden.Thestudywas fundedbyLaJunta deAndalucía, Spain, and afellowshipbythe SpanishMinistryofScienceand EducationtoV.M.F.
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