1、 AnEAV-HPInsertionin59FlankingRegionofSLCO1B3CausesBlueEggshellintheChickenZhepengWang.,LujiangQu.,JunfengYao.,XiaolinYang,GuangqiLi,YuanyuanZhang,JunyingLi,XiaotongWang,JirongBai,GuiyunXu,XuemeiDeng*,NingYang*,ChangxinWuNationalEngineeringLaboratoryforAnimalBreedingandKeyLaboratoryofAnimalGenetics,
2、Breeding,andReproductionoftheMinistryofAgriculture,ChinaAgriculturalUniversity,Beijing,ChinaAbstractThegeneticdeterminationofeggshellcolorationhasnotbeendeterminedinbirds.HerewereportthattheblueeggshelliscausedbyanEAV-HPinsertionthatpromotestheexpressionofSLCO1B3geneintheuterus(shellgland)oftheovidu
3、ctinchicken.Inthisstudy,thegeneticmaplocationoftheblueeggshellgenewasrefinedbylinkageanalysisinanF2chickenpopulation,andfourcandidategeneswithintherefinedintervalweresubsequentlytestedfortheirexpressionlevelsintheshell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was
4、 found to be the only oneexpressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencinganalysisshowedthatonlythealleleofSLCO1B3fromblue-shelledchickenswasexpressedintheuterusofheterozygoushens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion
5、 transporting polypeptide (OATP) family; and the OATPs,functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds,includingbilesaltinmammals.WesubsequentlyresequencedthewholegenomicregionofSLCO1B3anddiscoveredanEAV-HPinsertioninthe59flankingregio
6、nofSLCO1B3.TheEAV-HPinsertionwasfoundcloselyassociatedwithblueeggshellphenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell isassociated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the E
7、AV-HPinsertionisthecausativemutationfortheblueeggshellphenotype.TheinsertionwasalsofoundinanotherChineseblue-shelledbreedandanAmericanblue-shelledbreed.Inaddition,wefoundthattheinsertionsiteintheblue-shelledchickensfromAraucanaisdifferentfromthatinChinesebreeds,whichimpliedindependentintegrationeven
8、tsintheblue-shelledchickensfromthetwocontinents,providingaparallelevolutionaryexampleatthemolecularlevel.Citation: Wang Z, Qu L, Yao J, Yang X, Li G, et al. (2013) An EAV-HP Insertion in 59 Flanking Region of SLCO1B3 Causes Blue Eggshell in the Chicken. PLoSGenet9(1):e1003183.doi:10.1371/journal.pge
9、n.1003183Editor:GregoryS.Barsh,StanfordUniversitySchoolofMedicine,UnitedStatesofAmericaReceivedJune4,2012;AcceptedOctober28,2012;PublishedJanuary24,2013Copyright: 2013 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunr
10、estricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.Funding:ThisworkwassupportedinpartbygrantsfromNationalNatureScienceFoundation(31072024),ProgramsforChangjiangScholarsandInnovativeResearchinUniversity(IRT1191),NationalScientificSupportingProjectsofChi
11、na(2011BAD28B03and2008AA101002),ChinaAgricultureResearchSystems(CARS-41),andProgramforNewCenturyExcellentTalentsoftheMinistryofEducationofChina(NCET-09-0730).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.CompetingInterests:Theauthorshavedec
12、laredthatnocompetinginterestsexist.*E-mail:(NY);(XD).Theseauthorscontributedequallytothiswork.Introduction chickens are representative breeds laying blue eggs and showdominant inheritance as that in Araucana. However, the blueeggshellphenotypehasnotbeenfixedinthesethreebreedswhichstillproducebrowneg
13、gsatlowfrequency.Avian eggshell coloration is the result of crypsis or mimetismand plays important roles in filtering solar radiation andstrengthening the eggshell 1. Blue eggshell color has beenproposed as post-mating signals of female phenotypic quality totheir mates and is related to fitness of t
14、he offspring due to theantioxidant of biliverdin, a predominant pigment for blue eggs2,3.Blueeggshellscanbefoundnotonlyinsomewildbirds,e.g.eastern bluebird 4, blue-footed booby 5, and pied flycatcher6,butalsoindomesticbirdssuchasJapanesequail7,chickens8andducks9.Blueeggshellcolorexhibitsanautosomald
15、ominantinheritanceandeggslaidbyhomozygotesareadarkerbluethanthosefromheterozygotes (Figure 1A). In 1933, Punnett firstly reported thatblueorgreenshellappearanceoftheAraucanawasdeterminedbyasinglegeneticfactor,traditionallydenotedasoocyan(O)8.AseriesoflinkageanalysisinvolvingOhavebeenperformedwithOaf
16、firmativelymappedtotheshortarmofchromosome11216,and closely linked to ev1 and P which was identified as SRY (sexdetermining region Y)-box 5 (SOX5) 12,13,16,17. In the regionBrownandwhitearethetwomajoreggshellcolorsinchickens.Protoporphyrin-IX,biliverdin,andbiliverdinzincchelatearethemain pigments of
17、 the eggshell 10 and several blue egg layingbreedshavebeenreportedworldwide11,12.TheAraucana,anindigenousbreedfromChile,wasthefirstchickenbreeddescribedtolayblueeggs8,andhasbeenfrequentlyusedingeneticstudiesof the blue eggshell phenotype. In China, Dongxiang and Lushiaround ev1, two single nucleotid
18、e polymorphisms (SNPs)(rs15297163andrs15297165)werefoundtobehighlyassociatedwiththeblueeggshellphenotype18.A1.8Mbgenomicintervalharboring the O gene was defined in an F2 resource population19.ThelocalizationoftheOwasfurtherrefinedtothevicinityofss244244378 by linkage and association analysis 20. The
19、PLOSGenetics | www.plosgenetics.org 1 January2013 | Volume 9 | Issue 1 | e1003183GeneticBasisofChickenBlueEggshellwhite-shelledbreeds(O*N/O*N,6chickensperbreed)byreal-timePCR.Allblue-shelledchickensexpressedthegeneintheuteruswhilenon-blue-shelled chickens did not (Figure 1C). In addition,expression
20、of SCLO1B3 was 2 to 3 fold higher in homozygousblue-shelledchickensthaninheterozygousblue-shelledDongxiangandLushiindividuals(Figure1C).FluoscencelabeledcDNAinsituhybridization demonstrated that the transcripts of SLCO1B3 wereonlyexpressedintheuterusofblue-shelledbutnotbrown-shelledhens(Figure1D).Th
21、eseresultssuggestthatSLCO1B3isthecausativegeneforblueeggshellinthechicken.AuthorSummaryThe eggshell color of birds is of wide interest, but themolecular basis remained unknown until our discovery,reportedhere.Theblueeggshellisfoundnotonlyinwildbirdsbutalsoindomesticfowls.Inthisstudy,weidentifiedthat
22、blueeggshellinchickens fromdifferentgeographicalregions is caused by a ,4.2kb EAV-HP insertionin the 59flankingregionofSLCO1B3.TheEAV-HPinsertioninchickenis a derived mutation in domestic chickens. The geneticdeterminationofblueeggshellinotherbirdsrequiresfurtherinvestigation.WealsofoundthattheEAV-H
23、PinsertionsinthechickensfromChinaandAmericawereseparateintegrationevents,whichpresentsuswithaparallelmolecularevolutionexampledrivenbyartificialselection.Allele-specificexpressionofSLCO1B3We found a SNP (g.67334934 G.T) in exon 5 of SLCO1B3gene by sequencing the coding region and the SNP presentedco
24、mplete association with the blue eggshell phenotype in theDongxiang chicken by genotyping it in Dongxiang blue-shelledand brown-shelled chickens. With six heterozygous individualsproducedbymatingahomozygousDongxiangblue-shelledmalewith a White Leghorn female, theallelic expression of SLCO1B3genewasd
25、emonstratedbyRT-PCRanalysisandpyrosequencing.More than 95% of the transcripts expressed in the uterusoriginatedfromtheTallelecorrespondingtotheblue-shellallele(Figure1E).Thismeanstheexpressionofthegeneisregulatedbya cis-acting element. Surprisingly, its expression in liver is alsoallelespecific,and,
26、95%ofthetranscriptsinlivercomefromtheGallelewhichisnon-blue-shellallele(Figure1F).ss244244378isveryclosetothetwoSNPsreportedbyZhaoetal.18 with a physical distance of 0.12Mb implies that the regionaround the threeSNPs is mostlylike toharbor the blue eggshellgene.Combinedmappinginformationfromtraditio
27、nalbreedsandChileanvillagechickensallowedtheOtobefinemappedtotwosmall regions (Gga 1:67.2567.28Mb, Gga 1:67.2867.32Mb)21.Inthepresentstudywefoundthattheblueeggshellphenotypein chickens is caused by a retrovirus insertion in the 59 flankingregion of SLCO1B3 coding a membrane transporter OATP1B3whichc
28、ompoundsincludingbilesalt.is responsible for transporting amphipathic organicAnEAV-HPinsertioniscompletelyassociatedwithblueeggshellphenotypeResults We sequenced the genomic region of SLCO1B3 in order toreveal the potential causative mutation of the gene with 5 blue-shelled and 5 brown-shelled Dongx
29、iang chickens. Twenty-oneSNPs evenly covering the whole genomic region (,24kb) ofSLCO1B3weretakenforgenotypingin353chickensfrom3blue-shelledbreeds(Araucana,DongxiangandLushi)and9non-blue-shelled breeds. However, none of the SNPs was found to be incompletelinkagedisequilibriumwithblueeggshell(Table1)
30、.We subsequently cloned the 59UTR (GenBank accessionnumber: JN381032) of SLCO1B3 by 59 RACE in a blue-shelled(O*LC/O*LC)andabrown-shelled(O*N/O*N)Dongxiangchickenandanextra24bpswerefoundatthebeginningof59UTRendinblue-shelledDongxiangchicken(FigureS1).Wefurthersequenced5kbupstreamofthepromoterusing5b
31、lue-shelled(O*LC/O*LC)and5brown-shelled(O*N/O*N)Dongxiangchickens.A,4.2kbinsertionadjacentto59UTRcontainingtheextra24bpswasfoundin the blue-shelled but not in the brown-shelled chickens. Thesequence of the ,4.2kb insertion (GenBank accession number:JF837512) represents an incomplete retrovirus and s
32、hows 95.8%identitywiththesequenceoftheavianEAV-HPretrovirus(EMBLaccession number: AJ238124) 22. A typical proviral structureconsistsofgag,polandenvflankedbylongterminalrepeat(LTR),whicharearrangedintheorderof59LTR-gag-pol-env-LTR3922.Here,theinsertedretrovirusisabsentofthewholepolgeneandpartofgagand
33、env(Figure3A).Theretroviruswasintegratedintotheblue-shelledchickengenomeinaninvertedorientation(Figure3B)at Chr1: 6732464167324642. We also found that the EAVencompassed some promoter elements by sequence analysis,indicatingitsexpressionpromotionactivities(Figure3A).LinkageanalysisofChickenblueeggsh
34、ellgeneAlinkageanalysiswasperformedinanF2resourcepopulationsegregating for the O gene to refine the location of chicken blueshell gene in the present study. Eight molecular markers in thecandidateregionwereusedforlinkageanalysis(TableS1).Bytwo-point analysis, the O gene was mapped in the region betw
35、eenmarker L4 and L5 whichwere theclosest flanking markers to Owithrecombinationratebeingboth0.02(LOD=15.84)(Figure2).FifteenSNPmarkersbetweenL4andL5werefurthergenotypedintheF resourcepopulationtonarrowthemappingregionand2theOwasfinallylocatedina,120kbregionfrom67296991bpto67416784bponchromosome1onth
36、eUCSCchickengenome(May2006assembly)(TableS1)andnorecombinationwasfoundbetweentheblueeggshellphenotypeandthemarkerswithintheregion.SpecificexpressionofSLCO1B3inuterusofblue-shelledchickenTotally, four genes (SLCO1C1, SLCO1B3, LOC418189 andSLCO1A2) were found in the ,120kb interval by a MapViewsearch
37、(http:/www.ncbi.nlm.nih.gov/mapview/) (Table S2). Theuterus is where pigment is secreted to eggshell. We performedexpressionanalysisforthefourcandidategenesintheuterusofblue-shelled(n=16)andbrown-shelled(n=16)DongxianghensbyRT-PCR. We found that SLCO1B3 was the only gene expressedspecifically in the
38、 uterus of blue-shelled Dongxiang chickens(Figure 1B), thus we measured its expression in the uterus in 8blue-shelled(4O*LC/O*LCand4O*LC/O*N.O*LCisfortheblue-shellalleleinthetwoChinesebreeds,DongxiangandLushiChickenandO*Ndenotesthenon-blue-shellalleleorwild-typeallele)and4brown-shelled (O*N/O*N) Don
39、gxiang chickens, 6 blue-shelled (3O*LC/O*LC and 3 O*LC/O*N) and 6 brown-shelled (O*N/O*N)Lushichickens,and24chickensfromthreebrown-shelledandoneAwide-rangesurveyoftheEAV-HPinsertionwasperformedin705 chickens from 12 worldwide breeds and the F2 resourcepopulation (Table 2) using diagnostic PCR test.
40、The results thatthe EAV-HP insertion is completely associated with the blueeggshell phenotype provide strong evidence that the mutation iscausative.PLOSGenetics | www.plosgenetics.org 2 January2013 | Volume 9 | Issue 1 | e1003183GeneticBasisofChickenBlueEggshellFigure1.Theeggshellcolorandtheexpressi
41、onofSLCO1B3intheuterusofblue-shelledandnon-blue-shelledchickens.(A)Eggshellcolorsofhomozygousblue-shelled(O*LC/O*LC),heterozygousblue-shelled(O*LC/O*N)andbrown-shelled(O*N/O*N)ofDongxiangchickens.(B)TheexpressionanalysisofthefourgeneswhicharelocatedintherefinedregionbetweenmarkersL4andL5intheuteruso
42、fDongxiangblue-shelledhens(n=16)andDongxiangnon-blue-shelledhens(n=16)byRT-PCRs.DX-BS:blue-shelledDongxiang,DX-NBS:non-blue-shelledDongxiang.(C)Analysis of SLCO1B3 expression in the uterus. Expression data were presented as fold relative to heterozygous blue-shelled Dongxiang chickens(O*LC/O*N)bythe
43、comparativeCtmethod(22DDCt).SLCO1B3isexclusivelyexpressedinblue-shelledchickens,andtheamountofSLCO1B3transcriptsinhomozygousblue-shelledchicken(O*LC/O*LC)isapproximatelytwotothreefoldsofthatinheterozygousindividuals(O*LC/O*N).DX-BS:blue-shelledDongxiang,LS-BS:blue-shelledLushi,DX-NBS:non-blue-shelle
44、dDongxiang,LS-NBS:non-blue-shelledLushi.(D)MicrographsofcDNAinsituhybridizationforSLCO1B3mRNAintheuterusfromblue-shelledandnon-blue-shelledchickens.(E)DifferentialexpressionofSLCO1B3transcriptintheuterusfromblue-shelledheterozygotesusinggenomicDNA(gDNA)ascontrol.Thepolymorphicpositiong.67334934G.Twa
45、susedtomonitordifferentialexpressionusingpyrosequencing.TandGatthispositioncorrespondtoblue-shellandnon-blue-shellalleles,respectively.DuetotwoTsnexttotheSNPat39end,thepeaksofTintheschemacontainthreeTsincludingoneTfromblue-shellalleleandtwoTsfromnon-blue-shellallele.Thepercentexpressiononthepeaksfor
46、TandGaretheTorGatg.67334934G.T.(F)Summaryofthedetectionofdifferentialexpressioninuterusandliverfromsixheterozygotousblue-shelled(O*LC/O*N )birds.doi:10.1371/journal.pgen.1003183.g001IndependentEAV-HPinsertioneventsinblue-shelledchickensfromChinaandChilewe further sequenced the EAV-HP insertion regio
47、ns in ahomozygous Araucana and a homozygous blue-shelled Lushichicken.TheEAV-HPinsertionwasfoundinbothsamplesandthealignments of Araucana and Lushi to Dongxiang blue-shelledIn order to elucidate whether the blue-shelled chickens fromChinaandChilehavethesameoriginforthegenotypicmutation,PLOSGenetics
48、| www.plosgenetics.org 3 January2013 | Volume 9 | Issue 1 | e1003183GeneticBasisofChickenBlueEggshellFigure2.RefinedlocalizationofOonchickenchromosome1.Theblackbarrepresentstheshortarmofchickenchromosome1.NumbersinmiddlecolumnarerecombinationfractionsbetweenmarkersandO,thecorrespondingLODscoresaresh
49、owninthelastcolumn.Here,OisassignedtotheintervalofL4andL5atChr1:6729699167419904.Fourcandidategenesintheinterval,EAV-HPinsertionwithrecombinationfractionandLODintheparentheses,SRY(sexdeterminingregionY)-box5(SOX5)forpeacombphenotype17,ev1marker16areindicatedattheleftoftheblackbar.doi:10.1371/journal.pgen.1003183.g002chickenshowedtheidentityoftheinsertedEAV-HPbeingaround97%. Interestingly, the insertion sites in Araucana are differentfrom that in the two Chinese blue-shelled chickens. The breakpointforEAV-HPin
