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RESEARCHARTICLEOpenAccessDevelopmentofaESTdatasetandcharacterizationofEST-SSRsinatraditionalChinesemedicinalplant,Epimediumsagittatum(Sieb.EtZucc.)MaximShaohuaZeng1,2†,GongXiao1,2†,JuanGuo1,2,ZhangjunFei3,4,YanqinXu1,BruceARoe5,YingWang1*AbstractBackground:Epimediumsagittatum(Sieb.EtZucc.)Maxim,atraditionalChinesemedicinalplantspecies,hasbeenusedextensivelyasgenuinemedicinalmaterials.CertainEpimediumspeciesareendangeredduetocommercialoverexploition,whilesustainableapplicationstudies,conservationgenetics,systematics,andmarker-assistedselection(MAS)ofEpimediumisless-studiedduetothelackofmolecularmarkers.Here,wereportasetofexpressedsequencetags(ESTs)andsimplesequencerepeats(SSRs)identifiedintheseESTsforE.sagittatum.Results:cDNAsofE.sagittatumaresequencedusing454GS-FLXpyrosequencingtechnology.Therawreadsarecleanedandassembledintoatotalof76,459consensussequencescomprisingof17,231contigsand59,228singlets.About38.5%(29,466)oftheconsensussequencessignificantlymatchtothenon-redundantproteindatabase(E-value1e-10),22,295ofwhicharefurtherannotatedusingGeneOntology(GO)terms.Atotalof2,810EST-SSRsisidentifiedfromtheEpimediumESTdataset.TrinucleotideSSRisthedominantrepeattype(55.2%)followedbydinucleotide(30.4%),tetranuleotide(7.3%),hexanucleotide(4.9%),andpentanucleotide(2.2%)SSR.ThedominantrepeatmotifisAAG/CTT(23.6%)followedbyAG/CT(19.3%),ACC/GGT(11.1%),AT/AT(7.5%),andAAC/GTT(5.9%).Thirty-twoSSR-ESTsarerandomlyselectedandprimerpairsaresynthesizedfortestingthetransferabilityacross52Epimediumspecies.Eighteenprimerpairs(85.7%)couldbesuccessfullytransferredtoEpimediumspeciesandsixteenofthoseshowhighgeneticdiversitywith0.35ofobservedheterozygosity(Ho)and0.65ofexpectedheterozygosity(He)andhighnumberofallelesperlocus(11.9).Conclusion:AlargeESTdatasetwithatotalof76,459consensussequencesisgenerated,aimingtoprovidesequenceinformationfordecipheringsecondarymetabolism,especiallyforflavonoidpathwayinEpimedium.Atotalof2,810EST-SSRsisidentifiedfromESTdatasetand~1580EST-SSRmarkersaretransferable.E.sagittatumEST-SSRtransferabilitytothemajorEpimediumgermplasmisupto85.7%.Therefore,thisESTdatasetandEST-SSRswillbeapowerfulresourceforfurtherstudiessuchastaxonomy,molecularbreeding,genetics,genomics,andsecondarymetabolisminEpimediumspecies.BackgroundHerbepimedii,atraditionalChinesemedicinalherb,ispreparedfromtheaerialpartsofEpimediumspeciesofBerberidaceae,whichisabasaleudicotfamilycontainingenormousmedicinalplants.E.sagittatum(Sieb.EtZucc.)Maxim,togetherwithotherfourEpimediumspe-cies,E.brevicornuMaxim,E.pubescensMaxim,E.wush-anenseT.S.Ying,andE.koreanumNakai,islistedinChinesePharmacopoeia[1].Herbepimediicontainsvar-iousbioactivecomponentsandhasbeenutilizedexten-sivelyasatonicandantirheumaticmedicinalherbforthousandsofyearsinChina.Currently,Herbepimediiisstillwidelyusedtotreatmanydiseasessuchasimpo-tence,frequency/urgencyofurination,coronaryheartdisease,chronicbronchitis,andneurasthenia[2].In*Correspondence:[e-mail]†Contributedequally1KeyLaboratoryofPantGermplasmEnhancementandSpecialityAgriculture,WuhanBotanicalGarden,ChineseAcademyofSciences,Wuhan,Hubei430074,ChinaZengetal.BMCGenomics2010,11:94©2010Zengetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.addition,Epimediumspeciesarealsousedasgroundcoverplantsandornamentalplantsbecauseoftheabun-danceofflowercoloursandflowerpatterns.Therefore,Epimediumspeciespossessenormouspotentialsforcommercialapplication.ThemolecularresearchofEpi-medium,however,islaggedbehind.Firstly,atpresent,manystudiesfocusonextractingbioactivecomponentsfromEpimedium[3-5],butmetabolicpathwaysinvolvedinproducingthesebioactivecomponentsarestilllargelyunknown.Secondly,manystudieshaveattemptedtodistinguishtaxonomicallyEpimediumspeciesanddetectthegeneticdiversityofEpimedium[6-8].Butonlyafewofgenomicmarkershavebeenexploited,sofar,inourlabforstudyinggeneticdiversityofnaturalpopulations,including14,19,and12SSRsforE.sagittatum[9],E.koreanum[10],andE.brevicornu[11],respectively.Themostimportantthingisthat,accordingtoourknowl-edge,mostgenomicSSRsexploitedfailtotransferinotherEpimediumspecies.Thus,moremarkersareneededforin-depthunderstandingofthenaturaldiver-sityandfordevelopingstrategiesforsustainableutiliza-tionofEpimedium.Thirdly,althoughmanypapersreportedtaxonomicresultsofEpimediumusingdifferentmolecularmarkerssuchasRAPD[12],RFLP[12],AFLP[13],andrDNA[6,7,14],thetaxonomicalrelationshipsamongEpimediumspecies,especiallycloselyrelatedChinesespecies,arestillnotclearbecauseoftheshort-comingofmolecularmarkerswithlittlepolymorphicinformationorthelimitednumberofspeciessampledinformerstudies.Moreover,themorphologicalcharac-teristicsofE.sagittatumvarygreatlyamongdifferentpopulations,anditisstillnotknownwhetheritbelongstopolytypicspeciesoracomplexofcrypticspecies.Finally,accordin