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首页 SNP单倍型

SNP单倍型

SNP单倍型

chang
2012-12-28 0人阅读 举报 0 0 暂无简介

简介:本文档为《SNP单倍型pdf》,可适用于农林牧渔领域

METHODOLOGYARTICLEOpenAccessRapidgenebasedSNPandhaplotypemarkerdevelopmentinnonmodeleukaryotesusing’UTRsequencingTysonKoepke,ScottSchaeffer,VandhanaKrishnan,DerickJiwan,ArtemusHarper,MatthewWhiting,NnadozieOraguzieandAmitDhingra*AbstractBackground:Sweetcherry(PrunusaviumL),anonmodelcropwithnarrowgeneticdiversity,isanimportantmemberofsubfamilyAmygdoloideaewithinRosaceaeComparedtootherimportantmemberslikepeachandapple,sweetcherrylacksingeneticandgenomicinformation,impedingunderstandingofimportantbiologicalprocessesanddevelopmentofefficientbreedingapproachesAvailabilityofsinglenucleotidepolymorphism(SNP)basedmolecularmarkerscangreatlybenefitbreedingeffortsinsuchnonmodelspeciesRNAseqapproachesemployingsecondgenerationsequencingplatformsofferauniqueavenuetorapidlyidentifygenebasedSNPsAdditionally,haplotypemarkerscanberapidlygeneratedfromtranscriptbasedSNPssincetheyhavebeenfoundtobeextremelyutileinidentificationofgeneticvariantsrelatedtohealth,diseaseandresponsetoenvironmentashighlightedbythehumanHapMapprojectResults:RNAseqwasperformedontwosweetcherrycultivars,BingandRainierusinga’untranslatedregion(UTR)sequencingmethodyielding,assembledcontigsInordertotestourapproachofrapididentificationofSNPswithoutanyreferencegenomeinformation,over(,)ofthecontigswerescreenedfortheSNPsAtotalofcontigsfromthissetwereidentifiedtocontainhighqualitySNPsAsetofprimerpairsweredesignedtoamplifySNPcontainingregionsfromthesecontigsandhighresolutionmelting(HRM)analysiswasperformedwitheightimportantparentalsweetcherrycultivarsSixoftheparentcultivarsweredistantlyrelatedtoBingandRainier,thecultivarsusedforinitialSNPdiscoveryFurther,HRManalysiswasalsoperformedonseedlingsderivedfromacrossbetweentwooftheparentsOuranalysisresultedintheidentificationof()primersetsthatdemonstratedvariationamongthetestedgermplasmReassemblyoftheraw’UTRsequencesusingupgradedtranscriptomeassemblysoftwareyielded,contigscontainingputativeSNPsincontigsafterstringentfilteringContigswithmultipleSNPswerevisuallyparsedtoidentifyputativehaplotypesatlociincontigsConclusions:Thisapproach,whichleveragestheadvantagesofRNAseqapproaches,enabledrapidgenerationofgenelinkedSNPandhaplotypemarkersThegeneralapproachpresentedinthisstudycanbeeasilyappliedtoothernonmodeleukaryotesirrespectiveoftheploidyleveltoidentifygenelinkedpolymorphismsthatareexpectedtofacilitateefficientGeneAssistedBreeding(GAB),genotypingandpopulationgeneticsstudiesTheidentifiedSNPhaplotypesrevealsomeoftheallelicdifferencesinthetwosweetcherrycultivarsanalyzedTheidentificationoftheseSNPandhaplotypemarkersisexpectedtosignificantlyimprovethegenomicresourcesforsweetcherryandfacilitateefficientGABinthisnonmodelcrop*Correspondence:adhingrawsueduDepartmentofHorticulture,WashingtonStateUniversity,Pullman,WA,USAFulllistofauthorinformationisavailableattheendofthearticleKoepkeetalBMCGenomics,:http:wwwbiomedcentralcom©KoepkeetallicenseeBioMedCentralLtdThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http:creativecommonsorglicensesby),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycitedBackgroundSweetcherry(PrunusaviumL),anonmodelcrop,isanimportantnonclimactericmemberofsubfamilyAmygdoloideaewhereothermemberslikepeachandplumdemonstrateclimactericfruitripeningSweetcherryisadiploid(n=)andisestimatedtobeslightlylargerthanpeach,MB,SweetcherryunderwentarecentbreedingrelatedgeneticbottleneckthatreducedthediversitypresentinthegermplasmGeneticvariabilitycanbeutilizedtoscreenforresistancetodiseasesandimprovetheefficiencyofselectingdesirablegenotypesthroughbreedingespeciallyinsweetcherrywherenaturaldiversityislackingTypesofvariationatthenucleotidelevelare:microsatellitesorsimplesequencerepeats(SSRs),singlenucleotidepolymorphisms(SNPs),insertionsanddeletions(indels)andgenomicrearrangementsIdentificationofgeneticdiversityinspecieswhichlacksignificantgenomicresourceshastypicallybeenatimeconsumingandlaboriousprocessSSRmarkershavebeenusedextensivelyforpopulationgeneticsandgenomemappingstudiesinseveralmembersofRosaceae,SSRidentificationtechniquesaretypicallycostlyandtimeconsumingMostpublishedSSRsarelocatedintheintergenicregionsArecentstudyinPopulusattemptedtoidentifySSRsinexonsorexpressedgenefragmentsTheabundanceofmicrosatelliteswithinthecodingregionwasthreefoldlowerthanintergenicregionsand,whenpresent,microsatellitesdonotshowusefulallelicvariabilityFurther,theauthorsconcludedthatcandidategeneapproachfordevelopmentofmicrosatellitesmaynotbethebeststrategyWhileSSRsremaindifficulttodevelop,SNPidentificationandvalidationhasrapidlyimprovedinpastyearsmostlyduetoreductionofsequencingcostsPreviously,directsequencingofageneofinterestrelatedtosupernodulationwasusedtoidentifySNPsSimilarstudiesinnonmodelspecieslackingsuchresourcesrequiresequenceinformationfromrelatedspeciesSNPshavealsobeenusedforanchoringalinkagemapandbovinegenomeGanaletalreviewedrecentSNPidentificationmethodsincludingDNAarrays,ampliconsequencing,miningexistingESTresources,andusingsequencedatageneratedwithsecondgenerationsequencingtechnologiesComparedtoothermethods,resequencingapplicationsweredeterminedtoproduceahigherpercentageofvalidatedSNPs,whilenonreferencebasednextgenerationsequencing,ordenovo,approachesrequiredtheleastamountofapriorigeneticgenomicinformationAmajorcaveatofusingsecondgenerationsequencingdenovoistheabilitytoacquiresufficientdepthtoaccuratelyidentifySNPsTherefore,areducedrepresentationsequencingapproachwasrecommendedManyreducedrepresentationmethodsintegratinghighthroughputsequencingarediscussedbyDaveyetalandtheauthorsfurtherelaboratedontheutilityofSNPbasedmolecularmarkersContinuedimprovementsinsecondgenerationDNAsequencingtechnologieshaveincreasedtheabilitytoobtainsignificantsequencingdepthinarapidandcostefficientmanner,comparedtoSangersequencingapproachesBundocketal,performedampliconsequencingongenesofinterestwithtechnologytoproducealargenumberofreliableSNPsfromtwoparentsofaQTLmappingpopulationofsugarcanefindinghighsuccessratesforSNPverification()Recently,nextgenerationtechnologieshavebeenwidelyutilizedforsequencingtranscriptomesofvariousspeciesEvelandetalreportedaquantitativetranscriptomicsapproachbasedonselectivesequencingofthe’UTRofmRNAfromZeamaysTheirworkdemonstratedaclearabilitytoresolvetheexpressionofnearlyidenticalgenes(nucleotideidentity)basedonvariationinthe’UTR(nucleotideidentity)Throughcomparisonwithsequencesinmultiplemaizedatabases,oftheSNPsidentifiedbyEvelandetalwereconfirmedUseofa’UTRdirectedapproachexploitsthehighernumberofvariationsfoundinthe’UTRregioncomparedtothecodingregionofageneHighersequencevariation,combinedwithphysicallinkagetoaspecificgene,increasesthepotentialimpactof’UTRpolymorphismsinconnectinggeneticsandfunctionalgenomicsstudiesespeciallyinnonmodeleukaryotesThisisincontrasttocurrentapproacheswhereintergenicpolymorphismsareusedforscoringasegregatingphenotypewithouttheassociatedgenerelatedinformationThemethodpresentedhereutilizedthepositiveaspectsof’UTRsequencing,asareducedrepresentationapproach,tofacilitaterapidgenelinkedSNPidentificationInadditiontoidentifyingpolymorphisms,currentresearchinhumangenomicshasdemonstratedtheutilityofdevelopinghaplotypeinformationasawaytomorefullyunderstandgenotypetophenotyperelationships,especiallyincontextofhealth,diseaseandresponsetoenvironmentalcuesGenerally,haplotypesarecomprisedofallelicvariantsoneachofthetwochromosomesatthesamelocus,thoughthedefinitionandutilizationvariesinapplicationfromlinkingmultiplepolymorphismsacrossseverallocidowntomultiplepolymorphismsinasinglegeneAdditionally,haplotypedeterminationhasbeenaidedbyDNAstrandspecificorgenomicphasebasedinformationgeneratedusingsecondgenerationsequencingtechnologiessinceeachsequencingreadisfromonlyoneKoepkeetalBMCGenomics,:http:wwwbiomedcentralcomPageofhomologouschromosomeandnotaconsensusofthetwoSimilarly,nextgenerationRNAseqand’UTRsequencinghastheabilitytorevealhaplotypeswithinageneandthusenableidentificationofallelespecificsequenceanditsexpressionsimultaneouslyHerewepresentourapproachthatutilizes’UTRsequencingtorapidlydevelopSNPandhaplotypemarkersinsweetcherry,aspecieswithoutapublishedgenomesequenceandanonmodelcropThroughdenovoassemblyofgenerated’UTRsequencingreadsandstrictfiltering,weinitiallyidentifiedaputativesetofcontigscontainingSNPsPrimersetsdesignedtoamplifytheregionsofthesecontigswithputativeSNPsweredevelopedandusedforHighResolutionMelting(HRM)analysisamongeightcurrentlyutilizedparentalcultivarsofsweetcherryandhybridseedlingsderivedfromacrossbetweentwooftheparentalcultivars,respectivelyWedeterminedthatoutof()andoutof()ofthetestedprimerpairsareabletodetectgeneticvariabilityFromthesepolymorphicsites,haplotypeswereidentifiedfromcontigscontainingmultipleSNPsMethodsRNAExtractionandcDNApreparationTissuesamplesfromdevelopingfloralbudsoftwocommerciallyimportantcherrycultivars,BingandRainier,wereexcisedfromthetreesandflashfrozeninliquidnitrogenThefrozentissueswerepulverizeduniformlyinaSPEXSamplePrepFreezerMill(SPEXSamplePrep,Metuchen,NJ)forfivecycleseachwithcoolingfortwominutesandgrindingatcountspersecondforfourminutesTotalRNAfromeachsamplewasextractedusingtheRNeasyPlantDNAExtractionKit(Qiagen,Germany)FirststrandcDNAwasthensynthesizedusingtheAmbionaRNAsynthesiskitwithabiotinylatedpolyTBadaptorseeAdditionalFileforadaptorsequencesfor’UTRprofilingasdescribedbyEvelandetal()SecondstrandcDNAwascreated,cleavedwithMspI,andligatedtomodifiedAadaptorscontainingindexingtagsseeAdditionalFileforadaptorsequencesaspertheEvelandprotocolSequencingandassemblyThe’UTRlibrariesweresequencedaspertheFLXprotocol(Roche,USA)onasingleLRsequencingplateAftersequencing,theproducedreadswereprocessedusingacustomscriptseeAdditionalFiletoremovethemultiplexingbarcodeandrenameeachreadwithitsappropriatesamplenameattheendoftheheaderAllofthemodifiedreadswerethenassembledusingSeqManfromtheLasergenesuite(DNASTAR,Madison,WI)SNPIdentificationFormethoddevelopment,atotalof,contigswereexaminedforthepresenceofputativeSNPsusingLasergene’sSeqMan(DNASTAR,Madison,WI)ThehighconfidenceSNPshaveatleasttwoallelesrepresentedbyaminimumdepthofthreereadspernucleotidecallperallelePrimerpairsflankingpotentialSNPlociweredesignedusingthePRIMERprogramtoamplifybasepairampliconsThisyieldedprimersfromregionsofcontigsforHRManalysesPopulationVariationScreenEightsweetcherrycultivars:Bing,Chelan,EmperorFrancis,NewYork,Regina,Selah,StellaandCowicheusedasparentalmaterialintheWashingtonStateUniversity(WSU)SweetCherryBreedingProgram(Prosser,WA)wereusedtotestthepolymorphismsoftheidentifiedSNPlociacrossBingandRainiercultivarsForsegregantanalysis,seedlingsfromanFmappingpopulationofSelah×CowichewereusedLeavesoftheseaccessionswerecollectedfromtheWSUIrrigatedAgricultureResearchExtensionCenterinProsser,WAandDNAwasextractedfromdriedleavesusingaCTABextractionprotocolThereactionmixtureforHRManalysisconsistedofμLofeachprimer(μM),μLSYBR®Green,ngofgenomicDNAandautoclavednanopurewatertoatotalvolumeofμLTheCultivarpanelcomprisedofprimersetstestedonalleightparentalcultivarsandtheSeedlingpanelincludedprimerssetstestedononereactionofeachparent,CowicheandSelah,andoneofeachhybridseedlingAnalyseswereperformedontheLightCycler®System(RocheBranford,CT)usingthefollowingPCRcyclingandHRMconditionsInitialmeltingforminutesat°Cwasfollowedbycyclesof°Cforseconds,°Cforseconds,and°Cforseconds,thenheatedto°Cforminuteandcooledto°CHighResolutionMeltinganalysiswasthenautomaticallyinitiatedwherebytheampliconswereheatedfrom°Cto°CwithacquisitionsperdegreeAsthetemperatureslowlyincreased,thedyefluorescencewasrecorded,plottedandlateranalyzedusingtheLightCycler®GeneScanningSoftwareSincetheTmcanvarybasedontheHRMreactionconditions,curveshapeswerevisuallyexaminedandthenumberofdistinctcurveprofileswasidentifiedforeachprimersetSecondaryAssemblyandSNPreportingAftertheHRManalysis,asecondassemblyusingSeqManNGenv(DNASTAR,Madison,WI)wasperformedduetoitsimprovedalgorithmandtheresultswereusedforSNPreportingontheentiredatasetThisassemblywascompletedusingthedefaultparametersKoepkeetalBMCGenomics,:http:wwwbiomedcentralcomPageofforNGen’sdenovotranscriptomeassemblyof:match,matchsize,genomelengthMBThewholeSNPreportwasinitiallyfilteredtoretaintheHRMconfirmedSNPsusingaminimumtotaldepthofreadsatthepolymorphicbaseandatleastvariancefromtheconsensusFurtherfilteringintohighconfidenceSNPswasperformedbyscreeningforatleasttwoallelesrepresentedbyaminimumdepthofthreereadspernucleotidecallperalleleThisminimumdepthperalleleforeachSNPequalsorexceedsthepublisheddepthsusingeitherdata,orIlluminadata,Additionally,SNPsresultingfromthefirstorlastfivebasesofreadswererejectedThetransitionandtransversionsratio(Rvalue)wasdeterminedbysummingallofthetransitions(CTandAG)andtransversions(AC,AT,CG,andGT)HaplotypeIdentificationHaplotypeswereidentifiedvisuallybyanalyzingthecombinedtranscriptomeassemblygeneratedusingNGeninSeqMan(DNASTAR)SimilartotheSNPscreening,atleastthreereadsofanallelespanningtwoSNPlociwererequiredtolinkSNPsintoahaplotypeWhentwoormorehaplotypeswerepresentatonelocus,theyweredifferentiatedandrecordedasseparatehaplotypesfortheiruseashaplotypemarkersResultsandDiscussionMethodOverviewThegeneralmethodpresentedinthisstudyisbasedonfourstepsasoutlinedinFigureThefirststepofsamplepreparationinvolvesidentificationofappropriateindividualsacrosswhomgeneticpolymorphismneedstobedeterminedInourstudy,weusedtwocloselyrelatedsweetcherrycultivarstotestourapproachHowever,itisrecommendedthatphenotypicallydiverseindividualsshouldbechosenAdditionally,thenumberofindividualscanbeincreasedasdesiredkeepinginmindtheexpectedtranscriptomesizeandthenumberofsequencingreadsexpectedtobegeneratedbythenextgenerationsequencingplatformthatwillbeemployedfortranscriptomesequencinginstepThisparameteriscriticalforstrictfilteringofdataforidentificationofSNPsinstepTotalRNAneedstobeextractedfromtissueswhicharerepresentativeofthephenotypicdiversitybetweenthesamplesDevelopingreproductivebudsusedinthisstudywerederivedfromBingandRainiereachgraftedontotworootstocksMazzardandGiselaBingandRainiergraftedonGiselayieldedfruitthatwastomorethanthesamecultivarsgraftedonMazzardTheRNAisconvertedintocDNAandfurtherprocessedforselectionof’UTRsInstep,afterextensivequantificationofthe’UTRlibraries,samplesarepooledinequimolarratiosandsequencedusingnextgenerationsequencingplatformsAtthetime,weperformedpyrosequencingontheGSFLXinstrumentsinceitprovidedthelongestreadlengthsHowever,atpresent,suchamethodwouldbenefitgreatlyfromIlluminaorSOLiDplatformssincethereadlengthshavegreatlyimprovedDependingonthesequencingplatformtherawsequencedataneedstobepreprocessedbytrimmingoftagsandadaptorsequencespriortomovingtostepofdataprocessingwherethesequencedataisassembledWeusedtheNGenv(DNASTAR,Madison,WI)assemblerandtheoutputwasvisualizedusingSeqManwhichgeneratedaSNPreportThefinalsetofSNPswasselectedusingstrictparametersasoutlinedinthematerialsandmethodsInstep,putativeSNPsweretestedforvariabilityacrossparentalcultivarsandprogenyderivedfromacrossbetweentwocultivarsusingHRManalysisUtilizationofSNPsforscreeningvariabilityinpopulationhasbeenwelldocumentedinliteratureSubsequently,forSNPvalidation,barcodedampliconsequencingforaverylargenumberofmarkers(SNPorhaplotype)acrossalargearrayofprogenyinasegregatingpopulationorgeneticcollectionwouldbeanefficientapproachForsmallernumberofsamplesorforinitialconfirmationofvariation,techniquessuchasHRMmaybemoreappropriateasutilizedinthiscaseRapididentificationofgenelinkedpolymorphismsasproposedinthismethodcanfacilitateefficientGeneAssistedBreeding(GAB),genotypingandpopulationgeneticsstudiesinnonmodeleukaryotesSequencingandAssemblyof’UTRsPyrosequencingof’UTRlibrariesfromBingandRainieronasingleGSFLXsequencingplateproducedatotalof,reads(Table)ThereadshadanaveragelengthofbpwhichisasexpectedfromtheGSFLXsequencingplatformandthe’UTRlibrarypreparationThereadswereprocessedwithacustomscripttotrimindexsequencesandlabeltheheadersappropriatelyseeAdditionalFileTranscriptomeassemblyofthetrimmedsequenceswithSeqMan(LasergeneSuite,)yielded,contigsInitialSNPidentificationTotestourexperimentalapproach,analysisofasubsetoftheassembledcontigswasperformedtoidentifySNPswithinthedatasetThecontigswiththehighestnumberofreadsandcontigs,asproducedbySeqMan(LasergeneSuite,)wereanalyzedyieldingcontigsconta

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