Mol.Biol.Evol.15(11):1524 - PDF document

Mol.Biol.Evol.15(11):1524±1531.19981998bytheSocietyforMolecularBiologyandEvolution.ISSN:0737-4038TheMinisatelliteMSB1,intheFungusBotrytiscinerea,ProbablyMutatesbySlippageTatianaGiraud,DominiqueFortini,CarolineLevis,andYvesBrygooPathologieVeÂgeÂtale,InstitutNationaldelaRechercheAgronomique,Versailles,FranceAminisatellitewasidenti®edintheintronoftheATPsynthaseofthe®lamentousfungusBotrytiscinerea,andit EvolutionoftheMinisatelliteMSB11525ornamentals,bulbs,andfruits,particularlygrapes.Inapreviousstudy,weshowedthatB.cinereasexualreproductioninthe®eldandthatitcanbesub-dividedintoatleasttwospecies,B.transposa(Giraudetal.1997).OneofthemolecularmarkersusedforthisstudywastheATPsynthasegene,whosePCRproductshowsalengthpolymorphism.WeanalyzedtheminisatelliteMSB1,whichhasbeenfoundintheintronoftheATPsynthasegeneandisresponsibleforthevariationinthelengthofthisgene.Wesequencedtheminisatelliteinseveralstrainsofandafewotherspecies.Thisreportdescribesthevariationsinlength,sequence,andpresenceofMSB1andcomparesthesefeatureswiththoseofotherminisatellites.Lastly,wesuggesthowthevariabilityofMSB1mightbedriven.MaterialsandMethodsAtotalof44isolatesofB.cinereawereexamined:43ofthemwerecollectedfromgrapes(VitisviniferagrowinginChampagne,France,andtheremainingonewascollectedfromtomatoes.Oneisolateofeachofsixotherspeciesofwasalsoanalyzed(B.calthae,B.fabae,B.convulata,B.pelargoni,B.draytoni,B.alii),threeisolatesofSclerotiniahomoeocarpa,isolatesofSclerotiniasclerotiorum,and®veisolatesofSclerotiniaminor.Theisolateswerepuri®edbymono-sporeisolationandpreservedinparaf®noilat16C.Thefungiwereculturedonplatescontaining20mlNYme-dium(20g/lmalt,20g/lagar,2g/lyeastextract)fromaninoculumof1cmat25DNAExtractionThemyceliumwasculturedasdescribedbyDiolezetal.(1994).GenomicDNAwasisolatedbythemethodofMoÈlleretal.(1992).Ampli®cationwasdoneinatotalvolumeof50containing50ngfungalDNA,135ngofeachprimer,MdNTPs,and0.5UGibcoBRLpolymeraseintheprescribedbuffer.ThePCRused30cyclesof1minat95C,1minat60C,and2minat72C.ThereactionswereperformedinaPeltierThermalCycler200(MJResearch).TheEurogentecprimersusedwereTGGTGAAAGAGGTGACAACandTTTCAACGCA-GCATCAAGAforampli®cationoftheATPsynthasegene,andAAGTTGCTGGTTCCTTGAandGTTGCA-ACCGGCGTAGATtoamplifyitsintron.PCRproductswerepuri®edusingtheJetsorbgelextractionkit(BioprobeSystems)andsequencedusingtheABIPRISMDyeTerminatorCycleSequencingReadyReactionKitwithAmpliTaqDNAPolymerase(PerkinElmer)andtheABIPRISM310GeneticAn-alyser(AppliedBiosystems).Onestrandwassequencedforeachisolate,andonlysequencesofhighenoughqualityweretakenintoaccount;thesequencewascheckedtobesurethattherewasnoambiguitywhen-evertherewaspolymorphism.TheGenBankaccessionnumbersareAF020817,AF029687,AF029686,andSouthernBlotsBlottingwasdoneasdescribedinGiraudetal.(1997)usingapuri®edproductofPCRampli®cationoftheintronfromtheisolatewith11tandemrepeatsasTreeConstructionThetreesofthevariantswereconstructedusingtheheuristicsearchofPAUP3.1.1(Swofford1993).OnlyvariantswithoutdeletionsorinsertionsofafewbasepairsweretakenintoaccountforthePAUPsearch.Thefewothervariantsweretheneasilylocated,assumingthattheywerederivedfromthevariantwiththesamesignaturebutwithoutanydeletionorinsertion.IsolationandCharacteristicsofMSB1TheATPsynthaseofB.cinereastrain775wassequencedfrombothprimers.Newprimerswerethendesignedtoamplifytheintron,whichwasthense-quenced.Aminisatelliteof8tandemrepeatsof37bp,onehavingonly28bp,wasdetected(®g.1).Themini-satellitewasnamedMSB1,forthe®rstminisatelliteofTherepeatunitsequenceofMSB1didnothavethecoresequenceandwasAT-rich(62%intheconsen-susrepeatsequence).Theintronsof47isolatesofspeciesweresequencedtodeterminehowMSB1varied.Atotalof26allelesweredetected,eachcarrying6±8repeatsex-ceptforoneisolatewhichhad11repeatsandanotherwhichhad5repeats.Apriori,37differentrepeatunitoriginscouldbede®ned.We®rsttookthelast37bpastherepeatunit,sothatthe®rstrepeatwastheshorter.TheprecisesequencesoftherepeatsinMSB1variedgreatly.Thereweremanydifferentvariantsoftherepeatunitsequence(minisatellitevariantrepeats[MVRs]),whichdifferedfromoneanotherbypointmutations(basesubstitions,deletions,insertions),ortheinsertionofseveralbasepairs(variants).The51variantsidenti®edarelistedin®gure2.Aconsensustreeofthefull-lengthvariantsofMSB1,plustheconsensusrepeatunit,andaconsenustreeoftheshortervariantsaregivenin®gure3.Theconsensustreeofthefull-lengthvariantsshowstwomajorgroupsofvariants,bandc.Thevar-iantswerenamedaccordingtothetree:foreachdeeperbranching,theletterisdifferent(``b''or``c'';theshorterrepeatswerenamed``a''),foreachsecondseparationa®rstnumberwasgiven,asecondnumberindicatesathirddichotomy,etc.Thus,thenamesofthevariantsindicatetheirsimilarity.Forexample,thenamesofbrothervariantsdifferonlyintheirlastnumbers(e.g.,``b111''and``b112'').Somevariantswerefoundinasingleisolate,whileotherswerecommon,beingfoundinseveralspecies.TheallelesofMSB1areshownin®gure4byMVRmap.Thelargenumberofrepeatvariantswasusedtodetectastronglengthhomoplasy.Thus,21isolateshad 1526Giraudetal. .1.ÐSequencesoftheintronoftheATPsynthaseofB.cinereastrain775(GenBankaccessionnumberAF020817),theSm5strainofS.minor(GenBankaccessionnumberAF029686),theSD11strainofS.sclerotiorum(GenBankaccessionnumberAF029687),andtheS49strainofS.homoeocarpa(GenBankaccessionnumberAF020818).Basesthatareidenticalinatleasttwosequencesareshaded.Theminisatelliteregionisinboldanditalic,andrepeatsareindicatedbyarrows.Agapisindicatedby``.''.8repeats,buttherewere12distinctallelesthatdifferedbytheirvariants.Allexcept6ofthealleleswerefoundinsingleisolates.These6weremorewidelyrepresent-ed.Thevariantswerefoundinallallelesinthesamephysicalorder:therewasalwaysanavariant,followedbybvariants,followedbycvariants,andevenwithinthebandcvariantarrays,theorderwasthesameinallalleles:b111wasalwaysbeforeb115,b1beforeb2,c1beforec3,etc.Despitethefactthattheywereestablishedindependently,thisorderfollowedthetreeofthevari-ants:brothervariantswereoftenfoundatthesameplac-es,andagivenvariantwasalwaysnearitsmostsimilarvariant.avariantswerefoundinthe®rstposition,b11xvariantswerefoundinthesecondandthirdpositions,andc3xvariantswerealwaysinthelastpositionsandnearthec1andc2variants.Thesame-lengthallelesfol-lowedthesamemodel.Theallelewith5repeatsfol-lowedthemodel``abbcc,''allalleleswith6repeatsfol-lowedthemodel``abbbcc,''thealleleswith7repeatshadthestructure``abbbbcc''or``abbbccc,''thealleleswith8repeatshadthestructure``abbbbbcc''or``abbbbccc,''andlastly,theallelewith11repeatshadthestructure``abbbbbbbccc.''Inordertobesurethatthestrictphysicalorderofthevariantswasnotanartifactduetothearbitrarydef-initionoftherepeatunit,andtocheckthatthetreein-deedre¯ectedsomereality,thesameworkwasdonewithanotherrepeatunitde®nition(datanotshown).Thelastrepeatwaschosentobetheshortestineachallele,sotherepeatunitde®nitionwasthe®rst37bpofMSB1.Theconsensusofthemost-parsimonioustreesofthesenewvariantswasconstructed,andnamesweregiventothevariantsaccordingtothistreeaspreviouslyde-scribed.ThenewMVRmapsshowedthatvariantswereagaininthesamephysicalorderinallalleles,and,again,themostsimilarvariantswerethemostphysicallyclose.ThisindicatesthatthesetwofeaturesofMSB1donotdependonthede®nitionoftherepeatunit.Itmeansthat``signatures''intherepeatarefoundinthesameorderalongallalleles,andthatthemostsimilarsignaturesarealsothemostphysicallyclose.Welookedforpolymorphisminthe100-bp¯ank-ingregionsofMSB1in23isolates.Thesevenbasepairsthatwereinformativearerepresentedin®gure5.Inallisolates,thesevenpolymorphicbasepairswereassoci-ated:5isolateshadtheallelesH,I,J,K,L,andM,and18isolateshadtheallelesh,i,j,k,l,andm.Thisin-dicatesthatthewholeregionbehavesasasinglelinkagegroup.Thetotallackofrecombinationbetweenthe¯ankingregionscouldbeduetothespeciesbarrierbe-B.transposaB.vacuma.However,wefoundbothallelesinbothspecies.MSB1inOtherGeneraMSB1washybridizedwithgenomicDNAfromseveralisolatesbySouthernblotting,butonlyonelocusperstrain,theATPsynthasegene,wasdetected(datanotshown).No®ngerprintslikethosefoundwithcoreminisatelliteswereobtained.MSB1wasthenhybridizedwithgenomicDNAfromdifferentspecies.Thegenomesofseveralindividualsfromeachoffourspeciesoffun-gus(Magnaporthegrisea,Podosporaanserina,Fusar-iumoxysporum,Leptospheriamaculans)andfromArabidopsisthalianaDrosophilasimulanswereas-sayed.Nohybridizationsignalwasdetected.MSB1was EvolutionoftheMinisatelliteMSB11527 .2.ÐSequencesofthevariants.Thematchwiththeconsensusvariantisindicatedby``.'',agapisindicatedby``±'',andaninsertionisalignedbelowitsprecedingbasepairs. .3.ÐUnrootedtreesofthedifferentrepeatvariants:consensustreeoftheshortervariants(``a''variants)andconsensustreeofthe66most-parsimonioustreesofthefull-lengthvariants(``b''and``c''var-comparedwiththeGenBankdatabase,butnosigni®cantsimilaritywithanyothersequencewasfound.TheintronsoftheATPsynthasesfromtwoisolatesS.sclerotiorum,threeisolatesofS.minor,andthreeisolatesofS.homoeocarpawereampli®edandse-quenced(®g.1).Theintronwasshortinthese8isolatesandlackedMSB1.SomebasepairsateachendoftheintronweresimilartothesequenceofbutthecentralpartsoftheintronscompletelylackedMSB1andalsolacked36bpbeforeand8bpaftertheminisatellite.TherepeatunitsequencecouldnotbefoundinanypartoftheintronsoftheMSB1IsaNovelMinisatelliteThemostremarkableandunusualfeaturesofMSB1are(1)thefactthatvariantsareinthesameorderinallstrainsand(2)theparallelbetweenthisphysicalorderofthevariantsandtheirmostparsimonioustree.Thisstrictorderofvariantshasnotbeenfoundinanyotherwell-describedminisatellite.TheMVR-PCRtech-niqueshowedvariantscompletelymixed,andtheordersofthevariantswerecompletelydifferentinthealleles(e.g.,Jeffreysetal.1991).SlippageastheCauseoftheVariationsintheLengthofMSB1?Thelackof¯anking-regionexchange,thestrictphysicalorderofthevariants,anditscoincidencewith 1528Giraudetal. .4.ÐTheallelenumberforeachalleleofMSB1detectedandthenumberoftandemrepeats,MVRmap,andnumberofisolatesofB.vacuma(V),andotherspeciesof(O)thatcarryit.B.fabae,B.calthae,B.convulata,B.cinereaontomatoes. .5.ÐPolymorphisminthe¯ankingregionsofMSB1.Thesevenbasepairsthatarepolymorphicandinformativeareintheshadedboxes.``***''representstheminisatellite.In23isolates,norecombinationwasobservedbetweenthesesevenbasepairs.themostparsimonioustreeallsuggesthowtheinsta-bilityatthislocusisdriven.Thetotallackofrecom-binationin23isolatesbetweentheallelesofthe¯ankingregionsofMSB1(®g.5)clearlyshowsthatunequalcrossingovercannotbethemajorcauseofmutationinMSB1,althoughgeneticrecombinationisfrequentininChampagne(Giraudetal.1997).ThevariabilityofMSB1couldthenbeduetobeinterallelicconversionlikethatdecribedbyJeffreysetal.(1994)andMoncktonetal.(1994),conversionbe-tweentwosisterchromatids,orslippage.However,onlyamechanismthataddsorremovesasinglerepeatatatime,andaddsthisrepeatnearthemostsimilarone,canproducetheobservedconservedorderofvariants,withtheresemblingrepeatsbeingphysicallyclosetoeachother.Thisdoesnotseemconsistentwithaconversionmechanism.Toconservethesameorderofthevariantsinallallelesbyinterallelicconversion,wewouldhavetoassumethatthereisalwaysanalignmentbetweentwoidenticalvariants,thenaDNAbreakjustafterthisvari-ant,andtheninterallelicconversionofasinglerepeat.Furthermore,eventheseverystrongassumptionswouldchangetheorderofthevariantsiftherewasconversionbetweencertainalleles(e.g.,alleles3and22).Alltheconversioneventsdescribedsofarinminisatelliteshaveinvolvedseveralrepeats(e.g.,Jeffreysetal.1994;Moncktonetal.1994),evenforthemostsimplesomaticevents(JeffreysandNeumann1997).Moreover,con-versionshouldhomogenizerepeats,sothegreatnumberofvariantsofMSB1andthefactthatallvariantsaredifferentinalmostallallelesdonotsupportthismech-anism.Theconversionbetweentwosisterchromatidsassumesanothercomplicatedmechanism:thetwoallelecopiesshouldbeperfectlyaligned,withasinglerepeatshift;otherwisetherewouldbenochangeortheorderofthevariantswouldchange.Appelgren,Cederberg,andRannung(1997)suggestedanalignmentatthe5endbetweentwodifferentalleles,butifaperfectalign-mentofthe5¯ankingregionisconceivable,aconstantsingle-repeatshiftseemstobeveryspeculative.Thesimplestexplanationthat®tsallourresultsisthatmutationsleadingtoachangeinthenumberofrepeatsarerareandoccuronlybyone-repeatslippageonthesamechromatid.Thenewreplicatedstrandslipsandismispairedwiththehomologuouspartofthepre-cedingrepeat.Themutationsleadingtoachangeinthe EvolutionoftheMinisatelliteMSB11529 .6.ÐModelofthegenerationofvariabilityinMSB1byslippage. .7.ÐPhylogenetictreeofthespeciesanalyzed.Blackindi-catesthepresenceoftheminisatelliteMSB1.numberofrepeatsmustberaretoexplainthescarcityofallelesofdifferentlengthsandtoallowtimefornewrepeatstomutate,leadingtotheappearanceofmanynewvariants,eachoftenrestrictedtoasingleisolate.Thefactthatmostvariantsthatresembleeachotherareoftenfoundatthesameplaceandneartheirclosestvari-antalsopointstothismechanism,asillustratedin®gure6:variantsareduplicatedintandem;pointmutationsoccur,sothesonvariantisalwaysnearitsfathervariant.Thetreewouldthenreallybeaphylogenetictree.Theunresolvedbranchesofthevarianttreemayresultfromnewsonvariantsarisingindependentlyfromthesamefathervariant(e.g.,c1,c2,andc3variantsin®g.6).FootprintsofmutationsarestillvisibleintheMVRmaps(®g.4);e.g.,the11repeatsofthe26thalleleseemtobeduetofourtandemduplicationsoftheb116vari-ant.The®fthalleleseemstohavearisenfromthefourthallelebyasinglepointmutationinthelastrepeat.Thesix``abbbcc''repeatscanbegeneratedfromthe``abbcc''ofthe®ve-repeatallelesbytandemduplica-tionsofabvariant.Thepatterns``abbbbcc''or``abbbccc''followedbythealleleswithsevenrepeatscouldarisefromthesix-repeatpattern``abbbcc''bydu-plicationofaboracvariant.Itisnoticablethatbvariantsseemtobepreferentiallyduplicated.ThelengthofMSB1seemstobeundersomecon-straint.Our356isolatesfromChampagneincludednoalleleshorterthan6repeatsexceptforthesingleallelewith5repeats,andnoalleleswithmorethan8repeatsexceptforthesingle11-repeatallele.Theupperlimitcouldbeduetoaphysicalconstraint,suchastheintronbeingspliced.However,the11-repeatalleleexists,anditappearstobearecentallele,becauseits®veb116variantsdonotyethaveanymutations.Thisallele,alongwiththefactthatsomeminisatellitesareverylongeveninintrons,suggeststhattheupperconstraintcouldbeatimeconstraint.MSB1isfoundonlyinthegenus,andtheraremutationsmaynotyethavepro-ducedlongalleles.TheOriginofMSB1TheabsenceofMSB1fromthegenomesofgrisea,P.anserina,F.oxysporum,L.maculansfromtheintronoftheATPsynthaseofseveralspecies,oneofwhichisthefungusclosestto(CarbonandKohn1983),indicatesthatthismini-satelliteappearedveryrecently,inthebranchleadingtogenus(®g.7).ThelackofanysequenceintheintronofthatcouldresembletherepeatunitofMSB1,thelackofhybridizationatanyotherlocusintheB.cinereagenome,andthelackofanysimilarsequenceinpublisheddatabasesmaketheoriginofMSB1veryintriguing.MSB1musthavearisenafterthespeciationbetweenThefactthatthemostcommonvariantsarefoundindifferentspeciesindicatesthatitarosewellbeforethespeciationswithinthegenus.Minisatellitespresentinasinglegenus,oreveninasinglespecies,havebeenfoundinandin(GoodierandDavidson1998),andsomeminisatelliteshavebeenshowntobehighlyvariableinonlyonespe-cies(GrayandJeffreys1991).Suchlocicouldgivein-dicationsabouthowminisatellitesaregenerated.Severaldifferentmechanismshavebeenproposedtoexplainthemutantstructuresornaturalallelicpoly-morphismindifferentminisatellites.ThecharacteristicsofMSB1differfromthoseofallotherminisatellitesthathavebeenthoroughlyanalyzed.TheseoriginalfeaturesstronglysuggestthatMSB1mutatesbyslippage,al-thoughothermechanisms,suchasconversion,cannot 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