遺傳學(xué)英文教科書an introduction to genetic analysis第五章

Chapter5BasicEukaryoticChromosomeMapping根本真核生物染色體作圖KeyConcepts重要概念Twogenesclosetogetheronthesamechromosomepairdonotassortindependentlyatmeiosis.處于同一染色體上的兩個距離很近的基因不會在減數(shù)分裂中獨立別離。Recombinationproducesgenotypeswithnewcombinationsofparentalalleles.重組產(chǎn)生父本與母本等位基因的新結(jié)合的基因型。Apairofhomologouschromosomescanexchangesegmentsbycrossing-over.一對同源染色體可以通過交叉互換片段。Recombinationresultsfromeitherindependentassortmentorcrossing-over.重組產(chǎn)生于獨立別離與交叉互換。Genelocionachromosomecanbemappedbymeasuringthefrequenciesofrecombinantsproducedbycrossing-over.染色體上基因的定位作圖可以通過計算由交叉產(chǎn)生的重組頻率的方法來實現(xiàn)。Interlocusmapdistancesbasedonrecombinationmeasurementsareroughlyadditive.基于重組值測量方式的圖距確定中只是粗略計量。Theoccurrenceofacrossovercaninfluencetheoccurrenceofasecondcrossoverinanadjacentregion.交叉的發(fā)生會影響臨近區(qū)域的交叉再一次發(fā)生。Introduction簡介Wehavealreadyestablishedthebasicprinciplesofsegregationandassortment,andwehavecorrelatedthemwithchromosomebehaviorduringmeiosis.Thus,fromthecrossA/a;B/b×A/a;B/b,weexpecta9:3:3:1ratioofphenotypes.AswelearnedfromBridges'sstudyofnondisjunction(page76),exceptionstosimpleMendelianexpectationscandirecttheexperimenter'sattentiontonewdiscoveries.Justsuchanexceptionobservedintheprogenyofadihybridcrossprovidedthecluetotheimportantconceptsconsideredinthischapter.我們已經(jīng)建立起別離與分配的根本原那么，而且將此與染色體在減數(shù)分裂過程中的行為建立起聯(lián)系。因此，通過AaBb×AaBa雜交，推測預(yù)期的表現(xiàn)型比率為9：3：3：1。MESSAGEIngeneticanalysis,exceptionstopredictedbehaviorareoftensourcesofimportantnewinsights.Thediscoveryoflinkage相關(guān)發(fā)現(xiàn)Intheearly1900s,WilliamBatesonandR.C.Punnettwerestudyinginheritanceinthesweetpea.Theystudiedtwogenes:oneaffectingflowercolor(P,purple,andp,red)andtheotheraffectingtheshapeofpollengrains(L,long,andl,round).TheycrossedpurelinesP/P·L/L(purple,long)×p/p·l/l(red,round),andselfedtheF1P/p·L/lheterozygotestoobtainanF2.Table5-1showstheproportionsofeachphenotypeintheF2plants.TheF2phenotypesdeviatedstrikinglyfromtheexpected9:3:3:1ratio.Whatisgoingon?ThisdoesnotappeartobeexplainableasamodifiedMendelianratio.Notethattwophenotypicclassesarelargerthanexpected:thepurple,longphenotypeandthered,roundphenotype.Asapossibleexplanationforthis,BatesonandPunnettproposedthattheF1hadactuallyproducedmoreP·Landp·lgametesthanwouldbeproducedbyMendelianindependentassortment.Becausethesegenotypeswerethegametictypesintheoriginalpurelines,theresearchersthoughtthatphysicalcouplingbetweenthedominantallelesPandLandbetweentherecessiveallelespandlmighthavepreventedtheirindependentassortmentintheF1.However,theydidnotknowwhatthenatureofthiscouplingcouldbe.TheconfirmationofBatesonandPunnett'shypothesishadtoawaitthedevelopmentofDrosophilaasagenetictool.Aftertheideaofcouplingwasfirstproposed,ThomasHuntMorganfoundasimilardeviationfromMendel'ssecondlawwhilestudyingtwoautosomalgenesinDrosophila.Oneofthesegenesaffectseyecolor(pr,purple,andpr+,red),andtheotheraffectswinglength(vg,vestigial,andvg+,normal).Thewild-typeallelesofbothgenesaredominant.Morgancrossedpr/pr·vg/vgflieswithpr+/pr+·vg+/vg+andthentestcrossedthedoublyheterozygousF1females:pr+/pr·vg+/vg♀×pr/pr·vg/vg♂.Theuseofthetestcrossisextremelyimportant.Becauseoneparent(thetester)contributesgametescarryingonlyrecessivealleles,thephenotypesoftheoffspringrevealthegameticcontributionoftheother,doublyheterozygousparent.Hence,theanalystcanconcentrateonmeiosisinoneparentandforgetabouttheother.ThiscontrastswiththeanalysisofprogenyfromanF1self,wheretherearetwosetsofmeiosestoconsider:oneinthemaleparentandoneinthefemale.Morgan'sresultsfollow;theallelescontributedbytheF1femalespecifytheF2classes:Obviously,thesenumbersdeviatedrasticallyfromtheMendelianpredictionofa1:1:1:1ratio,andtheyindicateacouplingofgenes.Thetwolargestclassesarethecombinationspr+·vg+andpr·vg,originallyintroducedbythehomozygousparentalflies.Youcanseethatthetestcrossclarifiesthesituation.ItdirectlyrevealsthealleliccombinationsinthegametesfromonesexintheF1,thusclearlyshowingthecouplingthatcouldonlybeinferredfromBatesonandPunnett'sF1self.Thetestcrossalsorevealssomethingnew:thereisapproximatelya1:1rationotonlybetweenthetwoparentaltypes,butalsobetweenthetwononparentaltypes.Nowletusconsiderwhatmaybelearnedbyrepeatingthecrossingexperimentsbutchangingthecombinationsofallelescontributedasgametesbythehomozygousparentsinthefirstcross.Inthiscross,eachparentwashomozygousforonedominantalleleandforonerecessiveallele.AgainF1femalesweretestcrossed:Thefollowingprogenywereobtainedfromthetestcross:Again,theseresultsarenotevenclosetoa1:1:1:1Mendelianratio.Now,however,thelargestclassesarethosethathaveonedominantalleleortheotherratherthan,asbefore,twodominantallelesortworecessives.ButnoticethatonceagainthealleliccombinationsthatwereoriginallycontributedtotheF1bytheparentalfliesprovidethemostfrequentclassesinthetestcrossprogeny.Intheearlyworkoncoupling,BatesonandPunnettcoinedthetermrepulsiontodescribethissituation,becauseitseemedtothemthat,inthiscase,thenonallelicdominantalleles“repelled〞eachother—theoppositeofthesituationincoupling,wherethedominantallelesseemedto“sticktogether.〞Whatistheexplanationofthesetwophenomena:couplingandrepulsion?Morgansuggestedthatthegenesgoverningbothphenotypesarelocatedonthesamepairofhomologouschromosomes.Thus,whenprandvgareintroducedfromoneparent,theyarephysicallylocatedonthesamechromosome,whereaspr+andvg+areonthehomologouschromosomefromtheotherparent(Figure5-1).Thishypothesisalsoexplainsrepulsion.Inthatcase,oneparentalchromosomecarriesprandvg+andtheothercarriespr+andvg.Repulsion,then,isjustanothercaseofcoupling:inthiscase,thedominantalleleofonegeneiscoupledwiththerecessivealleleoftheothergene.ThishypothesisexplainswhyalleliccombinationsfromPremaintogether,buthowdoweexplaintheappearanceofnonparentalcombinations?Morgansuggestedthat,whenhomologouschromosomespairinmeiosis,thechromosomesoccasionallyexchangepartsinaprocesscalledcrossing-over.Figure5-2illustratesthisphysicalexchangeofchromosomesegments.Thetwonewcombinationsarecalledcrossoverproducts.Morgan'shypothesisthathomologsmayexchangepartsmayseemabitfarfetched.Isthereanycytologicallyobservableprocessthatcouldaccountforcrossing-over?WesawinChapter3thatinmeiosis,whenduplicatedhomologouschromosomespairwitheachother,twononsisterchromatidsoftenappeartocrosseachother,asdiagrammedinFigure5-3.Recallthattheresultingcross-shapedstructureiscalledachiasma.ToMorgan,theappearanceofthechiasmatavisuallycorroboratedtheconceptsofcrossing-over.(Notethatthechiasmataseemtoindicatethatitischromatids,notunduplicatedchromosomes,thatcrossover.Weshallreturntothispointlater.)NotethatMorgandidnotarriveatthisinterpretationoutofnowhere;hewaslookingforaphysicalexplanationforhisgeneticresults.Hisachievementincorrelatingtheresultsofbreedingexperimentswithcytologicalphenomenathusemphasizestheimportanceofthechromosometheoryasapowerfulbasisforresearch.MESSAGEChiasmataarethevisiblemanifestationsofcrossovers.Datalikethosejustpresented,showingcouplingandrepulsionintestcrossesandinF1selfs,arecommonlyencounteredingenetics.Clearly,resultsofthiskindareadeparturefromindependentassortment.Suchexceptions,infact,constituteamajoradditiontoMendel'sviewofthegeneticworld.MESSAGEWhentwogenesareclosetogetheronthesamechromosomepair,theydonotassortindependently.Theresidingofgenesonthesamechromosomepairistermedlinkage.Twogenesonthesamechromosomepairaresaidtobelinked.Itisalsopropertorefertothelinkageofspecificalleles:forexample,inoneA/a·B/bindividual,Amightbelinkedtob;awouldthenofnecessitybelinkedtoB.Thesetermsgraphicallyalludetotheexistenceofaphysicalentitylinkingthegenes—thatis,thechromosomeitself.Youmaywonderwhywerefertosuchgenesas“l(fā)inked〞ratherthan“coupled〞;theansweristhatthewordscouplingandrepulsionarenowusedtoindicatetwodifferenttypesoflinkageconformationinadoubleheterozygote,asfollows:Inotherwords,couplingreferstothelinkageoftwodominantortworecessivealleles,whereasrepulsionindicatesthatdominantallelesarelinkedwithrecessivealleles.Toascertainwhetheradoubleheterozygoteisincouplingorrepulsionconformation,aninvestigatormusttestcrossthedoubleheterozygoteorconsiderthegenotypesofitsparents.RecombinationInmoderngeneticanalysis,themaintestfordeterminingwhethertwogenesarelinkedisbasedontheconceptofrecombination.Recombinationisobservedinavarietyofsituationsbut,forthepresent,let'sdefineitinrelationtomeiosis.Meioticrecombinationisanymeioticprocessthatgeneratesahaploidproductwithagenotypethatdiffersfrombothhaploidgenotypesthatconstitutedthemeioticdiploidcell.Theproductofmeiosissogeneratediscalledarecombinant.Thisdefinitionmakestheimportantpointthatwedetectrecombinationbycomparingtheoutputgenotypesofmeiosisandtheparentalinputgenotypes(Figure5-4).Theinputgenotypesarethetwohaploidgenotypesthatcombinedtomakethegeneticconstitutionofthemeiocyte,thediploidcellthatundergoesmeiosis.MESSAGEInmeiosis,recombinationgenerateshaploidgenotypesdifferingfromthehaploidparentalgenotypes.Meioticrecombinationisapartofbothhaploidanddiploidlifecycles;however,detectingrecombinantsinhaploidcyclesisstraightforward,whereasdetectingthemindiploidcyclesismorecomplex.Theinputandoutputtypesinhaploidcyclesarethegenotypesofindividualsandmaythusbeinferreddirectlyfromphenotypes.Figure5-4canbeviewedassummarizingthesimpledetectionofrecombinantsinhaploidlifecycles.Theinputandoutputtypesindiploidlifecyclesaregametes.Becausewemustknowtheinputgametestodetectrecombinantsinadiploidcycle,itispreferabletohavepure-breedingparents.Furthermore,wecannotdetectrecombinantoutputgametesdirectly:wemusttestcrossthediploidindividualandobserveitsprogeny(Figure5-5).Ifatestcrossoffspringisshowntohavebeenconstitutedfromarecombinantproductofmeiosis,ittooiscalledarecombinant.Noticeagainthatthetestcrossallowsustoconcentrateononemeiosisandpreventambiguity.FromaselfoftheF1inFigure5-5,forexample,arecombinantA/A·B/boffspringcannotbedistinguishedfromA/A·B/Bwithoutfurthercrosses.Recombinantsareproducedbytwodifferentcellularprocesses:independentassortmentandcrossing-over.RecombinationbyindependentassortmentMendelianindependentassortmentisviewedwithregardtorecombinationinFigure5-6.Inatestcross,thetworecombinantclassesalwaysmakeup50percentoftheprogeny;thatis,thereis25percentofeachrecombinanttypeamongtheprogeny.Ifweobservearecombinantfrequencyof50percentinatestcross,wecaninferthatthetwogenesunderstudyassortindependently.Thesimplestinterpretationofsucharesultisthatthetwogenesareonseparatechromosomepairs.However,genesthatarefarapartonthesamechromosomepaircanactvirtuallyindependentlyandproducethesameresult.Recombinationbycrossing-overCrossing-overalsocanproducerecombinants.Anytwononsisterchromatidscancrossover.(WeshallshowproofofthisinChapter6.)Thereisnotacrossoverbetweentwospecificgenesinallmeioses,but,whenthereis,halftheproductsofthatmeiosisarerecombinant,asshowninFigure5-7.Meiosiswithnocrossoverbetweenthegenesunderstudyproducesonlyparentalgenotypesforthesegenes.Forgenesclosetogetheronthesamechromosomepair,thephysicallinkageofparentalallelecombinationsmakesindependentassortmentimpossibleandhenceproducesrecombinantfrequenciessignificantlylowerthan50percent(Figure5-8).WesawanexampleofthissituationinMorgan'sdata(page142),wheretherecombinantfrequencywas(151+154)÷2839=10.7percent.Thisisobviouslymuchlessthanthe50percentthatwewouldexpectwithindependentassortment.Therecombinantfrequencyarisingfromlinkedgenesrangesfrom0to50percent,dependingontheircloseness.Whataboutrecombinantfrequenciesgreaterthan50percent?Theansweristhatsuchfrequenciesareneverobserved,asweshallseeinChapter6.NoteinFigure5-7thatcrossing-overgeneratestworeciprocalproducts,whichexplainswhythereciprocalrecombinantclassesaregenerallyapproximatelyequalinfrequency.MESSAGEArecombinantfrequencysignificantlylessthan50percentshowsthatthegenesarelinked.Arecombinantfrequencyof50percentgenerallymeansthatthegenesareunlinkedonseparatechromosomes.Theremainderofthischapterfocusesmainlyonlinkedgenesandrecombinantsarisingfromcrossing-over.LinkagesymbolismOursymbolismfordescribingcrossesbecomescumbersomewiththeintroductionoflinkage.WecandepictthegeneticconstitutionofeachchromosomeintheDrosophilacrossasinthefollowingexample:whereeachlinerepresentsachromosome;theallelesaboveareononechromosome,andthosebelowareontheotherchromosome.AcrossoverisrepresentedbyplacinganXbetweenthetwochromosomes,sothatisthesameasWecansimplifythegenotypicdesignationoflinkedgenesbydrawingasingleline,withthegenesoneachsidebeingonthesamechromosome;nowoursymbolisButthisisstillinconvenientfortypingandwriting,solet'stipthelinetogiveusprvg/pr+vg+,stillkeepingthegenesofonechromosomeononesideofthelineandthoseofitshomologontheother.Wealwaysdesignatelinkedgenesoneachsideinthesameorder;itisalwaysab/ab,neverab/ba.Therulethatgenesarealwayswritteninthesameorderpermitsgeneticiststouseashorternotationinwhichthewild-typealleleiswrittenwithaplussignalone.Inthisnotationthegenotypeprvg/pr+vg+becomesprvg/++.Youmayseethisnotationinotherbooksorinresearchpapers.Aswehaveseeninearlierchapters,genesknowntobeondifferentchromosomepairsareshownseparatedbyasemicolon,forexample,A/a;B/b.Inthisbook,genesofunknownlinkageareshownseparatedbyadot,A/a·B/b.Now,ifwereconsidertheresultsobtainedbyBatesonandPunnett,wecaneasilyexplainthecouplingphenomenonbyusingtheconceptoflinkage.Theirresultsarecomplexbecausetheydidnotdoatestcross.However,wewillseelaterthatinfactitispossibletoderiveestimatednumbersforrecombinantandparentaltypesinadihybridcross.LinkageofgenesontheXchromosomeUntilnow,wehavebeenconsideringrecombinationofautosomalgenes.WhataretheconsequencesofnonsisterchromatidsoftheXchromosomecrossingoverbetweentwogenesofinterest?RecallthatahumanorDrosophilafemaleproducesmaleprogenyhemizygousforthegenesoftheXchromosome,sothegenotypeofthegametethatamothercontributestohersonisthesoledeterminantoftheson'sphenotype.Let'sconsideranexampleinwhichwefirstobservetheF1progenyfromthematingoftwoDrosophilafliesandthentheF2progenyfromintercrossingtheF1.Weuseherethefollowingsymbols:yandy+fortheallelesgoverningyellowbodyandbrownbody,respectively;wandw+forallelesforwhiteeyeandredeye;andYfortheYchromosome.IntheF1thenumbersofmalesinthephenotypicclassesare:BecausetheF2malesobtainonlyaYchromosomefromtheF1males,theseclassesrepresentperfectlytheproductsofmeiosisintheFfemales.Noticethatthisfacteliminatestheneedforatestcross;wecanfollowmeiosisinasingleparent,justaswecaninatestcross.Thetotalfrequencyoftherecombinantsinthisexampleis(43+22)÷14513=1.4percent.LinkagemapsThefrequencyofrecombinantsfortheDrosophilaautosomalgenesthatwestudied(prandvg)was10.7percentoftheprogeny—afrequencymuchgreaterthanthatforthelinkedgenesontheXchromosomejuststudied.Apparently,theamountofcrossing-overbetweenvariouslinkedgenesdiffers.Indeed,thereisnoreasontoexpectthatchromatidswouldcrossoverbetweendifferentlinkedgeneswiththesamefrequency.AsMorganstudiedmorelinkedgenes,hesawthattheproportionofrecombinantprogenyvariedconsiderably,dependingonwhichlinkedgeneswerebeingstudied,andhethoughtthatthesevariationsincrossoverfrequencymightsomehowindicatetheactualdistancesseparatinggenesonthechromosomes.Morganassignedthestudyofthisproblemtoastudent,AlfredSturtevant,who(likeBridges)becameagreatgeneticist.MorganaskedSturtevant,stillanundergraduateatthetime,tomakesomesenseofthedataoncrossing-overbetweendifferentlinkedgenes.Inonenight,Sturtevantdevelopedamethodfordescribingrelationsbetweengenesthatisstillusedtoday.InSturtevant'sownwords,“Inthelatterpartof1911,inconversationwithMorgan,Isuddenlyrealizedthatthevariationsinstrengthoflinkage,alreadyattributedbyMorgantodifferencesinthespatialseparationofgenes,offeredthepossibilityofdeterminingsequencesinthelineardimensionofachromosome.Iwenthomeandspentmostofthenight(totheneglectofmyundergraduatehomework)inproducingthefirstchromosomemap.〞AsanexampleofSturtevant'slogic,consideratestcrossfromwhichweobtainthefollowingresults:Theprogenyinthisexamplerepresent400femalegametes,ofwhich44(11percent)arerecombinant.Sturtevantsuggestedthatwecanusethepercentageofrecombinantsasaquantitativeindexofthelineardistancebetweentwogenesonageneticmap,orlinkagemap,asitissometimescalled.Thebasicideahereisquitesimple.Imaginetwospecificgenespositionedacertainfixeddistanceapart.Nowimaginerandomcrossing-overalongthepairedhomologs.Insomemeioticdivisions,nonsisterchromatidscrossoverbychanceinthechromosomalregionbetweenthesegenes;fromthesemeioses,recombinantsareproduced.Inothermeioticdivisions,therearenocrossoversbetweenthesegenes;norecombinantsresultfromthesemeioses.Sturtevantpostulatedaroughproportionality:thegreaterthedistancebetweenthelinkedgenes,thegreaterthechancethatnonsisterchromatidswouldcrossoverintheregionbetweenthegenesand,hence,thegreatertheproportionofrecombinantsthatwouldbeproduced.Thus,bydeterminingthefrequencyofrecombinants,wecanobtainameasureofthemapdistancebetweenthegenes(Figure5-9).Infact,wecandefineonegeneticmapunit(m.u.)asthatdistancebetweengenesforwhichoneproductofmeiosisin100isrecombinant.Putanotherway,arecombinantfrequency(RF)of0.01(1percent)isdefinedas1m.u.[Amapunitissometimesreferredtoasacentimorgan(cM)inhonorofThomasHuntMorgan.]Adirectconsequenceofthewayinwhichmapdistanceismeasuredisthat,if5mapunits(5m.u.)separategenesAandBwhereas3m.u.separategenesAandC,thenBandCshouldbeeither8or2m.u.apart(Figure5-10).Sturtevantfoundthistobethecase.Inotherwords,hisanalysisstronglysuggestedthatgenesarearrangedinsomelinearorder.Theplaceonthemap—andonthechromosome—whereageneislocatediscalledthegenelocus(plural,loci).Thelocusoftheeye-colorgeneandthelocusofthewing-lengthgene,forexample,are11m.u.apart.Therelationisusuallydiagrammedthisway:althoughitcouldbediagrammedequallywelllikethis:orlikethis:Usuallywerefertothelocusofthiseye-colorgeneinshorthandasthe“prlocus,〞afterthefirstdiscoverednon-wild-typeallele,butwemeantheplaceonthechromosomewhereanyalleleofthisgenewillbefound.Givenageneticdistanceinmapunits,wecanpredictfrequenciesofprogenyindifferentclasses.Forexample,intheprogenyfromatestcrossofafemaleprvg/pr+vg+heterozygote,weknowthattherewillbe11percentrecombinants,ofwhich51/2percentwillbeprvg+/prvgand51/2percentwillbepr+vg/prvg;oftheprogenyfromatestcrossofafemaleprvg+/pr+vgheterozygote,51/2percentwillbeprvg/prvgand51/2percentwillbepr+vg+/prvg.Thereisastrongimplicationthatthe“distance〞onalinkagemapisaphysicaldistancealongachromosome,andMorganandSturtevantcertainlyintendedtoimplyjustthat.Butweshouldrealizethatthelinkagemapisanotherexampleofanentityconstructedfromapurelygeneticanalysis.Thelinkagemapcouldhavebeenderivedwithoutevenknowingthatchromosomesexisted.Furthermore,atthispointinourdiscussion,wecannotsaywhetherthe“geneticdistances〞calculatedbymeansofrecombinantfrequenciesinanywayrepresentactualphysicaldistancesonchromosomes,althoughcytogeneticandmolecularanalysishasshownthatgeneticdistancesare,infact,roughlyproportionaltochromosomedistances.Nevertheless,itmustbeemphasizedthatthehypotheticalstructure(thelinkagemap)wasdevelopedwithaveryrealstructure(thechromosome)inmind.Inotherwords,thechromosometheoryprovidedtheframeworkforthedevelopmentoflinkagemapping.MESSAGERecombinationbetweenlinkedgenescanbeusedtomaptheirdistanceapartonthechromosome.Theunitofmapping(1m.u.)isdefinedasarecombinantfrequencyof1percent.Thestageofanalysisthatwehavereachedinourdiscussioniswellillustratedbylinkagemapsofthescrewworm(Cochliomyiahominivorax).Thelarvalstageofthisinsect—theworm—isparasiticonmammalianwoundsandisacostlypestoflivestockinsomepartsoftheworld.Ageneticsystemofpopulationcontrolhasbeenproposed,ofatypethathasbeensuccessfulinotherinsects.Toaccomplishthisgoal,anunderstandingofthebasicgeneticsoftheinsectisneeded,oneimportantpartofwhichistoprepareamapofthechromosomes.Thisanimalhassixchromosomepairs,andmappinghasbegun.Thejobofgeneralmappingstartsbyfindingandanalyzingasmanyvariantphenotypesaspossible.Theadultstageofthisinsectisafly,andgeneticistshavefoundphenotypicvariantsamongscrewwormflies.Theyfoundfliesofsixdifferenteyecolors,alldifferentfromthebrown-eyed,wild-typeflies,asFigure5-11ashows.Theyalsofoundfivevariantphenotypesforsomeothercharacters.Elevenmutantalleleswereshowntodeterminethe11variantphenotypes,eachatadifferentautosomallocus.PurelinesofeachphenotypewereintercrossedtogeneratedihybridF1s,andthentheseweretestcrossed.ThetestcrossrevealedthesetoffourlinkagegroupsshowninFigure5-11b.Noticethattheyeandcwlociareshowntentativelylinked,althoughtherecombinantfrequencyisnotsignificantlydifferentfrom50percent.Alinkageanalysissuchastheprecedingonecannotassignlinkagegroupstospecificchromosomes;thismustbedonebyusingthecytogenetictechniquestobeconsideredinChapter17.Inthepresentexample,suchcytogenetictechniqueshaveallowedthelinkagegroupstobecorrelatedwiththechromosomespreviouslynumberedasshowninFigure5-11b.Three-pointtestcrossSofar,wehavelookedatlinkageincrossesofdoubleheterozygotestodoublyrecessivetesters.Thenextlevelofcomplexityisacrossofatripleheterozygotetoatriplyrecessivetester.Thiskindofcross,calledathree-pointtestcross,illustratesthestandardapproachusedinlinkageanalysis.Weshallconsidertwoexamplesofsuchcrosseshere.First,wefocusonthreeDrosophilagenesthathavethenon-wild-typeallelessc(shortforscute,orlossofcertainthoracicbristles),ec(shortforechinus,orroughenedeyesurface),andvg(shortforvestigialwing).Wecancrosssc/sc·ec/ec·vg/vgtriplyrecessiveflieswithwild-typefliestogeneratetripleheterozygotes,sc/sc+·ec/ec+·vg/vg+.Weanalyzereco