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effectsofindividualQTLs(Reffayetal.2005;McIntyreetal.2005).Polyploidyisgoingtochallengetheapplicationofmarkersinsugarcanemorethaninanyothercrop,andimprovedbiometricalmethodsareneededtoextractfullinformationfromtheQTL-detectionexperiments.
MultiplexsegregationatQTLlocimaybepartlyresponsibleforphenotypicbuffering,whichisanimportantfactorinthesuccessofmanyautopolyploidcrops.Non-additivegeneactioninmultipledoseQTLsmayalsohavecontributedtoevolu-tionaryopportunities.Forexample,ifasinglecopyofagene/QTLisphysiologicallysuf?cient,theadditionalcopiesmaybefreetocollectmutations,oftenbecomingnonfunctional,perhapsoccasionallyresultinginadistinctivenewfunctionthatim-proves?tness.
21.3.3SyntenywithOtherGrasses
Duetoitshighpolyploidyandtheabsenceofdiploidcloserelatives,theadvantageofinvestigatingsyntenyconservationbetweensugarcaneandothergrasses,inpar-ticularwithothermembersoftheAndropogoneaetribe,wasrealizedearly.The?rstcomparisonsweremadewithmaize,whichhad,atthatstage,amoreadvancedmap.Gautetal.(2000)con?rmedsyntenytobeconserved(D’Hontetal.1994;Grivetetal.1994;Dufouretal.1997),althoughquiteperturbedbytheduplicatedstructureofthemaizegenomeandthepresenceofmanyrearrangements.Ricealsoshowedrelativelyglobalsimplesyntenyrelationshipswithsugarcanewith,however,manyrearrangementsexplainedbythelargedistancebetweenthetwospecies(Glaszmannetal.1997).Riceremainsaninterestingmodelforsugarcanebecausethesequenceofitsgenomeisavailable(InternationalRiceGenomeSequencingProject2005)andbecauselargenumbersofricemutantsarebeingcollected.Todate,ofthespeciesstudied,sorghumappearstohavethesimplestsyntenyrelationshipwithsugarcane(Grivetetal.1994;Dufouretal.1997;Glaszmannetal.1997;Guimar?esetal.1997;Mingetal.1998),andthusmoreadaptedtohelpwithsugarcanestudies(Asnaghietal.2000).CorrespondingQTLscontrollingplantheight,stalknumber,and?oweringwerefoundinsorghumandsugarcane(Mingetal.2002b;Jordanetal.2004).Theavailabilityoftheentiresorghumgenomesequenceinthenearfuture(SorghumGenomicsPlanningWorkshopParticipants2005)willbeofgreatinteresttosugarcanegenomics,inparticularformap-basedgeneisolation.
Co-linearityhasbeenemployedtohelpdevelopa?nemaparoundageneconferringresistancetobrownrust(Bru1),whichisthefocusofamapbasedcloningapproachusingthecultivarR570.Sorghumandriceregionsorthologoustothesugarcanetargetareawereidenti?edandmarkersderivedfromsorghumgenetics(Boivinetal.1999;Bowersetal.2003),physicalmaps(http://genome.arizona.edu/genome/sorghum.html),andacomparisonofsugarcanecDNAswiththericeor-thologoussequence(http://www.genome.arizona.edu/fpc/rice/),wereusedtosatu-ratethesugarcanemapinthetargetregion(Asnaghietal.2000,2004;D’Hontunpublisheddata).
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21.4BACLibraryDevelopmentandUtilization
Abacterialarti?cialchromosome(BAC)libraryof103,296clones,withameanin-sertsizeof130kbp,hasbeenconstructedforthesugarcanecultivarR570(Tomkinsetal.1999).Onthebasisofthemonoploidgenomesizeofsugarcanethecoverageisestimatedtobe14x.However,sincesugarcaneishighlypolyploidandheterozy-gous,thisrepresentsonlythecoverageof1.3xofthetotalgenomeofthissugarcanecultivar.
ThisBAClibraryiscurrentlybeingusedtodevelopaphysicalmapoftheregionbearingtherustresistancegeneBru1incultivarR570andtoperformcomparativegenomicstudieswithinsugarcaneaswellaswithothergrasses.
TwohomoeologousBACclones(97kband126kb),onederivedfromS.sponta-neumandonefromS.of?cinarum,correspondingtoaregionthathasalreadybeenstudiedinseveralcereals(Ilicetal.2003),weresequencedandcompared(Jannooetal.2007).TheresultsindicatedthatthetwoSaccharumspeciesdivergedby1.5–2myafromoneanotherand8–9myafromsorghum.Thetwosugarcanehomoeolo-goushaplotypesshowedperfectco-linearityandalsohighhomologyalongthenon-transcribedregions,apartfromtheinsertionofafewretro-transposableelements.Thegenedistributionhighlightedhighsyntenyandco-linearitywithsorghumandrice,andpartialco-linearitywitheachhomoeologousmaizeregion,whichbecameperfectwhenthesequenceswerecombined.This?rstanalysisofsugarcanehaplo-typeorganizationatthesequencelevelsuggestedthatthehighploidyinsugarcanedidnotinducegeneralizedreshapingofitsgenome,thuschallengingtheideathatpolyploidyquicklyinducesgeneralizedrearrangementofgenomes.Theseresultsalsoconsolidatedthefactthatsorghumisachoicemodelforsugarcane.
21.5FunctionalGenomics21.5.1ESTDevelopment
Thelargesugarcanegenome,inwhich,onaverage,eachsingle-copygeneisrep-resentedbytenalleles,representsachallengeforgeneticanalysis.Expressedse-quencetag”(EST)collectionsmaycontributesigni?cantlytoidentifycandidategenesassociatedwithimportantagronomicaltraits(i.e.,tolerancetoabioticandbi-oticstress,mineralnutrition,andsugarcontent,amongstothers)andmoregenerallytoproviderelevantinformationforfunctionalandevolutionaryanalyses.
SeveralsugarcaneESTcollectionshavebeendeveloped(CarsonandBotha2000,2002;Casuetal.2001,2003;Maetal.2004;Boweretal.2005),thelargestonebeingtheBraziliansugarcaneESTproject,SUCEST(Vettoreetal.2003).AllofthepubliclyavailablesugarcanesequenceESTswereassembledintotentativecon-sensussequences(virtualtranscripts),singletons,andmaturetranscripts,referredtoastheSugarcaneGeneIndex(SGI;http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=s_of?cinarum).TheSUCESTcollectionofESTswasassembled
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into43,141putativeuniquesugarcanetranscriptsreferredtoassugarcaneassembledsequences(SAS)http://sucest.lbi.ic.unicamp.br/public/;(Vettoreetal.2003).
AcomparativeanalysisbetweentheSASsandtheDNAandproteinsequencesfromthemodeleudicotyledonplantArabidopsisthaliana,modelmonocotyledonrice,andasetofotherangiospermswasundertaken.Threemainclassesofse-quenceswereidenti?ed,acoresetofeudicot-monocotsequencesthatmayrep-resentangiospermbasicfunctions(75%oftheSASs),monocot-speci?csequences(14%oftheSASs),andsequencesrestrictedtosugarcane(13.5%oftheSASs).Asigni?cantproportionofthemonocot-speci?csequenceswerefoundtorepresentfast-evolvingsequencesintegratedinmembersofconservedangiospermgenefami-lies.Thisobservationwasparticularlyrelevantsinceahighrateofevolutionmayberelatedtoafunctionaldiversi?cationthatcouldbeinvolvedinthedifferentiationofspeci?cevolutionarylineages.Newproteinarchitectureformedbymonocot-speci?cmotivesordomainsthatwererecruitedintoconservedeudicot-monocotproteinsandlongnon-codingRNA(～500nucleotides)werealsoidenti?edamongthemonocot-speci?csequences(Vincentzetal.2004;Vincentz,unpublishedre-sults).Thedivergencebetweenmonocotandeudicotmaythereforerelypartlyonfunctionaldiversi?cation(generatingnewproteinfunctions)fromduplicatedcopiesofconservedgenefamilies.Theextenttowhichthesugarcane-speci?cSASsrepre-sentnoveltiesrestrictedtothisorganismremainsunclear.
SeveralstudieshavedescribedtheSUCESTsequencesanddiscussedtheirputa-tiveroles,inparticularforgenesthatcorrespondtothegeneralmetabolismofsugar-cane,signalinggrowth,developmentandstressresponses(GrivetandArruda2001;Arruda2001).Over30%oftheidenti?edtranscriptscorrespondedtogeneswithnosequencesimilaritytoknowngenes.Theidenti?cationofnovelgenefunctionsinsuchacomplexorganismisaformidabletask.Ongoingeffortstoassociateputa-tivefunctionswiththesugarcanegenesincludegeneexpressionpro?lingoftissues,identi?cationofgenesassociatedwithsucrosecontentincultivarsshowingcontrast-ingBrixvalues,comparingdifferentvarietiessubmittedtobioticandabioticstress,aswellasmappingeffortstoassociategeneswithphenotypes,asdescribedabove.
21.5.2TissuePro?ling
TheuseofgenechipsandcDNAmicro-arraysallowsbothtemporalandspa-tialgeneexpressiondatatobeobtained.CustomdesignedcDNAmicro-arraysconstructedusingthecDNAcollectionfromtheSUCESTdatabase(http://sucest-fun.org),wereusedtodeterminethedistributionofgenetranscriptsinsugarcanetissuesandtode?netissue-speci?cactivitiesandubiquitousgenes.UsingcDNAmicro-arrayscontaining1,280distinctelements,theindividualgeneexpressionvariationofplantsgrowninthe?eldandtranscriptabundanceinsixplantorgans(?owers,roots,leaves,lateralbuds,1st[immature]and4th[mature]internodes),wasanalyzed(Papini-Terzietal.2005).Theexpressionof217geneswasfoundtobetissue-enriched,while153genesshowedexpressionlevelsthatwerehighly
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similarinallthetissuesanalyzed.Avirtualpro?lematrixwasconstructedwherethetissueexpressionlevelscanbecomparedamongst24tissuesamples.Mostofthegenescharacterizedcodedforsignaltransductioncomponents,hormonebiosynthe-sis,transcriptionfactors,stressandpathogenresponse-relatedgenes.AcatalogueofsugarcanesignaltransductionandotherregulatorygenescanbefoundattheSU-CASTDatabase(http://sucest-fun.org).Thedatabaseintegratesthegenecatalog,theinformationgeneratedbythephylogeneticcategorizationofthesugarcaneki-nome,tissuematrix,sequencingdata,andanalysesbytheSUCESTProject.Tissueexpressioninformationcanalsoaidintheidenti?cationofgenepromotersequences.
21.5.3ExploitationofESTResourcesforFunctionalAnalysis
21.5.3.1SugarSynthesis,Transport,andAccumulation
Sucrosesynthesizedinsugarcaneleavesisstoredintheculms.Sugarcanehasthestrikingabilityofaccumulatinghighlevelsofsucrosethatcanreachuptoabout0.7molarinmatureinternodes(Moore1995),whichcorrespondstoapproximately50%ofthestalkdryweight.Thisphysiologicalspecializationmakessugarcaneaninterestingmodelforstudiesonsugarsynthesis,transport,andaccumulation.
CarbohydratesaresynthesizedinsugarcaneleavesbyCO2?xationduringpho-tosynthesis(Moore1995;LunnandFurbank1999;GrofandCampbell2001).InC4plants,suchassugarcane,CO2is?xedbyphosphenolpyruvatecarboxylaseinmeso-phyllcells,producingoxaloacetate,whichisreducedtomalate.Inthebundlesheathcells,themalateisdecarboxylated,theCO2beingreleasedandre-?xedbyRubiscointhephotosyntheticcarbonreduction(PCR)cycle.Theresultingglyceraldehyde3-phosphatemoleculesarethesubstrateforamulti-steppathwayleadingtothesyn-thesisofsucrose,inwhichtheactivitiesoffructose-1,6-bisphosphatase(FBPase)andsucrose-phosphatesynthase(SPS)playamajorcontrolrole.Sucroseisthoughttobesynthesizedexclusivelyinthemesophyll,andthentransferredtophloemcells,whereitistransportedtothestemparenchymacells(GrofandCampbell2001).Besidestheseleafreactions,GrofandCampbell(2001)highlightedthreemajorratelimitingorco-limitingsteps:therateoftransporttothestalks,includingphloemloading;therateoftransportintotheparenchymacellsandintotheirvacuoles;andtherateofsucrosemobilizationusedforthevegetativegrowth.Itissupposedthatoncetheselimitationsaresolved,commercialyieldscouldbedoubled(GrofandCampbell2001andreferencescitedtherein).
Itisknownfromseveralplantspeciesthatsugartransportreliesonbothsym-plasticandapoplasticsteps(Patrick1997;Lalondeetal.2004).AnevaluationoftheSUCESTdatabaserevealedfull-lengthgenesencodingninemonosaccharideandfourdissacharidetransporters(Felix2006).Casuetal.(2003)performedanESTsurveycomparingtranscriptsfromimmatureandmatureinternodes.Severaltranscriptsencodingproteinshomologoustoknownsugartransporterswerefound,andallofthemweremoreabundantinthematureinternodes.Micro-arrayandnorthernblotanalysesshowedthataputativesugartransporter,type2a,washighly
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expressedinmatureinternodesandabsentinmatureleafandroot.However,thesugartransportedbythisproteinremainselusive.Raeetal.(2005)clonedthesugar-caneShSUT1gene,whichencodesatransporterthatmayplayaroleinthetransferofsucrosefromthevasculartissuetoparenchymacellsofinternodes.Sincesinkstrengthregulatesphotosynthesisinsugarcane(McCormicketal.2006),theevalu-ationofsugartransportergenesintransgenicsugarcaneplantswillcertainlyhelptoassesstheirroleinsugaraccumulationintheinternodes.
Toanalyzethegenesexpressionduringculmdevelopment,acollectionof7,409ESTsfrommaturingsugarcanestemsincombinationwithasmallercollection(1,089)ofESTsfromimmaturestems(Casuetal.2001,2003,2004)wereanalyzedbybio-informaticsandusingcDNAmicro-arrays,allowingfortheidenti?cationofgenesthatweredifferentiallyregulatedwithrespecttostemmaturity.Thesestud-iesindicatedthatgenesassociatedwithsucrosemetabolismwerenotabundantlyexpressedinstemtissuesandthatgenesrelatedtothesynthesisandcatalysisofsucroseweredown-regulatedasthestemmaturedandthesucroseconcentrationincreased.Inasimilarapproach,theuseofsugarcaneGeneChipsfromAffymetrix(Lockhartetal.1996)containingapproximately6,024distinctS.of?cinarumgenes,ledtothediscoverythatgenesinvolvedincellulosesynthesis,cellwallmetabolism,andligni?cationweredevelopmentallyregulatedduringculmmaturation(Casuetal.2007).
Geneactivityassociatedwithinternodedevelopmentwasalsocomparedbetweenanon-sucroseaccumulatinggenotypefromthespeciesS.robustumandtwohighsucroseaccumulatinggenotypes(anS.of?cinarumgenotypeandahybridculti-var)(Wattetal.2005).Usingnylonarrayscontaining88ESTs,geneexpressionvariationsassociatedwithstalkdevelopmentandthosepertainingtosucroseaccu-mulationwereinvestigated.Inmatureinternodesofallthreegenotypes,transcript-relativeactivitieswerefoundtodecreaseforthecellwallbiosynthesisgenesandincreaseforthesucrosemetabolism-relatedgenes.Themostnotabledifferenceswererepresentedbyincreasedactivityofsucrosesynthase-1andsucrosephos-phatase.MatureandimmatureculmsampleswerealsoanalyzedusingcDNAmicro-arrayscontaining1,228elementsincommonwiththearrayusedbyPapini-Terzietal.(2005)(mentionedabove),plusanadditional317elementsincluding229kinasesrepresentativesofthesugarcanekinome.Sucroseaccumulatinginternodes(sinktissues)werecollectedfrom?eldgrownplantscontrastingforBrix.Insomecases,sampleswerecollectedthroughouttheyear(Papini-Terzietal.2007).Genesidenti?edasdevelopmentallyregulatedduringculmmaturationincludedhormonesignaling(auxin,ethylene,jasmonate,salicylicacid),stressresponses,sugartrans-port,ligninbiosynthesisand?bercontent.
Toidentifygenesassociatedwithsucrosecontent,astrategyintroducedbyJansenandNap(2001),whichinvolvedthelarge-scaleanalysisofgeneexpressioninasegregatingpopulation,wasappliedtosugarcanepopulationssegregatedforsolublesolidscontent(Brix).UsingcDNAmicro-arrayscontaining4,715genes,62genes,mostlyunrelatedwithsucrosemetabolism,wereidenti?edasassociatedwithsucrosecontent(Casuetal.2005).Inasimilarwork,cDNAmicro-arrayscontaining1,545elementswereused,andover100genesfoundtobedifferentiallyexpressed