488A.D’Hontetal.
andgenomicinsituhybridization(GISH)(D’Hontetal.2002)clearlyshowthatS.barberiandS.sinensecultivarsaretheresultofinterspeci?chybridizationsbe-tweenrepresentativesofthetwogeneticgroupsoftheSaccharumgenus,S.spon-taneunononesideandS.of?cinarumorS.robustumontheother.SincetheS.barberiandS.sinensecloneshavesweetstalksandtheregionwheretheywereformerlycultivatedisoutsidethenaturaldistributionrangeofS.robustum,thesce-narioofBrandes(1956)providesthesimplestexplanationfortheirorigins:Sof-?cinarumcultivarswereprobablytransportedbyhumanstomainlandAsia,wheretheynaturallycrossedwithlocalS.spontaneumgivingrisetoS.barberiandS.sinenseinIndiaandChina,respectively.Itislikelythattheseclonesareearly-generationhybridsbecauseno,orveryfew,interspeci?cchromosomeexchangesweredetectedusingGISH(D’Hontetal.2002).Thiscontrastswiththeobservationsofhigherlevelsofinterspeci?cchromosomeexchangeinmoderncultivars.TheS.barberiandS.sinensecultivarsthatweretestedhavethemitochondrialhaplo-typeofS.of?cinarum,indicatingthatthisspecieswasthematernalparentandwildS.spontaneumthepaternalparentinthefoundingcrosses(D’Hontetal.1993).LowcopynuclearRFLPsuggeststhateachmorpho-cytogeneticgrouprepresentsasetofsomaticmutantsderivedfromasinglefoundinginterspeci?chybridevent(D’Hontetal.2002).ThePansahigroup,aliasS.sinense,isnotparticularlydistinctfromtheothergroupsaccordingtonuclearRFLPs.TheS.barberiandS.sinensecultivarsarethusallderivedfromsimilarprocessesinvolvinganinterspeci?chybridizationevent,followedbymorphologicalandgeneticradiationthroughmu-tation,whichmayhaveoccurredindifferentgeographicregionsofcontinentalAsia.
FewmoleculardataareavailablefortracingtheoriginofmarginalgroupofS.edule.ThisgroupisgrowninsubsistencegardensfromNewGuineatoFijiforitsedible,abortedin?orescence;itslarge,thick-stalkedcanescontainnosugar.Themitochondrialhaplotypehasbeenestablishedforasingleclone.ItwasthesameastheS.of?cinarum,S.barberi,andS.sinensecultivarsandmostoftheS.robus-tum(D’Hontetal.1993).AnindependentinvestigationbasedonchloroplastRFLPmarkersfromanothercloneledtoasimilarconclusion(Sobraletal.1994).ThesesparsedatasupportthehypothesisthatS.edulecorrespondstoaseriesofmutantclones,whichwereidenti?edinS.robustumpopulationsandwerepreservedbyhumans.
21.2GenomeStructureandMolecularDiversityofModern
Cultivars21.2.1ChromosomeStructure
Theoriginofmodernsugarcanecultivarsiswellknown.However,theirprecisegenomicstructurehasonlyrecentlybeenelucidated,thanksmainlytomolecu-larcytogenetics.Moderncultivarsarederivedfromseveralarti?cialinterspeci?c
21Sugarcane:AMajorSourceofSweetness,Alcohol,andBio-energy489
hybridizationsbetweenS.of?cinarum,usedasthefemale,andS.spontaneumand,toalesserextent,S.barberiasthepollendonor.F1hybridswerethenbackcrossedtoS.of?cinarumtorecoverahigh-sugar-producingtypespecies.Thisprocesswasacceleratedthroughtheselectionofhybridsderivedfromthe2ntransmissionofS.of?cinarumchromosomes(Bremer1961).Allpresent-daycultivarsarederivedfromtheinterbreedingofthese?rstinterspeci?chybrids.Altogether,itisestimatedthat19S.of?cinarumclones(fourwithhighfrequency),afewS.spontaneum(twowithhighfrequency)clones,andoneS.barbericlonewereinvolvedintheseinterspeci?ccrosses(Arceneaux1967).
Moderncultivarsarethushighlypolyploidandaneuploid,withabout120chro-mosomes.GISHstudiesofchromosomepreparationsdemonstratedthat15–25%oftheirchromosomeswereinheritedfromS.spontaneum,andthattherecombinationbetweenhomoeologouschromosomesispossible(D’Hontetal.1996;PiperidisandD’Hont2001;Cuadradoetal.2004).Incultivar‘R570’,forexample,10%ofthechromosomesareinheritedintheirentiretyfromS.spontaneum,80%areinheritedentirelyfromS.of?cinarum,and10%aretheresultofrecombinationbetweenchromosomesfromthetwoancestralspecies.Inaddition,asaconsequenceofthedifferentbasicchromosomenumbersofS.of?cinarumandS.spontaneum,twodis-tinctchromosomeorganizationscoexistincurrentcultivars.ThegenomestructureofatypicalmoderncultivarisrepresentedinFig.21.2.
S. spontaneumS. officinarumrecombinationFig.21.2SchematicrepresentationofthegenomeofmodernsugarcanecultivarsasdeducedfromFISHandGISHexperiments.Moderncultivarsarehighlypolyploidandaneuploidwitharound120chromosomes.Theyarederivedfrominterspeci?chybridizationbetweenS.of?ci-narum(2n=8x=80)andS.spontaneum(2n=5xto16x=40to128).10%to20%oftheirchromo-somesareinheritedintheirentiretyfromS.spontaneum(Greybars);70%to80%areinheritedentirelyfromS.of?cinarum(whitebars)andaround10%aretheresultofrecombinationbetweenchromosomesfromthetwoancestralspecies.Inaddition,asaconsequenceofthedifferentbasicchromosomenumbersofS.of?cinarum(x=10)andS.spontaneum(x=8),twodistinctchromo-someorganizationscoexistincurrentcultivars
490A.D’Hontetal.
Indiploids,genomesizesaregenerallygivenforthe(non-replicated)gameticgenomesize(1Cvalue),whichindiploidscorrespondstothesizeofthebasicsetofchromosomes(monoploidgenome).However,inpolyploidssuchassugarcane,thegameticgenomesize(1C)valuedoesnotcorrespondtothesizeofthebasicsetofchromosomes.Forthisreason,inpolyploidsitseemsmoreappropriatetorefertothegenomesizeof(non-replicated)somaticcells(2C)ortothesizeofthemonoploidgenome.
ThegenomesizeinS.of?cinarum(2n=8x=80)hasbeenestimatedas7.68pg,whichcorrespondsto7440megabasepairs(Mbp)forasomaticcellandto926Mbpforthemonoploidgenome(x=10)(D’HontandGlaszmann2001).ForaS.spon-taneumwith2n=8x=64,thegenomesizehasbeenestimatedas6.30pg,whichcorrespondsto6,080Mbpforasomaticcellandto760Mbpforthemonoploidgenome(x=8)(D’HontandGlaszmann2001).With760to926Mbp,thesizeoftheSaccharummonoploid(basic)genomeisroughlydoublethemonoploidgenomesizeofrice(389Mbp),similartothatofSorghumbicolorMoench(760Mbp),andsigni?cantlysmallerthanmaize(2500Mbp).Thegenomesizeofsomaticcells(2C)ofthetypicalmoderncultivarR570(2n=about115)hasbeenestimatedas10,000Mb(D’Hont2005).
21.2.2MolecularDiversity
LowcopynuclearRFLPanalysisshowedthatmodernsugarcanecultivarsarehighlyheterozygous,presentingmultipleallelesateachlocus(Luetal.1994b).DespitethelownumberofS.offcinaruminvolvedintheinterspeci?chybridizationattheoriginofmoderncultivars,morethan80%ofthemarkerspresentinthewholeS.of?cinarumsamplesstudiedbyJannooetal.(1999a)andSelvietal.(2005)werealsofoundinmoderncultivars.Thisisdueprobablytoahighheterozygosityrelatedtopolyploidy(Jannooetal.1999a).AlthoughthecultivarsappearcloselyrelatedtoS.of?cinarumclones,theminorchromosomecomplementinheritedfromS.spontaneumseemstoconstitutetheprincipalcomponentofcultivardiversity(Luetal.1994b;Jannooetal.1999a).Thesub-tropicalcultivarsseemtohavere-tainedalargernumberofS.spontaneummarkersthanthetropicalcultivars(Jannooetal.1999a;Selvietal.2005),re?ectingtheselectionfordifferentenvironmentsandresultinginnumerousthinnerstalksinsubtropicalregionsandthickerstalksintropicalregions.SSR(microsatellite)markersarenowroutinelyusedinbreedingprogramsforcultivaridenti?cationandprogenyvalidation.
21.2.3LinkageDisequilibrium
Thesmallnumberofmeioticdivisionssincethe?rstarti?cialcrossesthatgaverisetomoderncultivarsprovidedlittleopportunitytorecombinefounderchromosomes.Moreover,therewerenotmanyofthesechromosomes,astherewereonlyafew
21Sugarcane:AMajorSourceofSweetness,Alcohol,andBio-energy491
founderindividualsinvolvedintheoriginofmoderncultivars.Consequently,ahighleveloflinkagedisequilibriumisstillexpectedamongmoderncultivars.ThiswassuggestedbyLuetal.(1994b)andcon?rmedinasampleofMauritiancultivarsinwhichsomechromosomehaplotypesaresigni?cantlyconservedoverregionsaslongas10cM(Jannooetal.1999b).Thisisanimportant?ndingbecauseitmayof-feroriginalandpowerfulperspectivestoidentifyandlocategenesthatareinvolvedintraitsofinterest.Polyploidymaygreatlyhamperthistask,butthehugeleveloflinkagedisequilibrium,comparedtothatestimatedforthehumangenome,forexample,mayoffersomeadvantages.Inparticular,thedensityofmarkersneededforgeneticmappingmaybequitelow.RecentworkbasedonAFLPanddiversityarraystechnology(DArT)arepavingthewaytothistypeofapplication(Raboin,Pauquet,andButter?eldpersonalcommunication).
21.3GeneticMappingandSynteny21.3.1GeneticMaps
Polyploidydictatesparticularconstraintsformapping,whichhavebeentheoret-icallydevelopedbyWuetal.(1992).Whenpolyploidyishighandpairingispolysomicorirregular,suchasinsugarcane,multiplebandsareidenti?edbyaDNAprobeorapairofprimers,andalleleswithdifferentdosagelevelsegregate.Inthiscontext,allelesthatarepresentassinglecopiesaremuchmoreinformativefortheconstructionofgeneticmapsthananyothers.Usingmolecularmarkertech-nologiessuchasRAPD,AFLP,andRFLPmarkers,partialgeneticmapshavebeenproducedforS.spontaneum(daSilvaetal.1993,1995;Al-Janabietal.1993;Mingetal.1998S.of?cinarum(Guimar?esetal.1999;Mingetal.1998S.robustum(Guimar?esetal.1999;Mingetal.1998andmoderncultivars(D’Hontetal.1994;Grivetetal.1996;Hoarauetal.2001;Rossietal.2003;Aitkenetal.2005;Reffayetal.2005;Raboinetal.2006(http://tropgenedb.cirad.fr/).
Co-dominantmarkerssuchasRFLPandSSRcanrevealseveralallelesofthesamelocusandarethusveryusefulintheidenti?cationofco-segregatinggroupsthatcorrespondtohom(oe)ologouschromosomes.Developingasaturated,low-densitygeneticmapforsugarcanerequiresmuchmoreworkthanforadiploid;foragivenlevelofmoleculardiversity,theeffortrequiredtosimultaneouslydistinguishtenorsohaplotypesismuchgreaterthanthatneededtodistinguishonlytwo.Atthemoment,noneofthepublishedgeneticmapsofsugarcaneissaturated.Aboutonehalfofthegenomeisestimatedtobetaggedontothemostre?nedmaps(Rossietal.2003;Aitkenetal.2005).Asformapsofcurrentcultivars,markercoverageisuneven,withS.spontaneumchromosomesbeingcoveredmoredenselythanthoseofS.of?cinarum.
AtmeiosismainlybivalentsareobservedinS.of?cinarum,S.robustum,S.spon-taneum,andinterspeci?ccultivatedclones(reviewbySreenivasanetal.1987).Mappingdatasuggestthatpairingbehaviorprobablydoesnot?tanypre-established,
492A.D’Hontetal.
clear-cutscheme,suchascompletedisomyorcompletepolysomy.InS.robustum(MOL5829,2n=80),ahighproportionofpreferentialpairing(50%)inthefewco-segregationgroupsalreadyde?nedwasreported(Al-Janabietal.1994;Mingetal.1998).InS.of?cinarum,somepreferentialpairingwasalsoobserved,whereasnopreferentialpairingwasfoundinS.spontaneum(Al-Janabietal.1994;Mingetal.1998).InthecultivarR570,Grivetetal.(1996)andHoarauetal.(2001)ob-servedageneralpolysomywithseveralcasesofpreferentialpairingandsuggestedthepossibilityofcompletelocaldisomy.InR570,thepreferentialpairingdetectedconcernedchromosomesofS.of?cinarum,S.spontaneum,aswellasinterspeci?c-recombinantorigin.Jannooetal.(2004)tookadvantageofaparticularsinglecopyprobe(BNL12.06)revealing11allelesbyRFLPincultivarR570.TheydeterminedthedosesofthevariousBNL12.06RFLPallelesamong282progenyofR570andestimatedthemutualpairingfrequenciesamongthecorrespondinghomo-orho-moeologouschromosomesusingamaximumlikelihoodmethod.Theresultisanatypicalpicture,withpairingfrequenciesrangingfrom0to40%anddifferentialaf?nitiesleadingtotheidenti?cationofseveralchromosomesubsets.Ithighlightsacontinuousrangeofpairingaf?nitiesbetweenchromosomesandpinpointsastrongroleofindividualchromosomefeatures,partlyrelatedtotheirancestralorigin,inthedeterminationoftheseaf?nities(Jannooetal.2004).
21.3.2TaggingGenesofInterest
Animportantapplicationofgeneticmapsisthelocationonthegenomeoflocithatcontributetothevariationofphenotypictraits.Onlythreemajorgeneshavebeenmappeduntilnow,tworustresistancegenes(Daugroisetal.1996;Raboinetal.2006)andonegeneresponsibleforstalkcolor(Raboinetal.2006).Quanti-tativetraitloci(QTL)detectioniscomplicatedbythepotentialforsegregationofseveral(potentiallyupto12inamoderncultivar)allelesatalocusandbythelackofpreferentialpairing.Asaconsequence,differentparentalallelesarenotmutuallyexclusivealternatives.Forthesubsetofpolymorphicallelesthatshowsimplexseg-regationratios,theeffectofanallelecanbeestimatedfromtheaveragephenotypicdifferencebetweenthetwopossiblegenotypes(presenceversusabsence).
AQTLexperimentwasconductedwithtwointerspeci?cS.of?narumxS.spontaneumcrossestoinvestigatevariationinthesucrosecontentofthestalkpopulations(Mingetal.2001,2002a).Manyindependentsegregatingalleleswereidenti?edandcouldbeassignedtoeightdistinctloci.Inseveralcases,thepresenceofseveralallelesatalocusthatcontributedtothevariationofatrait,demonstratedthatstronginteractioneffectsbetweenallelescouldleadtoanimportantbufferingeffect.Large-scaleQTLmappingwasalsoconductedforthemoderncultivarR570(Hoarauetal.2002).TheeffectsofindividualQTLsweresmallforallofthetraitsinvestigated,alwaysaccountingforlessthan7%ofthephenotypicvariance,andthesizeoftheeffectswasnotconservedacrosscropcycles.AdditionalQTLexper-imentsusingmoderncultivarswereconducted,revealing,ingeneral,rathersmall