ContentslistsavailableatScienceDirect
Current
Plant
Biology
j ou rn a l h o m ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / c p b
The
repetitive
component
of
the
sunflower
genome
T.
Giordani,
A.
Cavallini,
L.
Natali
∗DepartmentofAgriculture,Food,andEnvironment,UniversityofPisa,ViadelBorghetto80,I-56124Pisa,Italy
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r
t
i
c
l
e
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n
f
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Articlehistory:
Received1February2014
Receivedinrevisedform19May2014
Accepted24May2014 Keywords: Genomecomposition Helianthus LTR-retrotransposons RepetitiveDNA Sunflower
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Thesunflower(Helianthusannuus)andspeciesbelongingtothegenusHelianthusareemergingasa modelspeciesandgenusforanumberofstudiesongenomeevolution.Inthisreview,wereportonthe repetitivecomponentoftheH.annuusgenomeatthebiochemical,molecular,cytological,andgenomic levels.Recentworkonsunflowergenomecompositionisdescribed,withemphasisondifferenttypes ofrepeatsequences,especiallyLTR-retrotransposons,ofwhichwereportonisolation,characterisation, cytologicallocalisation,transcription,dynamicsofproliferation,andcomparativeanalyseswithinthe genusHelianthus.
©2014TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Contents
1. Introduction... 45
1.1. Therepetitivecomponentofplantgenomes... 45
1.2. TheHelianthusgenus ... 46
2. TheHelianthusgenomestructure... 46
3. RepeattypesintheHelianthusgenome... 47
3.1. TheSUNREPdatabase... 47
3.2. LTR-retrotransposons... 48
3.3. Theso-called“Contig61”... 48
3.4. Tandemrepeats... 49
3.5. Otherrepeattypes... 50
4. RetrotransposonsintheevolutionoftheHelianthusgenus... 51
5. Retrotransposonsasmolecularmarkersinsunflower... 51
6. Conclusionsandperspectives... 52
Acknowledgements... 52
References... 52
1. Introduction
1.1. Therepetitivecomponentofplantgenomes
Eukaryotesshowlargevariationingenomesize,especiallyin higherplants.Angiospermgenomesize(1C)rangesfrom63Mbp inGenliseamargaretaeto150GbpinParisjaponica[1,2].Such differ-encesarisefromtwomainprocesses:polyploidyandamplification oftransposons andrelated sequences.Transposonamplification
∗ Correspondingauthor.Tel.:+390502216665;fax:+390502216661.
E-mailaddresses:lucia.natali@unipi.it,lnatali@agr.unipi.it(L.Natali).
has resulted in theaccumulation of many repeatedsequences (i.e., sequences that are identical or similarto sequences else-where in the genome but whose copy number is much larger than that possibly achieved through polyploidisation). Some repeats are considered to be non-functional, whereas others haveplayedkeyrolesintheevolutionofspecies[3].For exam-ple, the mutagenic action of transposons provides substantial increasesingeneticvariability[4].Transposonsalsocreatenovel functions by fine-tuning gene activity, resulting in phenotypic variation [5,6]. Although changes in the repetitive component have played major roles in the evolution of plant genomes, large datasets of repetitive DNA are available for such mono-cots as maize, rice, and barley, and a comprehensive analysis http://dx.doi.org/10.1016/j.cpb.2014.05.001
of the repetitive component is still lacking in many genera of dicots.
Amongdicotyledonousspecies,sunflowers(Helianthusspp.)are emergingasamodelsystemforgenomicresearchonadaptation andspeciation[7,8].Giventheextensivecharacterisationoftheir ecologyandgenomes,sunflowersareverysuitableforestablishing theoccurrenceofdifferenttypesofrepeats,theirtimingof amplifi-cationorreduction,andtherole(s)ofdifferentrepetitivesequences inthemaintenanceofauniquegenome.Exploringthediversityand evolutionarydynamicsoftransposableelementsinthesunflower (H.annuusL.)isofconsiderableimportanceforunderstandingthe evolutionaryhistoryofthis species,givenitslargegenomesize (3500Mbp)[9]andthewell-documentedcasesofretrotransposon amplificationwithinthegenus[10,11].
1.2. TheHelianthusgenus
TheAsteraceaefamilyisthelargestplantfamilyonEarth,with more than 24,000 described species, corresponding roughly to 10%ofallangiosperms[12].Asteraceaespeciesliveinanumber ofdifferentenvironments,includingforests, grasslands,deserts, wetlands, mountain tops, salt marshes, lawns, and agricultural fields[13].Theyincludeeconomicallyimportantcrops, wildflow-ers,valuablemedicinals,invasiveplants,andrangelandweeds[14]. Themostimportantcropisthecultivatedsunflower(H.annuusL.), whichranked14thin2012amongtheworld’sfoodcropsinterms ofareaharvested(http://www.fao.org/).
Helianthusincludes 49species, which arewidespread inthe continentalUnitedStates[15],althoughotherecotypesare assum-ingthestatus of thespecies[16].These speciesdiffer in many phenotypictraits,includingreproductivetiming,branching pat-terns,height[17,18],andespeciallyhabitatpreferences.Insome cases,species coexistinthesameenvironment,and interbreed-ing between species is very common [19], despite large-scale karyotypicdifferences[20,21]andhighlevelsofpollenandseed non-viabilityinthehybrids[22].
The described sunflower species, native to diverse environ-mentsthroughoutNorthAmerica,includeexamplesofallo-and
autopolyploids[23],ecologicallyisolatedsympatricandallopatric species[15],karyotypicallydivergentspecies[20,21,24],allopatric specieswithweakbarrierstogeneflow[15],andseveralhomoploid hybrids[25].Suchdiversityofspeciationmechanismsand barri-erstogeneflowmakesunfloweranidealmodelforunderstanding speciationandspeciesdivergence[26,27].
2. TheHelianthusgenomestructure
Sunflowergenomic DNAwas firststudied bythermal dena-turation and analysis of reassociation kinetics [28,29]. Fig. 1A showsthemeltingprofileandthefirstderivativecurveoftheDNA extractedfromrootsofseedlingsofaselfedsunflowerline.TheTm valueindicatesaGCcontentaround40%.Clear-cutshoulderswere observedbothonthelightandtheheavysidesofthederivative curves,indicatingthatminorspecificDNArepeatfamiliesoccurin thegenome[28,29].
Analysisofthereassociationcurves(Fig.1B)revealedthatthe genomeisorganisedintothreemainfractionsaccordingtotheir redundancy.Highlyrepeated(HR)sequencesaccountfor5%ofthe genome,mediumrepeated(MR)sequencesforaround60%,and theso-calleduniquesequencescomprisetheremaining35%ofthe genome[28,29].Thesameanalyses,performedondifferent sun-flowergenotypes,showeddifferences intherepetitivefractions (eitherHRorMR),reflectingvariationsinthegenomesize[28,29]. BiochemicalanalysesdidnotconsiderDNAsequencebutonly denaturation and reassociation kinetics of DNA. Therefore, the sequence composition of theisolated fractionwas not studied. Moreover,thoseanalysescouldnotevaluatetheoccurrenceinthe “unique”fractionofthegenomeofrareformsofrepeats,suchas retrotransposonremnants,thatwereexcludedfromtherepetitive component.
Therepetitivefractionofthesunflowergenomewas charac-terisedatthemolecularlevelusingaSanger-sequencedsmallinsert library[30].Thatlibraryprovidedafirstsetofsequences(1638,for atotalof954,517bp)thatwereused,incombinationwithslot-blot hybridisationandfluorescentinsituhybridisation(FISH),to ana-lysethecompositionofthegenomeintermsofrepeattypesand
Fig.1.(a)FirstderivativecurvesofthemeltingprofileofsunflowergenomicDNA.SmallshouldersindicatespecificA-TorG-Crichfamiliesofrepeats.(b)Reassociation
kineticsofthesameDNA(circles)andofDNAofE.coli(triangles).Horizontallinesseparategroupsofsunflowersequencesaccordingtotheirredundancy.
Table1
Percentagedistributionofdifferentfunctionalclassesofrepeatsinthesunflowergenome,basedondataofthesmallinsertSanger-sequencedlibrary[30]andmappingdata
ontheIlluminaplus454readsassemblies[40].Theobserveddifferencesaremainlyrelatedtothehugeamountofsequencedatapublishedbetween2010and2013:actually,
thepercentageofsequencesclassifiedasrepeatsbyNatalietal.[40]isnearlytwo-foldthatbyCavallinietal.[30].
Sequencetype Percentageinthesmallinsertlibrary[31] PercentageintheIlluminaplus454assemblies[39]
ClassI(retrotransposons) LTR-Copia 3.54 19.22
LTR-Gypsy 15.57 49.22
LTR-unknown 0.37 9.90
Non-LTR 0.43 0.71
Other/unclassified – 0.48
ClassII DNAtransposons 0.73 2.56
Tandemrepeats RibosomalDNA 1.16 0.35
Othertandemrepeats 0.37 0.60
Unknownrepeats 25.58 7.90
Total(classifiedasrepeats) 47.75 90.94
abundance.About62%ofthesequencesofthelibrarybelongedto therepetitivefraction,whileputativefunctionalgenesaccounted for4%.Thelargestcomponentwasmadeoflongterminalrepeat (LTR)retrotransposons (REs),especially of theGypsy superfam-ily.
Itappeared thatnotransposonfamilywasamplifiedtovery highlevelsinthesunflowerunlikeinotherspecies,inwhich spe-cificfamiliescanaccountforlargefractionsofthegenome[31–34]. Allknowntypesofrepetitiveelementswerefoundinthelibrary, althoughsomeclasses(i.e.,ClassIItransposableelements)were scarcelyrepresented.Onerepeatfamilyofunknownnature(the so-calledContig 61)wasshown tobethemostrepeatedinthe sunflowergenome[30].
DuetodramaticadvancementsofDNAsequencingtechnology in thepastdecade that have madesequencing and assembling a new genome more practical and much less expensive [35], thesunflowergenomeiscurrentlybeingsequenced,despiteits large size [36]. Next-generation sequencing technologies were usedtocharacterisetherepetitivecomponentof thesunflower genome.Statonet al. [37]have analysed approximately25% of thegenomeusing454randomsequencingandshowedthatthe sunflowergenomeis composedofmore than81% transposable elements, 77% of which were LTR-REs. The LTR-REs were also studiedin bacterialartificialchromosomes (BAC) clones, which resulteddisproportionatelycomposedofGypsyLTR-REs contain-ingachromodomain(“chromoviruses”)[38],withthemajorityof theintactchromovirusesshowingchromodomaintandem dupli-cations[37].
Inotherexperiments[39,40],Illuminaand454sequencing tech-nologieswereusedtoproduceawholegenomesetofsequencesof sunflowerbydifferentcomputationalassemblyapproaches. Map-ping a largesample of Illumina reads to this set of sequences (composedof 283,300 contigs)provided a pictureof sunflower genomecomposition[40].ConsideringthatIlluminareadsare sam-pledwithoutbiasforparticularsequencetypes,thepercentageof readsthatmatchedtoarepeatclassshouldindicatetheproportion ofthatclassinthegenome.Mappingresults,and,hence,sunflower genomecomposition,aresummarisedinTable1,inwhicha com-parisontoresultspreviouslyobtainedbyCavallinietal.[30]isalso reported.Thedifferentresultsobtainedfromanalysingthesmall insertlibraryaremainlyrelatedtothehugeamountofsequence datapublishedfrom2010to2013;specifically,thepercentageof sequencesclassifiedasrepeatsbyNatalietal.[40]isnearlytwo-fold thatbyCavallinietal.[30].
MappingresultsconfirmedthatLTR-REswerebyfarthemost abundantclassofsequencesinthesunflowergenome, account-ingforatleast79.53%ofthereadsmatchingthecontigs.Theratio betweenGypsy and CopiaREfrequencies in thewholegenome amounted to 2.29. This ratio is generally species-specific. The GypsytoCopiafrequencyratioisevenhigherinpapaya(5:1)[41], sorghum(4:1)[42],andrice(3:1)[43]comparedtosunflower.In
othercases,suchasinmaize[31]andolive[44],asimilarabundance ofthetwosuperfamilieswasobserved.Ingrapevine,anopposite trendwasfound,withCopiaelementsrepresentingtwo-foldmore thanGypsy[45].
3. RepeattypesintheHelianthusgenome
3.1. TheSUNREPdatabase
Adatabaseofrepetitivesequencesofsunflowerwasobtainedby subdividingthepreviouslydescribedwholegenomesetof assem-bled sequences[40] according totheaveragecoverage of each sequence.Thedatabase(SUNREP)isavailableattheDepartment ofAgriculture,Food,andEnvironmentofPisaUniversitywebsite ( http://www.agr.unipi.it/ricerca/plant-genetics-and-genomics-lab/sequence-repository.html).
The distribution of different sequence types in SUNREP is reportedinTable2.Around11%ofsequencesdidnotfindanyhits inthepublicdatabasesusedforannotation.Amongtheannotated sequencetypes,REsandespeciallyLTR-REswereclearlythemost represented.Ofthese,elementsbelongingtotheGypsysuperfamily were2.3-foldmorerepresentedthanthosebelongingtotheCopia superfamily.
TheassembledsequencesformingSUNREPwerealsoanalysed toestimatetheoccurrenceoffamilies(i.e.,composedofatleasttwo sequences)withineachrepeatclassorsuperfamily.Themost repre-sentedfamily,belongingtotheGypsyLTR-REsuperfamily,included only96sequences,andonlyfourGypsyfamilieswerecomposedof Table2
FunctionalpercentagedistributionofthesequencesintheSUNREPdatabase,as
reportedinNatalietal.[40].
Sequencetype Number(%)
ClassI(retrotransposons) Unclassified 192(0.40) LTR-Copia 8605(17.96) LTR-Gypsy 19,726(41.16) LTR-unknown 5636(11.76) Non-LTR 261(0.54) Pararetrovirus 11(0.02) ClassII(DNAtransposons) Unclassified 373(0.78) Tc1Mariner 5(0.01) hAT 67(0.14) Mutator 101(0.21) PIF-Harbinger 18(0.04) CACTA 64(0.13) Helitron 324(0.68) MITE 382(0.80) Tandemrepeats rDNA 84(0.18) Othertandemrepeat 385(0.80)
Putativegenes 483(1.01)
Unknownrepeats Unclassified 4739(9.89) Contig61-type 957(2.00)
Nohitsfound 5511(11.50)
morethan50sequences.Consideringthe30mostnumerous
LTR-REsfamilies,thevastmajoritybelongedtotheGypsysuperfamily.
Amongthe30mostnumerousDNAtransposons,themostcommon
familiesbelongedtothehelitronclass,followedbyputative
minia-tureinverted-repeattransposableelements(MITEs).Overall,the
numberofsequencescomprisingafamilywasgenerallylow,
con-firmingthattherearenotprominentrepeatfamiliesinthisspecies
[30,37].
3.2. LTR-retrotransposons
ThefirstREsequencefragmentofsunflowerwasisolatedbyPCR usingtheuniversalprimersdesignedfromtheCopia retrotranscrip-tasedomain[46].TheREcomponentofthesunflowergenomewas analysedindetailusingtwosequencesisolatedfromagenomic DNAlibrary,pHaS13andpHaS211:theformerrepresenting por-tionsoftheintegrasegeneofaGypsyretroelementandthelatter correspondingtoanRNase-HgeneofaCopiaretroelement[47,48]. Southernblottingpatternsobtainedbyhybridisingthetwoprobes torestrictedgenomicDNAfromdifferentHelianthusspeciesand fromotherAsteraceaespeciesshowedpHaS13andpHaS211were partsofdispersedrepeatsatleast8and7kbinlength,respectively. Insitu hybridisation experiments showed that sequences of both Gypsyand Copia superfamilies weredispersed throughout allchromosomesinH.annuus[30,47].However,Gypsysequences werelocalisedpreferentiallyatthecentromericregions,whereas Copia sequences were less represented or absent around the centromeres;onlyoneCopiasequencewasplentifulatthe chro-mosomeends.
AlthoughGypsyandCopiaLTR-REsarebyfarthemajor frac-tionofsunflowerrepetitiveDNA,largenumbersofsequenceswere identifiedasLTR-REs,ofwhichthesuperfamilycouldnotbe deter-mined[40,49].Suchelements,calledTRIMsorLARDs[50,51],are non-autonomous,usuallyspecies-specific,andlackanydistinctive protein-codingsequences.
Thelargenumber ofobserved REs indicatestheyhave been activelyreplicatingduringtheevolutionof H.annuus.Thelarge abundanceofGypsyelementscomparedtoCopiacanbeexplained bythreehypotheses,notmutuallyexcluding:(i)Gypsyelements havebeenmoreactiveduringsunflowerevolution;(ii)theyhave beenactivemorerecently,sothataremoreeasilyrecognisableby similaritysearches,havingbeensubjectedtofewermutations;and (iii)theyhavebeenlesspronetoDNAremoval.
TostudyREdynamicsindetail,itisnecessarythatfull-length elementsareavailable.Full-lengthREshaveabuilt-inmolecular clockusefulforestimatingtheirinsertiontimes,basedon sister-LTRdivergence.Infact,whenanREinsertsintothegenome,its LTRsareusually100%identical[52].Mutationsthenoccurwithin thetwoLTRs,andasmoretimepassessincetheinsertion,thelarger thegeneticdistancebetweenLTRs.Hence,theREinsertiontimecan beestimatedusinganucleotidesubstitutionratesuitableforsuch elementsthatisassumedtobehigherthanthatofgeneregions [53–55].
Sunflowerfull-lengthLTR-REswereidentifiedanalysinglarge sequencesbelongingtoBAClibraries[37,49,55].Inparticular,Buti etal.[49]performedafineannotationofthreeBACclones, iden-tifying18full-lengthLTR-REs.Insomecases,REsformednested structures,withoneelementinsertedintoanother,similartothose commonlyfoundinmaize[53].Analysisoftheinsertiontimeof full-lengthREsshowedthattheyinsertedrelativelyrecently(during thepast3millionyears).Gypsyelementsweregenerallyyounger thanCopia,thoughsomeCopiaelementswererelativelyyoung,as well(Fig.2)[49].Statonetal.[37]confirmedandextendedthese findings,showingthat mostintactLTR-REs likelyhave inserted sincetheoriginofH.annuus,providingfurtherevidencethatbiased
Fig.2.DistributionsofCopia,Gypsyandunknownfull-lengthelementsidentifiedin
threesequencedBACclonesaccordingtotheirestimatedinsertionages(millionsof
years,MYRS).Meaninsertionageforeachsuperfamilyisreportedinparentheses.
Redrawnfrom[49].
LTR-REactivityhasplayedamajorroleinshapingtheDNA land-scapeofthesunflowergenome.
That retrotransposition in sunflower has been and is prob-ably still occurringis also indicated by recent studiesshowing that sunflower LTR-REs are transcriptionally active [30,56−58]. Forexample,RT-PCRdatashowedthatbothCopiaandGypsyREs transcriptionoccurredinallanalysedsunflowerorgans(embryos, leaves, roots, and flowers). Transcription was apparently not inducedbyenvironmentalfactorsorcultureconditions.Analyses madeonprogeniesofaselfedlineshowedoneoutof64individuals exhibitedtheintegrationofanewelementintothegenome[57].
Similar results were obtained by Kawakami et al. [58]. In theirtranscriptionalassays,multiplelineagesofGypsyandCopia elements were found to be active in natural populations of H. annuus and H. petiolaris and in their natural interspecific hybrids.Inthesespecies,transcriptionalactivitywasnot associ-atedwithcopy number increases,suggestingtheoccurrence of strongpost-transcriptionalmechanismsofrepressionthatreduce themutagenicpotentialityofREtransposition[59].Infact,REscan inactivategenesafterinsertingwithinornearbygenesequences, asobservedinsunflowergenesencodinga 2-methyl-6-phytyl-1,4-benzoquinonemethyltransferase[60]andalipidtransferprotein [49].
Itis knownthat LTR-REsaresubjectedtobothamplification andremoval[61–63].TheREremovalisdriveninplantsbyDNA rearrangements,illegitimaterecombination,andunequal homol-ogousrecombinationvia anumber ofmechanisms,suchasthe repairofdouble-strandbreaks(non-homologousend-joining)and slipstrandmispairing[33,56,64–67].
The occurrence of unequal homologous recombination typi-callyproducetheso-calledsolo-LTRs.MappingIlluminareadsto asetofintactLTR-REsindicatedtheoccurrenceofnumerous solo-LTRsformanyofthetestedREfamilies(Fig.3).Natalietal.[40] suggestedthatmassiveamplificationoftheseelementsinthe sun-flowergenome waspartly counterbalancedbysubstantial DNA loss,especiallyrelatedtoGypsyelements.However,inotherstudies onsunflowerrepeats,solo-LTRshave beenfoundforCopia ele-ments,aswell[30,37].
3.3. Theso-called“Contig61”
Clusteringanalysisofsequencesintheshortinsertlibrary[30] producedmanycontigsrepresentingrepetitiveDNAsequences.Of these,thelongestcontig(Contig61)wasidentifiedasthemost repeatedinthesunflowergenome,witharedundancyof27,000 copiesperhaploidgenome,estimatedbyslot-blotand hybridisa-tion.
Fig. 3.Distribution of sunflower LTR-retrotransposons according to the ratio
betweenredundancies(measuredby averagecoverage)ofLTRsandinter-LTR
regions(datainclude9retrotransposonsanalysedbyCavallinietal.[30]and19
analysedbyNatalietal.[40]).
ManycontigssharinghighsequencesimilaritytoContig61were alsofoundintheSUNREPdatabase.Mappingthesecontigswitha largesetofIlluminareadsestablishedthatthisrepeataccountsfor 2.81%ofthegenome[40].
Southern analysis confirmed high redundancy of Contig 61 repeats, with heavy labelling and many bands. The use of isoschizomereswithdifferentsensitivities tocytosine methyla-tioninthetargetsiteshowedthatthisrepeatedelementishighly methylated[30].
When hybridising sequences belonging to Contig 61 to metaphasechromosomesofH.annuus,scatteredlabelling through-outallchromosomeswasobserved,indicatingwidedispersalof DNAsequences[30].
The occurrence and redundancy of sequences belonging to Contig 61 were alsostudied in species belongingto thegenus Helianthus,bothannualandperennial,andtootherAsteraceaeby slot-blothybridisation[30].InHelianthus, thehybridisation sig-nalswereasstrongasthoseobservedinsunflower;inperennial species,theywerestrongerthaninannuals.Theseresultsindicate thatamplificationofthissequenceshouldhaveoccurredinthe pro-genitoroftheHelianthusgenus,and,aftersplittingbetweenannuals andperennials,amplificationhasoccurredintheperennial ances-torand/orlossofsequenceshasoccurredintheannualancestor. RegardingtheotherAsteraceae,someofthesequencesbelongingto Contig61alsoshowedstronghybridisationsignalstoViguiera mul-tifloragenomicDNA.ThegenusViguieraisconsideredtheclosest relativetoHelianthus.BecauseContig61isalsoapparentlyhighly repeatedinViguiera,itispresumablethattheinitialamplification ofthisrepeatpredatestheoriginofthegenusHelianthus.
Sequence analysis of Contig 61 and of related sequences obtained after Illumina and 454 assemblies revealed no struc-turalfeaturethatcouldhelpintheclassificationofthisfamilyof sequences,which,therefore,awaitsfurthercharacterisationand whosenatureremainsunknown.
3.4. Tandemrepeats
Onlyafewtandemrepeatshavebeencharacterisedinthe sun-flowergenome.Infact,onlyasmallnumberofcontigscontaining
tandemrepeatswerefoundinSUNREP.Mappingthesecontigswith alargesetofIlluminareadsestablishedthatribosomalDNA(rDNA) andothertandemrepeatsaccountforonly0.35%and0.60%ofthe genome,respectively[40].
TherDNAwaslocalisedinchromosomesbyinsitu hybridisa-tion[68].Fourchromosomepairswerelabelledattheendoftheir shortarm.Ofthese,threepairs showedsecondaryconstrictions andstronghybridisationsignals.Twoothersignalswere puncti-formattheendoftheshortarmofachromosomepairwherea satellitewasneverobserved.Thesefindingsareinagreementwith thoseobtainedbySchraderetal.[69]regardingthenumberof satel-litepairsandthosethatbearrDNAbutcontrastwiththeirresults regardingthelabellingintensity,sincetheyobservedfourstrong andfourweaksignals.
Two other tandem repeats, the sequences HAG004N15 and HAG002P01,were isolatedin thesmallinsertlibrary and char-acterised.HAG004N15was1071bpinlengthandwasmadeup of three tandemly arranged repeats having a lengthof 368bp, which falls into the typical range of satellite DNAs [70]. The CAAAAorGAAAAmotifs,previouslyassociatedwiththe amplifi-cationofrepeatedsequences[71,72],werefoundbetweenrepeats. SouthernhybridisationwithHAG004N15asaprobeproducedthe expectedladderpatternafterdigestionofgenomicDNAwith cyto-sinemethylation-sensitiveenzymes,showingthatthissequenceis highlymethylated.Itscopynumberperhaploid(1C)genomewas estimatedtobe7800.
AfterinsituhybridisationofHAG004N15repeats[68],allthe chromosomes were labelled at discrete regions (Fig. 4). Using an image analyser, the labelling was precisely localised in the metaphasechromosomes, despite theirnumber (2n=34),small size, and similar morphology (Fig. 4). The FISH signals were observedattheendsofbothchromosomearmsinfourpairsandat theendofonlyonearmineightotherpairs.HAG004N15repeats werenotfoundinthecentromericchromosomalregions,exceptfor onepair,wherethehybridisationsignalsreachedthecentromere intheshortarm,which wasentirelylabelled.Signalswerealso observedattheintercalary(mostlysub-telomeric)regionsinall thepairs,inbotharmsineightpairs,andinonlyonearminthe otherninepairs.
ThechromosomallocalisationofrDNAandHAG004N15repeats formedapatternthatallowedallofthecomplementpairstobe distinguishedfromeachother[68].Thishybridisationpatternwas thesameindifferentsunflowergenotypes.
Theredundancy ofHAG004N15 and thechromosomal local-isationofHAG004N15 andofrDNAwerealsostudiedinannual andperennialspeciesofHelianthusandinotherAsteraceae[73]. HAG004N15sequences,whichdidnotshowamplificationinother AsteraceaeexceptViguieramultiflora, wereredundantin allthe Helianthus species tested, but theirfrequency wassignificantly higherinperennialscomparedtoannuals.Thesesequenceswere locatedattheendsandintercalaryregionsofallchromosomepairs ofannualspecies.Asimilarpatternwasfoundintheperennials,but ametacentricpairintheircomplementwasnotlabelled.Ribosomal geneswerecarriedontwochromosomepairsinperennialsandon threepairsinannuals,exceptforH.annuus,whererDNAlociwere onfourpairs.Thesefindingssupportthehypothesisthatthe sep-arationbetweenannualandperennialHelianthusspeciesoccurred throughinterspecifichybridisationinvolvingatleastonedifferent parent[74].However,genomicinsituhybridisationinH.annuus (usingDNAfromtheperennialH.giganteusasblockingDNA)failed torevealdifferentgenomicassetsinannualandperennialspecies. The othertandemrepeat isolatedin thesmall insertlibrary [30],HAG002P01,wasestimatedtobepresentin2600copiesper haploid genome.Using this sequenceas aprobe, FISHrevealed an interspersed distribution withpossible enhanced hybridisa-tionatthecentromericregionsofmanychromosomes,indicating
Fig.4. (a–c)MetaphaseplateofH.annuuslineHA89afterDAPIstaining(a)andhybridisationwithHAG004N15repeats(b;fluorescein)orpTa71ribosomalprobe(c;Cy3).
Numeralsindicatemembersofeachchromosomepair.Bar=10m(fromCeccarellietal.[68],CharacterisationofthechromosomecomplementofHelianthusannuusby
insituhybridisationofatandemrepeatedDNAsequence.Genome,50:429–434,©CanadianSciencePublishingoritslicensors);(d)Idiogramofthehaploidchromosome
complementshowingthedistributionofHAG004N15-relatedtandemrepeats(yellow)andofribosomalcistrons(red).Thechromosomesaresubdividedintotwogroups,
fouracrocentricandthirteenmeta-tosubmetacentricpairs,andarrangedwithineachgroupaccordingtothetotallength,accordingtoRaicuetal.[88]classification.(For
interpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
thatHAG002P01mayhavepreferentialparacentromeric localisa-tion.
3.5. Otherrepeattypes
Thenon-LTR-REswerepoorlyrepresentedintherepetitive frac-tionof the sunflowergenome, as frequentlyobserved in other plants. Putative DNA transposons accounted for a number of sequences.However,afractionofsuchsequenceswereclassified asDNAtransposonsonlybyvirtueofasequencesimilaritytothe shortdomainofthetransposasegene.AmongDNAtransposons,a prevalenceofMITEs[75]andhelitrons[5]wasobserved[40].
Interestingly, according to BLAST analysis, many repetitive sequences of the SUNREP database showed similarity to puta-tive protein-coding genes (Table 2) [40]. The most redundant gene family encodes the NBS-LRR class of proteins, receptors that recognisehighly variable pathogen effectors[76].Another redundantgenefamilyencodeDNAJproteins,whichfunctionin associationwithHsp70molecularchaperonestofacilitateprotein
folding and play an active role in regulating normal cellular events like protein degradation, morphogenesis, and cell cycle progression[77].Thethirdredundantgenefamilyisvery heteroge-neous,encodingproteinswithunspecifiedprotein-kinasedomains that areinvolved in the transductionof signals tobinding fac-tors,centromeres,andothereffectors.Inadditiontonon-specific kinases,serine/threonine/tyrosinekinasesalsowerefoundamong redundantgenefamilies.Finally,anotherredundantgenefamily encoding F-boxmotif-containing proteinswasidentifiedbythe presenceofproteininteractiondomainsthatbindubiquitination targets[78].
The occurrence of putative genes among repetitive DNA sequencesoftheSUNREPdatabasedeservesadditionalcomments. Insomecases,suchsequencesshowedsimilaritytogenefamilies alreadyknowntoberepeatedinplantgenomes,suchasNBS-LRR genes[76].In othercases,it islikely thatgeneportions encod-ingfunctionaldomains,andnotcomplete,full-lengthgenes,are apparentlyredundant.Forexample,F-boxproteinsincludealarge varietyoftypesthatshareashortmotif[78].Italsocouldbethatfor
otherassembledcontigs,agene(oragenefragment)liescloseto arepeatedsequence,andtheredundancyofthatcontigisrelated totherepeatedsequenceandnottothegenesequence.Itshould benotedthatSUNREPiscomposedofarelativelyhighnumberof putativehelitrons(Table2)knowntoincludeexonfragmentsin theirsequences[5],whichmightbepartiallyresponsibleforthe relativelyhighfrequencyofgenefragmentsinthedatabase.
4. RetrotransposonsintheevolutionoftheHelianthus genus
Southern blotting patterns obtainedby hybridising the first isolated Gypsy and Copia RE fragments (pHaS13 and pHAS211, respectively) [47] to restricted genomic DNA from different HelianthusspeciesandfromotherAsteraceaeshowedthatthese elementswereconservedinallHelianthusspeciesstudiedand,to alesser extent,in Tithoniarotundifolia,a speciesbelongingtoa genusstrictlyclosetoHelianthus[47,48].Comparablehybridisation patternswereobtainedinallHelianthusspeciesusingtheGypsy pHaS13asa probe.Conversely,thepatternsobtainedby hybri-dising theCopia pHAS211 clearly differentiated annual species fromperennials[47],attestingadifferentamplificationhistoryof thetwo superfamiliesof LTR-REsin theevolution ofthegenus Helianthus[30].
Asobserved in H.annuus, FISH analysis of Gypsy and Copia sequencesconfirmedscatteredlabellingthroughoutallmetaphase chromosomesalsoindifferentspeciesofHelianthus[48].However, preferentiallocalisationofGypsysequencesatcentromeric chro-mosomeregions wasobservedin allofthespecies.Conversely, Copiasequencesshowedpreferentiallocalisationatthe chromo-someendsonlyinH.annuus.
Theevolution of therepetitivecomponent in theHelianthus genusandinotherAsteraceaewasstudiedbycomparativeanalysis ofthehybridisationofgenomicDNAsisolatedfromthesespecies tothesunflowersmallinsertlibrary(Fig.5),whichrevealedsome similarityonlybetweenHelianthusspeciesandViguieramultiflora. RegardingREs,comparablehybridisationpatternswereobserved amongHelianthusspecies,whilenohybridisationoccurredusing otherAsteraceae.SuchresultsindicatethespecificityofREfamilies totheHelianthusgenus.
AninterestingexampleofhowREshaveaccompaniedthe evo-lutionofspecieswithinthegenusHelianthusconcernsthemassive amplificationoftransposableelementsafterinterspecific hybrid-isationoccurredinthree species ofsunflowers, H.anomalus,H. deserticola,andH.paradoxus[10].Allofthemoriginatedby hybrid-isationbetweentwoancestralspecies,H.annuusandH.petiolaris
Fig.5. ExamplesofhybridisationpatternsoflabelledgenomicDNAfromdifferent
speciesto36clonesofthesunflowersmall-insertlibrary,spottedinanon-regular
duplicatearrangement.Forfourclones,theputativeannotationisreported:forthree
ofthese,hybridisationisobservedintheDNAofthetwoHelianthusspeciesandin
Viguieramultiflora;rDNAcontainingcloneislabelledalsoinXanthiumstrumarium
DNA.Forthetechnicalproceduresee[30].
[25]andhavethesamechromosomenumber(n=17)andgenome sizesatleast50%largerthantheirparentalspecies.Suchgenome sizeincreaseispartiallyexplainedbyproliferationofGypsyand,to alesserextent,ofCopiaLTR-REsinhybrids[10,11].
In particular,Gypsy sequenceswerefirst shown tobemuch moreredundantinthethreehybrids[10].Ungereretal.[79]later demonstratedthatGypsyREsexistasmultiple,well-supported sub-lineagesinboththeparentalandhybridderivativespeciesandthat thesesequencesunderwentproliferationineachhybridspecies’ genome,occurringapproximately0.5–1millionyearsago.
In contrastto Gypsy,theburst of transpositionof CopiaREs variedamonghybridspeciesandwasmostpronouncedinH. para-doxus,aspeciesadaptedtosalineenvironments(H.anomalusand H.deserticolaarefoundindesert-likehabitats).Althoughexternal stressfactorsmaycontributetode-repressionoftransposable ele-ments[80],theassociationbetweenhabitatandREfrequencymay bepurelycoincidental.CopiaRElineagesunderlyingtheburstare ancientandpredatetheoriginofthesunflowergroup.However, themajority(70%)ofsequenceswerederivedfromasinglelineage ofelements,whichimplicatesarecentproliferationevent[79].
5. Retrotransposonsasmolecularmarkersinsunflower
TheLTR-REsinplantgenomeshavebeenemployedto gener-atemolecularmarkers [81,82].In fact,theubiquity,abundance, dispersion,anddynamismof theseelementsmakethem excel-lenttoolstoexploregeneticvariabilityevenwithinspecies[83]. ThemethodsgenerallyrelyonPCRamplificationbetweena con-served RE feature, most often the LTR, and another abundant,
Fig.6. IRAPfingerprintsobtainedwithLTR-Copiaspecificprimers[86]ineight
individuals(A–H)belongingtoawildaccession(USDA-PI597907)ofH.annuus.
Molecularweightmarker(M,GeneRulerDNALadderMix,Fermentas)wasalso
dispersed,conservedfeatureinthegenome.Thissecondsitemaybe arestrictionsiteadapterinsequence-specificamplified polymor-phism(SSAP)[84],amicrosatelliteinRE-microsatelliteamplified polymorphism(REMAP)[85],oranotherREininter-REamplified polymorphism(IRAP)[85].Thesemethodsidentifygeneticvariants relatedtotheinsertion/lossofaretroelementortoDNAsequence variations(nucleotidesubstitutionsorindels),whicharecommon withinREsbecauseoftheerror-pronemodeofretrotranscription usedbytheseelementsand extensiveDNA methylationwithin RE-richgenomeregions.
TheIRAPprotocolwasappliedwithinthegenusHelianthusto assessintraspecificvariabilitybasedonREsequencesamong 36 wildaccessionsand 26 cultivars of H.annuus and interspecific variability among 39 species of Helianthus [86]. Two groups of LTRs,onebelongingtoaCopiaretroelementandtheothertoan REofunknownnature,wereisolatedandsequenced,andprimers weredesignedtoobtainIRAPfingerprints.AnexampleofIRAP fin-gerprintsin plants ofa wildaccessionof H.annuus isreported in Fig.6. Thenumber ofpolymorphic bands and RE variability amongH.annuuswildaccessionsareashighasamongdifferent Helianthusspecies.Conversely,RE-relatedvariabilitywasreduced amongdomesticatedH.annuus.
LargeRE-relatedvariability wasalsofoundamongsunflower inbredlines[87].Itwasobservedthatbetween-linegenetic dis-tance correlated to heterosis in hybrids between those lines, suggesting that variations in the repetitive component of the genome,especiallyLTR-REs,affectthedisplayofheterosis.
6. Conclusionsandperspectives
Repetitive DNA and especially transposons apparently have a central role in the biology and evolution of plants. These sequencescanalsobeemployedtogeneratemolecularmarkers that have proved efficient in distinguishingeven similar geno-types.
TherepetitivecomponentoftheH.annuusgenomehasbeen studiedatthebiochemical, cytological,molecular,and genomic levels.Data are now availablethat indicate a genomemade of alargenumberof LTR-REs,especiallyof theGypsy superfamily. TheLTR-REs areapparently expressed, and theREburstseems tobe unexhausted as shown by therelatively recent insertion timesofmany REs.It isalsoworthnoting thelargenumber of LTR-REsofunknownnature,whichhavebeen(andpresumably are) active because of enzymes produced by intact retroele-ments.
Theconsequencesof suchREmobilisationareevident when observing theevolution of Helianthus interspecific hybrids that probablyhavecompletedtheirspeciationbecauseofchromosomal differentiationfromparentalspeciesduetoinsertionsof transpos-ableelements.
Manydataarealsoavailableonotherrepeatedsequences,such ashelitrons,whoseredundancyisrelativelyhighcomparedtothat foundinotherspecies.Allthesedatacanbecombinedtoproduce acollectionofsequencesthatwillbeusefulintheannotationof thesunflowergenomesequencewhenitiscomplete.Untilthen, theenormousmassofavailabledatawillbeusefulforcomparative studiesongenomeevolutioninagenus(Helianthus)thathasbeen studiedattheevolutionaryandecologicallevelsandespeciallyfor itsprotein-codingcomponents.ThecompletionoftheH.annuus wholegenomesequenceinconjunctionwiththeavailabilityofnew sequencingtechniquesallowingtheproductionofverylargeDNA sequences(i.e.,Pac-Biotechnology)willbeusefulforcomparative studiesamongHelianthusspeciesandaccessionsandforclarifying manyaspectsofgenomeevolutioninthisgenus.
Acknowledgements
ThisworkwassupportedbyPRIN-MIUR,Italy,Project “SUN-REP:caratterizzazionemolecolaredellacomponenteripetitivadel genomadigirasole”.
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