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Finemappingofui6.1,agametophyticfactorcontrollingpollen‐sideunilateral1
incompatibilityininterspecificSolanumhybrids2
3
WentaoLi*,SuzanneRoyer§,RogerT.Chetelat*4
5
*C.M.RickTomatoGeneticsResourceCenter6
Dept.ofPlantSciences7
OneShieldsAve.8
Davis,CA956169
10
§DepartmentofBiology11
ColoradoStateUniversity12
FortCollins,CO8052313
14
15
Genetics: Published Articles Ahead of Print, published on May 3, 2010 as 10.1534/genetics.110.116343
2
Runningtitle:unilateralincompatibilityinSolanum1
2
Correspondingauthor:3
RogerChetelat4
Dept.ofPlantSciences(stop3)5
OneShieldsAve.6
Davis,CA956167
tel.530‐752‐67268
fax.530‐752‐96599
11
Keywords:Solanumlycopersicum,Solanumlycopersicoides,Solanumpennellii,unilateral12
incongruity,self‐incompatibility13
14
15
3
Abstract1
Unilateralincompatibility(UI)isaprezygoticreproductivebarrierinplantsthat2
preventsfertilizationbyforeign(interspecific)pollenthroughtheinhibitionofpollen3
tubegrowth.Incompatibilityoccursinonedirectiononly,mostoftenwhenthefemale4
isaself‐incompatiblespeciesandthemaleisself‐compatible(the‘SIxSCrule’).Pistils5
ofthewildtomatorelativeSolanumlycopersicoides(SI)rejectpollenofcultivated6
tomato(S.lycopersicum,SC),butacceptpollenofSolanumpennellii(SCaccession).7
Expressionofpistil‐sideUIisweakenedinS.lycopersicumxS.lycopersicoideshybrids,8
aspollentuberejectionoccurslowerinthestyle.Twogametophyticfactorsare9
sufficientforpollencompatibilityonallotriploidhybrids:ui1.1onchromosome1(near10
theSlocus),andui6.1onchromosome6.Wereporthereinafine‐scalemapoftheui6.111
region.Recombinationaroundui6.1wassuppressedinlinescontainingashortS.12
pennelliiintrogression,butlesssoinlinescontainingalongerintrogression.More13
recombinantswereobtainedfromfemalethanmalemeioses.Ahigh‐resolutiongenetic14
mapofthisregiondelineatedthelocationofui6.1toapproximately0.128mapunits,or15
160kb.Identificationoftheunderlyinggeneshouldelucidatethemechanismof16
interspecificpollenrejectionanditsrelationshiptoself‐incompatibility.17
18
INTRODUCTION19
Floweringplantshaveevolvedseveralreproductivebarriersforpreventing20
illegitimatehybridizationwithrelatedspecies.Thesebarriersmaybeexpressedpre‐21
fertilizationand/orpost‐fertilization.Unilateralincompatibilityorincongruity(UI)isa22
pre‐fertilizationbarrierthatoccurswhenpollenofonespeciesisrejectedonpistilsofa23
4
relatedspecies,whilenorejectionoccursinthereciprocalcross(deNettancourt1977).1
Intheory,unilateralincompatibilityshouldreinforcespeciesidentityinnatural,2
sympatricpopulationsofrelatedtaxa.Thisbarrieralsoimpedestheeffortsofplant3
breederstotransfertraitsfromwildspeciesintorelatedcropplants.Forexample,the4
transferofcytoplasmictraitsfromspecieswithmaternally‐inheritedchloroplastsand5
mitochondriamaybepreventedbyunilateralcrossingbarriers.Nuclear‐encodedtraits6
mayalsobeinaccessibleifF1interspecifichybridsarebothmalesterileand7
incompatibleasfemaleparents(Chetelatetal.1989).8
IntheSolanaceae,unilateralincompatibilityisobservedincrossesbetween9
cultivatedtomato(Solanumlycopersicum,formerlyLycopersiconesculentum)andsome10
relatedwildspecies.Ingeneral,pistilsofthecultivatedtomatoactasa‘universal11
acceptor’,inthattheyfailtorecognizeandrejectpollenofothertomatospecies.Inthe12
reciprocalcrosses,pollenofS.lycopersicumisrejectedonstylesofvirtuallyallofthe13
green‐fruitedspecies,butnotonstylesofotherredororange‐fruitedspecies(reviewed14
byMutsclerandLiedl1994).Thispatternismostlyconsistentwiththe‘SIxSC’rule,15
whereinpollenofself‐compatiblespecies(includingcultivatedtomato)arerejectedon16
pistilsofself‐incompatible(SI)species,butnotinthereversedirection(Lewisand17
Crowe1958).ExceptionstotheSIxSCruleinthetomatocladeincludespeciesor18
populationsthathavelostself‐incompatibilitybutretaintheabilitytorejectpollenof19
tomato.ThisisthecaseforthefacultativeoutcrossingspeciesS.chmielewskii,the20
autogamousS.neorickii(formerlyL.parviflorum),aswellasmarginalSCpopulationsof21
normallySIspeciessuchasS.pennelliiandS.habrochaites(formerlyL.hirsutum).AnSC22
accessionofS.pennellii,LA0716,isexceptionalinhavinglosttheabilitytorejectself23
5
pollen,whileretainingtheabilitytoserveaspollenparentonstylesofSIaccessionsof1
thisspecies(andotherSIspecies,includingS.peruvianumandS.lycopersicoides)2
(Hardon1967;Quirosetal.1986;Rick1979).Inthisregard,S.pennelliiLA07163
conformstotheLewisandCrowemodelinthatitbehaveslikeatransitionalform4
lackingSIfunctioninthepistilbutnotinthepollen.5
Unilateralincompatibilitymayalsooccurincrossesbetweenpopulationsor6
racesofasinglespecies.InS.habrochaitesforexample,pollenfromSCbiotypeslocated7
atthenorthernorsouthernmarginsofitsgeographicrangeisrejectedonpistilsofthe8
central,SIpopulations(Martin1961,1963).Furthermore,pollenfromthenorthernSC9
groupisrejectedbystylesofthesouthernSCpopulations.YetpistilsofbothSCbiotypes10
areabletorejectpollenofcultivatedtomato.Thusthereappeartobeareatleast311
distinctunilateralcrossingbarriers,justwithinS.habrochaites,possiblyindicating12
differentpollentuberecognitionandrejectionsystems.TheF1NxShybridisSC,as13
expected,butSIplantsarerecoveredintheF2generation,suggestingthatthelossofSI14
occurredviaindependentmutationsinthenorthandthesouth(RickandChetelat15
1991).16
InterspecificF1hybridsbetweenSIwildspeciesandSCcultivatedtomatoare17
self‐incompatibleandrejectpollenofcultivatedtomato,indicatingbothtraitsareat18
leastpartiallydominant(Hardon1967;Martin1963;McGuireandRick1954).19
Interestingly,pollenoftheF1hybridsisincompatibleonpistilsofthewildspecies20
parent(i.e.includingotherindividualsofthesameaccessions,butwithnon‐matchingS‐21
alleles).Thisobservationsuggeststhattherearedominantfactorsfromcultivated22
tomatothatleadtopollenrejectiononstylesofthewildspecies,regardlessofthe23
6
pollengenotype.Thisapparentsporophyticeffectcontrastswiththepurely1
gametophyticnatureofpollenS‐specificityintheSolanaceae(deNettancourt1977).2
Earlystudiesoftheinheritanceofunilateralincompatibilityintomatosuggest3
theinvolvementofseveralgenescontrollingthepistilresponse,howeverthegenetics4
ofpollenresponseshavereceivedlittleattention.InF2S.habrochaites(NorthernSC5
accession)xS.habrochaites(CentralSIaccession),therejectionofpollenfromtheSC6
parentsegregatedasifcontrolledbyonetotwodominantgenesfromtheSIaccession7
(Martin1964).IncrossesofS.lycopersicumtobothSIandSCaccessionsofS.pennellii,8
theintra‐andinterspecificcrossingrelationswerelargelyconsistentwiththeLewis9
andCrowe(1958)modelofstepwisemutationattheSlocus(Hardon1967);therewas10
alsoevidenceofasecondbarrierintheSCS.pennelliiaccession.InF1andBC1hybrids11
ofS.lycopersicumxS.habrochaites,thesegregationofunilateralandself‐12
incompatibilitieswasconsistentwiththeactionoftwomajorgenes,withminor13
polygenesindicatedaswell(Martin1967).Morerecently,severalQTLunderlying14
pistil‐sideunilateralandself‐incompatibilitiesweremappedinBC1S.lycopersicumxS.15
habrochaites(BernacchiandTanksley1997);themajorQTLforbothformsofpollen16
rejectionwaslocatedatorneartheSlocusonchromosome1,whichcontrolsSI17
specificity(TanksleyandLoaiza‐Figueroa1985).18
Thereislittledataonpollen‐sideunilateralincompatibilityfactorsinthetomato19
clade,oranyothersystem.Ourpreviousworkidentifiedtwotothreegeneticlocifrom20
S.pennelliithatarerequiredforpollentoovercomeincompatibilityonpistilsofS.21
lycopersicumxS.lycopersicoidesorS.lycopersicumxS.sitienshybrids(Chetelatand22
DeVerna1991;Pertuzeetal.2003).OneofthesefactorsmappedtotheS‐locus,the23
7
othertwowereonchromosomes6and10.Inthissystemthefemaletesterstockswere1
eitherdiploidorallotriploidhybrids,thelattercontainingonegenomeofthewild,SI2
parent,plustwogenomesofcultivatedtomato;bothtypesofhybridsrejectpollenof3
cultivatedtomato.ThepollenparentswereeitherF1S.lycopersicumxS.pennelliior4
bridginglinesdevelopedbybackcrossingtheF1tocultivatedtomatoandselectingfor5
theabilitytoovercomestylarincompatibility.Intheprogeny,distortedsegregation6
ratioswereobservedinwhichtheS.pennelliialleleswerepreferentiallytransmitted,7
thusindicatinglinkagetogametophyticfactorsrequiredforacompatiblereaction.8
Thisexperimentalsystemhasseveraladvantagesfordetectingpollen9
(gametophytic)unilateralincompatibilitygenes.First,pollen‐expressedfactorsare10
readilydistinguishedfrompistilfactorsbecauseonlytheformershowlinkagetoS.11
pennelliispecificmarkers.Second,pollenrejectionisbyunilateral,notself‐12
incompatibility,sincebothspeciescontributingtothepollengenotype,S.lycopersicum13
andS.pennellii,areSC.Finally,aswedescribeherein,therejectionoftomatopollenby14
pistilsoftheinterspecifichybridsisweakenedbythedecreasingdosageoftheS.15
lycopersicoidesgenome,whichreducesthenumberofpollenfactorsrequiredfor16
compatibility.Thus,thegametophyticfactorsonchromosomes1and6(denoted17
hereinafterui1.1andui6.1),whenpresentinthesamepollen,aresufficientforfull18
compatibilityonpistilsofallotriploidinterspecifichybrids,whereastheyconferonly19
partialcompatibilityondiploidhybrids.20
Ouroverallobjectivesaretoidentifythegenesunderlyingboththechromosome21
1andchromosome6pollen‐specificunilateralincompatibilityfactorsfromS.pennellii22
8
andtodeterminethenatureoftheirinteraction.Towardsthisgoal,wereportherein1
thehigh‐resolutiongeneticandphysicalmappingoftheui6.1region.2
3
MATERIALSANDMETHODS4
Plantmaterials:TheparentalspeciesandaccessionsusedwereS.lycopersicum5
cultivars‘VendorTm‐2a‘(accessionLA2968),‘UC‐82B’(LA2801),and‘VF‐36’(LA0490),6
S. pennellii (LA0716), and S. lycopersicoides (LA1964, LA1991 and LA2951). Seed of7
these lines was obtained from the C.M. Rick Tomato Genetics Resource Center8
(http://tgrc.ucdavis.edu).Anallotriploidinterspecifichybrid(GH266,Ricketal.1986),9
containing two genomes of S. lycopersicum (cv. UC82B) and one genome of S.10
lycopersicoides (LA1964),was used as a female tester line to detect pollen unilateral11
incompatibility factors(Fig.1, ‘LLS’).Forcomparisonpurposes,pollinationtestswere12
alsocarriedoutwithpistilsofthe2xF1interspecifichybrid(90L4178=S.lycopersicum13
VF36xS. lycopersicoidesLA2951),andpureS. lycopersicoides(LA1991).Ourprevious14
studies(ChetelatandDeVerna1991)indicatedthattwopollenfactorsfromS.pennellii,15
located on chromosomes 1 and 6, were required and sufficient for overcoming16
incompatibility on styles of the allotriploid hybrid. The chromosome 1 factor, ui1.1,17
mappedclosetotheSlocuscontrollingself‐incompatibility.Thechromosome6factor,18
ui6.1,waslocatedontheshortarmofchromosome6.Thephenotypesofrecombinants19
aroundui6.1weredeterminedbytestpollinationsontotheallotriploidhybrid(Fig.1).20
Tofinemapui6.1, twoF2populationsweredevelopedfromS.pennellii‐derived21
bridging lines, obtained after several backcrosses from the wild species to S.22
lycopersicum (cv.s Vendor or UC‐82B) (Chetelat and DeVerna 1991, Fig. 1). Initial23
9
mappingofui6.1wasbasedonamappingpopulation (denoted ‘F2‐a’)of1,167plants1
derived from BC7F2 bridging lines heterozygous at both the ui1.1 and ui6.1 loci. To2
simplifyphenotypingatui6.1,asecondpopulation(‘F2‐b’)of1,920plantswasobtained3
from F2‐a individuals that were homozygous for the S. pennellii allele at ui1.1 and4
heterozygousatui6.1.5
Severe recombination suppression was observed in these two mapping6
populations.Toincreaserecombinationrates,wecreatedaheterozygoussubstitution7
line (SL‐6), carrying a nearly intact (~80 cM,VanWordragen et al. 1996)S. pennellii8
chromosome6 in thebackgroundof S. lycopersicum (Fig.6). TheheterozygousSL‐69
was then crossed as female (BC‐♀) or male (BC‐♂) to a bridging line which was10
homozygousfortheS.pennelliialleleatui1.1,andhomozygousfortheS. lycopersicum11
allele at ui6.1. This provided independent estimates of recombination frequency in12
femaleandmalegametes.13
Pollinationsandpollen tubegrowthstudies:All crosseswereperformed in14
the greenhouse using standard pollination techniques. The compatibility or15
incompatibilityofcrosseswasdeterminedbyvisualizingpollentubeswithinpistilsof16
the allotriploid hybrid 24 hr after pollination. The stainingmethodwas according to17
Martin(1959),exceptthatpistilswerefixedinethanol:glacialaceticacid(3:1)solution18
(insteadofFAA).Afterrinsingthreetimesinwater,pistilsweretransferredto8NNaOH19
for8hrtoclearandsoftenthetissue.Aftertriplerinsinginwater,thesoftenedpistils20
weresoaked inanilinebluesolution(0.1%anilineblue in0.1NK3PO4) for8to24hr.21
Observation and measurements of pollen tubes were carried out under UV light22
fluorescencemicroscopyusingaZeissAxioskop.Pollinationswerejudgedincompatible23
10
when the growth of all pollen tubes was arrested in the upper half of the style. In1
contrast, compatible pollinations resulted in at least some pollen tubes reaching the2
ovaries. For each cross, the length of the longest pollen tubes was measured and3
expressedasapercentageofthestylelength(fromstigmasurfacetobaseofstyle).At4
least threepistilswereexaminedpercross, fromwhich theaveragemaximumpollen5
tubelengthwascalculated.6
For imaging, pistilswere fixed in 3:1 ethanol : glacial acetic acid for 24 hr or7
more.Followingarinsewithdeionizedwater,thepistilsweresoakedovernightin10N8
NaOH,rinsedthreetimeswithdeionizedwater,andimmersedina0.005g/mlsolution9
ofAnilineBlueFluorochrome(BiosuppliesAustralia) in0.1MK2HPO4buffer,pH8.5.10
Pistils were stained overnight in the dark at room temperature. Stained pistils were11
mountedonglassslidesin50%glycerol,coveredwithaglasscoverslip,andflattened12
with gentle pressure on the cover slip. Observation and photography of pollen tubes13
werecarriedoutusingaLeicaDM5500B fluorescencemicroscopewithaDAPI filter.14
Images were captured with a Hamamatsu digital camera and IPLab software (BD15
Biosciences).Individualmicrographswereassembledintowholepistilmontagesusing16
AdobePhotoshopCS2.17
DNA isolations: A small scaleDNAextractionmethodwasadopted to isolate18
DNAfromthemappingpopulationsusing96‐wellmicrotiterplates.Seedsweretreated19
with50%householdbleachfor15min,rinsedthoroughlyunderrunningtapwater,and20
germinated on blotter paper (Anchor Paper, St. Paul,Minnesota) in acrylic sandwich21
boxes (10.8x10.8x3.4cm,HoffmanManufacturing, Jefferson,Oregon) for7‐10days.22
Seedlingswith fullyexpandedcotyledonsweretransplantedtosoil inplasticseedling23
11
traysandgrowninthegreenhouse.Twoorthreeweekslater,singleexpandingleaflets1
of about 1 cm2 in areawere collected fromeachplant into 96‐wellmicrotiter plates.2
Afteradding400µlofextractionbuffer(200mMTris‐HClpH7.5,250mMNaCl,25mM3
EDTA,0.5%SDSand~5%sodiummetabisulfite)and2 levitationstirballs (3.97mm,4
cat. no. VP725E, V&P Scientific Inc.), leaf tissues were ground on a paint shaker5
(SkandexSO‐10m,FluidManagementEurope)for8minandthenincubatedat65°Cfor6
onehour.Aftercooling,150µlofchilled5MKAcbufferwereadded,andcentrifugedat7
4,000RPM(3,000xg) for15min.Thesupernatant(200µl)was transferredtoanew8
96‐well microtiter plate. DNAwas precipitated by adding 100 µl 100% isopropanol,9
followedbycentrifugationat4,000RPMfor10min.Thesupernatantwaspouredout10
andpelletswerewashedonceusing200µlof70%ethanolandallowedtodryatroom11
temperature.Thepelletswereresuspendedin200µldistilledH2O.EachPCRreaction12
used2µl(100‐200ng)ofresuspendedDNAinatotalvolumeof20µl.13
CAPS markers: Initial mapping of ui6.1 relied on previously mapped CAPS14
markers obtained from the Solanaceae Genomics Network (SGN,15
http://solgenomics.net).Finescalemappingwasbasedonmarkersdevelopedfromthe16
sequences of BACs located in the candidate region, also downloaded from SGN. PCR17
primers of predicted amplification size ranging from 700 to 1000 base pairs were18
pickedoutusingthesoftwarePrimer3.PCRconditionswere94°Cfor2min,followed19
by 35 cycles of 94 °C for 30 sec, 55°C for 30 sec, and 72 °C for 2 min, with a final20
extensionof5minat72°C.Amplificationproductswereseparatelydigestedwitheight21
4‐bprecognizingrestrictionenzymes(AluI,HaeIII,HinfI,MboI,MseI,MspI,RsaI,and22
Taq I) to identify polymorphisms. A total of 142 markers were designed for the23
12
candidate region, of which 18 polymorphic markers were used in the final linkage1
analysis(TableS1).AllCAPSmarkerswereresolvedon2%agarosegels.2
Isolationandanalysisofrecombinants:Markeranalysisofthebridginglines3
indicated the size of the ui6.1 introgression from S. pennelliiwas ca. 32 cM on the4
EXPEN2000 referencemapof tomato (datanot shown).To identify theapproximate5
locationofui6.1, twomarkers,SSR47andT834locatedatpositions6.5and32cMon6
chromosome 6, were selected to genotype the first mapping population, F2‐a.7
Recombinantsidentifiedwiththesetwomarkerswerethensubjectedtocompatibility8
evaluation bymeasuring pollen tube growth on pistils of the allotriploid hybrid. The9
first round of screening indicatred that ui6.1 is closely linked but distal to marker10
SSR47.Therefore,aCAPSmarkernearertheendofchromosome6,T1928,wasusedin11
combinationwithT834toscreenallsubsequentmappingpopulations.Relativelyhigh12
recombinationfrequencieswereobservednearui6.1intwopopulations(BC‐♀andBC‐13
♂) derived from a S. pennellii substitution line. We focused on the recombinants14
identifiedamong1,824individualsofBC‐♀.Hundredsofpublicallyavailablemarkers,15
as well as markers developed from sequenced BACs and BAC ends, were used to16
conduct the linkage analysis. Based on themapping results from this phase, flanking17
markers73H07‐3andMiwerechosentoanalyzetherecombinantsbetweenT1928and18
T834fromtheotherpopulations,i.e.theF2‐a,F2‐b,BC‐♂andtheremainderoftheBC‐19
♀.Theadditionalrecombinantswerethentestedforcompatibilityontheallotriploid.20
Linkage analysis was based on the recombinants isolated from all mapping21
populations. Map distanceswere expressed in units of recombination frequency, i.e.22
13
(RF = # recombinants / total # gametes) x 100%, where the # gametes = (# BC1
progeny),or(2x#F2progeny).2
3
RESULTS4
Pollen tube growth in pistils of S. lycopersicoides and F1 hybrids with5
tomato:LikeotherSIwildtomatorelatives,pistilsofS.lycopersicoidesrejectpollenof6
cultivated tomato (S. lycopersicum, SC) (Table 1). No pollen rejection occurs in the7
otherdirection,consistentwiththeSIxSCrule.Rejectionoftomatopollenoccursearly8
duringgrowthontheS.lycopersicoidespistil,withpollentubesreachingnofurtherthan9
the stigma or uppermost portion of the style (<5%of style length). In contrast, self10
pollen tubes are arrested midway down the S. lycopersicoides style (~50% of style11
length).DiploidandallotriploidS. lycopersicumxS. lycopersicoideshybridsalsoreject12
pollenofcultivatedtomato,but therejectionoccurs lower in thestyle:~15%ofstyle13
length for the2xhybrid,and~50%forthe3xhybrid. Thus, the interspecifichybrids14
have weakened or delayed incompatibility responses compared to S. lycopersicoides;15
rejectionoftomatopollentubesontheallotriploidoccursinaboutthesamepositionas16
self pollen tubes on S. lycopersicoides. Pollen of S. pennellii (SC accession LA0716) is17
compatibleonstylesofS.lycopersicoidesandthediploidorallotriploidhybrids.18
Twopollenfactors,ui1.1andui6.1,arerequiredtoovercomestylarUI:We19
previously showed that two S. pennellii gametophytic factors, ui1.1 and ui6.1, are20
required for pollen to overcome the stylar incompatibility barrier of diploid and21
allotriploid S. lycopersicum x S. lycopersicoideshybrids (Chetelat and DeVerna 1991).22
Thesetwofactors,whenbredintoS.lycopersicum,conferfullcompatibilityonpistilsof23
14
theallotriploid(Table1).OnthepistilsofthediploidhybridorS.lycopersicoides,pollen1
containing both ui1.1 and ui6.1 penetrate further into the style than pollen lacking2
eitherfactor,yetdonotreachtheovaries.3
Toelucidatetheinheritanceofpollencompatibilityinthissystem,wegenotyped4
BC7F2progenyofbridginglinesheterozygousfortheS.pennelliiallelesatbothloci(the5
F2‐a population), usingmarkers SSR47 andT834on the short armof chromosome66
and two S‐locus flanking markers, TG184 and SSR98 on chromosome 1. Plants7
heterozygousorhomozygous for theS. pennelliialleles atonlyui1.1, oronlyui6.1,or8
both loci, in the genetic background of S. lycopersicum, were selected from this9
population.Compatibilityofpollenfromthevariousgenotypeswastestedonpistilsof10
theallotriploidhybrid.Thedistributionofmaximumpollentubelengthswasbimodal,11
withno intermediateplants, thuspollen compatibility canbe treated as a qualitative12
traitinthissystem(Fig.2).13
PollenofS. lycopersicumcv.VF‐36andbridging containinga singleS.pennellii14
factor (ui1.1 or ui6.1) were incompatible on pistils of the allotriploid, as the longest15
pollentubesgrewtoonlyhalfthelengthofthestyleafter24hours(Fig.3).Incontrast,16
bothS.pennelliiandtheui1.1+ui6.1bridginglineshowedacompatiblephenotype,with17
pollentubesreachingtheovaries.TheseresultsconfirmedthateitherS.pennelliifactor18
aloneisinsufficienttoovercomeincompatibility,andthatbotharerequiredtoelicita19
compatiblereaction.20
Thepollensidefactorsui1.1andui6.1donotconferUIorSI inthepistil:21
Importantly,thetwogametophyticfactorsfromS.pennelliidonotconferastylarUIor22
SI response. Bridging lines homozygous or heterozygous for the S. pennellii23
15
introgressions containingui1.1andui6.1 set fruit and seeds following selfpollination1
(datanotshown), indicatingtheyareSC.Furthermore,thebridginglinesset fruitand2
seed following pollinations with cultivated tomato, providing strong evidence that3
pistil‐sideandpollen‐sideUIresponsesarecontrolledbydifferentsetsofgenes.4
Maplocationofui6.1:Todeterminetheapproximategeneticlocationofui6.1,5
1,167F2‐aplantsweregenotypedwithmarkersSSR47andT834on theshortarmof6
chromosome 6. Recombinant plantswere genotypedwith two additionalmarkers in7
this interval, T892 and T507, and two Slocus markers, TG184 and SSR98 on8
chromosome1.Compatibility testsof the recombinantswerecarriedoutonpistilsof9
theallotriploid.Atotalof55recombinantswereidentifiedbetweenSSR47andT834,of10
which 47were tested for compatibility (Fig. 4). Thirteen recombinants lacking theS.11
pennelliialleleatui1.1showedanincompatiblephenotype,regardlessoftheirgenotype12
atui6.1.Theaverage‘maximum’pollentubelengthintheseplantswas48.2%ofstyle13
length.TherecombinantsheterozygousorhomozygousfortheS.pennelliialleleatui1.114
segregated for compatibility, depending on their genotype for ui6.1. The ten15
recombinantslackingtheS.pennelliialleleofSSR47wereincompatible,withanaverage16
pollen tube length of 47.4%,whereas the remaining 24 recombinants (i.e. those that17
wereatleastheterozygous)wereallcompatibleonthepistilsoftheallotriploid.18
Some F2‐a recombinants did not provide information on the map location of19
ui6.1. All 13 plants lacking the S. pennellii allele at ui1.1 locuswere incompatible, as20
expected, so they were uninformative regarding ui6.1. Recombinants that carried a21
parental(nonrecombinant)S.pennelliisegmentwerealsouninformative. Thesewere22
16
self‐pollinated andplants that carried only the recombinant segmentwere tested for1
compatibility.Theresultsshowedthatui6.1iscloselylinkedtomarkerSSR47(Fig.5).2
To avoid the problem of segregation for ui1.1, a second population (F2‐b),3
homozygous for theS. pennellii allele atui1.1 and segregating atui6.1,was analyzed.4
Thispopulationof1,920plantswasscreenedwithadistalmarker,T1928,andT834;5
recombinants were then genotyped with SSR47 and T892 (Fig. 5). A total of 866
recombinantswere identified, ofwhich31had crossoversbetweenT1928andT892.7
Onlythelatterweretestedforcompatibilityphenotypeontheallotriploid,sincetheF2‐8
a population indicated a location for ui6.1 closer to SSR47. Of the 31 tested9
recombinants, only 13were informative regarding the position ofui6.1, the other 1810
recombinants also carried a parental S. pennellii segment, and thus required an11
additionalgenerationtoobtainan informativephenotype. Theresults indicatedui6.112
liesbetweenT1928andT892(Fig.5).13
Recombinationnearui6.1 issuppressed: Recombination in theui6.1region14
wasstronglysuppressedinthetwoF2populationsderivedfromthebridginglines(Fig.15
6).Thetotalgeneticdistancesobservedinthetwopopulationswere2.35and2.32map16
units, betweenT834 and either SSR47 or T1928, respectively. These values are less17
thanonetenthoftherecombinationfrequenciesreportedforthesamemarkersonthe18
referencemap,EXPEN2000,basedonan interspecificF2S. lycopersicumxS.pennellii19
cross (http://solgenomics.net; Fulton et al. 2002). This degree of recombination20
suppressioncouldimpedethefinescalemappingandcloningofui6.1,sowedesigneda21
mapping strategy to elevate crossover frequency in this region. Our previous studies22
showed that recombination frequency is higher in lines with long introgressed23
17
segmentsorwholechromosomesubstitutions,relativetoshortintrogressions(Canady1
etal.2006).2
Wethereforetookadvantageofapartialsubstitutionline,SL‐6,whichcontainsa3
long S. pennellii introgression spanning most of chromosome 6 (Fig. 6). A line4
heterozygous for SL‐6 was crossed as female (BC‐♀) or male (BC‐♂) to a line5
homozygousfortheui1.1regionfromS.pennellii. Allplants inthesetwopopulations6
were screenedwith the flankingmarkers T1928 and T834 to identify recombinants,7
whichweregenotypedwithT892andSSR47.In2,946BC‐♀and672BC‐♂plants,2948
and29recombinants,respectively,werefound.TheBC‐♀datawerederivedfromtwo9
independent reciprocal crosses inwhich a homozygous SL‐6was crossed asmale or10
female to cultivar M‐82 to create the heterozygous stock; since no differences were11
seenbetweenthereciprocalcrosses(datanotshown),themappingdatawerepooled.12
ThegeneticdistancebetweenT1928andT834 in theBC‐♀populationwas9.98map13
units,orover four foldhigher thanobserved inF2‐aandF2‐b (Fig.5).Recombination14
frequencywassignificantlylowerinBC‐♂,butthetotaldistanceof4.32mapunitswas15
stillnearlytwicethatoftheF2populations. Theseresultsconfirmedthat longeralien16
introgressionsrecombinemorereadilythanshortones.Furthermore,recombinationin17
female gametes of SL‐6 heterozygotes (BC‐♀) was over twice that observed inmale18
gametes(BC‐♂).19
Heterogeneity for recombination rates along the chromosome: We also20
observed that recombination rates were not uniform along the chromosome. For21
example, the genetic distance between T1928 and T892 in the BC‐♀ populationwas22
4.60 map units, more than six times the distance in BC‐♂ (Fig. 5). In contrast, the23
18
relative increase in recombination between T892 and T834was only about 1.5 fold.1
Fortuitously,ui6.1was located inaregionof relativelyhighcrossover frequency(Fig.2
7).When additionalmarkerswere placed onto a geneticmap derived from selective3
genotypingof theBC‐♀ population, a regionof elevated recombinationwasobserved4
betweenmarkersU218000 andMi. The distance between these twomarkers on our5
mapwas2.13mapunits,morethanseventimesthevalueonthereferencemap(Fig.7).6
Finescalemappingofui6.1:Severalhundredplantswithcrossoversbetween7
T834 and T1928 or SSR47 were isolated from the four mapping populations. We8
initially focusedon the recombinants isolated from thepartialBC‐♀,whichproduced9
thehighestrecombination frequency.Theui6.1phenotypesof theseweredetermined10
by test pollinations on the allotriploid. Based on the mapping results, we used two11
closely flanking markers to further characterize the recombinants isolated from the12
otherpopulations.Theserecombinantswereselectivelyphenotyped,focusingonthose13
plantsmostlikelytofurtherresolvethepositionofui6.1.14
Our initial results showed ui6.1 is located betweenmarkers U218000 andMi15
(Fig.7),basedon39recombinantsinthisintervalfromthepartialBC‐♀population.To16
further resolve the location ofui6.1, hundreds ofmarkers based on the sequences of17
overlappingBACsweredeveloped.Ofthese,18polymorphicCAPSmarkers,developed18
from the sequences of twooverlappingBACs, C06HBa0250I21 andC06HBa0073H07,19
andtheendsequencesofLE_HBa00181H03(Fig.8,TableS1),wereused.Afinescale20
map of the ui6.1 region was constructed from these recombinants, and included six21
flankingmarkers(73H07‐3,73H07‐11,73H07‐13,73H07‐15,Miand181H03‐2),with22
onecrossoveroneachside(recombinant44‐32and50‐58)ofui6.1(Fig.8).23
19
The closely linked markers 73H07‐3 andMi were then used to genotype the1
recombinants from the other populations. Twomore recombinants, 19‐89 from F2‐b2
and 71‐61 from BC‐♀, were found in this region (Fig. 8). The compatibility of these3
recombinantswasevaluatedontheallotriploid(recombinant19‐89wastestedusinga4
homozygous F3 progeny). Linkage analysis using all the recombinants indicated that5
theintervalspanningui6.1comprisedapproximately0.128mapunits.Anestimatefor6
the corresponding physical distance was obtained from the prepublication draft7
genome sequence of tomato, made available by the International Tomato Genome8
Sequencing Consortium at http://solgenomics.net. This showed that the flanking9
markers73H07‐11andMiareseparatedby~160kb.10
11
DISCUSSION12
Relationshipbetweenunilateralandselfincompatibility:Abasicquestion13
aboutthenatureofprezygoticinterspecificreproductivebarriersinplantsistheir14
relationshiptoself‐incompatibility.AsdescribedbyLewisandCrowe(1958),15
unilateralcrossingbarriersoftenfollowthe‘SIxSCrule’,whereinpollenofaself‐16
compatiblespeciesisrejectedonstylesofarelatedself‐incompatiblespecies,butno17
rejectionoccursinthereciprocalcross.Thegeneralvalidityofthisruleinmanyplant18
families,includingtheSolanaceae,suggeststhatmutationattheS‐locusisanimportant19
elementofinterspecificbarriers.Thishypothesisissupportedbyexperimentsin20
NicotianainwhichectopicexpressionofS‐RNAse’sfromanSIspecies(N.alata)inthe21
backgroundofanSCspecies(N.tabacum)issufficienttoconferonthepistiltheability22
torejectpollenfromseveralSCspecies(Murfettetal.1995).Howeverthesame23
20
experimentsalsodemonstratedpollenrejectionpathwaysthatdidnotdependonS1
RNAseexpression.Thus,onthepistilside,theSlocusisstronglyimplicatedin2
interspecificpollenrejection,butothergeneticfactorsarealsoinvolved.Incrosses3
betweenS.lycopersicum(SC)andS.habrochaites(SI),severalindependentQTLwere4
detected,ofwhichtheSlocusregionhadthelargestphenotypiceffect(Bernacchiand5
Tanksley1997).6
Ontheotherhand,interspecificpollentuberejectiondiffersfromSIintiming7
andlocation.Pollentubesofcultivatedtomatoarerejectedintheupperportionofthe8
S.pennelliistyle(Hardon1967;Liedletal.1996),similartoourresultswithS.9
lycopersicoidespistils(the‘early’rejection).Incontrast,‘self’pollentubespenetrate10
furtherintothestyle.Thisearlyrejectionofinterspecificpollentubessuperficially11
resemblessporophyticSI,whereinselfpollenisrecognizedandrejectedonthestigma.12
Furtherevidenceforasporophyticcomponentofinterspecificincompatibilitycomes13
fromthecrossingrelationshipsofF1hybridsbetweencultivatedtomatoandSIwild14
relatives:backcrossestotheSIspeciesareonlypossiblewhentheF1’sareusedas15
femaleparent(Hardon1967;McGuireandRick1954).Inotherwords,ifthe16
compatibilityofpollenwerepurelydeterminedbygametophyticfactors,asignificant17
fractionofpollenfromtheF1hybridshouldbecompatibleonpistilsoftheSIspecies.18
Thus,expressionofinterspecificincompatibilityisgeneticallycomplexandmayinvolve19
morethanonemechanismforrecognizingandrejectingforeignpollen.20
Inthepresentstudy,wereportthefinemappingofui6.1,apollencompatibility21
factoronchromosome6thatinteractswithui1.1,locatedatorneartheSlocuson22
chromosome1oftomato.WeshowthatonlypollenbearingtheS.pennelliiallelesof23
21
bothui1.1andui6.1arecompatibleonstylesofS.lycopersicumxS.lycopersicoides1
hybrids.TheseresultsstronglyimplicatetheSlocusinpollen‐sideunilateral2
incompatibility.Theyalsoprovidethefirstgeneticevidence(toourknowledge)that3
factorsotherthantheproductoftheS‐locusarerequiredforpollenfunctioninself‐or4
interspecificincompatibility.5
Patternsofsegregationdistortioninvariousinterspecificcrossesoftomato6
supporttheseconclusions.Theyprovidefurtherevidencethatpollenbearingthe7
cultivatedtomatoallelesatui1.1andui6.1areselectivelyeliminatedonstylesof8
interspecifichybrids,butonlywhenthewildparentisSI.Forexample,apseudo‐F2S.9
lycopersicumxS.chilense(SI)population,obtainedbycrossingtwoindependentF1’s(to10
avoidself‐incompatibility)exhibitedadeficiencyofplantshomozygousfortheS.11
lycopersicumallelesofmarkersnearui1.1andui6.1(Graham2005).Thelikely12
explanationisthatpollenlackingeitherfactorfromS.chilenseareeliminatedonstyles13
oftheF1hybrid.Incontrast,interspecificF2’sbetweentomatoandS.chmielewskiiorS.14
pennelliiLA0716,bothofwhichareself‐compatibleyetrejecttomatopollen,showed15
normalMendeliansegregationformarkersnearui1.1andui6.1(Patersonetal.1991;16
http://solgenomics.net).MendeliansegregationisalsoobservedinterspecificF217
populationswhenbothparentsareSI,consistentwiththehypothesisthattheSIspecies18
eachcontainfunctionalallelesatui1.1andui6.1(AlbrechtandChetelat2009;Pertuzeet19
al.2002).TheseresultssuggesttheexistenceofanS‐RNAsedependentpathwayfor20
interspecificpollenrejectioninpistilsoftheSIspecies,aswellasanS‐RNAse21
independentmechanismintheSIandsomeSCspecies.22
22
Thus,boththegeneticevidenceandthedevelopmentaltimingofinterspecific1
pollenrejectionsupporttheexistenceofatleasttwopathways.Thefirst(‘early’)2
mechanismrejectstomatopollentubesinthestigmaandupperstylesonpistilsofSI3
andsomeSCspecies;pollencontainingtheui1.1andui6.1factorsdoesnotovercome4
thisbarrier.Thesecondmechanismcausespollentuberejectioninthemidpositionof5
stylesfrominterspecificSCxSIhybrids,andisovercomebypollencontainingui1.1and6
ui6.1.Wefoundevidenceofoverlapbetweenthesetwopathways:pollenofbridging7
linescontainingbothui1.1andui6.1growfurtherintopistilsofS.lycopersicoidesthan8
doespollenoftomatolineslackingeitherorbothfactors(10%vs.5%ofstylelength).9
Furtherresearchisclearlyneededtounderstandthegeneticandphysiologicalbasisof10
theseinterspecificpollenrejectionpathways.11
RelevancetootherSolanaceae:Webelieveourexperimentalsystemprovides12
ausefulmodelforstudyingunilateralincompatibilityinotherSolanaceae.Onthepistil13
side,thediploidandallotriploidS.lycopersicumxS.lycopersicoideshybridsareSIand14
rejectpollenofcultivatedtomato,indicatingthesetraitsareeffectivelydominant,as15
theyareinotherSolanaceaehybrids.Forexample,diploidF1hybridsbetween16
cultivatedtomatoandSIaccessionsofS.habrochaites,S.pennellii,S.peruvianum,orS.17
chilenseareallSIandrejectpollenofcultivatedtomato(Hardon1962;Martin1961,18
1963;McGuireandRick1954).Allotriploidscomposedoftwogenomesfromtomato19
andonefrompotato(S.tuberosum,SI)alsorejectpollenofS.lycopersicumandaccept20
pollenofS.pennelliiLA0716(Schoenmakersetal.1993),i.e.thesamecrossing21
relationshipswereportherein.Inothersolanaceousgenera,includingNicotiana22
23
(Murfettetal.1996)andCapsicum(Pickersgill1997),self‐andunilateral1
incompatibilityarealsodominantininterspecificSCxSIhybrids.2
Furthermore,iftheunderlyingpathwaysforpollentuberejectionbyunilateral3
incompatibilityareconserved,thentheresultswepresenthereinshouldberelevantto4
othersystems.Forexample,allotriploidS.lycopersicumxS.sitienshybridsalsoreject5
pollenofcultivatedtomato,andbridginglinescontainingui1.1andui6.1fromS.6
pennelliiarecompatible(Pertuzeetal.2003).Ininstanceswhereunilateral7
incompatibilityresultsfromthebreakdownofself‐incompatibility,therearelikelytobe8
commonmechanisms.Self‐incompatibilitysystemsarestronglyconservedwithinplant9
families,andrelatedfamiliesoftenhavethesamesystem.Forexample,allSImembers10
oftheSolanaceaeemploytheSRNAsebasedgametophyticsystem,underthecontrolof11
asingleSlocusdeterminingspecificity(deNettancourt1977,1997).WhiletheS12
RNAsesarehighlydivergent,eachallele(S1,S2,etc)isstronglyconserved,evenacross13
plantfamilies(IgicandKohn2001).14
AweakenedUIresponseintheallotriploidhybridsimplifiesthegenetics:15
TheabilitytorejectpollenofcultivatedtomatoiseffectivelydominantinpistilsofS.16
lycopersicumxS.lycopersicoideshybrids.However,expressionofthepistilresponsein17
thehybridsisweakerthaninthewildparent,intwoways.First,pistilsofthe3xhybrid18
arrestpollentubesofcultivatedtomatonearthemiddleofthestyle,andlackthe‘early’19
(i.e.stigmatic)pollentuberejectionphenotypeofS.lycopersicoidespistils,suggesting20
thelatterbarrierisadditiveorrecessive.Second,bridginglinescontainingboththe21
ui1.1andui6.1genesfromS.pennelliiwerefullycompatible(i.e.manypollentubesin22
theovaries)withpistilsoftheallotriploidhybrid,butincompatiblewiththediploid23
24
hybridorS.lycopersicoides.WehypothesizethatadditionalgeneticfactorsfromS.1
pennelliicontributetocompatibilitywiththesetwogenotypes;alocusonchromosome2
10wasidentifiedinanearlierstudy(ChetelatandDeVerna1991).Thus,theuseof3
allotriploidhybridsastesterstocksprovidesasimplergeneticsysteminwhichto4
isolatepollen‐sideunilateralincompatibilityfactors.Thismethodmayprovideauseful5
routeforgeneticanalysisofotherinterspecificincompatibilitysystems.6
Thecausesoftheweakenedexpressionintheallotriploidareunknown,but7
presumablyresultfromthereducedgenedosageoftheSIparent(i.e.onegenomeofS.8
lycopersicoides,vs.twogenomesoftheSCparent,S.lycopersicum).Wehypothesizethat9
thedifferentpathwaysforpollentuberejectionaredifferentiallyaffectedbygene10
dosageinthepistil.TheactionofafactoratorneartheS‐locusinpollenimpliesthat11
theS‐RNAsedependentpathwayisfullydominantinpistilsoftheallotriploidhybrid.12
However,the‘early’rejectionpathwaymaybeadditiveorrecessiveinhybrids.There13
aremanyexamplesofgene‐specificdosageeffectsinaneuploidsandtriploids.Genes14
thataredominantoverwildtypeinnormal(2x)heterozygotesmayshowreduced15
expressionintrisomicscontainingtwodosesofthewildtypeallele(KhushandRick16
1968).Someproteincodinglociareexpressedatlevelsproportionaltotheirgene17
dosage,whileotherlocimaintainexpressionatthediploidlevelviadosage18
compensation(Birchler1979;SmithandConklin1975;Tanksley1979).Inallotriploid19
hybridfish,geneexpressionisreducedtothediploidlevelthroughallele‐specificgene20
silencing(Palaetal.2008).Finally,trisomicdosagemayaltergeneexpressionthrough21
genicinteractions,forexampleifmodifierlociaremoresensitivetodosage(Gill1974).22
25
Towardsmapbasedcloningofui6.1:Todecipherthemolecularpathwaysof1
interspecificpollentuberejection,itisnecessarytoclonetheunderlyinggenes.2
Towardsthisgoal,wemappedtheui6.1genetoanintervalofca.0.128cMor160kbon3
theshortarmofchromosome6oftomato.Theefficiencyoflinkagemappingwas4
limitedinitiallybylowrecombinationcomparedtothereferencemapoftomato.5
However,thedegreeofrecombinationsuppressionvariedalongthechromosome,and6
crossoverfrequenciesclosetoui6.1werehigherthannormal.Thecausesof7
recombinationsuppression(orenhancement)inthisregionofthegenomeare8
unknown,butmayreflectthedegreeofdivergence(structuralorsequence)between9
theS.lycopersicumandS.pennelliichromosomes.Thetwospeciesshowcolinearityof10
chromosome6S,withnorearrangements(Petersetal.2009).InS.peruvianum,alarge11
paracentricinversiononchromosome6Swasresponsibleforrecombination12
suppressionaroundtheMigene(fornematoderesistance)incrossestotomato(Seahet13
al.2004).TheMilocusisclosetoui6.1,butwefoundnoevidenceforaninversioninS.14
pennellii.WhileafewBAC‐derivedmarkerswereinvertedinourgeneticmapofthe15
ui6.1regioncomparedtotheirorderonthetomatoBACsequence,theywereinaregion16
ofrelativelyhighrecombination.Thediscrepancysuggestsarearrangementmayhave17
occurredinthisparticularBAC.Furthermore,FISHmappingofmanyotherBACson18
chromosome6showedanarea(position0‐5cM)wherethelinearorderofmarkers19
predictedfromthegeneticmap(EXPEN2000)isinvertedrelativetotheiractual20
physicalarrangementalongthechromosome(Petersetal.2009),pointingtoalikely21
errorinthelinkagemap.22
26
Weobtainedmuchhigherrecombinationfrequenciesstartingwithanearly1
intactS.pennelliichromosome6,comparedtotheshorterintrogressedsegments2
presentinthebridginglines.Thisagreeswithourpreviousobservationsofapositive3
correlationbetweencrossoverfrequencyandthelengthofalienchromosomesegments4
(Canadyetal.2006).Higherrecombinationrateswereseeninfemalegametesthanin5
malegametes,ashasbeenreportedbefore(deVicenteandTanksley1991;Rick1969).6
Thus,despiteanoverallsuppressionofrecombinationinthisregion,the7
efficiencyofmappingcouldbemaximizedbychoiceofstartingmaterialsandthe8
directionofthecross.Inthisway,thechromosomalpositionofui6.1wasrefinedfrom9
arelativelyimpreciseinterval,encompassingtheentireshortarmofchromosome6,to10
only0.128mapunits(160kb).Weanticipatethatisolationoftheui6.1genewillshed11
lightonthemechanismofpollentuberejectioninunilateralincompatibility,andits12
relationshiptoself‐incompatibility.Fromapracticalstandpoint,amorecomplete13
understandingofthegeneticfactorsunderlyinginterspecificcrossingbarriersmay14
eventuallyenablewidercrosses.Forexample,overcomingtherejectionofS.15
lycopersicumpollenonpistilsofrelatedSIspeciesmightfacilitatethedevelopment16
cytoplasmicmalesterilityintomato.17
18
Acknowledgements19
TheauthorsgratefullyacknowledgePeterMarch,KatieSmith,andtheC.M.Rick20
TomatoGeneticsResourceCenterstaffforprovidingseedofkeygenotypes,andfor21
growingandmaintainingplantsinthegreenhouse.WethankShehMayTamforhelpful22
discussions.BACsequenceswereobtainedfromhttp://solgenomics.net.Fundingfor23
27
thisresearchwasprovidedbygrantDBI0605200fromtheNationalScience1
Foundation,PlantGenomeProgram.2
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controlledbyasinglemajorlocusonchromosome1inLycopersiconperuvianum.13
Proc.Natl.Acad.Sci.USA82:5093‐5096.14
VANWORDRAGEN,M.F.,R.L.WEIDE,E.COPPOOLSE,M.KOORNNEEF,andP.ZABEL,15
1996Tomatochromosome6:ahighresolutionmapofthelongarmand16
constructionofacompositeintegratedmarker‐ordermap.Theor.Appl.Genet.17
92:1065‐1072.18
WEIDE,R.,M.F.VANWORDRAGEN,R.K.LANKHORST,R.VERKERK,C.HANHARTetal.,19
1993Integrationoftheclassicalandmolecularlinkagemapsoftomato20
chromosome6.Genetics135:1175‐1186.21
32
Table1.PollentubegrowthfollowingcompatibleandincompatiblepollinationsofSolanumlycopersicoidesandinterspecific1
Solanumhybrids.Pollencompatibilityisindicatedbythelengthofthelongestpollentubeexpressedasapercentageofthe2
totallengthofthestyleandstigma(avalueof100%indicatesacompatiblecross).Valuesaretheaverages(±SE)ofatleast33
replicatepistilsforeachcross.Onthefemaleside,thegenotypeswereS.lycopersicoides,anddiploidandallotriploidF1S.4
lycopersicumxS.lycopersicoideshybrids.Onthemaleside,crossesweremadewithpollenfromS.lycopersicoides,S.5
lycopersicum,S.pennellii,andbridginglinescontainingtheui1.1and/orui6.1factorsfromS.pennelliiintrogressedinto6
cultivatedtomato.ThesegenotypesaredescribedinMaterialsandMethods.7
PistilGenotype
PollenGenotype
LengthofLongestTube(%ofstyle)
LocationofRejection
#TubesinOvaries
S.lycopersicoidesLA2951 S.lycopersicoidesLA2951(self)S.lycopersicoidesLA2951(sib)
50.2±2.76100
stylen/a
0>50
S.pennelliiLA0716 100 n/a 4.9±2.91 S.lycopersicumcv.VF‐36 5.6±1.88 stigma 0 S.lycopersicum(ui1.1) 5.3±0.67 stigma 0 S.lycopersicum(ui6.1) 4.7±1.52 stigma 0 S.lycopersicum(ui1.1+ui6.1) 10.9±0.96 stigma/style 0S.lycopersicumxS.lycopersicoides S.lycopersicoidesLA2951 100 n/a >50(diploidhybrid90L4178) S.pennelliiLA0716 100 n/a >50 S.lycopersicumcv.VF‐36 16.6±1.86 style 0 S.lycopersicum(ui1.1) 13.7±0.71 style 0 S.lycopersicum(ui6.1) 15.3±3.14 style 0 S.lycopersicum(ui1.1+ui6.1) 42.3±14.5 style 0
S.lycopersicumxS.lycopersicoides S.lycopersicoidesLA2951 100 n/a >50(allotriploidhybridGH266) S.pennelliiLA0716 100 n/a >50
33
S.lycopersicumcv.VF‐36 47.8±4.93 style 0 S.lycopersicum(ui1.1) 45.9±6.40 style 0 S.lycopersicum(ui6.1) 53.0±11.4 style 0 S.lycopersicum(ui1.1+ui6.1) 100 n/a >501
34
FigureLegends1
Figure1.CrossingschemeusedtodevelopS.lycopersicumxS.lycopersicoides2
interspecifichybridsandbridginglinescontaininggametophyticunilateral3
incompatibilityfactorsfromS.pennellii.L=genomeofS.lycopersicum,S=genomeofS.4
lycopersicoides.PollenofS.lycopersicumisrejectedonpistilsofS.lycopersicoidesand5
itsinterspecifichybridswithcultivatedtomato,whereas‘bridginglines’containing6
specificgametophyticfactorsfromS.pennelliiarecompatible.7
8
Figure2.HistogramofpollentubelengthsfollowingpollinationsofallotriploidS.9
lycopersicumxS.lycopersicoideshybrids(♀)withbridginglinessegregatingfortwo10
pollencompatibilityfactorsfromS.pennellii.Pollentubelengthsaremeasuredfrom11
thepositionwherethemajorityoftubesarearrested,excludingoutliertubesthatmay12
growalittlefurther(seeFigure3).Measurementsweretaken24hoursafter13
pollination.14
15
Figure3.PollentubegrowthonpistilsofallotriploidS.lycopersicumxS.lycopersicoides16
hybridspollinatedbydifferentspeciesorgenotypes.Pistilswerefixed24hoursafter17
pollination,andstainedwithanilinebluetovisualizepollentubes.Fortheincompatible18
crosses,arrowheadsindicatethezoneofthestylewheregrowthofmosttubesstopped,19
andarrowsindicatethelongestpollentube.Thebridginglinegenotypescontainedone20
orbothpollencompatibilityfactors(ui1.1,ui6.1)fromS.pennelliiintrogressedintothe21
backgroundofS.lycopersicum.22
23
35
Figure4.Genotypesofrecombinantsrecoveredintheui6.1regioninF2progenyof1
bridginglienscontainingboththechromosome1and6factors.Thecompatibilityor2
incompatibilityofrecombinantswasdeterminedbypollinationsontopistilsofthe3
allotriploidS.lycopersicumxS.lycopersicoideshybrid.Openbars=homozygousforthe4
S.lycopersicumalleles,grey=heterozygous,andblack=homozygousfortheS.pennellii5
alleles.IntheabsenceoftheS.pennelliichromosome1factor,allplantselicitan6
incompatiblereaction,regardlessoftheirgenotypeatui6.1.Recombinantsthatwere7
heterozygousfortheparentalS.pennelliiintrogressionwereprogenytestedto8
determinethephenotypeofui6.1.9
10
Figure5.Comparisonofrecombinationfrequenciesnearui6.1indifferentmapping11
populations.TheF2mapsarebasedonrecombinationwithinarelativelyshort(32cM)12
S.pennelliisegment,whereastheBCmapsarederivedfromalongersegment(~80cM)13
inasubstitutionline.ThereferencemapisthetomatoEXPEN2000map14
(http://solgenomics.net).Geneticdistancesareinunitsofpercentrecombination,or,15
ontheEXPEN2000map,incentiMorgans.OntheF2‐bandBCmaps,ui6.1isshownat16
itsapproximatelocationbecausenotallplantsinthesepopulationswerephenotyped.17
18
Figure6.Crossingschemeusedtooptimizetherateofrecombinationaroundui6.1.A19
substitutionlinecontaininganearlyintactS.pennelliichromosome6(vanWordragen20
etal.1996)wascrossedtotomatocv.M‐82.Theresultinghybrid,heterozygousforthe21
pennelliichromosome,wasbackcrossedasmale(BC‐♂)orfemale(BC‐♀)parenttoa22
linehomozygousfortheSlocusregionfromS.pennellii.Recombinantswereidentified23
36
bymarkeranalysisinthetwoBCpopulations,thentestedforcompatibilityofpollenon1
pistilsoftheallotriploidhybrid.2
3
Figure7.Geneticmapoftheui6.1regionontheshortarmofchromosome6.Results4
arebasedonanalysisofprogenyfromthesubstitutionline,usedasfemaleparent(BC‐5
♀),crossedtoalinehomozygousfortheS.pennelliifactoronchromosome1.Markers6
arefromtheSGNdatabase(http://solgenomics.net).Theshadedregionofthe7
chromosomesindicatesazoneofelevatedrecombinationintheprogenyofSL‐68
relativetotheEXPEN2000referencemap.9
10
Figure8.Finescalegeneticandphysicalmapoftheui6.1region,andphenotypesof11
recombinantswithcrossoversintheflankingintervals.ThepositionofthreeBACsis12
shownrelativetoafinescalemapoftheregionbasedontheanalysisofrecombinants.13
Foreachmarkerinterval,thepercentrecombinationandthenumberofrecombinants14
obtainedfromallmappingpopulationsareshown.MarkerswerederivedfromtheS.15
lycopersicumBACsequences.Thegenotypesoffourrecombinants(solid=homozygous16
S.pennellii,hatched=heterozygous,open=homozygousS.lycopersicum)andtheir17
pollenphenotypesonpistilsoftheallotriploid(I=incompatible,C=compatible)are18
shown.19
map pollen UI factors from S. pennellii
Phenotyping and marker analysis
S. lycopersicum ×
Allotriploid hybrid (LLS)
colchicine
Allotetraploid hybrid (LLSS)
S. lycopersicum (LL) S. lycopersicoides (SS)×
F1 hybrid (LS)
Backcrosses &selection
Bridging lines
S. lycopersicum × S. pennellii
Pollen Tube Length (% of style)
30 40 50 60 70 80 90 100 110
Nu
mb
er o
f P
lan
ts
0
5
10
15
20
25
S. pennelliiS. lycopersicum ui1.1 + ui6.1 ui1.1 ui6.1
bridging lines
1 mm
Pollen Genotypes
Compatibility on LLS: I C
2No. of recombinants: 4 6 5 3 2 4 5 52 2 1 6
I I I I C C C C CI I
chr. 1
chr. 6
SSR47
T892
T507
T834
orS
TG184
SSR98
T1928
SSR47
T892
T507
T834
6.5
7.5
11.0
7.0
ui6.1SSR47
T892T507T834
0.430.901.02
T1928
SSR47T892T834
0.550.261.51
T1928
SSR47T892
T834
3.62
0.98
5.38
T1928
T892
T834
0.74
3.58
EXPEN 2000
Short Introgression
Long IntrogressionBC-♀
F2-a F2-bBC-♂
ui6.1
ui6.1
ui6.1
×
×
SL-6M-82
BC-♀
♀ ♂
ui1.1
BC-♂
♀ ♂
ui1.1
×
chr.1 chr.6 chr.1 chr.6
T1928
C2_At3g25120U218000MiSSR48SSR47
C2_At4g01900
U295750
T892
5.2
0.30.50.5
1.5
4.5
1.5
T1928
C2_At3g25120U218000
Mi, ui6.1SSR48SSR47C2_At4g01900U295750T892
1.10
2.13
0.330.060.220.540.22
EXPEN 2000
BC-♀
C06HBa0250I21 C06HBa0073H07
250I
21-1
4 25
0I21
-11
250I
21-1
0 25
0I21
-7
250I
21-2
7
73H
07-2
8 73
H07
-22
73H
07-2
1 73
H07
-17
1 73
H07
-8
1 73
H07
-7
73H
07-5
4
73H
07-3
3
73H
07-1
5 73
H07
-13
73H
07-1
1
1
ui6.
1
Mi
181H
03-2
7 / 6705
181H
03-1
LE_HBa00181H03
3
0.230 0.034 0.034 0.034 0.136 0.102 0.094 0.230 % Recombination:
# Recombinants:
19-89 44-32
71-61
50-58
ICII
![Interspecific Interactions Simple Assignment 2ak6ehl[1]](https://img.dokumen.tips/doc/110x75/577cc9cb1a28aba711a4a461/interspecific-interactions-simple-assignment-2ak6ehl1.jpg)
