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RESEARCHOpenAccessHigh-densitymappingofquantitativetraitlociforgrain-weightandspikeletnumberinriceDong-MinKim1,4,Hyun-SookLee1,Soo-JinKwon2,MarkEdwardFabreag1,Ju-WonKang1,Yeo-TaeYun3,Chong-TaeChung3andSang-NagAhn1*AbstractBackground:Highgrainyieldisoneofthemostimportanttraitsrequiringimprovementinricebreedingprograms.
Consequently,thegeneticbasisofspikeletsperpanicle(SPP)andgrainweight(TGW)havereceivedmuchresearchfocusbecauseoftheirimportanceinriceyield.
Results:Inthisstudy,IL28,whichisanearisogenicline(NIL)developedbyintrogressingchromosomalsegmentsofthecultivar'Moroberekan'intothecultivar'Ilpumbyeo',showedasignificantincreaseinthenumberofspikeletsperpanicle(SPP)and1,000-grainweight(TGW)comparedtotherecurrentparent,Ilpumbyeo.
Quantitativetraitlocus(QTL)analysisin243F2plantsderivedfromacrossbetweenIL28andIlpumbyeoindicatedthatbothqSPP6andqTGW6arelocatedintheintervalRM3430–RM20580.
Followingsubstitutionmappingwith50F3:4:5lines,qSPP6wasmappedtoa429-kbintervalbetweenRM20521andInDel-1,whileqTGW6wasmappedtoa37.
85-kbintervalbetweenInDel-1andSNP–3basedonthejaponicagenomesequence.
ThisresultindicatesthatqSPP6andqTGW6aredifferentgenes.
YieldtrialswithsubstitutionlinesindicatedthatlinesharboringthehomozygousMoroberekansegmentatboththeqSPP6andqTGW6regionshowedsignificantlyhighergrainyieldthanIlpumbyeo.
Conclusion:BecausetheMoroberekanallelesforSPPandTGWhavebeenshowntobebeneficialinthegeneticbackgroundofIlpumbyeo,boththeqSPP6andqTGW6allelesmightprovevaluableinimprovingriceyields.
CloselylinkedSSRmarkersareexpectedtofacilitatethecloningofgenesthatunderlietheseQTLs,aswellaswithmarker-assistedselectionforvariationinSPPandTGWinricebreedingprograms.
Keywords:Rice;Spikeletsperpanicle;Grainweight;QTL;Linkage;NearisogeniclineBackgroundRice(OryzasativaL.
)istheworld'smostimportantcerealfoodcrop.
Theanticipatedrapidincreaseintheglobalhu-manpopulation,whichisexpectedtoreach9.
1billionby2050,mightgenerateseriousfoodshortageproblems.
Moreover,variousfactors,suchaswaterscarcity,soilsalin-ity,disease,climatechange,andreducedarableland,willexacerbatefoodshortagesinthenext50years(Khush1999andKhush2005;Zhang2007).
Therefore,researchersarefocusingonincreasingexistingcropgrainyieldlevels.
Grainweight,spikeletsperpanicles,andthenumberofpaniclesperplantarethemostimportantcomponentsofgrainyield.
However,thegeneticanalysisofthesethreeyieldcomponentsisdifficult,becausethesetraitsarecontrolledbymultiplegenes,inadditiontobeinginfluencedbytheenvironment.
Therefore,theadventofmolecularmapsforrice(Causseetal.
1994)andquantitativetraitlocus(QTL)analysisapproacheshavefacilitatedtheanalysisofthesequantitativetraits.
Genes/QTLsforspikeletsperpanicle(SPP)havebeenreportedinpopulationsderivedfrominter-specificcrosses(Xiongetal.
1999;Thomsonetal.
2003;Suhetal.
2005;Lietal.
2006andLinhetal.
2008),indica-indicacrosses(Linetal.
1996;Zhuangetal.
1997),andinter-subspecificcrosses(Yamagishietal.
2002;Andoetal.
2008).
Todate,afewQTLsassociatedwithSPPshavebeendetected,includingGn1a,APO1,DEP1,andOsSPL14.
Gn1acon-trolsgrainnumberinrice,andwasfoundtoencodecytokininoxidase/dehydrogenase(OsCKX2),whichisanenzymethatdegradesthephytohormonecytokinin(Ashikarietal.
2005).
HigherexpressionofOsSPL14duringthereproductivestageofricepromotespanicle*Correspondence:ahnsn@cnu.
ac.
kr1DepartmentofAgronomy,CollegeofAgriculture&LifeSciences,ChungnamNationalUniversity,Daejeon305-764,SouthKoreaFulllistofauthorinformationisavailableattheendofthearticle2014Kimetal.
;licenseeSpringer.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
Kimetal.
Rice2014,7:14http://www.
thericejournal.
com/content/7/1/14branchingleadingtohighergrainyield(Miuraetal.
2010).
DEP1,whichalterspaniclearchitecture,andhenceincreasesgrainyield,hasalsobeenidentified(Huangetal.
2009).
Anotherapproachtoidentifythegenesrelatedtopaniclearchitectureisthroughtheanalysisofmutantsthatalterpaniclestructure.
Thecharacterizationoftheaberrantpanicleorganization1(apo1)mutantinricerevealedthatAPO1positivelycontrolsspikeletnumberbysuppressingtheprecociousconversionofinflores-cencemeristemstospikeletmeristems(Ikedaetal.
2007).
Ookawaetal.
(2010)alsoreportedthataneariso-geniclinecarryingAPO1enhancedculmstrengthandincreasedspikeletnumberperpanicle.
Grainweight(GW)isanothercharacteristicthatistar-getedtoenhancericeyield.
ManyQTLsforgrainsizehavebeenidentifiedinricepopulationsbasedoncrossesbetweendivergentcultivarsoraccessions(Linetal.
1996;Cuietal.
2003;Ishimaru2003;Zhangetal.
2009).
Severalgenesthatregulategrainweighthavebeenidentified,in-cludingGS3,GS5,GW5,GW2,andTGW6.
GW2isaQTLforricegrainwidthandweight,whichencodesapreviouslyunknownRING-typeE3ubiquitinligasethatnegativelyregulatescelldivisionbytargetingitssubstrate(s)fordegradationbytheubiquitin-proteasomepathway(Songetal.
2007).
Ishimaruetal.
(2013)foundthatwhentheIAA-glucosehydrolasegenestopsfunctioning,TGW6enhancesgrainweightthroughpleiotropiceffectsonsourceorgans,andleadstosignificantyieldincreases.
Numerousstudieshavereportedthatonegenomicre-gionharborsmorethantwoindependentgenesthatarecloselylinked,oragenewithpleiotropiceffectonsev-eraltraits(Xieetal.
2008;Jinetal.
2009;Louetal.
2013;Jietal.
,2014).
Xieetal.
(2008)reportedthattwoQTLs,sn9.
1andgw9.
1,for1,000-grainweightandspikeletsperpaniclewereidentifiedinthe37.
4-kbinter-valflankedbymarkersRM24718andRM30005.
TwoQTLsactedinanadditivemannerinthisregion,result-ingintheNILwiththeO.
rufipogonallelesexhibitingsignificantlyhigherSPPandTGWvaluescomparedtotherecurrentparent,Hwaseongbyeo(Xieetal.
2008).
However,itremainsunclearwhethereitherofthetwocloselylinkedQTLs,oronepleiotropicQTL,areassoci-atedwiththeobservedvariation.
Alarge-sizedpopula-tioncombinedwithhigh-densitymappingisrequiredtodeterminewhethertargettraitsarecontrolledbycloselylinkedQTLs,oronepleiotropicQTL.
Inpreviousstud-ies(Juetal.
2008),IL28wasdeveloped,whichisanintrogressionlinederivedfromacrossbetweenthecul-tivarsIlpumbyeoandMoroberekan,showinghigherSPPandTGWvaluescomparedtoIlpumbyeo.
Kimetal.
(2013)reportedthataQTLforspikeletsperpaniclewaslocatedbetweenRM20521andRM20572usingF2:3pop-ulationsderivedfromacrossbetweenIlpumbyeoandIL28.
ThepresentstudywascarriedouttodetectanovelQTLforTGW,andtoclarifyitsrelationshiptotheqSPP6QTLusingF3:4:5populations.
ResultsCharacteristicsofIL28Inourpreviousstudy(Kimetal.
2013),thegeneticmapofIL28wasconstructedusing134markersthatexhib-itedpolymorphismbetweenIlpumbyeoandMorobere-kan,with2Moroberekanintrogressionsegmentsbeingdetectedonchromosomes4and6.
Thephenotypicevaluationofagronomictraitsfortheintrogressionline,IL28,andtherecurrentparentwasconductedattwolo-cations,DaejeonandYeasan,in2013.
Comparisonof10agronomictraitsbetweenIL28andIlpumbyeoobtainedinthecurrentstudyareshowninTable1andFigure1.
Theresultsindicatedthattherewerehighlysignificantdifferences(P<0.
01)inpaniclelength(PL),secondarybranchnumber(SBN),spikeletsperpanicle(SPP),firstnodewidth(FNW),secondnodewidth(SNW),and1,000-grainweightbetweenIL28andIlpumbyeo,whilenosignificantdifferencewasobtainedfortillernumber(TN)andculmlength(CL).
BecauseIL28hasalargeendospermandheaviergrain,weinvestigatedthegrainmilk-fillingrateinIL28andIlpumbyeo.
Nodifferencewasobservedineitherendospermfreshweightordryweightat3dand6dafterfertilization(Figure2).
Incomparison,boththefreshanddryweightofIL28weresignificantlyhigher(P<0.
01)comparedtothoseofIlpumbyeoat11dafterfertilization.
Thesedifferencesreachedamaximum~25dafterfertilization,atwhichpointthefreshanddryweightoftheendospermofIL28were11.
2%and13.
4%highercomparedtoIlpumbyeo,respectively.
Thus,anincreaseingrainweightinIL28whichisassociatedwithhighergrainmilk-fillingratemightbeduetotheMoroberekansegmentonchromo-some6consideringthatnoQTLforgrainwidthandweightwasdetectedintheMoreberekansegmentonchromosome4.
TraitvariationoftheF2populationInourpreviousstudy(Kimetal.
2013),fourtraits(paniclelength,numbersofprimarybranchesandsecondarybranches,andspikeletsperpanicle)exhibitedcontinuousandnormaldistributionsinthe243F2plants.
The250-grainweightofIlpumbyeoandIL28was5.
51and6.
14,re-spectively.
Inalltraitsmeasured,transgressiveplantswithhigherorlowermeanvaluescomparedtoeitherparentwereobserved.
Thecorrelationbetweenspikeletsperpan-icleandgrainweightwasnotsignificant(r=0.
102).
QTLanalysisThreesignificantQTLsforpaniclelength,secondarybranchnumber,andspikeletsperpanicleweredetected.
TheseQTLsoccurredinthesameregion,neartheSSRKimetal.
Rice2014,7:14Page2of11http://www.
thericejournal.
com/content/7/1/14markerRM3430(Kimetal.
2013).
Inaddition,theQTLforgrainweightwasdetectedinthesameregion,onchromosome6.
TheQTLforgrainweightexplained8.
3%ofphenotypicvariance.
TheMoroberekanallelesincreasedalltraitvaluesofpaniclelength,secondarybranchnumber,spikeletsperpanicleandgrainweightatthisregion.
SubstitutionmappingofqTGW6Toconfirmandnarrowdownthetargetregioncontain-ingqTGW6,1,120F2plantsderivedfromacrossbe-tweenIlpumbyeoandIL28weregenotypedwith4additionalmarkersnearthetargetregion(RM20512,RM20521,RM20562,andRM20572)andonemarker(RM551)locatedonchromosome4.
Atotalof44F2homozygousplantswithdifferentcross-overbreak-pointsbetweenRM7269andRM20653onchromosome6wereselectedandselfedtoproduceF3:4:5lines.
Geno-typeanalysisindicatedthatthese44F2plantsdidnothaveMoroberekansegmentonchromosome4.
Accord-ingtothemarkergenotype,the44F3:4:5linesweredi-videdinto11groups,andusedforthesubstitutionmappingofqTGW6(Figure3).
ThemeanphenotypicvaluesofeachtraitforeachF3:4:5linewascomparedtothoseofIlpumbyeoandIL28attheP<0.
05level.
Inapreviousstudy,theQTLsforSPP,PL,SBM,FNW,andSNWwereconsistentlymappedbetweenRM20521andRM20572,whichare680-kbapart(Kimetal.
2013).
FortheqTGW6locus,groupsD,E,F,G,H,I,andJshowedsignificantlyhighervaluesthanIlpumbyeofor1,000-grainTable1Comparisonof10agronomictraitsbetweenIL28andIlpumbyeoattwolocationsin2013TraitsMean±S.
D.
DaejeonDifferenceAandBYeasanDifferenceAandBIlpumbyeo(A)IL28(B)Ilpumbyeo(A)IL28(B)Daystoheading115113NS113112NSGrainweight/5plants(g)175189**#174190**#Paniclelength(cm)22.
3±1.
324.
3±2.
0**22.
5±1.
625.
4±1.
8**Spikeletsperpanicle157±11182±13**152±14185±15**Secondarybranch25.
3±4.
432.
2±1.
9**28.
3±3.
434.
2±2.
2**Firstnodewidth(mm)4.
3±0.
25.
2±0.
2****Secondnodewidth(mm)5.
3±0.
26.
2±0.
2****1,000grainweight(g)21.
6±0.
222.
9±0.
3**21.
5±0.
323.
1±0.
2**Amylosecontent(%)18.
1±0.
118.
3±0.
1NS18.
2±0.
118.
2±0.
1NSProteincontent(%)5.
5±0.
15.
8±0.
1NS5.
8±0.
15.
7±0.
1NSDatawerefromthetrial2013inDaejeonandYeasan.
ComparisonoftwolineswascarriedoutatDaejeonforthreeyearsfrom2011to2013,andtheresultswerenearlythesame.
Onlythe2013datafromDaejeonarepresented.
-Notevalutated#**SignificantatP<0.
01,NS:notsignificant.
(a)(b)(c)IlpumbyeoIL28IlpumbyeoIL28IlpumbyeoIL28Figure1PhenotypedifferencebetweenIlpumbyeoandIL28in(a)panicle,(b)grainsize(scalebar:3mm),and(c)nodewidth(scalebar:3mm).
III,IV,andVindicatethethird,fourth,andfifthnodes,respectively.
Kimetal.
Rice2014,7:14Page3of11http://www.
thericejournal.
com/content/7/1/14weight,whereasnosignificancedifferencewasdetectedbetweenIlpumbyeoandgroupsA,B,C,andKin2011,2012,and2013(Figure3a).
Thus,qTGW6waslocateddownstreamofRM20521andupstreamofRM20580.
GroupDshowedsignificantlyhighervaluesforSPPaf-fectedbyqSPP6andforTGWcomparedtoIlpumbyeo,whilegroupJwassignificantlydifferentfromIlpumbyeoforTGWbutnotSPP.
ThisresultindicatesthatqSPP6andqTGW6aredifferentgenes.
ComparisonofthegenotypesofrecombinantsdelimitedtheqTGW6betweenmarkersRM20562andRM20572,basedonthefindingthattheTGWofbothDandJgroupsweresignificantlydifferentfromthatofIlpum-byeo.
TheregionbetweenthetwomarkersRM20562andRM29572forgroupsDandJsharedcommonsegmentharboringofqTGW6.
TofurtherconfirmthecommonsegmentsharedbygroupsDandJ,wege-notypedtheF3linesusingfourmarkers(oneInDelmarkerandthreeSNPmarkers)locatedbetweenRM20562andRM20572.
ThegenotypesshowedthatqTGW6wasdefinedina37.
85-kbregionbetweenInDel-1andSNP3(Figure3b).
EvaluationofthegrainshapetraitTodeterminewhichgrainshapetraitsareassociatedwithincreaseingrainweight,threegrainshapetraits(specifically,grainlength[GL],width[GW],andthickness[GT])wereevaluatedforthetwopar-entsandF5lines(Figure3).
ThemeanphenotypicvaluesofeachF5linewerecomparedtothoseofIlpumbyeoandIL28attheP<0.
05level.
TheGLofIlpumbyeoandIL28were4.
88mmand5.
13mm,re-spectively.
TheGWvaluesforIlpumbyeoandIL28were2.
93mmand3.
15mm,respectively.
TheGLandGWvaluesofIL28weresignificantlyhighercomparedtoIlpumbyeo(P<0.
05),whereasnosig-nificantdifferencewasdetectedforGT.
TheGLandGWofthegroupswithqTGW6(D,E,F,G,H,I,andJ)weresignificantlydifferentfromthoseofthegroupswithoutqTGW6(A,B,C,andK).
Thisfind-ingindicatesthatqTGW6variationiscausedbyvariationinGLandGWatqTGW6.
GrainweightwassignificantlycorrelatedwithGLandGW(P<0.
01forbothcharacteristics),butnotwithGT(r=0.
06).
(a)(b)daysafterpollination05101520253035357911131517192123252729313305101520253579111315171921232527293133daysafterpollinationNIL(IL28)IlpumbyeoNIL(IL28)IlpumbyeoFigure2CharacterizationofgrainfillinginIlpumbyeoandIL28.
(a)Time-courseofendospermfreshweightincrease.
(b)Time-courseofendospermdryweightincrease.
Dataaremean±s.
d.
(n=7to10plants)inaandb.
Kimetal.
Rice2014,7:14Page4of11http://www.
thericejournal.
com/content/7/1/14Cross-sectionofthecentralpartofthespikelethullGiventhatthehulloftheIL28spikeletwaswiderthanthatofIlpumbyeobeforefertilization,thecross-sectionofthecentralpartofthespikelethullforgroupsDandJwasanalyzedincomparisontoIlpumbyeotoinvestigatetheoriginsoftheobservedsizedifferences(Figure4).
TheouterglumecelllayerofgroupsDandJcontainedsubstantiallymorecells(16.
5%and15.
3%,respectively)comparedtothatofIlpumbyeo,withonlya1.
3%and1.
4%increaseincelllength,respectively.
Thesedatain-dicatethattheincreasedwidthofgroupDandJspikelethullsmainlyresultsfromanincreaseincellnumber,ratherthancellsize,indicatingthatqTGW6maybein-volvedintheregulationofcelldivision.
ImpactoftheQTLclusteronyieldperplantElevengroupscontaining44F5lineswereusedfortheyieldtrials,togetherwithIlpumbyeoandIL28in2013(Figure3).
IL28hadsignificantlyhighergrainyieldcom-paredtoIlpumbyeo.
IL28produced8%highergrainyieldonaveragecomparedtoIlpumbyeo.
AllgroupswiththeMoroberekansegmentatbothqSPP6andqTGW6(i.
e.
,groupsD,E,F,G,H,andI)hadsignifi-cantlyhighergrainyieldvaluescomparedwiththe162b139b157b21.
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2bSPPTGW(g)(2011)(2012)(2011)(2012)P1P2A(5)B(4)C(2)D(5)E(5)F(4)G(5)H(4)I(2)Group&J(5)RM7269RM162RM3567RM20512RM20521RM3430RM20562RM20572RM20580RM3307RM20653RM20562RM20572LOC_Os06g45560LOC_Os06g45550LOC_Os06g45570LOC_Os06g45580APO1(2013)27.
56527.
57227.
58627.
603DJ27.
560InDel-1SNP1SNP2SNP327.
82837.
85kb(2013)GL(mm)GW(mm)GT(mm)YD(g)RM461K(3)Physicaldist.
address(Mb)(b)300-kbqTGW6RM7269RM20653(a)(c)(1,806bp)TGAATGIlpumbyeo,NipponbareCMoroberekanATyrosineCystein828(6,383bp)TAAATGIlpumbyeo,NipponbareAMoroberekanGAlanineThreonine2,267qSPP6(Kimetal.
2013)Figure3DevelopmentofahighdensitymapofqTGW6andstructuresofcandidategenesforqTGW6.
(a)GraphicalrepresentationofF3:4:5linesandafinescalemapofthetargetregiononchromosome6.
Apo1locatedbetweenRM3430andRM20562mightbeallelictoqSPP6detectedinthisstudy.
WhiteandblackportionsofthegrapharehomozygousIlpumbyeo,homozygousMoroberekan,respectivelyanddottedregionsarewherecrossing-overoccurred.
Genotypesofthe44linesweredoublecheckedatF3generation.
Thetabletotherightofthegraphicalgenotypesindicatesmeanvaluesoftraits.
NumbersfollowedbythesameletterineachcolumnarenotsignificantlydifferentatP=0.
05basedonDuncan'smultiplerangetest.
&No.
in()indicatethenumberofF3lines.
P1:Ilpumbyeo,P2:IL28.
(b)HighdensitymapofqTGW6usingtwogroups(DandJ)andfourmarkers.
Fourgeneswerepredictedinthetarget37.
85-kbregion.
Thearrowheadsindicatethepositionofasinglenon-synonymousnucleotidesubstitution.
(c)Structuresoftwogenes,Loc_Os06g45550andLoc_Os06g45560.
Loc_Os06g45550consistsoffiveexons(blackboxes)andfourintronsandLoc_Os06g45560consistsof8exonsand7introns.
Numbersonthestructureoftwogenesshowthepositionofnon-synonymousnucleotidesubstitutionsfromthestartcodon.
Kimetal.
Rice2014,7:14Page5of11http://www.
thericejournal.
com/content/7/1/14groupswithouttheMoroberekansegmentatbothqSPP6andqTGW6(i.
e.
,groupsA,B,C,andK)andIlpumbyeo(Figure3).
Also,theyieldperplantofgroupJwithqTGW6andwithoutqSPP6washigherthanthatofgroupCandIlpumbyeoatP<0.
1(P=0.
068,P=0.
067,respectively),andthisresultimpliesthecontributionofqTGW6totheyieldincrease.
However,wecouldnotde-terminetheeffectofqSPP6onyieldduetothelackofline(s)onlywiththeMoroberekansegmentatqSPP6.
CandidategenesforqTGW6qTGW6wasdefinedina37.
85-kbregionbetweenInDel-1andSNP3.
Onthebasisofavailablesequenceannotationdatabases(http://www.
gramene.
org),therearefourpredictedgenesinthetargetregion.
Thefunc-tionalannotationsofthefourgenesareasfollows:Loc_Os06g45550(fiveexonsandfourintrons)andLoc_Os06g45560(eightexonsandsevenintrons)areretrotransposonproteinputativeexpressedgenes;Loc_Os06g45570(oneexon)isaVQdomain-containingproteinputativeexpressedgene;Loc_Os06g45580(oneexon)isaRING-H2zincfingerputativeproteinexpressedgene(Figure3).
Wecomparedthegenomicsequencesoftheopenreadingframe(ORF)andpro-moterregionofthefourgenesbetweenIlpumbyeoandMoroberekan.
Comparisonofthegenomicsequenceoftwogenes(Loc_Os06g45550andLoc_Os06g45560)amongNipponbare,Ilpumbyeo,andMoroberekanre-vealedtheexistenceofasinglenon-synonymousSNPinthethirdandfourthexonofLoc_Os06g45550andLoc_Os06g45560,respectively.
Thenon-synonymousSNPatthethirdexonofLoc_Os06g45550ischaracter-izedbythenucleotideCytosineinIlpumbyeoandthenucleotideAdenineinMoroberekan.
ThechangeinthenucleotidebasefromCytosinetoAdenineresultedinmissensemutationfromcysteinetotyrosine.
Thenon-synonymousSNPatthefourthexonofLoc_Os06g45560,ischaracterizedbythenucleotideAdenineinIlpumbyeoandthenucleotideGuanineinMoroberekan.
ThenucleotidesubstitutionA(adenine)toG(guanine)inLoc_Os06g45560resultedinamissensemutationfromThreoninetoAlanine.
NosequencevariationinopenreadingframesandthepromoterregionoftwogeneswasdetectedforLoc_Os06g45570andLoc_Os06g45580amongthethreevarieties,Nipponbare,Ilpumbyeo,andMoroberekan.
IlpumGroupDGroupJ01234567891011IlpumNIL-DNIL-JCelllength(um)1000110012001300140015001600IlpumNIL-DNIL-JCellnumberabcabbaaaFigure4Histologicalanalysesofspikelethull3daysbeforeheadinginIlpumbyeo,groupD,andgroupJ.
(a)Cross-sectionofspikelethull.
Upper:spikelets(scalebar,3mm).
Low:cross-sectionofspikelethull(scalebar,500um).
Dottedlineindicatespositionofcross-section(b)Magnifiedviewofspikelethullcross-sectionboxedina.
(c)ComparisonoftotalcellnumberandmeancelllengthintheouterglumecelllayersofspikelethullofIlpumbyeo,groupD,andgroupJ.
(n=5to8spikelets).
NumbersfollowedbythesameletteroneachboxarenotsignificantlydifferentatP=0.
05basedonDuncan'smultiplerangetest.
Kimetal.
Rice2014,7:14Page6of11http://www.
thericejournal.
com/content/7/1/14DiscussionTherehasbeenmuchdebateastowhethersimilargen-omiclocationsofQTLsthataffectdifferenttraitsarecausedbythepleiotropyofasinglegeneorthetightlinkageofseveralgenesthatindividuallyinfluencespe-cifictraits.
Thequestionofpleiotropyversustightlink-agemightberesolvedbyusingalarge-sizepopulationcombinedwithhigh-densitymapping,becausetwolinkedQTLssegregateindependentlyinNIL-F2:3popu-lations.
Recently,severalQTLscontrollingthesameormultipletraitshavebeenconfirmedtobecloselylinkedinasmallgenomicregion.
Forexample,thericephoto-periodsensitivitygene,Hd3wasoriginallydetectedasaheadingdate-relatedquantitativetraitlocusthatislocalizedonchromosome6intheF2population(Yanoetal.
1997).
Forthefine-scalemappingofHd3,high-resolutionlinkageanalysisusingalargesegregatingpopulationderivedfromadvancedbackcrossprogenybetweenthejaponicaricevariety,Nipponbareandtheindicavariety,Kasalath,wascarriedout.
Asaresultofthehigh-resolutionlinkagemappingofHd3,twotightlylinkedloci,Hd3aandHd3b,whichpromotefloweringundershort-dayconditionsandinhibitheadingunderlong-dayconditions,wereidentifiedintheHd3region(Monnaetal.
2002).
Louetal.
(2013)mappedtwoQTLs(qSPP5andqTGW5)forspikeletsperpanicleand1,000-grainweighttothesameregiononchromosome5.
SubstitutionmappingwiththeBC5F3andBC5F4pop-ulationsderivedfromacrossbetweenQTL-NILandtherecurrentparent,demonstratedthattwotightlylinkedQTLs(qSPP5andqTGW5)weredifferent.
SeveralQTLswithlargepleiotropiceffectsonmultipletraitshavealsobeenfoundandcloned(APO1,Ghd7,andGhd8orDTH8).
Forinstance,Huaetal.
(2002)reportedthataQTLclusterinfluencesmultipletraits(namely,grainperpanicle,plantheight,andheadingdate)onchromosome7inaprimarypopulation.
High-resolutionmappingwithalarge-sizedsegregatingpopulationandcom-plementationtestsrevealedthatthesethreetraitsarecontrolledbyasinglelocus,designatedasGhd7(Xueetal.
2008).
TheoriginaltargetofthisstudywasqSPP6,whichwasmappedonthelongarmofchromosome6(Juetal.
2008).
Intheprocessofthefine-scalemapping,theQTLforadditionalyieldcomponents(includingpaniclelength,nodewidth,and1,000-grainweight)werecon-sistentlydetectedinthesameregion.
Atotalof243F2plantsderivedfromacrossbetweenNIL(IL28)andIlpumbyeoforgeneticanalysiswasnotlargeenoughtoclarifytherelationshipbetweenSPPandTGW.
Tocon-firmthepreciselocationofthetwoQTLs,substitutionmappingwascarriedout.
Followingsubstitutionmap-pingwith44F3:4:5lines,qSPP6wasmappedtoa431-kbintervalbetweenRM20521andSNP1,whileqTGW6wasmappedtoa37.
85-kbintervalbetweenInDel-1andSNP3,basedonthejaponicagenomesequence.
There-fore,thedevelopmentofNILandsubstitutionmappinghasenabledustomapSPPandTGWatthefine-scale.
NoTGW-associatedQTLwasdetectedneartheqTGW6QTLinthisstudy,indicatingthatqTGW6isanovelQTLforTGW.
AmongthefourpredictedgenesinthetargetregionbetweenInDel-1andSNP-3(37.
85-kb),Loc_Os06g45570encodesaVQdomain-containingprotein.
VQdomainsarebelievedtobecriticalforthedefenseresponsetopathogensbyinteractingwithWRKYtranscriptionfactors(Taoetal.
2011).
Loc_Os06g45580encodestheRING-H2zincfingerprotein.
Recentstudiesreportedacriticalroleoftheubiquitinpathwayingraindevelopmentinrice(Songetal.
2007;Wengetal.
2008).
GW2,amajorQTLforricegrainwidthandweighten-codesanewRING-typeE3ubiquitinligase.
GW5,anothermajorQTLunderlyingricewidthandweight,islikelytoactintheubiquitine-proteasomepathwaytoregulatecelldivisionduringcelldivision(Wengetal.
2008).
qTGW6exhibitedcommonfunctionforgrainwidthwithGW2andGW5inthatitwasassociatedwithincreasedcellnumbers,awiderspikelethullandanincreasedgrainmilk-fillingrate.
ThisraisesapossibilitythatLoc_Os06g45580isacandidateforqTGW6.
However,nosequencevariationinopenreadingframesandthepromoterregionsoftwogenes,Loc_Os06g45570andLoc_Os06g45580wasob-servedbetweenIlpumbyeoandMoroberekanandthisresultseemstosuggestthattheyarenotcandidategenesforqTGW6.
Theothertwogenesinthetargetregionareretrotransposonproteins,Loc_Os06g45550andLoc_Os06g45560.
Loc_Os06g45560wastranscribedinseedlingandembryo/endosperm25daysafterpollin-ationbutLoc_Os06g45550wasnottranscribedbasedontheavailablegeneexpressiondatabase(RGAP,http://rice.
plantbiology.
msu.
edu/expression.
shtml).
Morethan40%ofricegenomeiscomposedofrepetitivesequencesandtransposableelements(Goffetal.
2002).
ArecentstudyshowedthattheexpressionofTos17,acopia-likeretrotransposonwasaffectedbyoverexpres-sionordown-regulationofseveralriceSUVH(Su(var)3–9homologs)genesandRNAiplantswithdown-regulatedSDG728,ariceSUVHgeneshowedareducedseedsizephenotype(Qinetal.
2010).
ThisraisesapossibilitythatarelationshipbetweenalteredseedmorphologyandoverexpressionofTos17mightexist.
Interestingly,ouralignmentanalysisofGW5a.
asequenceusingBLASTPprogramshowedthatGW5proteinhashighhomologywithtworetrotrans-posonproteinsencodedbyLOC_Os08g26160.
1andLOC_Os05g47720.
1(datanotshown),althoughithasbeenreportedthattheproteinencodedbytheGW5ORFhadnosequencehomologywithanyproteinsofknownmolecularfunction(Wengetal.
,2008).
GW5Kimetal.
Rice2014,7:14Page7of11http://www.
thericejournal.
com/content/7/1/14proteinshowed56%homologytotheretrotransposonproteinwith194a.
aencodedbyLOC_Os08g26160.
1and47-50%homologiestothetransposonproteinwith411a.
a.
byLOC_Os05g47720.
1.
NumerousIndelsandsingle-nucleotidepolymorphisms(SNPs)weredetectedbetweenNipponbare(japonica)and9311(indica)andIndelsaremainlycausedbytransposons(Fengetal.
2002).
IndelsandSNPsinsomekeydomesticationgenesincludingqSH1andGW2areresponsibleformorphologicalchangesoccurredduringdomestication(Konishietal.
2006;Songetal.
2007).
Also,thediffer-enceingrainwidthbetweenjaponicaandindicaculti-varsisassociatedwitha1212-bpdeletionharboringtheGW5ORFwhichwaspossiblycausedbytransposons(Wengetal.
2008).
Theseobservationssupportourhy-pothesisthattheproteinencodedbytheGW5ORFmightbearetrotransposonandLoc_Os06g45560isacandidateforqTGW6.
However,thepresenceofSNPsintheupstreamregionsofthegenesbetweentwopar-entssuggeststhepossibilitythatalong-distanceregula-tionisinvolvedincontrollingtheqTGW6(datanotshown).
Konishietal.
(2006)reportedthatthecasualmutationinqSH1underlyingtheseedshatteringwasfoundinthe12-kbupstreamregionfromtheORFofthegeneandcausedtheabsenceoftheabscissionlayerinNipponbare.
Additionalfinemappingexperimentsareunderwaytodeterminethefunctionalrelationshipbe-tweenqTGW6andGW5incontrollingseeddevelopment.
Also,loss-of-functionmutantlinesofLoc_Os06g45560willbeevaluatedforgrainmorphologytraitsandthegeneexpression.
Theseinformationsonthemolecularmechan-ismofqTGW6wouldbenecessarytogetmoreinsightsintothemechanismofgraindevelopment.
AlthoughthecandidategeneofqTGW6anditsgen-eticmechanismwasnotelucidatedhere,wefoundthattheMoroberekanchromosomalsegmentatthisregion(atropicaljaponica)harborsQTLsforyieldcomponenttraitsleadingtoanyieldincreaseinjaponicacultivar.
YieldtrialsconfirmedthatintrogressionlinescontainingtheMoroberekansegmentattheqSPP6andqTGW6lociproducedsignificantlyhigheryieldsthanIlpumbyeo(P<0.
05).
ThegrainyieldperplantinIL28was8%higherthanthatofIlpumbyeo.
Moroberekanallelesinthetargetregiononchromosome6hadafavorableeffectonyield,byincreasingthenumberofspikeletsperpanicleand1,000-grainweight,inadditiontolodgingtolerancebyin-creasingthenodewidth,whereasithadnonegativeeffectonheadingdateandplantheight.
Thus,theqSPP6andqTGW6allelesarepotentiallyvaluableforimprovingriceyield.
ItisproposedthatNILscontainingthetargetseg-mentassociatedwithpositiveQTLsfromMoreberekanshouldbedevelopedfromthispopulation,andevaluatedinawiderangeofenvironments,toassesstheinteractionbetweenQTLsandtheenvironment.
TightlylinkedSSRmarkersareexpectedtofacilitatethecloningofgenesunderlyingtheseQTLs,inadditiontomarker-assistedse-lectionforvariationintheSPPandTGWinricebreedingprograms.
ConclusionInthisstudy,wedemonstratedthattwoQTLs,qSPP6forspikeletsperpanicle(SPP)andqTGW6for1,000-grainweight(TGW)aretightlylinkedonchromosome6.
BecauseMoroberekanallelesfortheSPPandTGWhavebeenshowntobebeneficialinthegeneticback-groundofIlpumbyeo,theqSPP6andqTGW6allelesmayprovevaluableforimprovingtheyieldpotentialofjaponicaricecultivars.
TightlylinkedSSRmarkersareexpectedtofacilitatethecloningofgenesunderlyingtheseQTLs,inadditiontomarker-assistedselectionforvariationinSPPandTGWinricebreedingprograms.
MethodsPlantmaterialsInapreviousstudy(Kimetal.
2013),IL28wasshowntoexhibitahighernumberofspikeletsperpanicleandheaviergraincomparedtotherecurrentparent,andcontainedtheMoroberekanchromosomalsegmentattheqSPP6regiononchromosome6;hence,itwasse-lectedforthefine-scalemappingofqSPP6inthecurrentstudy.
IL28wascrossedwiththecultivarIlpumbyeo,withtheresultingF1plantsbeingself-pollinatedtoob-tainanF2population(234plantsin2009and1,150plantsin2010).
ToconfirmqSPP6,44F2homozygousplantswithdifferentcross-overbreakpointsbetweenRM7269andRM20653wereselectedandselfedtopro-duceF3linesforthephenotypingandsubstitutionmap-pingofqSPP6.
Intheprocessofthefine-scalemappingofqSPP6,aQTLfor1,000-grainweight(TGW)wasconsistentlydetectedinthesameregion.
ToconfirmthepreciselocationofqSPP6andqTGW6,these44F3lineswereadvancedtoF4andF5lines,whichwereusedtoevaluatespikeletsperpanicle,grainweight,grainmorphologytraits,andgrainyield.
FieldtrialandtraitevaluationAtotalof243F2plantsand1150F2plants(2009and2010),and41F3:4:5lines(2011,2012,and2013)andtheparentallinesweregrownattheexperimentalfieldofChungnamNationalUniversity,Daejeon,Korea.
In2009,the243plantsandtheparentswereplantedatdis-tancesof30*15cm,andevaluatedforgrain(brownrice)weight(150outof243plantswereusedtoevaluategrainweight).
In2010,forthesubstitutionmappingofqTGW6,1,150F2plantsweregenotypedwithfouradditionalmarkersbeinglocatedbetweenRM7269andRM20653(RM20512,RM20521,RM20652,andRM20572)andonemarker(RM551)locatedonchromosome4.
TheKimetal.
Rice2014,7:14Page8of11http://www.
thericejournal.
com/content/7/1/14experimentusing44F3:4:5linesderivedfrom44F2plantsfollowedacompletelyrandomizedblockdesignwithtworeplications,onerowperplot,and25plantsperrowin2011,2012,and2013.
Themiddle10plantsfromeachlinewerechosenfortheevaluation,andtheaverageofthemea-surementswasusedforthephenotypeofeachlineforthespikeletsperpanicle(SPP)and1,000-grainweight(TGW).
SPPweremeasuredbyaveragingthethreemajorpaniclesperplants.
Grainswithhullswereallowedtodrynaturallyafterharvesting,andpartialorunfilledseedswereremovedbysoakinggrainsinwater.
Fullyfilledseedswerere-driedinanovenat30°Cfor24h.
TheTGWwasevaluatedbymeasuringtheweightof250randomlyselected,de-hulledgrainsperplant(10plantsperline).
In2013,grainlength(GL),grainwidth(GW),grainthickness(GT),andyieldperplants(YD)weremeasured.
Thegrainlength(GL),grainwidth(GW),andgrainthickness(GT)weremeasuredfor50grains(brownrice)perplant(10plantsperline)usinga150-mmVerniercaliper(MitutoyoCorp.
,Japan).
Yieldperplant(YD)wasmeasuredbyaveragingthegrainyield(g)of10plantsthatwererandomlyselectedfromthecenterofeachplotperblock.
The1,000-grainweightandtheyieldperplantwerecorrectedforthe12%grainmoisturecontent.
DNAextractionandSSRanalysisDNAwasextractedfromtheleaftissueoftheF2popula-tionusingachloroform-basedDNAextractionprotocol(Causseetal.
,1994).
A20-μLreactionmixturewasused,containing5.
0μL(5ng/μL)oftemplateDNA,0.
1μl(5Unit/μL)Taqpolymerase,0.
8μldNTP(2.
5mMeach),Forward+Reverseprimer1μl(10pmoleach),2.
0μl10*PCRbuffer(10mMTris–HClPH8.
3,50mMKCl,1.
5mMMgCl2,0.
1%gelatin),and11.
1μltriple-distilledwater.
AmplificationwasachievedusingaThermoCycler(Bio-Rad)basedonthestep-cycleprogramsetfordenatur-ationat94°Cfor5min,subsequentdenaturationwasper-formedat94°Cfor1min,annealingat55°Cfor1min,extensionat72°Cfor1min;steps2to4wererepeatedforatotalof35cycles,withafinalextensionstepat72°Cfor5min.
PCRproductswererunon4%polyacrylamidede-naturinggelsfor1–2hat1800–2000V,andmarkerbandswererevealedbysilverstaining(Panaudetal.
1996).
TheorientationoftheSSRmarkerswasbasedontheSSRmaps(McCouchetal.
2002).
DuetothelackofmarkersshowingpolymorphismintheregionbetweenRM20562andRM20572,additionalgenotypingoftheF3lineswascon-ductedwithfourtargetedSNPsandoneInsertion/Delitionmarker.
TodetecttheSNPsandInDelsofIlpumbyeoandMoroberekan,resequencingwithNGS(Next-GenerationSequencing)wasconductedusingthetwoparentsaccordingtoJeongetal.
(2013).
ThroughthecomparisonofgenomicsequencescorrespondingtotheregionbetweenRM20562andRM20572,atotalof28polymorphisms(14SNPs,and14Insertion/Deletions)weredetectedbetweenIlpumbyeoandMoroberekan.
Thepolymorphismswereassayedbythedirectsequencingofthefourregions.
InformationontheprimersusedispresentedinTable2.
Theprimerswerede-signedaccordingtotheNipponbaresequence(http://rgp.
dna.
affrc.
go.
jp/E/IRGSP/Build5/build5.
html).
ThefirstInDel,hereafterreferredtoasInDel-1,occursatthe27,565,774thpositionbasedontheNipponbaresequence(www.
gramene.
org),andischaracterizedbynucleotide-(1bpdeletion)inIlpumbyeoandnucleotideAinMoroberekan.
ThefirstSNP,SNP-1,thatoccursatthe27,572,767thposition,ischaracter-izedbynucleotideGinIlpumbyeoandnucleotideAinMoroberekan.
ThesecondSNP,SNP-2,thatoccursatthe27,586,202thposition,ischaracterizedbynucleotideAinIlpumbyeoandnucleotideGinMoroberekan.
ThethirdSNP,SNP-3,thatoccursatthe27,629,080thposition,ischaracterizedbynucleotideAinIlpumbyeoandnucleotideCinMoroberekan.
StatisticalanalysisStatisticalanalysiswasperformedusingQgenesoftwareversion2.
30forMacintosh(Nelson1997)andSAS(SASInstitute).
Singlepointanalysis(SPA)wasperformedtodeterminetheeffectofeachmarkeroneachtrait.
InSPA,aQTLwasconfirmedifthephenotypewasassoci-atedwithamarkerlocusatP<0.
005,orwithtwoadja-centmarkerlociatP<0.
01.
TheproportionoftotalphenotypicvariationexplainedbyeachQTLwascalcu-latedasanR2value,fromtheregressionofeachmarker/phenotypecombination.
QTLsweremappedatafine-scalebycomparingthephenotypicmeansoftwogeno-typicclassesofrecombinants(IlpumbyeohomozygoteandMoroberekanhomozygote)withinthetargetregionbyusingtheSASstatisticalsoftwarepackage.
HistologicalanalysisParaffin-embeddedsectionsofspikeletsampleswerepreparedaccordingtoLietal.
(2009),withminorTable2PrimersequencesofnewlydevelopedSNPmarkersMarkernameForwardprimer(5'-3')Reverseprimer(5'-3')PositionProductsize(bp)InDel-1GAGAAAGTGACCCTCGTTTAGTCAAGACATGACACTACGATTGC27,565,623–27,565,899276SNP1CCATATGATTAAATGGGAGGCTCACATAGGCACTGCCAAGGTTC27,572,630–27,572,896266SNP2ACGGACGCCTGCTATAGGGACACCAGAGGCGATCTTCTTC27,585,994–27,586,360366SNP3GAATATCACGATCAAAGAACTTGGTTACTCTAATATGATAATATGCC27,603,470–27,603,822352Kimetal.
Rice2014,7:14Page9of11http://www.
thericejournal.
com/content/7/1/14modifications.
PlantmaterialswerefixedinFAA(50%ethanol,5%glacialaceticacidand5%formaldehyde)andstoredat4°Cfor24h.
Thefixedspikeletsweredehydratedbysoakingthemfor6hinagradientetha-nolseries(60%,70%,80%,90%),andwerethenincu-batedin100%ethanolovernight.
DehydratedspikeletswereembeddedinParaplast(Sigma).
Tissuesections(8μmthick)werecutusingarotarymicrotome,trans-ferredontogelatin-coatedglassslides,anddriedat42°Cfor1day.
Thesectionswerede-paraffinizedwith100%xylenefor5min,followedbysoakingfor2minin50%ethanol/50%xylen,100%ethanol,andsterilewater.
Thesampleswerestainedwithtoluidinebluefor30s,andwashedtwicewithsterilewater.
Thesampleswerethensoakedfor2minin70%ethanol,80%ethanol,90%ethanol,and100%ethanol.
Finally,thesampleswerecleanedbysoakingthemtwicein100%xylen.
Thesec-tionswerephotographedunderanOlympusBH2-RFCAusingaU-PMTVCcamera.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsDKparticipatedinphenotyping,genotyping,draftingthemanuscript.
HL,MFandJKparticipatedinphenotyping.
SKparticipatedinsequencing,primerdesignandgenotyping.
SAconceivedofthestudy,draftedproposalandcorrectedmanuscript.
Allauthorshavereadandapprovedthemanuscript.
AcknowledgementsThisworkwassupportedbytheKoreaResearchFoundationGrantfundedbytheKoreanGovernment(MOEHRD,BasicResearchPromotionFund)(KRF-2010-0024118)andtheNext-GenerationBiogreen21Program(PlantMolecularBreedingCenterNo.
PJ008136),RuralDevelopmentAdministration,RepublicofKorea.
Authordetails1DepartmentofAgronomy,CollegeofAgriculture&LifeSciences,ChungnamNationalUniversity,Daejeon305-764,SouthKorea.
2NationalAcademyofAgriculturalScience,RuralDevelopmentAdministration,Suweon441-707,SouthKorea.
3ChungnamAgriculturalResearchandExtensionServices,Yesan340-861,SouthKorea.
4Presentaddress:DepartmentofVarietyTesting,KoreaSeed&VarietyService,MinistryofAgriculture,FoodandRuralAffairs,Kimcheon740-220,SouthKorea.
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rufipogon.
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ProcNatlAcadSciUSA104:16402–16409ZhangY,LuoL,LiuT,XuC,XingY(2009)FourriceQTLscontrollingnumberofspikeletsperpanicleexpressedthecharacteristicsofsingleMendeliangeneinnearisogenicbackgrounds.
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TheorApplGenet95:799–808doi:10.
1186/s12284-014-0014-5Citethisarticleas:Kimetal.
:High-densitymappingofquantitativetraitlociforgrain-weightandspikeletnumberinrice.
Rice20147:14.
Submityourmanuscripttoajournalandbenetfrom:7Convenientonlinesubmission7Rigorouspeerreview7Immediatepublicationonacceptance7Openaccess:articlesfreelyavailableonline7Highvisibilitywithintheeld7RetainingthecopyrighttoyourarticleSubmityournextmanuscriptat7springeropen.
comKimetal.
Rice2014,7:14Page11of11http://www.
thericejournal.
com/content/7/1/14
Consequently,thegeneticbasisofspikeletsperpanicle(SPP)andgrainweight(TGW)havereceivedmuchresearchfocusbecauseoftheirimportanceinriceyield.
Results:Inthisstudy,IL28,whichisanearisogenicline(NIL)developedbyintrogressingchromosomalsegmentsofthecultivar'Moroberekan'intothecultivar'Ilpumbyeo',showedasignificantincreaseinthenumberofspikeletsperpanicle(SPP)and1,000-grainweight(TGW)comparedtotherecurrentparent,Ilpumbyeo.
Quantitativetraitlocus(QTL)analysisin243F2plantsderivedfromacrossbetweenIL28andIlpumbyeoindicatedthatbothqSPP6andqTGW6arelocatedintheintervalRM3430–RM20580.
Followingsubstitutionmappingwith50F3:4:5lines,qSPP6wasmappedtoa429-kbintervalbetweenRM20521andInDel-1,whileqTGW6wasmappedtoa37.
85-kbintervalbetweenInDel-1andSNP–3basedonthejaponicagenomesequence.
ThisresultindicatesthatqSPP6andqTGW6aredifferentgenes.
YieldtrialswithsubstitutionlinesindicatedthatlinesharboringthehomozygousMoroberekansegmentatboththeqSPP6andqTGW6regionshowedsignificantlyhighergrainyieldthanIlpumbyeo.
Conclusion:BecausetheMoroberekanallelesforSPPandTGWhavebeenshowntobebeneficialinthegeneticbackgroundofIlpumbyeo,boththeqSPP6andqTGW6allelesmightprovevaluableinimprovingriceyields.
CloselylinkedSSRmarkersareexpectedtofacilitatethecloningofgenesthatunderlietheseQTLs,aswellaswithmarker-assistedselectionforvariationinSPPandTGWinricebreedingprograms.
Keywords:Rice;Spikeletsperpanicle;Grainweight;QTL;Linkage;NearisogeniclineBackgroundRice(OryzasativaL.
)istheworld'smostimportantcerealfoodcrop.
Theanticipatedrapidincreaseintheglobalhu-manpopulation,whichisexpectedtoreach9.
1billionby2050,mightgenerateseriousfoodshortageproblems.
Moreover,variousfactors,suchaswaterscarcity,soilsalin-ity,disease,climatechange,andreducedarableland,willexacerbatefoodshortagesinthenext50years(Khush1999andKhush2005;Zhang2007).
Therefore,researchersarefocusingonincreasingexistingcropgrainyieldlevels.
Grainweight,spikeletsperpanicles,andthenumberofpaniclesperplantarethemostimportantcomponentsofgrainyield.
However,thegeneticanalysisofthesethreeyieldcomponentsisdifficult,becausethesetraitsarecontrolledbymultiplegenes,inadditiontobeinginfluencedbytheenvironment.
Therefore,theadventofmolecularmapsforrice(Causseetal.
1994)andquantitativetraitlocus(QTL)analysisapproacheshavefacilitatedtheanalysisofthesequantitativetraits.
Genes/QTLsforspikeletsperpanicle(SPP)havebeenreportedinpopulationsderivedfrominter-specificcrosses(Xiongetal.
1999;Thomsonetal.
2003;Suhetal.
2005;Lietal.
2006andLinhetal.
2008),indica-indicacrosses(Linetal.
1996;Zhuangetal.
1997),andinter-subspecificcrosses(Yamagishietal.
2002;Andoetal.
2008).
Todate,afewQTLsassociatedwithSPPshavebeendetected,includingGn1a,APO1,DEP1,andOsSPL14.
Gn1acon-trolsgrainnumberinrice,andwasfoundtoencodecytokininoxidase/dehydrogenase(OsCKX2),whichisanenzymethatdegradesthephytohormonecytokinin(Ashikarietal.
2005).
HigherexpressionofOsSPL14duringthereproductivestageofricepromotespanicle*Correspondence:ahnsn@cnu.
ac.
kr1DepartmentofAgronomy,CollegeofAgriculture&LifeSciences,ChungnamNationalUniversity,Daejeon305-764,SouthKoreaFulllistofauthorinformationisavailableattheendofthearticle2014Kimetal.
;licenseeSpringer.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
Kimetal.
Rice2014,7:14http://www.
thericejournal.
com/content/7/1/14branchingleadingtohighergrainyield(Miuraetal.
2010).
DEP1,whichalterspaniclearchitecture,andhenceincreasesgrainyield,hasalsobeenidentified(Huangetal.
2009).
Anotherapproachtoidentifythegenesrelatedtopaniclearchitectureisthroughtheanalysisofmutantsthatalterpaniclestructure.
Thecharacterizationoftheaberrantpanicleorganization1(apo1)mutantinricerevealedthatAPO1positivelycontrolsspikeletnumberbysuppressingtheprecociousconversionofinflores-cencemeristemstospikeletmeristems(Ikedaetal.
2007).
Ookawaetal.
(2010)alsoreportedthataneariso-geniclinecarryingAPO1enhancedculmstrengthandincreasedspikeletnumberperpanicle.
Grainweight(GW)isanothercharacteristicthatistar-getedtoenhancericeyield.
ManyQTLsforgrainsizehavebeenidentifiedinricepopulationsbasedoncrossesbetweendivergentcultivarsoraccessions(Linetal.
1996;Cuietal.
2003;Ishimaru2003;Zhangetal.
2009).
Severalgenesthatregulategrainweighthavebeenidentified,in-cludingGS3,GS5,GW5,GW2,andTGW6.
GW2isaQTLforricegrainwidthandweight,whichencodesapreviouslyunknownRING-typeE3ubiquitinligasethatnegativelyregulatescelldivisionbytargetingitssubstrate(s)fordegradationbytheubiquitin-proteasomepathway(Songetal.
2007).
Ishimaruetal.
(2013)foundthatwhentheIAA-glucosehydrolasegenestopsfunctioning,TGW6enhancesgrainweightthroughpleiotropiceffectsonsourceorgans,andleadstosignificantyieldincreases.
Numerousstudieshavereportedthatonegenomicre-gionharborsmorethantwoindependentgenesthatarecloselylinked,oragenewithpleiotropiceffectonsev-eraltraits(Xieetal.
2008;Jinetal.
2009;Louetal.
2013;Jietal.
,2014).
Xieetal.
(2008)reportedthattwoQTLs,sn9.
1andgw9.
1,for1,000-grainweightandspikeletsperpaniclewereidentifiedinthe37.
4-kbinter-valflankedbymarkersRM24718andRM30005.
TwoQTLsactedinanadditivemannerinthisregion,result-ingintheNILwiththeO.
rufipogonallelesexhibitingsignificantlyhigherSPPandTGWvaluescomparedtotherecurrentparent,Hwaseongbyeo(Xieetal.
2008).
However,itremainsunclearwhethereitherofthetwocloselylinkedQTLs,oronepleiotropicQTL,areassoci-atedwiththeobservedvariation.
Alarge-sizedpopula-tioncombinedwithhigh-densitymappingisrequiredtodeterminewhethertargettraitsarecontrolledbycloselylinkedQTLs,oronepleiotropicQTL.
Inpreviousstud-ies(Juetal.
2008),IL28wasdeveloped,whichisanintrogressionlinederivedfromacrossbetweenthecul-tivarsIlpumbyeoandMoroberekan,showinghigherSPPandTGWvaluescomparedtoIlpumbyeo.
Kimetal.
(2013)reportedthataQTLforspikeletsperpaniclewaslocatedbetweenRM20521andRM20572usingF2:3pop-ulationsderivedfromacrossbetweenIlpumbyeoandIL28.
ThepresentstudywascarriedouttodetectanovelQTLforTGW,andtoclarifyitsrelationshiptotheqSPP6QTLusingF3:4:5populations.
ResultsCharacteristicsofIL28Inourpreviousstudy(Kimetal.
2013),thegeneticmapofIL28wasconstructedusing134markersthatexhib-itedpolymorphismbetweenIlpumbyeoandMorobere-kan,with2Moroberekanintrogressionsegmentsbeingdetectedonchromosomes4and6.
Thephenotypicevaluationofagronomictraitsfortheintrogressionline,IL28,andtherecurrentparentwasconductedattwolo-cations,DaejeonandYeasan,in2013.
Comparisonof10agronomictraitsbetweenIL28andIlpumbyeoobtainedinthecurrentstudyareshowninTable1andFigure1.
Theresultsindicatedthattherewerehighlysignificantdifferences(P<0.
01)inpaniclelength(PL),secondarybranchnumber(SBN),spikeletsperpanicle(SPP),firstnodewidth(FNW),secondnodewidth(SNW),and1,000-grainweightbetweenIL28andIlpumbyeo,whilenosignificantdifferencewasobtainedfortillernumber(TN)andculmlength(CL).
BecauseIL28hasalargeendospermandheaviergrain,weinvestigatedthegrainmilk-fillingrateinIL28andIlpumbyeo.
Nodifferencewasobservedineitherendospermfreshweightordryweightat3dand6dafterfertilization(Figure2).
Incomparison,boththefreshanddryweightofIL28weresignificantlyhigher(P<0.
01)comparedtothoseofIlpumbyeoat11dafterfertilization.
Thesedifferencesreachedamaximum~25dafterfertilization,atwhichpointthefreshanddryweightoftheendospermofIL28were11.
2%and13.
4%highercomparedtoIlpumbyeo,respectively.
Thus,anincreaseingrainweightinIL28whichisassociatedwithhighergrainmilk-fillingratemightbeduetotheMoroberekansegmentonchromo-some6consideringthatnoQTLforgrainwidthandweightwasdetectedintheMoreberekansegmentonchromosome4.
TraitvariationoftheF2populationInourpreviousstudy(Kimetal.
2013),fourtraits(paniclelength,numbersofprimarybranchesandsecondarybranches,andspikeletsperpanicle)exhibitedcontinuousandnormaldistributionsinthe243F2plants.
The250-grainweightofIlpumbyeoandIL28was5.
51and6.
14,re-spectively.
Inalltraitsmeasured,transgressiveplantswithhigherorlowermeanvaluescomparedtoeitherparentwereobserved.
Thecorrelationbetweenspikeletsperpan-icleandgrainweightwasnotsignificant(r=0.
102).
QTLanalysisThreesignificantQTLsforpaniclelength,secondarybranchnumber,andspikeletsperpanicleweredetected.
TheseQTLsoccurredinthesameregion,neartheSSRKimetal.
Rice2014,7:14Page2of11http://www.
thericejournal.
com/content/7/1/14markerRM3430(Kimetal.
2013).
Inaddition,theQTLforgrainweightwasdetectedinthesameregion,onchromosome6.
TheQTLforgrainweightexplained8.
3%ofphenotypicvariance.
TheMoroberekanallelesincreasedalltraitvaluesofpaniclelength,secondarybranchnumber,spikeletsperpanicleandgrainweightatthisregion.
SubstitutionmappingofqTGW6Toconfirmandnarrowdownthetargetregioncontain-ingqTGW6,1,120F2plantsderivedfromacrossbe-tweenIlpumbyeoandIL28weregenotypedwith4additionalmarkersnearthetargetregion(RM20512,RM20521,RM20562,andRM20572)andonemarker(RM551)locatedonchromosome4.
Atotalof44F2homozygousplantswithdifferentcross-overbreak-pointsbetweenRM7269andRM20653onchromosome6wereselectedandselfedtoproduceF3:4:5lines.
Geno-typeanalysisindicatedthatthese44F2plantsdidnothaveMoroberekansegmentonchromosome4.
Accord-ingtothemarkergenotype,the44F3:4:5linesweredi-videdinto11groups,andusedforthesubstitutionmappingofqTGW6(Figure3).
ThemeanphenotypicvaluesofeachtraitforeachF3:4:5linewascomparedtothoseofIlpumbyeoandIL28attheP<0.
05level.
Inapreviousstudy,theQTLsforSPP,PL,SBM,FNW,andSNWwereconsistentlymappedbetweenRM20521andRM20572,whichare680-kbapart(Kimetal.
2013).
FortheqTGW6locus,groupsD,E,F,G,H,I,andJshowedsignificantlyhighervaluesthanIlpumbyeofor1,000-grainTable1Comparisonof10agronomictraitsbetweenIL28andIlpumbyeoattwolocationsin2013TraitsMean±S.
D.
DaejeonDifferenceAandBYeasanDifferenceAandBIlpumbyeo(A)IL28(B)Ilpumbyeo(A)IL28(B)Daystoheading115113NS113112NSGrainweight/5plants(g)175189**#174190**#Paniclelength(cm)22.
3±1.
324.
3±2.
0**22.
5±1.
625.
4±1.
8**Spikeletsperpanicle157±11182±13**152±14185±15**Secondarybranch25.
3±4.
432.
2±1.
9**28.
3±3.
434.
2±2.
2**Firstnodewidth(mm)4.
3±0.
25.
2±0.
2****Secondnodewidth(mm)5.
3±0.
26.
2±0.
2****1,000grainweight(g)21.
6±0.
222.
9±0.
3**21.
5±0.
323.
1±0.
2**Amylosecontent(%)18.
1±0.
118.
3±0.
1NS18.
2±0.
118.
2±0.
1NSProteincontent(%)5.
5±0.
15.
8±0.
1NS5.
8±0.
15.
7±0.
1NSDatawerefromthetrial2013inDaejeonandYeasan.
ComparisonoftwolineswascarriedoutatDaejeonforthreeyearsfrom2011to2013,andtheresultswerenearlythesame.
Onlythe2013datafromDaejeonarepresented.
-Notevalutated#**SignificantatP<0.
01,NS:notsignificant.
(a)(b)(c)IlpumbyeoIL28IlpumbyeoIL28IlpumbyeoIL28Figure1PhenotypedifferencebetweenIlpumbyeoandIL28in(a)panicle,(b)grainsize(scalebar:3mm),and(c)nodewidth(scalebar:3mm).
III,IV,andVindicatethethird,fourth,andfifthnodes,respectively.
Kimetal.
Rice2014,7:14Page3of11http://www.
thericejournal.
com/content/7/1/14weight,whereasnosignificancedifferencewasdetectedbetweenIlpumbyeoandgroupsA,B,C,andKin2011,2012,and2013(Figure3a).
Thus,qTGW6waslocateddownstreamofRM20521andupstreamofRM20580.
GroupDshowedsignificantlyhighervaluesforSPPaf-fectedbyqSPP6andforTGWcomparedtoIlpumbyeo,whilegroupJwassignificantlydifferentfromIlpumbyeoforTGWbutnotSPP.
ThisresultindicatesthatqSPP6andqTGW6aredifferentgenes.
ComparisonofthegenotypesofrecombinantsdelimitedtheqTGW6betweenmarkersRM20562andRM20572,basedonthefindingthattheTGWofbothDandJgroupsweresignificantlydifferentfromthatofIlpum-byeo.
TheregionbetweenthetwomarkersRM20562andRM29572forgroupsDandJsharedcommonsegmentharboringofqTGW6.
TofurtherconfirmthecommonsegmentsharedbygroupsDandJ,wege-notypedtheF3linesusingfourmarkers(oneInDelmarkerandthreeSNPmarkers)locatedbetweenRM20562andRM20572.
ThegenotypesshowedthatqTGW6wasdefinedina37.
85-kbregionbetweenInDel-1andSNP3(Figure3b).
EvaluationofthegrainshapetraitTodeterminewhichgrainshapetraitsareassociatedwithincreaseingrainweight,threegrainshapetraits(specifically,grainlength[GL],width[GW],andthickness[GT])wereevaluatedforthetwopar-entsandF5lines(Figure3).
ThemeanphenotypicvaluesofeachF5linewerecomparedtothoseofIlpumbyeoandIL28attheP<0.
05level.
TheGLofIlpumbyeoandIL28were4.
88mmand5.
13mm,re-spectively.
TheGWvaluesforIlpumbyeoandIL28were2.
93mmand3.
15mm,respectively.
TheGLandGWvaluesofIL28weresignificantlyhighercomparedtoIlpumbyeo(P<0.
05),whereasnosig-nificantdifferencewasdetectedforGT.
TheGLandGWofthegroupswithqTGW6(D,E,F,G,H,I,andJ)weresignificantlydifferentfromthoseofthegroupswithoutqTGW6(A,B,C,andK).
Thisfind-ingindicatesthatqTGW6variationiscausedbyvariationinGLandGWatqTGW6.
GrainweightwassignificantlycorrelatedwithGLandGW(P<0.
01forbothcharacteristics),butnotwithGT(r=0.
06).
(a)(b)daysafterpollination05101520253035357911131517192123252729313305101520253579111315171921232527293133daysafterpollinationNIL(IL28)IlpumbyeoNIL(IL28)IlpumbyeoFigure2CharacterizationofgrainfillinginIlpumbyeoandIL28.
(a)Time-courseofendospermfreshweightincrease.
(b)Time-courseofendospermdryweightincrease.
Dataaremean±s.
d.
(n=7to10plants)inaandb.
Kimetal.
Rice2014,7:14Page4of11http://www.
thericejournal.
com/content/7/1/14Cross-sectionofthecentralpartofthespikelethullGiventhatthehulloftheIL28spikeletwaswiderthanthatofIlpumbyeobeforefertilization,thecross-sectionofthecentralpartofthespikelethullforgroupsDandJwasanalyzedincomparisontoIlpumbyeotoinvestigatetheoriginsoftheobservedsizedifferences(Figure4).
TheouterglumecelllayerofgroupsDandJcontainedsubstantiallymorecells(16.
5%and15.
3%,respectively)comparedtothatofIlpumbyeo,withonlya1.
3%and1.
4%increaseincelllength,respectively.
Thesedatain-dicatethattheincreasedwidthofgroupDandJspikelethullsmainlyresultsfromanincreaseincellnumber,ratherthancellsize,indicatingthatqTGW6maybein-volvedintheregulationofcelldivision.
ImpactoftheQTLclusteronyieldperplantElevengroupscontaining44F5lineswereusedfortheyieldtrials,togetherwithIlpumbyeoandIL28in2013(Figure3).
IL28hadsignificantlyhighergrainyieldcom-paredtoIlpumbyeo.
IL28produced8%highergrainyieldonaveragecomparedtoIlpumbyeo.
AllgroupswiththeMoroberekansegmentatbothqSPP6andqTGW6(i.
e.
,groupsD,E,F,G,H,andI)hadsignifi-cantlyhighergrainyieldvaluescomparedwiththe162b139b157b21.
3c21.
6b22.
8b4.
88c2.
93b2.
13a35.
0b182a159a182a23.
8a22.
9a24.
4a5.
13a3.
16a2.
15a37.
8a167b135b153b21.
8c21.
7b23.
1b5.
00b2.
90b2.
13a35.
4b171ab141b160b22.
3c21.
5b22.
9b5.
02b2.
95b2.
12a35.
1b164b143b155b21.
8c21.
6b23.
1b5.
00b2.
94b2.
12a35.
5b182a158a174a22.
9ab22.
6a24.
4a5.
09a3.
09a2.
12a37.
2a181a156a176a22.
9ab22.
6a24.
3a5.
10a3.
08a2.
12a37.
1a187a156a179a23.
1ab22.
7a24.
2a5.
09a3.
10a2.
10a37.
4a188a159a178a23.
2ab22.
9a24.
2a5.
10a3.
11a2.
12a37.
5a185a164a182a23.
1ab22.
8a24.
5a5.
12a3.
11a2.
12a37.
6a179a157a177a23.
0ab22.
9a24.
4a5.
12a3.
11a2.
12a37.
6a163b142b162b23.
5a22.
9a24.
2a5.
09a3.
13a2.
13a36.
4ab161b144b158b21.
9c21.
5b23.
0b5.
01b2.
92b2.
14a35.
2bSPPTGW(g)(2011)(2012)(2011)(2012)P1P2A(5)B(4)C(2)D(5)E(5)F(4)G(5)H(4)I(2)Group&J(5)RM7269RM162RM3567RM20512RM20521RM3430RM20562RM20572RM20580RM3307RM20653RM20562RM20572LOC_Os06g45560LOC_Os06g45550LOC_Os06g45570LOC_Os06g45580APO1(2013)27.
56527.
57227.
58627.
603DJ27.
560InDel-1SNP1SNP2SNP327.
82837.
85kb(2013)GL(mm)GW(mm)GT(mm)YD(g)RM461K(3)Physicaldist.
address(Mb)(b)300-kbqTGW6RM7269RM20653(a)(c)(1,806bp)TGAATGIlpumbyeo,NipponbareCMoroberekanATyrosineCystein828(6,383bp)TAAATGIlpumbyeo,NipponbareAMoroberekanGAlanineThreonine2,267qSPP6(Kimetal.
2013)Figure3DevelopmentofahighdensitymapofqTGW6andstructuresofcandidategenesforqTGW6.
(a)GraphicalrepresentationofF3:4:5linesandafinescalemapofthetargetregiononchromosome6.
Apo1locatedbetweenRM3430andRM20562mightbeallelictoqSPP6detectedinthisstudy.
WhiteandblackportionsofthegrapharehomozygousIlpumbyeo,homozygousMoroberekan,respectivelyanddottedregionsarewherecrossing-overoccurred.
Genotypesofthe44linesweredoublecheckedatF3generation.
Thetabletotherightofthegraphicalgenotypesindicatesmeanvaluesoftraits.
NumbersfollowedbythesameletterineachcolumnarenotsignificantlydifferentatP=0.
05basedonDuncan'smultiplerangetest.
&No.
in()indicatethenumberofF3lines.
P1:Ilpumbyeo,P2:IL28.
(b)HighdensitymapofqTGW6usingtwogroups(DandJ)andfourmarkers.
Fourgeneswerepredictedinthetarget37.
85-kbregion.
Thearrowheadsindicatethepositionofasinglenon-synonymousnucleotidesubstitution.
(c)Structuresoftwogenes,Loc_Os06g45550andLoc_Os06g45560.
Loc_Os06g45550consistsoffiveexons(blackboxes)andfourintronsandLoc_Os06g45560consistsof8exonsand7introns.
Numbersonthestructureoftwogenesshowthepositionofnon-synonymousnucleotidesubstitutionsfromthestartcodon.
Kimetal.
Rice2014,7:14Page5of11http://www.
thericejournal.
com/content/7/1/14groupswithouttheMoroberekansegmentatbothqSPP6andqTGW6(i.
e.
,groupsA,B,C,andK)andIlpumbyeo(Figure3).
Also,theyieldperplantofgroupJwithqTGW6andwithoutqSPP6washigherthanthatofgroupCandIlpumbyeoatP<0.
1(P=0.
068,P=0.
067,respectively),andthisresultimpliesthecontributionofqTGW6totheyieldincrease.
However,wecouldnotde-terminetheeffectofqSPP6onyieldduetothelackofline(s)onlywiththeMoroberekansegmentatqSPP6.
CandidategenesforqTGW6qTGW6wasdefinedina37.
85-kbregionbetweenInDel-1andSNP3.
Onthebasisofavailablesequenceannotationdatabases(http://www.
gramene.
org),therearefourpredictedgenesinthetargetregion.
Thefunc-tionalannotationsofthefourgenesareasfollows:Loc_Os06g45550(fiveexonsandfourintrons)andLoc_Os06g45560(eightexonsandsevenintrons)areretrotransposonproteinputativeexpressedgenes;Loc_Os06g45570(oneexon)isaVQdomain-containingproteinputativeexpressedgene;Loc_Os06g45580(oneexon)isaRING-H2zincfingerputativeproteinexpressedgene(Figure3).
Wecomparedthegenomicsequencesoftheopenreadingframe(ORF)andpro-moterregionofthefourgenesbetweenIlpumbyeoandMoroberekan.
Comparisonofthegenomicsequenceoftwogenes(Loc_Os06g45550andLoc_Os06g45560)amongNipponbare,Ilpumbyeo,andMoroberekanre-vealedtheexistenceofasinglenon-synonymousSNPinthethirdandfourthexonofLoc_Os06g45550andLoc_Os06g45560,respectively.
Thenon-synonymousSNPatthethirdexonofLoc_Os06g45550ischaracter-izedbythenucleotideCytosineinIlpumbyeoandthenucleotideAdenineinMoroberekan.
ThechangeinthenucleotidebasefromCytosinetoAdenineresultedinmissensemutationfromcysteinetotyrosine.
Thenon-synonymousSNPatthefourthexonofLoc_Os06g45560,ischaracterizedbythenucleotideAdenineinIlpumbyeoandthenucleotideGuanineinMoroberekan.
ThenucleotidesubstitutionA(adenine)toG(guanine)inLoc_Os06g45560resultedinamissensemutationfromThreoninetoAlanine.
NosequencevariationinopenreadingframesandthepromoterregionoftwogeneswasdetectedforLoc_Os06g45570andLoc_Os06g45580amongthethreevarieties,Nipponbare,Ilpumbyeo,andMoroberekan.
IlpumGroupDGroupJ01234567891011IlpumNIL-DNIL-JCelllength(um)1000110012001300140015001600IlpumNIL-DNIL-JCellnumberabcabbaaaFigure4Histologicalanalysesofspikelethull3daysbeforeheadinginIlpumbyeo,groupD,andgroupJ.
(a)Cross-sectionofspikelethull.
Upper:spikelets(scalebar,3mm).
Low:cross-sectionofspikelethull(scalebar,500um).
Dottedlineindicatespositionofcross-section(b)Magnifiedviewofspikelethullcross-sectionboxedina.
(c)ComparisonoftotalcellnumberandmeancelllengthintheouterglumecelllayersofspikelethullofIlpumbyeo,groupD,andgroupJ.
(n=5to8spikelets).
NumbersfollowedbythesameletteroneachboxarenotsignificantlydifferentatP=0.
05basedonDuncan'smultiplerangetest.
Kimetal.
Rice2014,7:14Page6of11http://www.
thericejournal.
com/content/7/1/14DiscussionTherehasbeenmuchdebateastowhethersimilargen-omiclocationsofQTLsthataffectdifferenttraitsarecausedbythepleiotropyofasinglegeneorthetightlinkageofseveralgenesthatindividuallyinfluencespe-cifictraits.
Thequestionofpleiotropyversustightlink-agemightberesolvedbyusingalarge-sizepopulationcombinedwithhigh-densitymapping,becausetwolinkedQTLssegregateindependentlyinNIL-F2:3popu-lations.
Recently,severalQTLscontrollingthesameormultipletraitshavebeenconfirmedtobecloselylinkedinasmallgenomicregion.
Forexample,thericephoto-periodsensitivitygene,Hd3wasoriginallydetectedasaheadingdate-relatedquantitativetraitlocusthatislocalizedonchromosome6intheF2population(Yanoetal.
1997).
Forthefine-scalemappingofHd3,high-resolutionlinkageanalysisusingalargesegregatingpopulationderivedfromadvancedbackcrossprogenybetweenthejaponicaricevariety,Nipponbareandtheindicavariety,Kasalath,wascarriedout.
Asaresultofthehigh-resolutionlinkagemappingofHd3,twotightlylinkedloci,Hd3aandHd3b,whichpromotefloweringundershort-dayconditionsandinhibitheadingunderlong-dayconditions,wereidentifiedintheHd3region(Monnaetal.
2002).
Louetal.
(2013)mappedtwoQTLs(qSPP5andqTGW5)forspikeletsperpanicleand1,000-grainweighttothesameregiononchromosome5.
SubstitutionmappingwiththeBC5F3andBC5F4pop-ulationsderivedfromacrossbetweenQTL-NILandtherecurrentparent,demonstratedthattwotightlylinkedQTLs(qSPP5andqTGW5)weredifferent.
SeveralQTLswithlargepleiotropiceffectsonmultipletraitshavealsobeenfoundandcloned(APO1,Ghd7,andGhd8orDTH8).
Forinstance,Huaetal.
(2002)reportedthataQTLclusterinfluencesmultipletraits(namely,grainperpanicle,plantheight,andheadingdate)onchromosome7inaprimarypopulation.
High-resolutionmappingwithalarge-sizedsegregatingpopulationandcom-plementationtestsrevealedthatthesethreetraitsarecontrolledbyasinglelocus,designatedasGhd7(Xueetal.
2008).
TheoriginaltargetofthisstudywasqSPP6,whichwasmappedonthelongarmofchromosome6(Juetal.
2008).
Intheprocessofthefine-scalemapping,theQTLforadditionalyieldcomponents(includingpaniclelength,nodewidth,and1,000-grainweight)werecon-sistentlydetectedinthesameregion.
Atotalof243F2plantsderivedfromacrossbetweenNIL(IL28)andIlpumbyeoforgeneticanalysiswasnotlargeenoughtoclarifytherelationshipbetweenSPPandTGW.
Tocon-firmthepreciselocationofthetwoQTLs,substitutionmappingwascarriedout.
Followingsubstitutionmap-pingwith44F3:4:5lines,qSPP6wasmappedtoa431-kbintervalbetweenRM20521andSNP1,whileqTGW6wasmappedtoa37.
85-kbintervalbetweenInDel-1andSNP3,basedonthejaponicagenomesequence.
There-fore,thedevelopmentofNILandsubstitutionmappinghasenabledustomapSPPandTGWatthefine-scale.
NoTGW-associatedQTLwasdetectedneartheqTGW6QTLinthisstudy,indicatingthatqTGW6isanovelQTLforTGW.
AmongthefourpredictedgenesinthetargetregionbetweenInDel-1andSNP-3(37.
85-kb),Loc_Os06g45570encodesaVQdomain-containingprotein.
VQdomainsarebelievedtobecriticalforthedefenseresponsetopathogensbyinteractingwithWRKYtranscriptionfactors(Taoetal.
2011).
Loc_Os06g45580encodestheRING-H2zincfingerprotein.
Recentstudiesreportedacriticalroleoftheubiquitinpathwayingraindevelopmentinrice(Songetal.
2007;Wengetal.
2008).
GW2,amajorQTLforricegrainwidthandweighten-codesanewRING-typeE3ubiquitinligase.
GW5,anothermajorQTLunderlyingricewidthandweight,islikelytoactintheubiquitine-proteasomepathwaytoregulatecelldivisionduringcelldivision(Wengetal.
2008).
qTGW6exhibitedcommonfunctionforgrainwidthwithGW2andGW5inthatitwasassociatedwithincreasedcellnumbers,awiderspikelethullandanincreasedgrainmilk-fillingrate.
ThisraisesapossibilitythatLoc_Os06g45580isacandidateforqTGW6.
However,nosequencevariationinopenreadingframesandthepromoterregionsoftwogenes,Loc_Os06g45570andLoc_Os06g45580wasob-servedbetweenIlpumbyeoandMoroberekanandthisresultseemstosuggestthattheyarenotcandidategenesforqTGW6.
Theothertwogenesinthetargetregionareretrotransposonproteins,Loc_Os06g45550andLoc_Os06g45560.
Loc_Os06g45560wastranscribedinseedlingandembryo/endosperm25daysafterpollin-ationbutLoc_Os06g45550wasnottranscribedbasedontheavailablegeneexpressiondatabase(RGAP,http://rice.
plantbiology.
msu.
edu/expression.
shtml).
Morethan40%ofricegenomeiscomposedofrepetitivesequencesandtransposableelements(Goffetal.
2002).
ArecentstudyshowedthattheexpressionofTos17,acopia-likeretrotransposonwasaffectedbyoverexpres-sionordown-regulationofseveralriceSUVH(Su(var)3–9homologs)genesandRNAiplantswithdown-regulatedSDG728,ariceSUVHgeneshowedareducedseedsizephenotype(Qinetal.
2010).
ThisraisesapossibilitythatarelationshipbetweenalteredseedmorphologyandoverexpressionofTos17mightexist.
Interestingly,ouralignmentanalysisofGW5a.
asequenceusingBLASTPprogramshowedthatGW5proteinhashighhomologywithtworetrotrans-posonproteinsencodedbyLOC_Os08g26160.
1andLOC_Os05g47720.
1(datanotshown),althoughithasbeenreportedthattheproteinencodedbytheGW5ORFhadnosequencehomologywithanyproteinsofknownmolecularfunction(Wengetal.
,2008).
GW5Kimetal.
Rice2014,7:14Page7of11http://www.
thericejournal.
com/content/7/1/14proteinshowed56%homologytotheretrotransposonproteinwith194a.
aencodedbyLOC_Os08g26160.
1and47-50%homologiestothetransposonproteinwith411a.
a.
byLOC_Os05g47720.
1.
NumerousIndelsandsingle-nucleotidepolymorphisms(SNPs)weredetectedbetweenNipponbare(japonica)and9311(indica)andIndelsaremainlycausedbytransposons(Fengetal.
2002).
IndelsandSNPsinsomekeydomesticationgenesincludingqSH1andGW2areresponsibleformorphologicalchangesoccurredduringdomestication(Konishietal.
2006;Songetal.
2007).
Also,thediffer-enceingrainwidthbetweenjaponicaandindicaculti-varsisassociatedwitha1212-bpdeletionharboringtheGW5ORFwhichwaspossiblycausedbytransposons(Wengetal.
2008).
Theseobservationssupportourhy-pothesisthattheproteinencodedbytheGW5ORFmightbearetrotransposonandLoc_Os06g45560isacandidateforqTGW6.
However,thepresenceofSNPsintheupstreamregionsofthegenesbetweentwopar-entssuggeststhepossibilitythatalong-distanceregula-tionisinvolvedincontrollingtheqTGW6(datanotshown).
Konishietal.
(2006)reportedthatthecasualmutationinqSH1underlyingtheseedshatteringwasfoundinthe12-kbupstreamregionfromtheORFofthegeneandcausedtheabsenceoftheabscissionlayerinNipponbare.
Additionalfinemappingexperimentsareunderwaytodeterminethefunctionalrelationshipbe-tweenqTGW6andGW5incontrollingseeddevelopment.
Also,loss-of-functionmutantlinesofLoc_Os06g45560willbeevaluatedforgrainmorphologytraitsandthegeneexpression.
Theseinformationsonthemolecularmechan-ismofqTGW6wouldbenecessarytogetmoreinsightsintothemechanismofgraindevelopment.
AlthoughthecandidategeneofqTGW6anditsgen-eticmechanismwasnotelucidatedhere,wefoundthattheMoroberekanchromosomalsegmentatthisregion(atropicaljaponica)harborsQTLsforyieldcomponenttraitsleadingtoanyieldincreaseinjaponicacultivar.
YieldtrialsconfirmedthatintrogressionlinescontainingtheMoroberekansegmentattheqSPP6andqTGW6lociproducedsignificantlyhigheryieldsthanIlpumbyeo(P<0.
05).
ThegrainyieldperplantinIL28was8%higherthanthatofIlpumbyeo.
Moroberekanallelesinthetargetregiononchromosome6hadafavorableeffectonyield,byincreasingthenumberofspikeletsperpanicleand1,000-grainweight,inadditiontolodgingtolerancebyin-creasingthenodewidth,whereasithadnonegativeeffectonheadingdateandplantheight.
Thus,theqSPP6andqTGW6allelesarepotentiallyvaluableforimprovingriceyield.
ItisproposedthatNILscontainingthetargetseg-mentassociatedwithpositiveQTLsfromMoreberekanshouldbedevelopedfromthispopulation,andevaluatedinawiderangeofenvironments,toassesstheinteractionbetweenQTLsandtheenvironment.
TightlylinkedSSRmarkersareexpectedtofacilitatethecloningofgenesunderlyingtheseQTLs,inadditiontomarker-assistedse-lectionforvariationintheSPPandTGWinricebreedingprograms.
ConclusionInthisstudy,wedemonstratedthattwoQTLs,qSPP6forspikeletsperpanicle(SPP)andqTGW6for1,000-grainweight(TGW)aretightlylinkedonchromosome6.
BecauseMoroberekanallelesfortheSPPandTGWhavebeenshowntobebeneficialinthegeneticback-groundofIlpumbyeo,theqSPP6andqTGW6allelesmayprovevaluableforimprovingtheyieldpotentialofjaponicaricecultivars.
TightlylinkedSSRmarkersareexpectedtofacilitatethecloningofgenesunderlyingtheseQTLs,inadditiontomarker-assistedselectionforvariationinSPPandTGWinricebreedingprograms.
MethodsPlantmaterialsInapreviousstudy(Kimetal.
2013),IL28wasshowntoexhibitahighernumberofspikeletsperpanicleandheaviergraincomparedtotherecurrentparent,andcontainedtheMoroberekanchromosomalsegmentattheqSPP6regiononchromosome6;hence,itwasse-lectedforthefine-scalemappingofqSPP6inthecurrentstudy.
IL28wascrossedwiththecultivarIlpumbyeo,withtheresultingF1plantsbeingself-pollinatedtoob-tainanF2population(234plantsin2009and1,150plantsin2010).
ToconfirmqSPP6,44F2homozygousplantswithdifferentcross-overbreakpointsbetweenRM7269andRM20653wereselectedandselfedtopro-duceF3linesforthephenotypingandsubstitutionmap-pingofqSPP6.
Intheprocessofthefine-scalemappingofqSPP6,aQTLfor1,000-grainweight(TGW)wasconsistentlydetectedinthesameregion.
ToconfirmthepreciselocationofqSPP6andqTGW6,these44F3lineswereadvancedtoF4andF5lines,whichwereusedtoevaluatespikeletsperpanicle,grainweight,grainmorphologytraits,andgrainyield.
FieldtrialandtraitevaluationAtotalof243F2plantsand1150F2plants(2009and2010),and41F3:4:5lines(2011,2012,and2013)andtheparentallinesweregrownattheexperimentalfieldofChungnamNationalUniversity,Daejeon,Korea.
In2009,the243plantsandtheparentswereplantedatdis-tancesof30*15cm,andevaluatedforgrain(brownrice)weight(150outof243plantswereusedtoevaluategrainweight).
In2010,forthesubstitutionmappingofqTGW6,1,150F2plantsweregenotypedwithfouradditionalmarkersbeinglocatedbetweenRM7269andRM20653(RM20512,RM20521,RM20652,andRM20572)andonemarker(RM551)locatedonchromosome4.
TheKimetal.
Rice2014,7:14Page8of11http://www.
thericejournal.
com/content/7/1/14experimentusing44F3:4:5linesderivedfrom44F2plantsfollowedacompletelyrandomizedblockdesignwithtworeplications,onerowperplot,and25plantsperrowin2011,2012,and2013.
Themiddle10plantsfromeachlinewerechosenfortheevaluation,andtheaverageofthemea-surementswasusedforthephenotypeofeachlineforthespikeletsperpanicle(SPP)and1,000-grainweight(TGW).
SPPweremeasuredbyaveragingthethreemajorpaniclesperplants.
Grainswithhullswereallowedtodrynaturallyafterharvesting,andpartialorunfilledseedswereremovedbysoakinggrainsinwater.
Fullyfilledseedswerere-driedinanovenat30°Cfor24h.
TheTGWwasevaluatedbymeasuringtheweightof250randomlyselected,de-hulledgrainsperplant(10plantsperline).
In2013,grainlength(GL),grainwidth(GW),grainthickness(GT),andyieldperplants(YD)weremeasured.
Thegrainlength(GL),grainwidth(GW),andgrainthickness(GT)weremeasuredfor50grains(brownrice)perplant(10plantsperline)usinga150-mmVerniercaliper(MitutoyoCorp.
,Japan).
Yieldperplant(YD)wasmeasuredbyaveragingthegrainyield(g)of10plantsthatwererandomlyselectedfromthecenterofeachplotperblock.
The1,000-grainweightandtheyieldperplantwerecorrectedforthe12%grainmoisturecontent.
DNAextractionandSSRanalysisDNAwasextractedfromtheleaftissueoftheF2popula-tionusingachloroform-basedDNAextractionprotocol(Causseetal.
,1994).
A20-μLreactionmixturewasused,containing5.
0μL(5ng/μL)oftemplateDNA,0.
1μl(5Unit/μL)Taqpolymerase,0.
8μldNTP(2.
5mMeach),Forward+Reverseprimer1μl(10pmoleach),2.
0μl10*PCRbuffer(10mMTris–HClPH8.
3,50mMKCl,1.
5mMMgCl2,0.
1%gelatin),and11.
1μltriple-distilledwater.
AmplificationwasachievedusingaThermoCycler(Bio-Rad)basedonthestep-cycleprogramsetfordenatur-ationat94°Cfor5min,subsequentdenaturationwasper-formedat94°Cfor1min,annealingat55°Cfor1min,extensionat72°Cfor1min;steps2to4wererepeatedforatotalof35cycles,withafinalextensionstepat72°Cfor5min.
PCRproductswererunon4%polyacrylamidede-naturinggelsfor1–2hat1800–2000V,andmarkerbandswererevealedbysilverstaining(Panaudetal.
1996).
TheorientationoftheSSRmarkerswasbasedontheSSRmaps(McCouchetal.
2002).
DuetothelackofmarkersshowingpolymorphismintheregionbetweenRM20562andRM20572,additionalgenotypingoftheF3lineswascon-ductedwithfourtargetedSNPsandoneInsertion/Delitionmarker.
TodetecttheSNPsandInDelsofIlpumbyeoandMoroberekan,resequencingwithNGS(Next-GenerationSequencing)wasconductedusingthetwoparentsaccordingtoJeongetal.
(2013).
ThroughthecomparisonofgenomicsequencescorrespondingtotheregionbetweenRM20562andRM20572,atotalof28polymorphisms(14SNPs,and14Insertion/Deletions)weredetectedbetweenIlpumbyeoandMoroberekan.
Thepolymorphismswereassayedbythedirectsequencingofthefourregions.
InformationontheprimersusedispresentedinTable2.
Theprimerswerede-signedaccordingtotheNipponbaresequence(http://rgp.
dna.
affrc.
go.
jp/E/IRGSP/Build5/build5.
html).
ThefirstInDel,hereafterreferredtoasInDel-1,occursatthe27,565,774thpositionbasedontheNipponbaresequence(www.
gramene.
org),andischaracterizedbynucleotide-(1bpdeletion)inIlpumbyeoandnucleotideAinMoroberekan.
ThefirstSNP,SNP-1,thatoccursatthe27,572,767thposition,ischaracter-izedbynucleotideGinIlpumbyeoandnucleotideAinMoroberekan.
ThesecondSNP,SNP-2,thatoccursatthe27,586,202thposition,ischaracterizedbynucleotideAinIlpumbyeoandnucleotideGinMoroberekan.
ThethirdSNP,SNP-3,thatoccursatthe27,629,080thposition,ischaracterizedbynucleotideAinIlpumbyeoandnucleotideCinMoroberekan.
StatisticalanalysisStatisticalanalysiswasperformedusingQgenesoftwareversion2.
30forMacintosh(Nelson1997)andSAS(SASInstitute).
Singlepointanalysis(SPA)wasperformedtodeterminetheeffectofeachmarkeroneachtrait.
InSPA,aQTLwasconfirmedifthephenotypewasassoci-atedwithamarkerlocusatP<0.
005,orwithtwoadja-centmarkerlociatP<0.
01.
TheproportionoftotalphenotypicvariationexplainedbyeachQTLwascalcu-latedasanR2value,fromtheregressionofeachmarker/phenotypecombination.
QTLsweremappedatafine-scalebycomparingthephenotypicmeansoftwogeno-typicclassesofrecombinants(IlpumbyeohomozygoteandMoroberekanhomozygote)withinthetargetregionbyusingtheSASstatisticalsoftwarepackage.
HistologicalanalysisParaffin-embeddedsectionsofspikeletsampleswerepreparedaccordingtoLietal.
(2009),withminorTable2PrimersequencesofnewlydevelopedSNPmarkersMarkernameForwardprimer(5'-3')Reverseprimer(5'-3')PositionProductsize(bp)InDel-1GAGAAAGTGACCCTCGTTTAGTCAAGACATGACACTACGATTGC27,565,623–27,565,899276SNP1CCATATGATTAAATGGGAGGCTCACATAGGCACTGCCAAGGTTC27,572,630–27,572,896266SNP2ACGGACGCCTGCTATAGGGACACCAGAGGCGATCTTCTTC27,585,994–27,586,360366SNP3GAATATCACGATCAAAGAACTTGGTTACTCTAATATGATAATATGCC27,603,470–27,603,822352Kimetal.
Rice2014,7:14Page9of11http://www.
thericejournal.
com/content/7/1/14modifications.
PlantmaterialswerefixedinFAA(50%ethanol,5%glacialaceticacidand5%formaldehyde)andstoredat4°Cfor24h.
Thefixedspikeletsweredehydratedbysoakingthemfor6hinagradientetha-nolseries(60%,70%,80%,90%),andwerethenincu-batedin100%ethanolovernight.
DehydratedspikeletswereembeddedinParaplast(Sigma).
Tissuesections(8μmthick)werecutusingarotarymicrotome,trans-ferredontogelatin-coatedglassslides,anddriedat42°Cfor1day.
Thesectionswerede-paraffinizedwith100%xylenefor5min,followedbysoakingfor2minin50%ethanol/50%xylen,100%ethanol,andsterilewater.
Thesampleswerestainedwithtoluidinebluefor30s,andwashedtwicewithsterilewater.
Thesampleswerethensoakedfor2minin70%ethanol,80%ethanol,90%ethanol,and100%ethanol.
Finally,thesampleswerecleanedbysoakingthemtwicein100%xylen.
Thesec-tionswerephotographedunderanOlympusBH2-RFCAusingaU-PMTVCcamera.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsDKparticipatedinphenotyping,genotyping,draftingthemanuscript.
HL,MFandJKparticipatedinphenotyping.
SKparticipatedinsequencing,primerdesignandgenotyping.
SAconceivedofthestudy,draftedproposalandcorrectedmanuscript.
Allauthorshavereadandapprovedthemanuscript.
AcknowledgementsThisworkwassupportedbytheKoreaResearchFoundationGrantfundedbytheKoreanGovernment(MOEHRD,BasicResearchPromotionFund)(KRF-2010-0024118)andtheNext-GenerationBiogreen21Program(PlantMolecularBreedingCenterNo.
PJ008136),RuralDevelopmentAdministration,RepublicofKorea.
Authordetails1DepartmentofAgronomy,CollegeofAgriculture&LifeSciences,ChungnamNationalUniversity,Daejeon305-764,SouthKorea.
2NationalAcademyofAgriculturalScience,RuralDevelopmentAdministration,Suweon441-707,SouthKorea.
3ChungnamAgriculturalResearchandExtensionServices,Yesan340-861,SouthKorea.
4Presentaddress:DepartmentofVarietyTesting,KoreaSeed&VarietyService,MinistryofAgriculture,FoodandRuralAffairs,Kimcheon740-220,SouthKorea.
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