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第 28卷 第 5期 作 物 学 报 Vol.28,No.5
2002年 9月 650～653页 ACTAAGRONOMICASINICA pp.650～653 Sept.,2002
ConstructionofRFLPLinkageMapandIdentificationofQTLsControlling
SpikeletandPollenSterilityinRice*
WANGChun-Ming1 HideshiYasui2 AtsushiYoshimura2 ZhaiHu-Qu1 WanJian-Min1①
(1StateKeyIaboratoryforCropGenetics&GermplasmEnhancement,NanjingAgriculturalUniversity,Nanjing210095,China;2Facultyof
Agriculture,KyushuUniversity,Fukuoka812-8581,Japan)
Abstract Quantitativetraitlocus(QTL)analysishasbeencarriedouttoidentifygenesconferring
spikeletsterilityandpolensterilityinrice.Asafirststep,anRFLPlinkagemapbasedontherecombi-
nantinbredlines(RILs)wasconstructed.125RILs(F10)derivedfromacrossbetweenaJaponicacultivar
Taichung65andanIndicacultivarARC10313weredeveloped.TheRFLPmapcontained113wel-dis-
persedRFLPmarkers.Totalmaplengthwas1462.4cM LinkagearrangementoftheRFLPmarkerswas
ingoodagreementwiththatofthepreviouslyconstructedmaps.63backcrossF1lines(BF1)derivedfrom
RILswereusedasasegregatingpopulationforQTLanalysis.ThreeQTLsforF1spikeletsterilitywere
detectedonChromosome1,7and11.Amongthem,aQTLneartheRFLPmarkerR1789(chromosome
7)wasdetectedatsignificantlevelP=0.0001,andoneQTLforF1polensterilitywasalsodetectednear
R1789marker.Rf4onchromosome7isknownasfertilityrestorergene.AboveQTLsdetectedonchro-
mosome7inthisstudyappearedtocorrespondtoRf4locus.
Keywords RFLP;Spikeletsterility;Polensterility;QTLanalysis;Rice
水稻 RFLP连锁图谱的构建及控制小穗不育和花粉不育的 QTL分析
王春明1 安井秀2 吉村醇2 翟虎渠1 万建民1①
(1南京农业大学作物遗传与特异种质创新国家重点实验室,南京农业大学水稻研究所,江苏南京,210095;2日本国立九州大学农学部,日本
福冈 812-8581)
摘 要 用台中 65(粳稻)/ARC10313(籼稻)的重组近交系(F10)构建了RFLP连锁图谱,含 113个分布均匀的标记。作
成的图谱覆盖全基因组,全图总长 1462.4cM,图中标记位置与所使用的参照图谱基本符合。利用该重组自交家系材
料与亲本台中 65回交得到 BF1家系,用于对小穗不育和花粉不育的 QTL分析,检测出 3个小穗不育和 1个花粉不育
QTL,且有一个小穗不育位点和花粉不育位点重叠于第 7条染色体的标记 R1789处。已知的恢复基因 Rf4与该座位对
应。正在建立该座位的近等基因系以进行基因精确定位和克隆。
关键词 水稻;RFLP;小穗不育;花粉不育;QTL
中图分类号:S511 文献标识码:A
RFLPmarkersareusedindistantlyrelated
crossesandquantitativetraitloci(QTLs)canbe
normalydetectedinthemappingpopulation.With
RFLPmarkers,wel-saturatedlinkagemapbased
onrecombinantinbreedlines(RILs)ofricewas
constructedinthispaper.Sterilityisamajorbarri-
* 基金项目:(Foundationitem):教育部优秀骨干教师基金和国家留学基金委员会资助。 Thestudywassupportedinpartbyfounda-
tionforkeyteachers,ministryofeducationandconductedinKyushuUniversity,Japanduringthevisitingresearchbythefirstauthor
supportedbyChinaScholarshipCounsel(CSC).
作者简介:王春明(1967-),江苏,博士,专业:水稻遗传和育种
① 通讯作者。 (Authorforcorrespondence.) E-mail:wanjm@mail.njau.edu.cn Tel.(025)4396516
Receivedon(收稿日期):2001-11-21,Acceptedon(接受日期):2001-12-30
erforutilizingaliengermplasm forricebreed-
ing[2～4].Varyingdegreesofhybridsterilityare
commonlyseen in crossesbetween Indica and
Japonicavarieties.UsingtheRILsandtheirback-
crosslines,theQTLsassociatedwith spikelet
sterilityandpolensterilityinricewassought.
1 Materialsandmethods
1.1 ConstructionofRFLPlinkagemap
125F10linesdevelopedbySSD(single-seed-
decent)wereplantedandDNAoftheseRILswas
extracted forRFLP linkagemap construction.
RFLP probes (Harushima 1998, Tusnematus
1996[1,6])weresuppliedbyRiceGenomeProgram,
Japan.RFLPbetweentheparentsweresurveyed
using six enzymes (BamHⅠ,BglⅡ,DraⅠ,
EcoRⅠ,EcoRⅤ andHindⅢ).113markerswere
selectedtoconstructthelinkagemap.
1.2 Spikeletsterilityobservation
IndicacultivarARC10313wascrossedtoa
JaponicacultivarTaichung65asthefemaleparent.
63RILswerebackcrossedtoTaichung65in1999
andthe63backcrossF1(BF1)lineswereplantedin
2000.Spikeletsterilitywascalculatedastheper-
centageofemptyspikeletsovertotalspikeletsper
panicleforeachoftheRILsandBF1.
1.3 Pollensterilityobservation
28RILswerecrossedtoTaichung65in2000
andthesebackcrossF1(BF1)lineswereplantedin
2001.WithBF1in2001,polensteriltywasob-
servedfromantherscolectedfromspikeletsat1to
2daysbeforeanthesisandstoredin70% ethanol
andestimatedasthepercentageofpolengrains
thatcouldbestainedwithaceticcarmine.QTL
analysiswasconductedusingQGENE (Nelson
1997),asoftwareforDNA-marker-basedgenetic
analysisonMacintosh[5].
2 Results
2.1 ConstructionofRFLPlinkagemap
TheRFLPmapbasedontheRILswasshown
inFig.4.Itincluded113wel-dispersedRFLP
markers.Sincemarkerscloselylinkedtothetermi-
nalendsinthepreviousmaps(Harushima1998,
Tsnematsu1996)wereusedinthisstudy,thepre-
sentmapcoveredwholegenome.Totalmaplength
was1462.4cM.LinkageoftheRFLPmarkers
wasingoodagreementwiththatofthepreviously
constructedmaps[1,6].
2.2 Spikeletsterility
AmongtheRILs,spikeletsterilitywasfound
andthefrequencydistributionwasshowninFig.
1.Though4QTLsforRILsterilityweredetect-
ed,theeffectsoftheQTLswerelittle(Table2),
thereforethespikeletsterilitymayberesultedfrom
F1steriltiy.
Fig.1 Frequencydistributionfor
RILspikeletsterility
FrequencydistributionforBF1spikeletsterili-
tyin2000wasshowninFig.2.ThreeQTLsfor
BF1spikeletsterilityweredetectedonChromosome
1,7and11usingBF1population(Fig.4,Table
3).ARC10313alelesintheseregionsreducedthe
spikeletfertilityontheheterozygousconditions.
Amongthem,theQTL neartheRFLPmarker
R1789(chromosome7)wasdetectedatsignificant
levelP=0.0001.Intheheterozygouscondition,
ARC10313alelearoundtheRFLPmarkerR1789
causedlowspikeletsterilityby9.4%.
Fig.3 FrequencydistributionforBF1polensterility
1565期 WANGChun-Mingetal.:ConstructionofRFLPLinkageMapand……
2.3 BF1pollensterility
FrequencydistributionforBF1polensterility
in2001wasshowninFig.3.OneQTLforF1
polen sterility werealso detected nearR1789
markeronChromosome7(Fig.4,Table4)using
BF1populationin2001.ARC10313aleleinthis
regionreducedthepolenfertilityontheheterozy-
gousconditionby10.7%.
Fig.4 AnRFLPmapbasedontherecombinantpopulation(Taichung65/ARC10313)
3 Discussions
Thelinkagemappresentedherecanbeapplied
toanalysisotheragronomictraitsasitcoversade-
quatelythericegenome.Strongsegregationdistor-
tiontoARC10313wasobservedinchromosome3,
6,9,10and11.AttheKy11andC607loci,less
than10% ofRIL plantswerehomozygousfor
Taichung65 aleles. The segregation distortion
maycauseaproblemasitwilbedifficulttoelimi-
nateARC10313alelesfromtheseregions.
TheQTLnearR1789onChromosome7was
detectednotonlyconferringspikeletsterilitybut
alsopolensterilityinthisstudy.Rf4(Zhanget
al.1997)onchromosome7isknownasfertility
restorergene.QTLdetectedinthisstudyappeared
tocorrespondtothisfertilityrestorationlocus.
ThissterilityQTLwasthefirstonedetectedusing
theRILsderivedfrom Indica/Japoninavariety
cross.MoreBF1linesandnear-isogeniclinesare
nowunderconstructionforfurtherQTLanalysis
andgenemapping.
Table1
===
RFLPlocishowingsegregationdistortion
RFLPloci(Chr.) AA1) aa2) Total x2 RFLPloci(Chr.) AA1) aa2) Total x==
2
C563(3) 23 97 120 45.63 C== 607(6) 8 112 120 90.13
R2778(3) 14 109 123 73.37 R== 2638(9) 49 74 123 5.08
XNpb182(3) 14 110 124 74.32 C== 609(9) 49 75 124 5.45
XNpb144(3) 29 94 123 34.35 C== 1263(9) 49 75 124 5.45
C1677(3) 27 91 118 34.71 XNpb== 108(9) 46 74 120 6.53
R19(3) 46 74 120 6.53 XNpb== 293(9) 44 74 118 7.62
XNpb48(3) 49 75 124 5.45 XNpb== 333(10) 42 79 121 11.31
XNpb27(6) 39 79 118 13.55 R== 1629(10) 38 73 111 11.03
S1520(6) 46 72 118 5.72 C== 1166(10) 47 74 121 6.02
XNpb12(6) 48 74 122 5.54 XNpb== 52(11) 36 70 106 10.90
XNpb135(6) 25 97 122 42.49 S== 723(11) 41 62 103 4.28
Ky11(6) 5 120 125 105.80 G1465(11) 94 21 115 46.33
1)AA:NumberofplantswithTC65/TC65genotype;2)aa:NumberofplantswithARC10313/ARC10313genotype;(x2(1,0.05)=3.84)
256 作 物 学 报 28卷
Table2 QTLsdetectedforRILspikeletsterility
RFLPlociNearQTL Chromosome AA1) (N) aa2) (N) F P Additive3)
S1520 chr6 95.93 45 92.79 72 7.38 0.0076 1.8
XNpb27 chr6 95.9 39 93.04 78 5.61 0.0195 1.4
C955 chr1 95.07 74 92.48 47 5.23 0.024 1.3
R727 chr8 95.4 55 93.08 62 4.43 0.0375 1.2
1)AA:homozygousforTaichung65alele. 2)aa:homozygousforARC10313alele. 3)Additive:AdditiveeffectofTaichung65
alele.
Table3 QTLsdetectedforBF1spikeletsterility
RFLPlociNearQTL Chromosome AA1) (N) Aa2) (N) F P Additive3)
R1789 chr7 93.36 34 83.91 18 17.93 0.0001 9.4
XNpb179 chr11 92.06 26 87.2 28 4.2 0.0456 4.8
XNpb346 chr1 91.64 38 87.05 18 4.15 0.0466 4.6
1)AA:homozygousforTaichung65alele. 2)Aa:heterozygote. 3)Additive:Additiveeffectofhomozygousalele.
Table4 QTLsdetectedforBF1polensterility
RFLPlociNearQTL Chromosome AA1) (N) Aa2) (N) F P Additive3)
R1789 chr7 88.71 14 67.25 14 8.96 0.006 10.7
1)AA:homozygousforTaichung65alele. 2)Aa:heterozygote. 3)Additive:Additiveeffectofhomozygousalele.
References
[1] HarushimaH,M.yano,A.shomuraetal.Ahigh-density
ricegeneticlinkagemapwith2275markersusingasingleF2
population.Genetics,1998,148:479～494
[2] Ikehashi,H,H.Arakietal.GeneticsofF1sterilityinthere-
motecrossesofrice.ProceedingoftheinternationalRiceGe-
neticsSymposium.IRRI,ManilaPhilipines,1986,119～333
[3] J.Wan,Y.yamaguch,H.kato,etal.Twonewlociforhy-
bridsterilityincultivatedrice(OryzasativaL.)Theor.Ap-
pl.Genet,1996,92:183～190
[4] K.Doi,K.Taguchi,A.yoshimuraetal.Anewlocusaffect-
ing
highF1polensterilityfoundinbackcrossprogeniesofJaponi-
cariceandAfricarice.RiceGeneticsNewsleter,1998,15:
149～151
[5] Nelson,JCetal.QGENE:softwareformarker-basedge-
nomicanalysisandbreeding.MolecularBreeding,1997,3:
239～245
[6] TsunematsuH,YoshimuraA.,HarushimaY.,etal.RFLP
frameworkmapusingrecombinantinbredlinesinrice.Breed-
ingScience,1996,46:279～284
[7] ZhangTS,BharajY,LuSSetal.MappingoftheRf-3nu-
clearfertility-restoringgeneandRFLPmarkers.TheorAppl
Genet,1997,94:27～33
3565期 WANGChun-Mingetal.:ConstructionofRFLPLinkageMapand……