mrg系列孟山都ssr

1、TheApplGe(1988)76:815829?9SpringerVerlag1988MolecularmappingofricechromosomesS.R.McCouchG.KochertZ.H.YuZ.Y.WangG.S.KhushW.R.CoffmanS.D.TanksleyDepartmentofPlantBreedingBiometry252EmersonHallCneliUniversityIthacaNY14853US

2、AReceivedMarch281988AcceptedJune21988CommunicatedbyF.SalaminiSummary.WerepttheconstructionofanRFLPgeicmapofrice(yzasativa)chromosomes.Themapiscomprisedof135locicrespondingtoclonesedfromaPstIgenomiclibrary.Thismolecularma

3、pcovers1389cMofthericegenomeexceedsthecurrentclassicalmapsbymethan20%.ThemapwasgeneratedfromFzsegregationdata(50individuals)fromacrossbetweenanindicajavanicaricecultivar.Primarytrisomicswereusedtoassignlinkagegroupstoeac

4、hofthe12ricechromosomes.SeventyeightpercentoftheclonesassayedrevealedRFLPsbetweenthetwoparentalcultivarsindicatingthatricecontainsasignificantamountofRFLPvariation.Strongcrelationsbetweensizeofhybridizingrestrictionfragm

5、entslevelofpolymphismindicatethatasignificantproptionoftheRFLPsinricearegeneratedbyionsdelections.Thisconclusionissupptedbytheoccurrenceofnullallelesfsomeclones(presumablycreatedbyiondeletionevents).OnecloneRG229hybridiz

6、edtosequencesinboththeindicajavanicagenomeswhichhaveapparentlytransposedsincethedivergenceofthetwocultivarsfromtheirlastcommonancestprovidingevidencefsequencemovementinrice.Asabyproductofthismappingprojectwehavediscovere

7、dthatriceDNAislessCmethylatedthantomatomaizeDNA.Ourresultsalsosuggestthenotionthatalargefractionofthericegenome(approximately50%)issinglecopy.Keywds:yzasativaMolecularmarkersRFLPGeneticmapTrisomicsDNAmethylationPermanent

8、address:DepartmentofBotanyUniversityofGegiaAthensGA30602USADepartmentofPlantBreedingInternationalRiceResearchInstituteLosBanosPhilippinesOffprintrequeststoS.D.TanksleyIntroductionRiceinmanyrespectsisanidealmonocotspecies

9、fgeicmolecularstudies.Itisdiploid(2n=24)hasarelativelysmallgenome(C=0.6pg)canberegeneratedfromprotoplaststransfmedbyexogenousDNA(BentSmith1976Yamadaetal.1985LzGobel1986Uchimiyaetal.1986).Inadditionitisthemostwidelygrownc

10、ropplantwldwidebeingastaplefoodfmethanhalfthewldspopulation(CoffmanJuliano1987).Finallythereisavastreservoirofgermplasm(200000accessions)ofbothdomesticatedwildricesmaintainedbyriceresearcherswldwide.Geicstudiesoverthepas

11、t70yearshaveledtotheestablishmentofalinkagemapofricechromosomesbasedonmphologicalmarkersmerecentlyisozymemarkers(Kinoshita1986KushSingh1986Wuetal.1987Ranjhanetal.1988).Whilethesemapshaveprovidedimptantinsightsintothegeic

12、scytologyofricetheyembodyinherentlimitationscommontoclassicalgeicmaps.Themajityofthemarkersconsistofmphologicalmutantlociwhichareundesirableinbreedingpopulations.Inadditionallelesoftheselociarenmallyrecessiveseriouslylim

13、itingthetypesofcrossesinwhichtheycanbeutilizedsuccessfully.Molecularlinkagemapshavebeenarebeingdevelopedinseveralcropspeciesincludingtomato(BernatzkyTanksley1986aTanksleyetal.1987)maize(Helentjaris1987Burretal.1988)lettu

14、ce(Lryetal.1987)potato(Bonierbaleetal.1988).ThesemapsbasedlargelyonclonedDNAsequenceshaveanumberofinherentadvantagesoverclassicalmapsmayprovidenewopptunitiesfapplicationsingeicsbreeding.Sincevariationisdetecteddirectlyat

15、theDNAleveltheallelesbehaveinacodominantmanner817valueswerecalculatedfpairsofmarkerswhichdeviatedfromindependentasstment(P90%ofthesignalinalanewasaccountedfbyonetwodiscretebs(Fig.1a)(2)multiplecopysequences(m.c.)where3me

16、bswerepresentnosinglebaccountedflessthan20%methan80%ofthesignal(Fig.1cdf)(3)repeatedsequenceswherethesignalinalaneappearedasasmearwithnodiscretebsifnosinglebwithinthesmearaccountedfmethan20%ofthetotalsignalinthelane(Fig.

17、1e).Afterdefiningthesecategieswithdensitometricscansmostprobescouldbeclassifiedbyeyebasedonautadiograms.Whenfilterswerewashedatmediumstringency(1x0.5xSSC65~58%ofthecloneswereclassifiedassinglecopy20%asmultiplecopy22%asre

18、peated(Fig.2).Atthisstringencyitisestimatedthattheminimumlevelofsequencehomologybetweenduplexes.is80%(Beltzetal.1983).Athighstringency(0.05?0.02xSSC)where99%sequencehomologyisrequiredfmaintainingduplexfmation85%oftheclon

19、eswereclassifiedassinglecopy12%multiplecopy3%repeatedsequences(Fig.2).Hencefthreferencestocopynumberwillrefertomediumstringencyunlessspecifiedotherwise.ComparisonofricePstIlibrarywithsimilarlibrariesintomatomaizeevidence

20、flowerlevelsofCmethylationinrice.ThelibraryusedinthisstudywasconstructedwiththerestrictionenzymePstIamethylationsensitiveenzymewhichrecognizesthesequence5CTGCAG3issensitivetocytosinemethylationinthe5location(NelsonMcClel

21、l1987).Clonesrepresentedinthelibraryusedinthisstudyarelikelytobefromregionsofthegenomewhicharenotmethylatedatthissite.Ithasbeenobservedinmaize(Burretal.1988)tomato(Tanksleyetal.1987)thatsuchundermethylatedareasareenriche

22、dfsinglecopysequencespossiblysincetheyrepresentcodingregions(Burretal.1988).ItisofinterestthentocomparethepercentagesinglecopyversusmultiplecopyrepeatedsequenceprobesgeneratedbyPstIlibrariesinthesethreespecies.Ricetomato

23、havesimilarsizegenomes(0.60.7pgofDNArespectively)(BentSmith1976IyengarSen1978Galbraithetal.1983)bothhaveahaploidcomplementof12chromosomes.Intomato40%ofclonesderivedfromlibrariesconstructedfromshearedDNAfromtheCmethylatio