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Fundam.appl.Nemawl.,1992,15Cl),35-41.

DNAhybridizationprobesforstudying

theaffinities ofthreeMeloidogynepopulations

Vancouver,B.C.,CanadaV5A1S6.

Acceptedforpublication19January1991.

Summary- AdirectanalysisofthegenorypeofMeloidogynespp.wasdoneusingDNArestrictionfragmentlengthpolymor

phisms plasmid.Randomlycloned

Analysis

thethreepopulationsrangefrom2.1 ago. directe restriction. fragments d'ADNclonésauhasardsontmarquésaup J2 populationsappartenantà

L'analysedesbandesmises

fragments populations surlabasedesdonnèestirèesde d'unancêtrecommunily a 2à4millionsd'années.

TheadvancesinrecombinantDNAtechnologynow

al., 1988)

Globodera(Burrows&Perry,1988)andBur

saphelenchus(Websterelal.,1990).

TheDNAhybridizationprobestonematodetotal

genomic

DNAprovideatooltorevealtheRFLP'sand

al.,

1986)orRFLP'sofmitochondrialDNA(Powers&

inggenomic

DNAandtheaffinitiesofMeloidogyne

neticaffinities betweenthesespecies.

Materialsandmethods

CULTUREANDEXTRACTIONOFNEMATODES

McClure)andM.arenanarace1andM.javanica

greenhouseonpottedtomato byDr.B.A.Ebsary. 63
ings ona25llm-poresieve,resuspendedin0.1MNaCI and0.05M K 2 P0 4 (pH6.0)buffer(Brenner,1974)and storedinliquidnitrogenuntilneeded.

DNAISOLATION

Onemlofeggsfromeachspecieswerefrozenin

gestedwith6ml of1.0%proteinaseK.TheDNAwas extractedbythephenol-chloroforrn method(Curranel

EDTA(1XTE)buffer.RNAwasdigestedbythe

addition of100DNase-freeRNasesolutiontoa finalconcentration of10andincubatedatroom theaddition of8 Mammoniumacetatetoafinal concentration of2M,andthen2volumesof95%

PROBEISOLATIONANDLABELLING

thefollowing manner.OneoftheDNAofM. andligatedinto0.2 ofXbalcutplasmidpVZ1DNA.

CloningSystems)

thatcontainsanextendedpolylinker segmentinsertedintotheEcoRIsite (Henikoff& Fred

HutchinsonCancerResearchCenter,Seattle,

21
\8 15 .12 g f u.

01234567891011121314151617181920

SizoofClonodDNA(kb)

Meloidogyneincognitarace

#3.TheDNAofM.incognita race3wasdigestedwith

Xba1andligatedintaXbA1cut

ctedbythealkalinelysismini-preparation.

The94plasmids

determined. 36
intacompetentE.coliJM83,andselectedonNZY

Iyl-D-galactopyranoside,160

!J.g/mlisopropyl-D-thioga lactopyranoside and100!J.g/mlampicillin.Thebacteria containingplasmidswith nematodeDNAfragments, wereisolated andplasmidDNAwasextractedbythe alkalineIysis mini-preparationmethod(Maniatiselal., electrophoresis andthesizeofeachinsertdeterrnined (Fig. randomlytouseasprobes.Theseplasmids,each

0.3-0.5

DNA,wereradiolabelledwith(a_

32
p)dATP translation(Rigby elal.,1977).Theselabelledprobes (6x 10 5 cpmpermlhybridizationsolution)wereboiled for

15min,putonicefor5minandthenaddedtothe

prehybridizedfilterforhybridization.

SOUTHERNBLOTANDHYBRIDIZATION

TotalDNA,includinggenomicDNAandmitochon

Eco recommendations.Digested

DNAwasfractionatedin

0.7 bluedyefronthadmoved18cm.TheDNAonthegel wasnickedin0.25N

HCIfor15min,denaturedin0.5N

1 MNaOAC!0.02MNaOHfor60minandtransferred

to0.45 poresizeBioTraceNTnitrocellulosefilter baked undervacuumfor2.5hat80oc. 5 X

SSPE(1XSSPEis180mMNaCI,10mM

NaH 2 P0 4 .H 2

0and1mMEDTA(pH7.4)),0.3%SDS

(sodiumdodecylsulfate)and5 XDenhardtssolution were incubatedat68oCfor24handwashedfourtimes in0.2X

SSPE,0.2%SDSat65oCfor1h.Theabove

minimizefalsehybridization.

Thewashedfilterswereair

dried, andautoradiographedat-70oCwithKodak

X-OmatARP-Kx-rayfilmwithapairofCronexin

tensifyingscreens.

Results

TheclonedXbalDNAfragmentsofM.incognita

hybridizationare showninTable1.

Fundam.appl.NemalOl.

genomicONA inkilobases(kb). gyne bandsarefrom0.16ta24.0kb.Themostnotable

EcoRI(Fig.5).

genomicONAfromeachofthenematodespecies wasdigested withEco RI,fractionated in0.7%agarosegelandtransferred 5 in race3;A,M.arenariarace1;J,M.javanica. JA .c.24 Il ,(,4.9 .c.3.5 .(,2.7 .c.1.6 1- -el

ProbeSizeProbeSize

Name inKbNameinKb pMi12.40pMi

174.30

pMi2

1.20pMi181.91

pMi30.50pMi192.50 pMi4

1.43pMi2015.00

pMi5

1.70pMi212.35

pMi6

3.41pMi2212.00

pMi72.00pMi232.14 pMi88.82pMi245.55 pMi96.50pMi251.50 pMi105.10pMi261.35 pMiII14.50pMi272.20 pMi

121.59pMi282.65

pMi

1313.50pMi2913.00

pMi

143.61pMi302.70

pMi

153.10pMi311.15

pMi169.35pMi321.55 CIES

MostoftheclonedM.incognitaDNAfragments

severalrestriction fragmentlengthpolymorphisms about3.3kb,andoneat2.7kbthatoccursinM. nicahasauniquebandat3.5kb.

AutoradiographsofEcoRI-digestedtotalDNA

probedwithJ2plabelledDNAfromplasmidpMi10 (Fig.

3A)andpMi3(Fig.3B)showdiscretespecies

32

Vol.15,no1 -1992

37
A B J A .c:.24 "-4.9 "-4.5 .(,2.0 JA1 <24 <14 <5.1 <2.0 andM.arenariaandprobepMi3detectsonlyM.incognitaunderhybridizationconditionsof5 XSSPE,0.3%SDSand5 X

J,M.javanica.

ingwith

32plabelledprobes,wasusedtodeterminethe

relationshipbetween j\1eloidogynespecies. digestions ofM.incognita,M.arenariaandM.javanica wascalculatedusingtheformulaF=2Nxy/{N x +N) (Ney&Li,1979;Nei,1987);whereN,=numberof 38
M. incognitaandM.javanicaitis0.52. Based ments,thenucleotide substitutionratebetweenspecies wascalculatedbythe formulad= -(2/r)lnG(Nei,

1987);whered=thenumberofnucleotidesubsti

gnized percleavagesiteandGisamaximumlikelihood estimate mula,

F=c;J/(3- 2G).Theresuitsshowthattherate

Fundam.appl.NemalOl.

li> '"5 .c o a: :;;3 .c E :J Z 2

M.Incognlta

•M.srensris oM./Bv.nlca nica is2.12±0.35,betweenM.arenariaandM. incognita itis3.49±0.42andbetweenM.incognitaand

M.javanicaitis3.77±0.45.

to arithmeticmeansdiscussedin

Nei(1987).

9101112 13 14 15

Number01HybrldizedBands

arenaria divergedinrelativelyrecenttimes.

DNAfragmentsisolated

fromonespecies ofMeloidogynehybridizedpartiallywitheach 30
270

24I!IM.incognita

21

0M.arenaria

0M.javanica

18 15D u c: D 12 :J 9 u. 6 3 aa

3 6 912 15 182124

51ze01HybrldlzedBands(kb)

mentsforthreeMeloidogynespecieswith

32probes.Total

Eco

BioTrace

differentprobes underthehybridizationconditionsdescribed inthetext. species. bandsare around1to8kilobases.

Vol.15,nO1 -1992

Discussionandconclusion

differentiatesbetweenthepopulations ofthreeMeloido gyne speciesandhelps identifytheirrelationships.Ofthe

94randomlycloned

DNAfragments,themolecularsize

rangedfrom0.33to19.54kb andmost(76%)of thefactthat

MeloidogynegenomecouIdbecutevery

1.5-6.5

kpwiththe6-baserestrictionenzymeXba tionenzymeEcoRI andhybridizedwiththeseprobes, ofmost range istheexpectedresult,givena 6basecuttershould be foundevery4 6 onaverage.Approximately69%ofthe usefuldifferencesinthe numberofrestrictionfrag ments.Nevertheless, ourresultsshowthatspecieswithin this probes. using lizingthe serepetitivebandsinatotalgenomicdigeston anagarosegelcanbeovercomebyusingprobesas shownby

CurranandWebster(1987).Inourdata,DNA

ThepMi10probehybridizedtoDNAfromtheM.

incognita andM.arenariapopulationsbutjustweaklyto probe large thiskind ofspeciesdifferentiation. 39

J1.06M.arenaria

----'O"'.7'->6<---=t .L---'-1"'.8:=.2M.incognita three distance method(PUMA)fromtheestimatesofthepercent representsthe percentageofnucleotidedivergenceinbase substitutions. banding ofprobepMi9couldbeofthetypeassociated withrepetitive

DNA,suchastransposableelementsor

multiplegenefamilies.

TherepetitiveregionofpMi9

variesbetweenspeciesand isapotentiallyusefulprobe forgenomicanalysis.

Onotherhand,thepMi10and

pergenome. flectedintheirhostspecificity andgeographicaldis tribution,canbeidentifiedbyusing randomprobes.

Analysis

ofrestrictionendonudeasegeneratedDNA ciesthere isconsiderabledifferenceingenotype.These and alsotodemonstratetaxonomierelationships.

Among32randomlyseleeted

pMiprobestherewere30 probeswhichhybridizedtothreespecies ofMeloidogyne andshowedmore thanoneband.Underthehybridiza occurifthesequenceshaddivergedbymorethan about

1982),Meloidogynespecies

mustbepolyploidratherquotesdbs_dbs28.pdfusesText_34

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