林育研報 Bull.For.TreeBreed.CenterN0.18 , 2002 -33-40 一

InheritanceofChloroplastandMitochondrialDNAinInterspecificHybridbetweenPinusthunbergiiandP. 1nαssoni αnα

TeijiKondo(l), YoshihikoTsumura(2)andTakayukiKawahara(3)

Summary:InheritanceofchloroplastDNA(cpDNA)andmitochondrialDNA(mtDNA)wぉ investigated ininterspecific

hybridofPinus.TotalDNAsofsixteenhybridplantsbetweenP.thunbergiiandP.massonianaweredigestedwithEcoRI

andhybridizedwithtobaccocpDNA,pTB8.FifteenplantshadthesameSouthernhybridizationpatternsasthemale

parentspecies,P.massonianabutoneplantshowedafaintpa 抗em. TheDNAsof 仕Ie hybridwerealsodigestedwithApal

andhybridizedwithmtDNA, coxI.FifteenplantshadthesameSouthernhybridizationpatternsasthefemaleparent

species, P.thungergiibutonehybirdshowedafaintpa 抗em. ItwasconcludedthatcpDNAisinheritedpaternallyand

mtDNAisinheritedmaternallyin 出is hybrid. 百Iere weremoreinter-andintra-specificpolymorphismsinmtDNAthanin

cpDNA.ThepartialsequencesofcoxIgeneinP.thunbergiiwereclarified.

1 Introduction

Paternalinheritanceofanomalousleafcolorwasfirstly

reportedinCryptomeriajaponicausingcrossedprogeny

byOhbaetal.(1971).AsDNAanalysistechniqueshave

beendeveloped , paternalinheritaneofchloroplastDNA

(cpDNA)wasclarifiedfirstlyinPseudotsugamenziesii

(Nealeetal.1986).Thenseveralreportswerereleased

concerninginheritanceoforganelleDNAinconiferous

speciesandcpDNAisshowedtobeinheritedpaternally

inLarix, (Szmidtetal.1987) , Picea (Stineeta1.1989,

StineandKeathley1990, Suttonetal.1991) , Pinus

(Wagnereta1.1987, 1989,NealeandSederoff1989, Dong

etal.1992) , Sequoia (Nealeeta1.1989) , Calocedrus

(ト~eale etal.1991), Chamaecypan's(Kondoetal.1998).

Ontheotherhand,mitochondrialDNA(mtDNA)inherｭ

itedmaternallyinLarix(DeVernoeta1.1993) , Picea

(Suttoneta1.1991) , Pseudotsuga (MarshallandNeale

1992)andPinus(NealeandSederoff1989,Wagneretal.

1991)andinheritedpaternallyinSequoia(Nealeetal.

1989) , Calocedrus(Nealeeta1.1991)andChamaecyparis

(Kondoeta1.1998).Mostreportsconcerningtheinheritｭ

anceoforganelleDNAdealedwithPinaceae.

WeinvestigatedinheritanceofcpDNAandmtDNAusｭ

inginterspecifichybridbetweenP.thunbergiiandP.

massoniana.Thishybridisresistanttopinewoodnem 仕

todewhichhasprevailedmostpartsof]apanexcept

Hokkaido. 官le leafcolorof 出efemaleparent, P.thunbergii

wasdarkgreenandthatofthemaleparent, P.massoniana

(1) KyushuRegionalBreedingOffice,ForestTreeBreedingCenter

2320Suya , Nishigoshi, Kikuchi, Kumamoto861 ・1102 japan

林木育種センター九州育種場

(2) ForestryandForestProductsResearchInstitute

1Mastunosato, Kukiz品<i ， lbar紘i 305-8687japan

森林総合研究所

(3) HokkaidoResearchCenter,ForestryandForestProductsResearchInstitute

7Hitsujigaoka,Toyohira, Sapporo, Hokk出do 062・0045japan

森林総合研究所北海道支所

-34 一 林木育種センター研究報告 第 18 号

wasyellowishgreen.Astheleafcolorofthehybridwas

yellowishgreen, itwasreportedthattheleafcolorwas

inheritedpaternallyinthishybrid(SasakietaJ.1981).

Itmightbehelpfulforthisphenomenontounderstand

theinheritancemodeoforganelleDNAinthishybrid.

2 Materialsandmethods

2.1 Plantmaterials

Twointerspecifichybridfamiliesbetween 臥TO plustrees

ofP.thunbergiiandP.massonianawereused;Kitasouma

2XP.massoniana, andMizunami4XP.massoniana.

Thenumbersoftheprogenywere7and9, respectively.

TheyweregrowninForestTreeBreedingCentersituｭ

atedinIbarakiPrefecture.Asthemaleparentplantswere

notclearorabsentintheseinterspecificcrossing, differｭ

entindividualswereusedasthemaleparentspeciesin

thisexperiment.SixindividualsofP.massonianawere

usedtoexamineintraspecificpolymorphisms.

2.2 DNAisolationandSouthernblothybridization

Fivegofleaveswerefrozeninliquidnitrogenand

groundwithIwatanigrinder (I FM ・-150). Theground

powderwasadded20mlofice-coldacetoneandfiltered

throughfilterpaper(ToyoRosiNo.2)usingaspirator.

ThentotalDNAwasisolatedaccordingtotheCTABproｭ

cedure(MurrayandThompson1980).

DNAsweredigestedwithrestrictionen 勾引児島 and apｭ

proximately4μg ofeachsamplewasfractionatedon0.8

%agarosegel.DNAswereblottedtonylonmembranes

andhybridizedwithprobesusingDIGsystem

(BoehringerMannheim)accordingtothemanufacture's

instruction.pTB8cloneoftobaccowasusedforcpDNA

probeandcoxIgeneamplifiedbyPCRwasusedfor

mtDNAprobe.Primersweredesignedbasedonthenucleｭ

otidesequencedatafromKemmereretal.(1989).Two

oligonucleotideprimers,5'-CGATGGCTGTTCTCCACTM

♂ (forward) and5'-ATCTGGATMTCTGGMTGC ♂

(reverse)wereusedforcoxI(KondoetaJ.1998).

2.3 peRamplificationandsequencingofcoxIgene

PCRamplificationwasperformedasfollows:reaction

mixtures (100μ 1) contained10m MTris-HCI, pH8.3,

50mMKCI ,2mMMgCI2,0.1%TritonX・100 ， 0.01 %gelaｭ

tin , 0.1mMdNTP , 100pmolofeachprimer, 600ngof

templateDNAsofKitasouma2, and5unitofTaqpolyｭ

merase(BoehringerMannheim).Amplificationwas

carriedoutfor1minof94DC , followedby35cyclesof1

minof94DC , 1minat55DC ,and2minat72DC ,withfinal

1minincubationat72DCwithanairthermo ・cycler 1605

(IdahoTechnology).ηle amplifiedDNAswerefractionｭ

atedon0.85%agarosegelandapproximately1300bpprodｭ

uctwasdissectedandpurifiedbyGenepure(NipponGene

Co.).官 lis DNAwasusedasatemplateforthesequencｭ

ingreactionwhichwasperformedusingdyeterminator

sequencingkit(AppliedBiosystems)andthenanalysed

bya373Sautomaicsequencer(AppliedBiosystems).As

thetemplatewastoolongtosequenceatonetime,other

primersweredesignedusingOligosoftware(NBII

Genovus, Inc.).

3 Results

3.1 PaternalinheritanceofcpDNAs

TheDNAsofbothparentspeciesweredigestedwi 出 16

restrictionen 勾明記 s， ApaI, BamHI, BglIl , EcoRI, EcoRV,

HaeIlI, HindIII, HinfI, Kpnl , Mspl , PstI, P日lIl， Seal , Smal ,

XbaIand 沼101 ， andhybridizedwitheachoftwotobacco

cpDNAprobes,pTB8andpTB15.Onlyinthecombinaｭ

tionofEcoRIandpTB8therewasadiffernecebetween

parentspeciesinSouthernhybridizationpatternsandno

polymorphismsweredetectedamong6individualsofP.

massonianainthiscombination.TheDNAofeachhybrid

individualwasdigestedwithEcoRIandhybridizedwith

InheritanceofchloroplastandmitochondrialDNAininterspecifichybridbetween.1守'n us thunbergiiandP.massoniana -35-

pTR8.All15hybridindividualsshowedthesameSouth- massonianabutoneindividualofMizunami4X P.

emhybridizationpa 枕ems asthemaleparentspecies, P. massonianashowedafaintpa 抗em (Fig.1A,R).

Fig.1.SouthernhybridizationpatternofcpDNAinPinus.

A.Eco-RIdigestsofKitasouma2(P.thunbergii ・) XP.massoniana.Lane1,Femaleparent,Kitasouma2;Lanes2・8 ，

Hybridprogeny;Lane9,Maleparent, P.massoniana.

B.EcoRIdigestsofMizunami4(P.thunbergii) XP.massoniana.Lane1,Femalep訂ent ， Kitasouma2;Lanes2-10,Hybridprogeny;Lane9, Maleparent, P.massoniana.

3.2 MaternalinheritanceofmtDNA andXhoI ,andhybridi ヌed withmtDNAprobe, eoxI.There

宵le DNAsofbothparentspeciesweredigestedwith15 werecleardifferencesbetweenparentspeciesinSouth-

restrictionen 勾mes ， ApaI, RgnI , DraI, HeoRI, HeoRV, emhybridizationpa 仕ems intworestrictionen 勾刊 es ， ApaI,

HaeIII , HindIII , HinfIII , KpnI , MspI , PstI, PvuII, SaJI,XbaI BgJII,andnopolymorphismsweredetectedamong6in-

-36- 林木育種センター研究報告 第 18 号

dividualsofP.massonianainthesecombinations.There individualsshowedthesameSouthernhybridizationpat-

werealsosomedifferencesbetweenparentspeciesin6 ternsasfemaleparent, P.thunbergiibutoneindividualof

restrictionen 勾刊 es ， DraT, EcoRV, HindIII , KpnT , Pstl,and Mizunami4XP.massonianashowedafaintpattern(Fig.

XhoI.TheDNAofeachhybridindividualwasdigested 2A, B).Thisindividualwassameonewhichhadfaint

withApaIandthenhybridizedwithcoxI.All15hybrid patterninthecaseofcpDNA.

Fig.2.Southernhybridizationpa 此em ofmtDNAinPinus.

A.ApaIdigestsofKitasouma2(P.thunbergii)XP.massoniana.Lane1，印刷 III/LambdaDNAsizemarker;

Lane2,Femaleparent,Kitasouma2;Lanes3-9,Hybridprogeny;Lane10,Maleparent,P.massoniana.B.ApaldigestsofMizunami4(P.thunbergii)XP.massoniana. Lane1,HindIII/LambdaDNAsizemarker;

Lane2,Femaleparent,Kitasouma2;Lanes3・11 ， Hybridprogeny;Lane12,Maleparent,P.massoniana.

Inheritanceofchloroplastandmitρchondrial DNAininterspecifichybridbetween. 日'n us thunbergiiandP.massoniana ー 37 一

3.3 SequenceofcoxI cytochromeoxidasesubunitI ,Solanumtuberosummito ・

ThepartialsequencesofcoxIwere1325bplongand chondrialcoxIgene,Raphanussa 白rvus mitochondrialcox

showninFig.3.Thissequencehad90%identitywithsoy- IgeneforcytochromecoxidasesubunitI , Oenothera

beancytochromeoxidasesubunitIgene,peamitochon- mitochonodrialcoxIgeneforcyotochoromeonthedata-

drialcoxIgeneforcytochromeoxidasesubunit, baseGenBankbyBLAST.

LycopersiconesculentummitochondrialcoxIgenefor

5 ・ 50

CGTGGCTGTTCTCCACTAACCACAAGGATATAGGGACTCCACAπ 仁AATC

1ω

TTCGGTGCCA 甘GCTGGAGTAATGGGCACATGCTTCTCAGTACCAATTCG

150

TATGGAATTAGCA 仁AACCCGGCGAT 仁AAA 廿ζTTGGTGGGAATCATCAAζ

2ω

CTζATAATGTGTTAATAACGGCTCACGCTTCT 仁CAATGATCCCTTTTATG

250

GTTATGCCGGCGGTGATAGGTGGATCTGGTAAπGGT 仁CGTTCCGATTCC

300

TATAGGTGCACCTGACATGGCATTT 仁CACGATTGAATAATA 廿CCATCCC

350

GGTTGTTGCCACCTTCGCTGTTGζTCC 仁ATTAAGCCCAGCCTCGGTAGAA

400

GTGGGTAGCGGCACTGGGTGGACGGT 仁TATCCGCαCTAAGTGGTATTA 仁

450

CAGTCATTCCGGAGGAGCTGCTGATCCAGCGA 廿TCTAGTCCT 仁ATCTAT

5ω

CAGGTGTTTCATCCAπTCAGGTTCTATCAATCTCATAA 仁TACTATCCCC

550

AAζATGCG 仁GGGCCTGGAATGACTATGCATAGATCACCC 仁TA廿TGTGCG

600

GT 仁CGTTCCAGTGACAGCA 廿仁 CTA 仁TCTTATCATCACTTCCGGTA 仁CGG

650

仁AGGGGCAATTACCATGTTATCAA 仁TGAT 仁GAAGCTTTAATACAACCTTT

7ω

TTCGATC 仁TGCTGGAGGGGGAGACCCGATA 廿ATACCAGCATCTζTTTCG

750

GTTCT 仁仁 GGTCATCCAGAGGTGTATATTCCCA 廿CTGCCCGGAπCGGTA

8ω

TCAπAGTCATATCGTATCGACTTTTTCGGGAAAACCGGTA 廿CGGGTAT

850

αAGGCATGGTTTATGαATGATCAGTAπGGTGTTCCTGGA' 廿TCCTGT

9悌

T仁GGGCTCATCATATGTTTACTGTGGGζTCAGACGTTGATACGCGTGCTC

950

ACTCTACCGCAGCTACCATGATCATAGCTGTζζCCACTGGAATCAAAATC

1000

TTTAGTCGGATCGCTACCATGTGGGGAGGTT 仁GATACGATACAAAA 仁A仁仁

1050

CATGTTAC 廿GCTGCAGGGTCCATCTTTCCGTCCACCATAGGAGGACTCA

11ω

CTGGAATAGTCCTGGCAAATCζTGGGCTAGACA 廿GCTζTG 仁ATGATACT

1150

CAπATGTGGTTGCACATTCCCA 廿ATGTACTTTCTATGGGAG 仁ζGTTTC

1200

TGCTT 仁AT 仁TG 仁AGGA 廿TCACTTαGGGTGGGTAAAATCCCTGGTCGAA

1250

仁ATACCCTGAAACTTTAGGTCAAATCCATCTT 仁GGATζACTCTTTTCGGG

13ω

GTGAATT 仁GAC 仁TTCTTT 仁仁 ζATGCATTTCTTGGGGCTTTCGGGTATGC 仁

1325bp

ACGTCGCAπ 仁CAGA 廿ATCCAGAT

3 ・

Fig.3.PartialsequenceofcoxIgeneinPinusthunbergii.

ooqο 林 木 育 種 セ ン タ ー 研 究 報 告第 1 8号

4.Discussion

Therewereseveralrepo 吋s concerningtheinheritance

oforganelleDNAinconiferousspeciesandpaternalinｭ

heritanceofcpDNAisconsistent.Inthehybridbetween

P.thunbergiiandP.massoniana,weobservedsamemode

ofinheritance,paternalinheritanceofcpDNA.mtDNAwas

inheritedmaternallyinPinusintworepo 吋s. Wagneretale

(1991)detectedpaternalleakageofmtDNAin6outof

84progenyinPinus.Wedidn'tdetectsuchleakagebut

perfectmaternalinheritanceofmtDNA.Paternalinheritｭ

anceofleafcolorwasreportedbySasakietal.(1981)

usingthehybridbetweenthesameparentspecies, P.

thunbergiiandP.massoniana.Astheydidnotuserecipｭ

rocalcrossingfamilies,ithasbeenstillambiguousabout

theinheritancemodeofleafcolor.Therewasthepossibilｭ

itythatthisphenomenonwasaffectedbythedominant

nucleargene, whichcontrolledtheleafcolorofthemale

parent, P.massoniana.Electronmicroscopyobservations

alsosuggestedmaternalinheritanceofmtDNAinPinus

(Willemse1974,ChesnoyandThomas1971).

cpDNAoftheparentspecieswasdigestedwith16reｭ

strictionen 勾相官 s andtherewasadifferencebetweenthe

parentspeciesonlyinonerestrictionen 勾rrne. InmtDNA,

thereweredifferencesbetweentheparentspeciesin8

outof15restrictionen 勾nnes. InChamaecyparis,wealso

observedhigherdegreeofinterspeci 自cpolymorphismin

mtDNAthanincpDNA(Kondoetal.1998).Nealeand

Sederoff(1989)foundnointraspecificPOlymOI 下hism of

cpDNAinPinustaedausingthecombinationsof6restricｭ

tionen 勾rrnes and6probesofpetuniabutfoundpolymorｭ

phisminmtDNA.Wechecked3individualsofPinus

thunbergiiand6individualsofP.massonianaandfound

sametrendofintraspecificpolymorphism.Although 出ere

wasnointraspecificpolymorphismincpDNA,polymorｭ

phismwasfoundinmtDNAofbothspecies. Asthere

aremorepolymo 叩hisms inmtDNA,intraspecificpolymor-

phismwillbeavailabletoclari か the inheritanceofmtDNA

inotherconiferousspecies.

Acknowledgement

WethankMr.M.Sasaki,JapanForestTreeBreeding

Association, forprovidingthehybridsofPinus, Mr.K.

Yoshimura,ForestryandForestProductsResearchInstiｭ

tute , forDNAprimerdesignand Prof.M.Sugiura,

NagoyaUniversity,forsupplyingtobaccocpDNAclone.

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-40 一 林木育種センター研究報告第 18 号

クロマツとタイワンアカマツとの種間雑種における葉緑体およびミトコンドリア DNAの遺伝様式

近藤頑二(1)・津村義彦(2) ・河原孝行(3)

要旨:クロマツとタイワンアカマツとの種間雑種における葉緑体DNAおよびミトコンドリア DNAの遺伝様式を調

べた。雑種 15個体から全DNA を単離し、葉緑体DNAについては、制限酵素 EcoRIで切断後、タバコの葉緑体DNA

の断片pTB8 をプローブにしてサザンハイブリダイゼーションを行ったところ、 14個体はすべて父親であるタイワ

ンアカマツと同じバンドパターンを示したが、 1 f固体は明瞭なバンドを示さなかった。ミトコンドリアDNAについ

ては、制限酵素 ApaI で切断後、 PCR で増殖した coxI の遺伝子をプローブにしてサザンハイブリダイゼーションを

行ったところ、 14個体はすべて母親であるクロマツと同じバンドパターンを示したが、 1 個体は明瞭なバンドが出

なかった。以上のことから、この雑種においては、葉緑体DNA は父性遺伝し、ミトコンドリア DNA は母性遺伝し

ていると考えられた。また、 PCR で増殖したクロマツの coxI 遺伝子の一部の塩基配列を明らかにした。

(1) 林木育種センタ一九州育種場

干 861 ・1102 熊本県菊池郡西合志町大字須屋 2320

(2) 森林総合研究所

干 305 ・8687 茨城県稲敷郡茎崎町松の里 1

(3) 森林総合研究所北海道支所

干 062 -0 045 北海道札幌市豊平区羊ケ丘 7