Molecular Ecology Notes (2003) doi: 10.1046/j.1471-8286 ｡2003.00345.x

PRIMER NOTECharacterization of 12 PolymorPhic nlicrosatelliteloci

in the JaPanese bush warbleＴＣｅ廿iad却houe

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Abstrad

Japanesebush warblers/ Ce抒紬訴μ/zo17らare a colnnlonsPecies in JaPan andhave aPOly'

gynousbreedingsystem.lnthebreedinggroundsomemaleshavetheirownterritorieswith

morethan onefen!ale. N4ale floaters are also not uncomnlon in the breeding ground. TO

understand breeding strategy in this sPeciesﾀﾞexactParentage should be elucidated. ln order

to obtain a tool for this PurPose, we isolated 34 microsatellite loci from a genomic library

in this species and develoPed primers for 12 1oci. These Primers were tested in the JaPanese

bush warbler and successfully amPlified. ln analyses of 49 unrelated individuals， allelic

numbersranged flro11!t゛o to 22バllld obsel″゛edheteio7ygosity (Ho)ranged from 0.27to0.854

e゛cePt folrt゛o lod (Cdi29 and Cdi35a)with Ho ＜0.1.

瓦印Jo�s:Cdtia d加h皿ＯａPanesebush warblers, microsatellite, Primer

沿cど治�8/X昭四回卯2バ？面面打だ印治�2SS印加涌ぽ2卯2パ7ぼ印f�28S叩tembeT2002

JaPanese bush warblers (Cdtia d巾h皿e)are distributed

along the eastem rim of the Eurasian continent and the

JaPanese islands andare oneof the best known sPedes in

JaPan because of their unmistakablesongs(Hamao 1997).

They are residents or short'distant migrants on Honshu

(main)island in JaPan. ln early sPring, males begin to sing

in wintering sites，such as bushes， Parks， and backyards

of lowlandareas and most of them migrate to higher

mountainsides and breed in bamboo bushes or grass

meadows.At the field sitein Chichibu, Honshu island, we

observed their behaviourand colleded blood samPles for

horn!one nleasurernent after caPture with mist nets. We

found that males sing from late March through August in

the breedingarea and cirmlating testosterone is found

high during this Period (Wada d�.1999).The bush

゛arblers oftelltake a Polygynousbreeding strategy if the

resource is abundant. ln the breeding ground some males

have their own territorieswith more than one female. Male

floaters are also not uncommon in the breeding ground.

Territorial males occasionally change their territories

during a旨eedingseason. To 1111derstand breeding stmtegy

in this sPedes, exad Parentage should be eluddated.

CorresPondence: R. 0tsuka.Te1.＆Fax:十81-47-300-7125; E-mail:

rotsuka.1as＠tmd.ac.jP

　As faras vveknow， no nudear markers have been sPe-

cifically develoPed for bush warblers. Nishiu面ｄ屁

(1996)have develoPed Primers for great reed warblers

(AcToc叩hlusaTundiceus),but they did not show any Positive

result with bush warblers. Thus we dedded to construct a

genomic library and develoP Primers for the sPedes. DNA

for library constructionvvasobtained from blood samPles

ofbirdscaPturedinChichibu.GenomicDNAwasdigested

using Sau3AI. Digested DNA fragments ranging from 400

to 800 bP were ligated into PUC118BamHI/BAP (Takara)

and transformed into Esch�chia eo10M109 Electro-Cells

(Takara)by eledroPoration. Colonies were transferred into

Positively charged nylon membrane (Biodyne B, Pan)and

tho hybiidi7ed ゛ith (GT)15(GA)15(AAAC)5(AAAG)5，

1abelled with digoxygenin using DIG Oligonudeotide

3'-End Labeling Kit (Roche).Hybridized Probes vvere

detected using DIG Nucleic Acid Detection Kit (Roche).

0ut of aPProximately 10 000 coloniesscreened，wefound

34 Positive colonies, whichxveresequenced using BigDye

Termillator Cyde Sequendng FS Ready Reaction Kit

(APPlied Biosystems)on an ABI 310. 0uto04パ2 clones

contained a microsatellite sequence， and Primers xvere

designed for each locus.

　Polyn!erase　chain reaction(PCR)amPlifications

were Performed using Takara PCR ThermaI Cycler MP.

Each reaction was carried out in a total volume of 7.5μL，

⑥2003 Blackwel1 Publishing Ltd

Table 1 Characterization oH2 microsatellite loci in the JaPanese bush warbleTCettia di坤o四

　　　　　　RePeat

Locus　sequence

Cdil　　TDG(TGCG)2(TG)12

Cdi2　(TTTG)11

Cdi8　(AAAAAc)3AAA(AAAc)5

Cdi10(CAAA)、...(CAAA)2

Cdi25　(GT)5(AT)21

Cdi29　　(CAAA)2CATA(CAAA)5

Cdi31　(AG)11

Cdj32　　(CAAA)む..(CAAA)5

Cdi35a(TTrrGG)4

Cdi38（CT）!2

Cdi39（TC）12

Primer sequences (5'to 3')

UPPer: Forward， Lower:Reverse

ＨＥＸ一GTACAGGTrF]7LX:X3ACAGTG

CAGTTCCCTCTTCTCCTA

FAM 一TATCAGTTGCCAGGAGGG

CTGCTCATCACCACCCAA

FAM －ACACCCATAGGCAATAGCA

八

(゜Ｏ

Size

(bP)

No.

alleles

57　157　22

57　203　14

57　298　17

53　196　　9

57　149　19

57　161　　2

57　116　　6

53　210　　7

57　244　　3

57　　96　　7

57　110　　8

53　183　　6

Size

range

ＰＲＩＭＥＲ ＮＯＴＥ ４５

　　　　　　ＨＷＥ

ＨＯ　ＨＥ ｔｅｓｔ

Ａｃｃｅｓｓｉｏｎ

ｎｕｍｂｅｒ

154-194　0.73　0.94　Pく0.01　　AB089166

182－202　0.乃　0.87　Pく0.01　　AB089167

272-295　0.73　0.92　P＜0.01　　AB089168

152-198　0.85　0.74　P＝0.0529　AB089169

105-149　0.77　0.93　P＜0.01　　AB089170

158-162　0.04　0.04　NS

103-119　0.54　0.6　NS

AB089171

AB089172

195－209　0.27　0.75　P＜0.01　　AB089173

240-247　0.02　0.25　Pく0.01　　AB089174

87-101　0.76　0.71　NS

98-110　0.48　0.56　NS

AB089175

AB089176

180-185　0.31　0.58　P＜0.01　　AB089177

and Cdi41バhere are significant differences between召o

andHE.SuchadeviationfromHVVEmaybeaconsequence

of inbreeding， of a substructuring of the samPleorof the

Presence of null alleles｡

　Wexvereable to use the 12 Primers for analysis of Par‘

entage. However， several lod may be unsuitable for this

P11rPose becauseof their low variability (esPedally Cdi29

and Cdi35a)or Possible existence of null allele(e･g.Cdi41).

These POlymorPhic lod were tested in fourother related

sPedes on thesan!e conditions.ln short-tailed bush war-

blerCettia sq皿mdc叩s， Taczanowski's grasshoPPer warbler

Locustella Pleskei and ljima's willow warbleTP㈲11oscoPus

1坦皿e，no lod were a°Plified.�theμeat reed waゐ1er

AcTocephalus arlmdinaceus， Cdi38 was amPlmed and Poly-

morPhic， bllt the other ll microsatemte lod failed to be

amPlified.

Acknowledgements

We thank the students who worked in the fieldat Chichibu to col-

1ect samPles， and the members of DePartment of Zoology，

Nationa1ScienceMuseumTokyofortheirkindsuPPortduringthe

laboratory work. Wearealso grateful to Daichi Saito for kindly

teaching the cloning techniques.ThisstudywassuPPorted in Part

ＴＥＴ一

FAM－GCCrrGCAAGATGGTGGT

CAGTGGATGGCAAGGTG

TET －GGCAAGAAGTGTCAGAAC

CATTCATTCCCCTCTCTC

TET一GAA7[で:;AGTGAGCTGGGTC

CTAGGCACTCTTGGACAG

HEX－｢r？CCAAGCACTAAGACTG

TGCACAAAGCCAACACAGG

TET－GAGCAArrGGCAGrFrCTACC

GCAGGAACAGGAGGAAr[Xﾆ;

FAM －ACATCTTCGGCACGGCT

ＨＥＸ

Cdi41　(GTTTV..(AG)5AAGG(AG)4　FAM－AGATrrrccAGGTCAT7りGG

　　　　　　　　　　　　　　　　　　　　　　rrrGACTAATCTCIでICIGC

Ta:optjmized annealing temperature. Size:the sizeof originaldone. Ho:observedheterozygosily.HE:expectedheterozygosity.HWEtest:

differencesbetween Ho and HF calculated byGENEPOP.NS: not significant.

containing 20 ng of temPlate DNA, 10 mM Tris-HC1, 50mM

KC1, 2.5 mM MgCI2， 0.25 mM of each nudeotide, 2.5 Pmol

of ead! Primer， and 0.45 u�tsT凹Polyn!erase(Takara).

PCRcydesvvereas follows: 3 min at 94 oC, 30 cydes of 30

s at 94 °C，30 s at the oPtimized annealing temPerature

(Table l),45sat72゜C,and 72 ･C for 10 min Each forward

(orreverse)Primer was labelled at the y-end with either 6-

FMT， TET OH‾IEx by the suPPlier (APPlied Biosystems).

PCR Products were sized using GENEscAN software on ABI

310 according to the manufacture's instructions. ln order to

check PolymorPhism and assigll the hetem7ygosity of the

lod, 49 Presumably unrelated adult individuals caPtured

inChichibuxvereanalysed.The Primer Pair for each micro-

satellite locus amPlified PCR Products of aPProPriate

length and were PolymorPhidor the JaPanesebush war-

bler(Table l).Weused GENEPOP for analysis of these data

(Raymond＆Rousset 1995).ThevvINDoxへ/s-based comPuter

Progran! GENEPOP vvas nlade Publidy available and is now

a widely used tool by molecular ecologists.0bserved hetero'

zygosities(Ho)at five lod (Cdi10，Cdi29，Cdi31，Cdi38

゛111dCdi39)are not significantly different from exPected

heterozygosity(HE)under Hardy-Weinberg equilibrium

(HWE)(Table l).AtCdil,Cdi2,Cdi8,Cdi25,Cdi32,Cdi35a

○2003 Blackwe11 Publishing Ltd, Mok�訂E印/o詐No治,3,44－46

46 PRIMER NOTE

by a sPedal grant-in-aid f()rscientific research from Tokyo Medi-

cal and Denta1 University to MW 訂1d by a grant (N0.13740448)

from the Grant-in-Aid 柘r Encouragement of Young Scientists of

the Ministry of Education, Sdence and Culture ooaPan.

References

Hamao S (1997)1卯皿esc Bush W肝心削i引叩凹cse）･P. 63.Bunichi

　Sogo Press,Tokyo･

Nishiumil,Yamagishi S, Maekawa H,ShimodaC(19％)Patemal

　exPenditure is rdated to brood sex ratio in Polygynousgreat

　reed warblers. BehaiJioγalEcok〕gFlnd Sociobiolo9,39,2U－217.

Raymolld M，Rousset F (1995)GENEPOP version l.2: POPulatioll

　genetics software for exact tests andecurnenicism･徊回回/吋

　Herediり，86,248-249.

Wada M, Shimizu T, KobayashいtaL(1999)Behavioral and hor，

　monalbasisofPolygynousbreedinginmalebushwarblers(Cd-

　山山･姉one).GelleTalandC四尹m7面ｄ訥面�皿/県y,116,422－432.

⑥2003 Blackwel1 Publishing Ltd,Λ/1池?a/訂石印/四yN討鴎3,44－46