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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
R.0TSUKA,゛十I.NISHIUMI十andM.WADA゛
゛COUege of LibeyaI Arts and Sdences,T(≒o Medical ad Datal U咄zrs向,2-S-30 K油回心i, M・‘j(au-shi,C�k272-0827d叩回,
†D伊�n�吋Zo歯泄,N凶aslSdaaMsam,3-23-IHy政lmin一油o,S油涵h-ku,T(勿o169-00731叩n
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ダ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.
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exPenditure is rdated to brood sex ratio in Polygynousgreat
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Raymolld M,Rousset F (1995)GENEPOP version l.2: POPulatioll
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⑥2003 Blackwel1 Publishing Ltd,Λ/1池?a/訂石印/四yN討鴎3,44-46
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