Molecula Ecology No es (2001)
1
, 120–122
© 2001 Blackwell Science L d
Blackwell Science, L d
PRIMER NOTE
The isola ion o mic osa elli e loci in he Medi e anean
ui ly
Ce a i is capi a a
(Dip e a: Teph i idae) using
a bio in/s ep a idin en ichmen echnique
D. G. CASEY and A. M. BURNELL
Ins i u e o Bioenginee ing & Ag oecology, Biology Depa men , Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland
Abs ac
The Med ly (
Ce a i is capi a a
) is a polyphagous dip e an pes which has sp ead om
No h A ica o he coun ies o he Medi e anean Basin and has also in aded opical and
sub opical egions h oughou he wo ld. Colonizing popula ions ypically possess low
le els o gene ic a iabili y. Mic osa elli es p o ide an e ec i e means o in es iga ing he
popula ion s uc u e o such gene ically depaupe a e popula ions, howe e , mic osa elli e
ma ke s adi ionally equi e a long phase o de elopmen in new axa. We used a bio in/
s ep a idin cap u e echnique o isola e mic osa elli es di ec ly om
C. capi a a
genomic
DNA and we desc ibe he e he iden i ica ion o se en polymo phic mic osa elli e ma ke s
in
C. capi a a
.
Keywo ds
:
Ce a i is capi a a
, en ichmen p o ocol, Med ly, mic osa elli e
Recei ed 18 No embe 2000; e ision accep ed 4 Janua y 2001
The Medi e anean ui ly (Med ly)
Ce a i is capi a a
, a
polyphagous mul i ol ine pes o g ea economic
impo ance, in aded Spain om No h A ica o e
150 yea s ago (Hagen
e al
. 1981). The Med ly has since
sp ead o mos o he coun ies o he Medi e anean Basin
and has also colonized opical and sub opical egions
h oughou he wo ld. To manage his pes , i is impo an
o ind gene ic ma ke s sui able o de e mining he
geog aphical o igin o
C. capi a a
popula ions in ading
new a eas. Because o ounde e ec s and gene ic
bo lenecks, colonizing popula ions ypically possess low
le els o gene ic a iabili y. Mic osa elli es p o ide an
e ec i e means o in es iga ing he popula ion s uc u e o
such gene ically depaupe a e popula ions. We ha e used a
bio in/s ep a idin cap u e echnique (Re se h
e al
. 1997;
Ga dne
e al
. 1999) o isola e mic osa elli es di ec ly om
C. capi a a
genomic DNA and we ha e iden i ied se en
polymo phic mic osa elli e ma ke s ha a e sui able o
he analysis o he gene ic s uc u e and gene low s udies
in
C. capi a a
popula ions.
C. capi a a
genomic DNA was isola ed using s anda d
phenol/chlo o o m ex ac ion wi h RNase (20
µ
g/mL)
diges ion (Mania is
e al
. 1989). Fi e
µ
g o Med ly genomic
DNA we e diges ed in a olume o 50
µ
L wi h 10 uni s o
Mbo
I (P omega) o 5 h a 37
°
C, ollowed by hea inac i a-
ion o
Mbo
I a 65
°
C o 30 min. The oligonucleo ides,
linke A, 5
′
-GGGTAGGATGGGGGATGGG-3
′
(1.6 nmol)
and linke B, 5
′
-GATCCCCATCCCCCATCCTACCC-3
′
(1.6 nmol) we e mixed and hea dena u ed o 5 min a
95
°
C in a o al olume o 60
µ
L con aining 50 m
m
T is-
ace a e pH 7.5, 10 m
m
magnesium ace a e, 50 m
m
po assium ace a e, and allowed o cool slowly o e nigh
o oom empe a u e o gene a e he double-s anded
Mbo
I adap e .
This adap e (0.53 nmole) was liga ed o 5
µ
g o
Mbo
I
diges ed genomic DNA in a olume o 100
µ
L con aining
30 m
m
T is-HCl pH 7.8, 10 m
m
MgCl
2
, 1 m
m
ATP, 10
µ
g
bo ine se um albumin (P omega) and 40 uni s T4 DNA
Ligase (P omega). Excess adap e molecules and low
molecula weigh genomic DNA we e emo ed by cen i-
uging he liga ion eac ion h ough a Mic on 50 il e
(Amicon®) a 16 100
g
o 30 s. Cleaned elu an was eco -
e ed by in e ing he sample ese oi and spinning a
16 100
g
o a u he 30 s in o a 1.5-mL ube. Adap e
liga ed DNA was hyb idized o 1
µ
g (15 nmol) o bio-
inyla ed p obe in a o al olume o 100
µ
L con aining 50
µ
L
o 2
×
binding and washing (B & W) bu e (10 m
m
Co espondence: A. M. Bu nell. Fax: + 353 1 7083845; E-mail:
[email p o ec ed]
men_038. m Page 120 F iday, Augus 24, 2001 8:43 AM
PRIMER NOTE
121
© 2001 Blackwell Science L d,
Molecula Ecology No es
, 1, 120–122
T is-HCl, pH 7.5; 1 m
m
EDTA, 2.0
m
NaCl: Dynal). The
bio ynla ed p obes used we e 5
′
-(AC)
10
GAGC[Bio in]A-
3
′
, 5
′
-(AG)
10
GCAC[Bio in]A-3
′
and 5
′
-(TGC)
10
AGCG-
[Bio in]A-3
′
. Following hea dena u a ion o 5 min a
95
°
C, he hyb idiza ion mix u e was apidly cooled o he
app op ia e hyb idiza ion empe a u e (AC
10
and AG
10
,
50
°
C; TGC
10
, 55
°
C). In a sepa a e ube 100
µ
L o Dyna-
beads® M-280 S ep a idin (Dynal®) we e washed h ee
imes in 100
µ
L o B & W bu e . The hyb idiza ion eac ion
was added o he p epa ed bead mix and incuba ed wi h
gen le agi a ion a he hyb idiza ion empe a u e o
30 min. The cap u ed agmen s we e washed h ee imes
in 100
µ
L o 1
×
SSC a oom empe a u e ollowed by h ee
washes in 100
µ
L o 1
×
SSC a 30
°
C. Cap u ed agmen s
we e elu ed om he beads by hea ing o 5 min a 95
°
C
and we e pu i ied using a Mic on 50 il e (Amicon®).
The mic osa elli e en iched e en a e was polyme ase
chain eac ion (PCR) ampli ied in a 50-
µ
L PCR eac ion
con aining 1
×
PCR bu e (P omega), 4 m
m
MgCl
2
, 10 m
m
dNTPs, 10 pmol o linke A and 1 uni o
Taq
polyme ase
(P omega). PCR was ca ied ou in a Pe kin-Elme 2400
The mal cycle wi h one cycle o dena u ing a 94
°
C o
5 min ollowed by 35 cycles o 95
°
C o 45 s, 63
°
C o 45 s
and 72
°
C o 90 s, ending wi h one cycle o 72
°
C o
10 min. PCR p oduc s we e cloned using he TOPO TA
cloning sys em® (In i ogen). Recombinan clones we e
es ed o mic osa elli e epea sequences by a h ee-p ime
PCR ampli ica ion es (Ga dne
e al
. 1999). P oduc s om
clones ha yielded wo o mo e bands in he h ee-p ime
es we e pu i ied using he S a aP ep™ ki (S a agene)
and we e sequenced on a ABI P ism® 310 Gene ic ana-
lyse . The esul s o he h ee-p ime es s and DNA
sequencing a e p esen ed in Table 1. PCR p ime s we e
designed o eigh
C. capi a a
mic osa elli e loci and se en
o hese loci we e polymo phic (Table 2). App oxima ely
20 indi iduals om i e
C. capi a a
popula ions we e
Table 1 The esul s o he PCR h ee-p ime es s and DNA
sequencing analyses on ecombinan clones o Ce a i is capi a a
genomic DNA gene a ed using a bio in/s ep a idin magne ic
en ichmen p o ocol
P obe
To al(AC)10 (AG)10 (TGC)10
No. o clones es ed
in h ee-p ime PCR es
19 9 5 33
No. o clones yielding
wo o mo e PCR bands
13 3 2 18 (54.5%)
No o sequenced clones
which con ained
mic osa elli es*
13 3 2 18 (100%)
*Recombinan clones which yielded wo o mo e bands in he
h ee-p ime es we e sequenced.
Table 2
Cha ac e is ics o se en mic osa elli e loci o
Ce a i is capi a a
Locus Mo i
P ime sequence (5
′−
3
′
).
F: o wa d, R: e e se
Size*
(bp)
Allele size
ange (bp)
T
a
(
°
C) D
A
H
O
H
E
GenBank
Accession no.
dccap1 (
CA
)
2
CTGC
(
CA
)
4
F:
ACATACACACTGACATCCGCTAAGT
R:
CCAATAACGACGACAATCACC
152 277–281 56 3 0.63 0.43 AF267491
dccap2 (
TGCCGC
)
2
(
TGC
)
11
CAC
(
TGC
)
2
F:
GCAACAACAAAGCAAAGCAA
R:
ATCGGGGTAACGGCTGAGTA
214 288–312 58 4 0.41 0.33 AF267489
dccap4 (
AT
)
4
(
CA
)
8
F:
CTAGGGAACCCTGGGGGAGG
R:
CTTCCCTTTATGCCCGTATGTAT
184 284–344 58 4 0.51 0.45 AF267494
dccap5 (
AC
)
2
TA
(
TG
)
3
AT
(
TG
)
5
C
(
TG
)
6
TC
(
TG
)
3
F:
GCAATGAAAGCAAGCAACAA
R:
GCCGTGAAAGGTGAATGAC
223 336–344 56 3 0.16 0.35 AF267287
dccap6 (
AT
)
2
AG
(
AT
)4(AC)2(AT)2(AC)3F: AGCCTGTTTTGACCAACGTC
R: CGTCACTTAGCGGATGTTCAG
164 287–229 58 4 0.58 0.4 AF267493
dccap1.1 (TA)2TG(TA)2CATG(TA)2CAT(AC)2GTC(TG)4F: TGCCAATAACGACGAAATC
R: AGCCGAAGAATTGGCATTTA
152 277–279 56 2 0.6 0.44 AF267492
dccap9 (TGCCGC)2(TGC)4TAC(TGC)2CGC(TGC)2F: AGTGTCTGAAAACACAACAGCAAC
R: GTTGTATTGTTGCACGAGGATATG
239 306–324 58 4 0.5 0.46 AF267490
The locus name, epea mo i , p ime sequence, annealing empe a u e, sequenced allele size and GenBank accession no. o mic osa elli e loci isola ed a e gi en. The numbe o dis inc alleles
(DA) and le els o he e ozygosi y (HO = obse ed p opo ion o he e ozygo es, HE = expec ed p opo ion o he e ozygo es) a e based on da a om 20 indi iduals om i e popula ions.
men_038. m Page 121 F iday, Augus 24, 2001 8:43 AM
122 PRIMER NOTE
© 2001 Blackwell Science L d, Molecula Ecology No es, 1, 120–122
assessed. Genomic DNA om single lies was isola ed
using he DNeasy™ Tissue Ki (Qiagen). Mic osa elli e loci
we e ampli ied in 25 µL PCR eac ions con aining 50 ng o
C. capi a a genomic DNA, 1 × PCR bu e (P omega)
(10 mm T is-HCl pH 8.3, 50 mm KCl), 2.5 mm MgCl2,
200 µm o each dNTP, 10 pmol o each p ime and 1 uni o
Taq polyme ase (P omega). Ampli ica ions we e ca ied
ou in a Pe kin-Elme 2400 The mal cycle wi h one cycle o
dena u ing a 94 °C o 5 min ollowed by 35 cycles o 45 s
a 95 °C, 45 s a he p ime -speci ic annealing empe a u e
(Table 2), 72 °C o 90 s, ending wi h one cycle o 72 °C
o 10 min. P oduc s we e elec opho esed on s anda d
sequencing gels (6% ac ylamide, 8 m u ea, in 1 × TBE) and
isualized using he Sil e Sequence™ DNA S aining
Sys em (P omega).
Analyses o gene ic di e si y we e ca ied ou using
genepop so wa e (Raymond & Rousse 1995). Numbe s
o dis inc alleles anged om one o ou pe locus wi h
obse ed and expec ed he e ozygosi ies anging om
0.32 o 0.63 (Table 2). Null alleles we e iden i ied in he
locus dccap5. The o e all gene ic di e si y (Nei 1987)
ound in his s udy (GD = 0.51) is compa able wi h alues
epo ed in Medi e anean C. capi a a popula ions by
Bonizzoni e al. (2000). These se en mic osa elli e loci a e
cu en ly being used o analyse gene low and mu a ion
p ocesses in C. capi a a popula ions om he Medi e a-
nean Basin.
Acknowledgemen s
We hank Michael Ga dne o p o iding a copy o his en ichmen
p o ocol p io o publica ion, Unn Re se h o he linke sequence
and Thomae Kakouli-Dau e o Ce a i is capi a a genomic DNA.
The esea ch was suppo ed by he Eu opean Communi y (P ojec
FAIRPL 96–1972).
Re e ences
Bonizzoni M, Malac ida AR, Gugliemino CR, Gomulski LM,
Gaspe i G, Zheng L (2000) Mic osa elli e polymo phism in he
Medi e anean ui ly Ce a i is capi a a. Insec Molecula Biology,
9, 251–261.
Ga dne MG, Coope SJB, Bull CM, G an WN (1999) Isola ion o
mic osa elli e loci om a social liza d Ege nia s okesii, using a
modi ied en ichmen p ocedu e. Jou nal o He edi y, 90, 301–304.
Hagen KS, William WW, Tassen RL (1981) Medi e anean ui ly:
The wo s may be ye o come. Cali o nia Ag icul u e, 35, 5–7.
Mania is T, F i sch EF, Samb ook J (1989) Molecula Cloning: a
Labo a o y Manual. Cold Sp ing Ha bou Labo a o y P ess,
New Yo k.
Nei M (1987) Molecula E olu iona y Gene ics. Columbia Uni e si y
P ess, New Yo k.
Raymond M & Rousse F (1995) genepop (Ve sion 1.2): popula ion
gene ics so wa e o exac es s and ecumenicism. Jou nal o
He edi y, 86, 248–249.
Re se h UH, Fangan BM, Jakobsen KS (1997) Hyb idisa ion
cap u e o mic osa elli es di ec ly om genomic DNA. Elec o-
pho esis, 18, 1519–1523.
men_038. m Page 122 F iday, Augus 24, 2001 8:43 AM