new 24 polymorphic dna microsatellite loci for the major malaria vector anopheles darlingi and...
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TECHNICAL NOTE
New 24 polymorphic DNA microsatellite loci for the majormalaria vector Anopheles darlingi and transpecies amplificationwith another anophelines
G. N. Lima • J. S. Batista • K. M. Formiga •
F. W. Cidade • M. S. Rafael • W. P. Tadei •
J. M. M. Santos
Received: 13 April 2010 / Accepted: 17 April 2010 / Published online: 19 May 2010
� Springer Science+Business Media B.V. 2010
Abstract Anopheles darlingi is a major human malaria
vector in the Neotropics. Twenty-four polymorphic
microsatellite loci were isolated and characterized in 21–32
individuals collected in Coari (Amazonas, Brazil). The
number of alleles per locus ranged from 4 to 11 (average of
7.667). The observed heterozygosity (HO) varied between
0.037 and 0.833 (average of 0.500), while the expected
heterozygosity (HE) ranged from 0.177 to 0.871 (average
of 0.723). Thirteen loci showed a significant deviation from
HWE. No linkage disequilibrium was found between the
loci.
Keywords Anopheles darlingi � Microsatellites �Malaria vector � Amazon Basin
Anopheles (Nyssorhynchus) darlingi is the major and the
most anthropophilic and endophagic malaria vector in the
Brazilian Amazon Basin (Tadei et al. 1998). It is also a
significant vector in other countries in South America such
as Peru, Colombia and Suriname (Vittor et al. 2006).
Although morphology and genetics variation had been
described suggesting that A. darlingi is a species complex
(Freitas-Sibajev et al. 1995), morphometric and genetic
analyses using isozymes, RAPD and ITS2 demonstrated
the existence of a single species (Manguin et al. 1999).
Studies using mitochondrial and nuclear DNA markers
provide support for moderate level of population genetic
structure (Mirabello and Conn 2006; Scarpassa and Conn
2007; Mirabello et al. 2008).
Eight microsatellite loci were characterized (Conn et al.
2001) but the development of new markers would improve
genetic population studies, gene mapping and QTL anal-
ysis in this vector. This study reports the isolation and
characterization of new 24 microsatellite markers for A.
darlingi and cross-amplification in three con-generic
species.
A genomic library enriched with microsatellite DNA of
A. darlingi was constructed (Billotte et al. 1999) from the
Genomic DNA extracted (Wilkerson et al. 1995) using a
pool of 10 A. darlingi adult specimens newly hatched and
unfed collected in Coari, Amazonas, Brazil. The DNA was
digested with RsaI restriction enzyme (Invitrogen), and
linked to RSA21 and RSA25 adapters. Microsatellite DNA
fragments were selected by hybridization with (CT)8 and
(GT)8 repeats biotin-linked probes and recovered with
streptavidin-linked particles (Promega). Selected fragments
were linked into a pGEM-T vector (Promega), transformed
into Escherichia coli XL1-blue competent cells and inoc-
ulated into plates with X-Gal/IPTG/LB agar. After the
growth at 37�C the white colonies were transferred onto
G. N. Lima � M. S. Rafael � W. P. Tadei � J. M. M. Santos (&)
Laboratorio de Vetores Malaria e Dengue, Instituto Nacional de
Pesquisas da Amazonia, Coordenacao de Pesquisas em Ciencias
da Saude (CPCS), Avenida Andre Araujo, No. 2936, Petropolis,
Manaus, AM CEP 69060-001, Brazil
e-mail: [email protected]
J. S. Batista � K. M. Formiga
Instituto Nacional de Pesquisas da Amazonia, Coordenacao
de Pesquisas em Biologia Aquatica (CPBA), Avenida Andre
Araujo, No. 2936, Petropolis, Manaus, AM CEP 69060-001,
Brazil
J. S. Batista � K. M. Formiga
Laboratorio Tematico de Biologia Molecular (LTBM), Instituto
Nacional de Pesquisas da Amazonia, Coordenacao de Pesquisas
(COPE), Avendia Andre Araujo, No. 2936, Petropolis, Manaus,
AM CEP 69060-001, Brazil
F. W. Cidade
Departamento de Genetica e Evolucao, Centro de Biologia
Molecular e Engenharia Genetica, Universidade Estadual de
Campinas, CP 6010, Barao Geraldo, Campinas, SP CEP
13083-970, Brazil
123
Conservation Genet Resour (2010) 2:205–209
DOI 10.1007/s12686-010-9237-y
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erse
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ence
s(50 -
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Siz
era
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DH
OH
EP
-HW
EF
IS(f
)
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a03
GU
05
98
66
(AC
) 12
FF
AM
:AG
AG
AG
CT
AA
TG
CG
GT
TG
GT
C2
89
25
3–
28
30
.58
80
.83
90
.42
90
.61
80
.02
10
.31
1
R:A
CG
TT
CC
TC
TA
CT
CC
GA
AA
GC
Ad
a06
GU
05
98
68
(CA
) 8F
FA
M:T
TA
AT
CA
CT
TG
CG
AC
AG
AC
C3
07
96
–1
18
0.5
91
0.8
65
0.5
67
0.6
66
0.1
01
0.1
51
R:G
AC
TC
CA
TT
CC
TT
GA
AC
CA
Ad
a09
GU
05
98
69
(GT
) 9F
FA
M:G
TC
AT
CA
TC
GT
CG
TC
GG
AA
T3
07
14
2–
15
40
.71
50
.92
30
.83
30
.76
00
.71
9-
0.0
98
R:C
TG
CA
AC
CA
GC
GA
GT
TC
TT
AC
Ad
a10
GU
06
53
63
(AC
) 10
FF
AM
:GC
AA
CA
GA
CC
AG
AC
CA
GA
CA
T2
74
a1
42
–1
86
0.1
68
0.3
95
0.0
37
0.1
77
0.0
00
*1
.00
0
R:C
CT
GG
AC
GC
TC
TG
TG
CG
C
Ad
a17
GU
06
53
64
(CA
) 10
CG
(CA
) 8F
HE
X:G
GG
TA
GT
AA
AG
CA
AC
TG
AA
GC
C2
91
0a
16
6–
20
80
.84
00
.96
60
.65
50
.87
10
.00
0*
0.2
51
R:C
TA
CA
GC
AA
GC
GA
AG
GG
AA
G
Ad
a18
GU
06
53
65
(AC
) 9T
(CA
) 5F
HE
X:G
AC
AC
TC
CG
CA
CT
CT
CT
TC
AC
24
8a
24
0–
27
20
.78
40
.95
20
.41
70
.82
60
.00
0*
0.5
01
R:G
CT
TG
CC
CA
TA
AC
TC
TC
AC
C
Ad
a20
GU
06
53
66
(GT
) 10
FF
AM
:AG
CA
AT
AT
GT
TC
CC
GA
CA
GC
27
93
02
–3
32
0.7
98
0.9
62
0.7
41
0.8
33
0.0
29
0.1
13
R:C
GG
CT
TC
TA
AA
TG
AC
TC
CT
AG
C
Ad
a21
GU
06
53
67
(TG
) 8F
FA
M:G
GT
AG
TC
CG
AG
AG
GA
GA
GG
TG
31
7a
28
0–
31
00
.81
10
.94
60
.54
80
.84
60
.00
0*
0.3
56
R:G
CA
GG
AC
AA
AA
CC
AA
TC
TG
C
Ad
a22
GU
06
53
68
(CA
) 8F
FA
M:G
GC
TT
CC
GT
CT
TC
TT
CT
AT
TC
C2
48
a1
58
–1
86
0.7
03
0.9
31
0.2
92
0.7
50
0.0
00
*0
.61
6
R:G
TC
CT
TA
CG
CA
CG
GT
TT
CT
C
Ad
a23
GU
06
53
69
(AC
) 7F
HE
X:A
CT
CG
TT
CG
TG
CT
CT
GT
CA
CT
31
81
24
–1
68
0.6
69
0.9
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0.6
13
0.7
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0.3
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0.1
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AG
TG
TG
TT
GT
GT
CC
TC
A
Ad
a24
GU
06
53
70
(AC
) 9F
FA
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TG
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CA
CA
TG
GA
AG
CG
TA
G2
85
21
5–
22
50
.60
60
.84
50
.64
30
.67
30
.01
00
.04
5
R:G
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AT
GG
AA
GA
AG
Ad
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GU
06
53
71
(CA
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FA
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TC
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TG
TT
CT
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CT
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CC
26
9a
30
4–
33
20
.80
60
.95
00
.26
90
.84
50
.00
0*
0.6
86
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AC
TG
GT
TC
TG
GT
TC
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G
Ad
a27
GU
06
53
72
(CA
) 8F
FA
M:A
GC
GG
AT
CT
AC
CT
AC
GG
GT
TA
32
10
11
9–
16
30
.78
90
.95
60
.78
10
.82
40
.62
50
.05
3
R:C
GC
TA
TC
AG
CA
TC
AT
CA
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G
Ad
a29
GU
12
00
62
(CG
T) 1
5F
FA
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AA
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AG
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CT
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C2
77
66
–8
40
.70
60
.90
10
.59
30
.76
20
.00
0*
0.2
26
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GA
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AC
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Ad
a30
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00
63
(GT
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CC
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CT
CA
C3
07
31
8–
33
10
.60
00
.82
70
.63
30
.65
40
.00
0*
0.0
32
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AT
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AC
AC
Ad
a32
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12
00
64
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C) 6
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GT
AT
GT
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GA
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31
71
80
–1
98
0.6
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0.8
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0.7
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0.8
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.08
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Ad
a33
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12
00
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(GT
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EX
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0–
15
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.95
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.51
60
.83
00
.00
0*
0.3
82
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TC
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a37
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CC
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–2
65
0.6
78
0.8
81
0.3
33
0.7
41
0.0
00
*0
.55
4
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GA
AA
AG
G
206 Conservation Genet Resour (2010) 2:205–209
123
microplates with a HM/FM medium to grow overnight.
Plasmid DNA was extract (Sambrook et al. 1989) from 96
insert and bi-directionally sequenced on an ABI 3130
(Applied Biosystems) using T7 and SP6 primers and the
v3.1 Big Dye terminator 3.1 kit (Applied Biosystems).
The assemble were performed with 77 clones sequences
and edited using STADEN package (Staden 1996). The
124 microsatellite were identified in 73 non-redundant
clone sequences and the 69 primers’ pairs were designed
with the WEBSAT program (Martins et al. 2009). A M13
sequence tail was added in the 50 end of each forward
primer following a labeling protocol (Schuelke 2000).
The microsatellite fragments were amplified in 10 ll
containing 10–50 ng of genomic DNA, each forward and
M13 Label primer (FAM or HEX) at 0.4 lM, reverse
primer (0.8 lM), each dNTP (200 lM), MgCl2 (1.5 mM),
1X PCR buffer and 0.5U of goTaq DNA Polymerase
(Promega). PCR was carried out with two main steps: the
denaturation (68�C, 1 min; 94�C, 30 s) followed by 30
cycles of 30 s at 93�C, 35 s at 60�C, 40 s at 68�C; the
second step consisted of 15 cycles following: 25 s at 93�C,
35 s at 53�C, 30 s at 72�C, and a final extension at 72�C for
30 min. PCR products were visualized on a MegaBACE
1000 (GE Healthcare) and allele sizes were scored using
ET-550 ROX (GE Healthcare) analyzed on FRAGMENT
PROFILER v1.2 (GE Healthcare) program.
Polymorphisms in 24 microsatellite loci were evaluated
in 21–32 individuals of A. darlingi collected in Coari. The
descriptive statistics and linkage disequilibrium (LD) were
inferred using FSTAT v2.9.3.2 (Goudet 2002), Polymor-
phism Information Content-PIC (Botstein et al. 1980) was
estimated with MSTOOLS v3 (Park 2001) and the test for
Hardy–Weinberg Equilibrium (HWE) was perform using
GENEPOP v4 (Raymond and Rousset 1995). The number
of alleles per locus ranged from 4 (Ada10, Ada37 e Ada39)
to 11 (Ada63), with an average of 7.667. The observed
heterozygosity (HO) ranged between 0.037 and 0.833
(Ada10–Ada09, respectively) with a mean of 0.500, while
the expected heterozygosity (HE) ranged from 0.177 to
0.871 (Ada10 and Ada17, respectively) with an average of
0.723. The PIC ranged between 0.168 (Ada10) and 0.840
(Ada17), with a mean of 0.679. Thirteen loci showed a
significant deviation from HWE after the Bonferroni
Correction (Rice 1989). This deviation may be due to
sampling effect or the presence of null alleles as suggested
by Microchecker v2.2.3 program (Van Oosterhout et al.
2004) (Table 1). LD was not detected between all pairs of
loci following sequential Bonferroni Correction. The Dis-
criminating Power (D) (Jones 1972) was estimated for
each loci and ranged from 0.395 (Ada10) to 0.966
(Ada17), with an average of 0.888. The value of FIS ranged
from -0.098 (Ada9) to 1.000 (Ada10) with a mean of
0.328.Ta
ble
1co
nti
nu
ed
SS
Rlo
cus
Gen
ban
kac
cess
ion
no
.R
epea
tm
oti
fP
rim
erse
qu
ence
s(50 -
30 )
NA
Siz
era
ng
e(b
p)
PIC
DH
OH
EP
-HW
EF
IS(f
)
Ad
a39
GU
12
00
67
(GT
) 13
FH
EX
:GA
TC
GC
AG
TA
GC
TG
AA
AG
TC
G2
34
27
1–
28
70
.47
40
.85
50
.30
40
.53
20
.02
80
.43
4
R:G
AA
TA
TC
GC
GG
TG
GA
TC
AG
Ad
a40
GU
12
00
68
(TC
) 19
FF
AM
:TA
CT
AC
TG
AT
TG
GC
GC
TC
CT
G2
59
20
1–
23
50
.56
40
.88
30
.56
00
.59
80
.11
70
.06
5
R:A
CT
AC
GG
GT
CC
TC
TC
GT
GT
TC
Ad
a41
GU
12
00
69
(TG
) 10
FF
AM
:CG
CT
GA
GA
AC
AT
TG
GG
TA
GT
C3
11
02
75
–3
01
0.6
94
0.9
13
0.5
81
0.7
37
0.1
37
0.2
15
R:G
TG
GT
AC
TG
CG
AG
GA
TC
AA
AG
Ad
a48
GU
12
00
70
(GG
T) 4
FH
EX
:CG
AC
GG
TG
AA
CT
GA
AC
TC
G2
97
a2
65
–2
83
0.6
76
0.8
65
0.3
45
0.7
27
0.0
00
*0
.53
0
R:C
AC
TC
GT
GG
GA
AC
TG
CT
TT
C
Ad
a60
GU
90
84
93
(AA
G) 9
FF
AM
:GC
AT
AT
AG
CC
CC
TT
TT
CC
TC
C2
18
a2
33
–2
54
0.7
78
0.9
60
0.2
86
0.8
22
0.0
00
*0
.65
8
R:C
TG
CC
GT
CT
CG
TG
TT
TA
GT
GT
Ad
a63
GU
12
00
71
(GT
) 9F
HE
X:T
GT
TG
CC
TT
GA
CT
AT
CC
TT
TT
G2
21
11
77
–2
13
0.7
78
0.9
33
0.2
27
0.8
15
0.0
00
*0
.72
6
R:T
AT
TC
GT
TG
TG
TT
GT
GT
TC
GC
Th
ean
nea
lin
gte
mp
erat
ure
inal
llo
ciw
as6
0�C
.N
:n
um
ber
of
ind
ivid
ual
ssc
reen
ed;
A:
nu
mb
ero
fal
lele
s;a:
pre
sen
ceo
fa
nu
llal
lele
;b
p:
bas
ep
airs
;P
IC:
po
lym
orp
his
min
form
atio
nco
nte
nt;
D:
dis
crim
inat
ing
po
wer
;H
O:
ob
serv
edh
eter
ozy
go
sity
;H
E:
exp
ecte
dh
eter
ozy
go
sity
;P
-HW
E*
:d
epar
tssi
gn
ifica
ntl
yfr
om
HW
Eaf
ter
Bo
nfe
rro
ni
corr
ecti
on
and
FIS
:in
bre
edin
gco
effi
cien
t
Conservation Genet Resour (2010) 2:205–209 207
123
All the 24 polymorphic markers were tested for cross-
amplification in three Anopheles species (Table 2). Eight
loci were amplified in all three species and 17 loci
amplified in at least one species. The number of markers
amplified ranged from 10 (A. rangeli) to 15 (A. benarro-
chi). Fourteen loci are polymorphic in at least one species
and ranged between eight (A. rangeli) to 13 loci (A.
triannulatus).
The 24 polymorphic microsatellites developed can be
used as efficient markers for investigating population
genetic and genome mapping of A. darlingi and others
Anopheles species.
Acknowledgments This work was supported by FAPEAM/PIPT,
CTPetro-Rede malaria/CNPq and PROCAD-Amazonia-INPA/UNI-
CAMP/UFRGS/CAPES (023/2006). The authors thank Anete Souza,
Adna Souza and Tatiana Campos (CBMEG/UNICAMP) for help in
genomic microsatellites library construction, Vera Val (LEEM/INPA)
for help sequencing on the ABI 3130, the technicians in malaria and
dengue/INPA laboratory for help in capture and identification of
mosquitoes and the LTBM/INPA where a great amount of this work
was perform. GNL was supported by PosGrad/FAPEAM masters
fellowship.
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Table 2 Cross-species amplification of 24 Anopheles darlingimicrosatellite markers loci
SSR locus A. triannulatus* A. rangeli* A. benarrochi*
Size range
(bp)
A Size range
(bp)
A Size range
(bp)
A
Ada03 9 9 9
Ada06 85–103 3 103 1 83–103 3
Ada09 151–153 2 9 143–153 2
Ada10 9 9 9
Ada17 9 9 9
Ada18 9 9 9
Ada20 300–332 3 320–332 2 310–332 2
Ada21 9 9 9
Ada22 176–182 2 176–182 2 176–182 2
Ada23 124–126 2 124–126 2 120–124 2
Ada24 243–253 5 237–247 2 237–247 2
Ada25 275–295 2 295–307 2 9
Ada27 124–154 2 134–158 2 126–130 2
Ada29 9 9 82–100 2
Ada30 9 9 335 1
Ada32 9 9 199 1
Ada33 88–138 2 114–144 2 88–98 3
Ada37 192–234 4 182–212 3 192–212 2
Ada39 9 9 9
Ada40 204–216 3 212 1 9
Ada41 262–284 5 9 264–300 2
Ada48 9 9 9
Ada60 9 9 233 1
Ada63 143–189 2 9 187 1
* PCR annealing temperature as in Table 1. N: 5 for all species, A:
estimated number of alleles and 9: no amplification
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