MEC802.fm Page 2141 Saturday, December 18, 1999 2:05 PM
Molecular Ecology (1999) 8, 2141– 2152
PRIMER NOTES
Blackwell Science, Ltd
Isolation and characterization of
microsatellites in Theobroma cacao L.
1000
Graphicraft
Limited, Hong Kong
C. LANAUD, A. M. RISTERUCCI,
I . P I E R E T T I , M . FA L Q U E , A . B O U E T
and P. J . L . L A G O D A
CIRAD — BIOTROP, Avenue Agropolis, BP 5035, 34032 Montpellier cédex,
France
Keywords: breeding, cocoa, diversity, identification, microsatellites
Received 9 May 1999; revision accepted 19 July 1999
Correspondence: C. Lanaud. Fax: +33 (0) 467615605; E-mail:
[email protected]
Theobroma cacao L. (2n = 2x = 20) is a Sterculiaceae native
from central and south America. Three main genetic groups
may be distinguished: Criollo, Forastero and their hybrid
form, Trinitario.
Molecular markers, mainly restriction fragment length
polymorphisms (RFLP) and random amplified polymorphic
DNA (RAPD) have been applied to evaluate genetic resources and refine T. cacao classification (Laurent et al. 1993;
N’Goran et al. 1994). Several hypotheses were advanced for
the origin of the Criollo group, the first domesticated cacao,
and the one that gives the finest chocolate. Microsatellites
would be particularly useful to better understand the origin
and domestication of Criollo.
To isolate cocoa microsatellites, we screened several
different libraries. A partial PstI genomic library was constructed by cloning total DNA in puc18, and had an insert
size ranging between 0.5 and 2 kb (Laurent et al. 1993). Two
hundred and seventy-five unique inserts were screened with
(TC)10 (AC)10 (GC)8 (AT)15, for detection of simple sequence
repeats (SSR). Inserts were amplified from the cloning vector
using universal primers (M13 forward and M13 reverse),
electrophoresed, transfered onto Hybond N+ membranes
and probed with [γ 32P]-dATP end-labelled oligonucleotides.
Libraries enriched separately for GA and GT were also constructed according to a modified version of Karagyozov et al.
(1993). Five-hundred inserts from each library were probed
with [γ 32P]-dATP end-labelled (GA)15 and (GT)15.
After hybridization with (TC) 10 and (AC)10, 4% and
1.5%, respectively, of the genomic clones from the PstI
library were positive. No positive signals were obtained
after hybridization with (GC)8 or (AT)15. Nevertheless, the
presence of microsatellites such as (AT)n were revealed
after sequencing some clones, demonstrating the difficulties
that occur during hybridization with these autocomplementary oligonucleotides.
Approximately 90% of the sequenced positive candidate
clones contained GA or GT repeats. Perfect or imperfect SSR
were observed, and the number of perfect repeats varied
from two to 28. Primers were defined in the flanking regions
of the SSR using the software oligo (Rychlik 1992). Not all of
© 1999 Blackwell Science Ltd
the candidate clones could be used to define primers due to
the redundancy of some, or to the extreme position of SSR
in the DNA fragment.
Microsatellite polymorphisms were screened on several
genotypes of cocoa belonging to the different groups of T.
cacao. PCR amplification was performed in a MJ Research
PTC 100 thermal cycler, in a 20-µL reaction containing 1 U of
Taq polymerase (Eurobio), 10 ng of cocoa DNA, 0.2 mm dNTP
mix, 2 mm MgCl2, 50 mm KCl, 10 mM Tris-HCl (pH 8.3) and
2 pmol primer (5′ end-labelled with [γ 33P]-dATP). These were
overlaid with a drop of ultra-pure mineral oil. The samples
were denaturated at 94 °C for 4 min, and subjected to 32
repeats of the following cycle: 30 s at 94 °C, 1 min at 46 °C
or 51 °C, and 1 min at 72 °C. They were then kept at 4 °C
prior to analysis. After adding 20 µL of loading buffer (98%
formamide, 10 mm EDTA, bromophenol blue, xylene cyanol),
the mix was denaturated at 92 °C for 3 min and kept at
70 °C. Four µL of each sample was loaded in a 5% polyacrylamide sequencing gel with 7.5 m urea in 0.5% TBE buffer
and electrophoresed at 55 W for 1 h 40 min. The gel was
dried for 30 min at 80 °C and exposed overnight to X-ray
film (Fuji RX).
For approximately 45% of candidate clones, primers generating a polymorphic amplified product could be defined,
equal to 23 microsatellites as defined in Table 1. In the analysed samples, the observed mean heterozygosity per locus
varies from 0.14 to 0.66 (mean 0.46) and the number of alleles
per loci from 2 to 13 (mean 5.6). The results could be compared
with previous diversity studies realized with nine isozymes
(Lanaud 1987) and 31 RFLP probes (Laurent et al. 1993) on
larger populations (300 and 180 genotypes, respectively)
where a mean number of alleles per locus of 3.3 and 2.4
was observed, with 5 and 4 alleles observed as maximum
in each case.
These 23 microsatellites were also tested with the same
PCR conditions on one sample of eight other species more
or less botanically distant from T. cacao but belonging to
the same family (Sterculiaceae) (Table 2). In order to confirm
the presence of the microsatellite, amplification products
were probed with (GA)15 and (GT)15. The membranes were
exposed for 2 h to X-ray film. Approximately 50% of the
primers defined in T. cacao could amplify a clearly visible
DNA fragment that contains a microsatellite in species
belonging to the same genus Theobroma or to the closely
related genus Herrania. Six primer pairs could also amplify
microsatellite-containing fragments of Cola nitida. In some
cases, no signal was observed in spite of the presence of
an amplified DNA fragment that had the same size as the
amplified cocoa fragment. This could reflect the total absence
of microsatellite, the presence of very small microsatellites in
homologous DNA fragments, or the presence of imperfect
microsatellites that prevented the hybridization with (GA)15
and (GT)15.
The large polymorphism revealed by these first microsatellites
MEC802.fm Page 2142 Saturday, December 18, 1999 2:05 PM
2142 P R I M E R N O T E S
Table 1 Characteristics of 23 cocoa microsatellites
Marker
name
EMBL
accession
number
mTcCIR1
Y16883
mTcCIR2
Y16978
mTcCIR3
Y16977
mTcCIR4
Y16979
mTcCIR6
Y16980
mTcCIR7
Y16981
mTcCIR8
Y16982
mTcCIR9
Y16983
mTcCIR10
Y16984
mTcCIR11
Y16985
mTcCIR12
Y16986
mTcCIR13
Y16987
mTcCIR15
Y16988
mTcCIR16
Y16989
mTcCIR17
Y16990
mTcCIR18
Y16991
mTcCIR19
Y16992
mTcCIR21
Y16994
mTcCIR22
Y16995
mTcCIR24
Y16996
mTcCIR25
Y16997
mTcCIR26
Y16998
mTcCIR28
Y16999
Primer sequence (5′– 3′)
Ta(°C)
Size of
cloned
allele (bp)
GCAGGGCAGGCTCAGTGAAGCA
TGGGCAACCAGAAAACGAT
CAGGGAGCTGTGTTATTGGTCA
AGTTATTGTCGGCAAGGAGGAT
CATCCCAGTATCTCATCCATTCAGT
CTGCTCATTTCTTTCATATCA
CGACTAAAACCCAAACCATCAA
AATTATTAGGCAACCCGAACTT
TTCCCTCTAAACTACCCTAAAT
TAAAGCAAAGCAATCTAACATA
ATGCGAATGACAACTGGT
GCTTTCAGTCCTTTGCTT
CTAGTTTCCCATTTACCA
TCCTCAGCATTTTCTTTC
ACCATGCTTCCTCCTTCA
ACATTTATACCCCAACCA
ACAGATGGCCTACACACT
CAAGCAAGCCTCATACTC
TTTGGTGATTATTAGCAG
GATTCGATTTGATGTGAG
TCTGACCCCAAACCTGTA
ATTCCAGTTAAAGCACAT
CAGTCTAACAAAGGTGAG
TGCCCCACTTGACAACTA
CAGCCGCCTCTTGTTAG
TATTTGGGATTCTTGATG
ACCTTCACCAGCTCACC
TAAATTCTACTAGCAAATTACC
AAGGATGAAGGATGTAAGAGAG
CCCATACGAGCTGTGAGT
GATAGCTAAGGGGATTGAGGA
GGTAATTCAATCATTTGAGGATA
CACAACCCGTGCTGATTA
GTTGTTGAGGTTGTTAGGAG
GTCGTTGTTGATGTCGGT
GGTGAGTGTGTGTGTTTGTCT
ATTCTCGCAAAAACTTAG
GATGGAAGGAGTGTAAATAG
TTTGGGGTGATTTCTTCTGA
TCTGTCTCGTCTTTTGGTGA
CTTCGTAGTGAATGTAGGAG
TTAGGTAGGTAGGGTTATCT
GCATTCATCAATACATTC
GCACTCAAAGTTCATACTAC
GATCAATCAGAAGCAAACACAT
TAAAGCAGCCTACCAAGAAAAG
51
143
(CT)14
51
254
(GA)3N5(AG)2GG(AG)4
46
249
51
Repeat structure
N
18
No. of
alleles
HO
HE
3
0.5
0.62
8
3
0.55
0.51
(CT)20(TA)21
18
6
0.54
0.77
259
(TCTCTG)2(TC)8
14
3
0.66
0.45
46
231
(TG)7(GA)13
24
8
0.54
0.57
51
160
(GA)11
24
6
0.42
0.75
46
301
(TC)5TT(TC)17TTT(CT)4
24
5
0.33
0.55
51
274
(CT)8N15(CT)5N9(TC)10
18
4
0.45
0.59
46
208
(TG)13
18
4
0.56
0.71
46
298
(TC)13
24
9
0.46
0.81
46
188
(CATA)4N18(TG)6
24
10
0.62
0.87
46
258
(AG)13
14
4
0.33
0.47
46
254
(TC)19
24
10
0.62
0.84
46
308
(TC)9N37(TC)13
14
3
0.14
0.45
51
271
(GT)7N4(GA)12
14
3
0.5
0.87
51
345
(GA)12
24
8
0.46
0.72
46
376
(CT)28
18
6
0.58
0.72
46
157
(TC)11N5(CA)12
18
6
0.47
0.68
46
289
(TC)12N146(CT)10
14
4
0.29
0.43
46
198
(AG)13
18
4
0.35
0.31
46
153
(CT)21
24
13
0.42
0.84
46
298
(TC)9C(CT)4TT(CT)11
14
6
0.41
0.67
46
336
(TC)8
4
2
0.43
0.41
Ta, annealing temperature used; HO, observed mean heterozygosity; HE, expected heterozygosity.
isolated in cocoa confirm their usefulness for mapping and
diversity studies, as well as for identification purposes. This
could be applied to confirm the conformity of germplasm
or the genetic origin of cocoa beans sent to chocolate
manufacturers from various tropical countries. The potential
use of some primers defined in T. cacao to reveal polymorphism in species of the same family also represents a
major interest to the management of genetic resources of
species that have not been well studied because of few
available means.
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141– 2152
MEC802.fm Page 2143 Saturday, December 18, 1999 2:05 PM
P R I M E R N O T E S 2143
Table 2 Screening of the 23 microsatellites isolated in Theobroma cacao and studied on eight different species belonging to the same family
(Sterculiaceae) than T. cacao
Microsatellite mTcCIR
1
2
3
4
6
7
8
9
10
11
12
13
15
16
17
18
19
21
22
24
25
26
28
T. cacao
T. grandiflora
T. microcarpa
T. mamosum
T. bicolor
T. angustifolia
H. umbratica
H. cuatrescasana
Cola nitida
+
a
+
+
a
+
a
a
+
–
+
+
+
+
+
+
+
a
+
a
a
a
a
a
–
a
–
+
+
+
a
+
a
a
+
+
+
–
–
–
–
–
–
–
–
+
a
a
a
a
a
+
+
–
+
–
a
a
a
–
a
–
+
+
+
+
+
+
+
+
+
+
+
+
+
a
–
a
+
+
–
+
+
+
+
a
+
a
a
a
+
+
+
+
+
+
a
a
–
+
+
+
+
+
+
+
+
–
+
+
+
a
–
–
–
–
–
+
–
a
–
–
–
a
a
–
+
+
+
+
+
+
+
+
+
+
+
+
–
a
–
–
–
a
+
+
+
+
+
+
+
a
a
+
+
+
–
+
–
+
+
–
+
+
+
+
+
+
+
a
+
+
–
–
–
–
–
–
–
–
+
+
+
+
+
–
a
a
–
+
+
+
+
+
+
–
+
a
+
+
+
+
+
a
+
+
–
+, corresponds to a microsatellite locus-specific amplification; –, corresponds to a non specific or no amplification;
a, corresponds to an amplification, but without detection of microsatellites after hybridization.
Acknowledgements
We wish to thank Dr B. Vosman for helpful discussions on microsatellite production and Dr C. Kaye for English corrections.
References
Karagyozov L, Kalcheva ID, Chapman VM (1993) Construction
of random small-insert genomic libraries highly enriched
for simple sequence repeats. Nucleic Acids Research, 21, 3911–
3912.
Lanaud C (1987) Nouvelles données sur la biologie du cacaoyer
(T. cacao L.): diversité des populations, système d’incompatibilité, haploïdes spontanés. Leurs conséquences sur
l’amélioration génétique de cette espèce. Université de Paris XI,
centre d’Orsay, doctorat d’état.
Laurent V, Risterucci AM, Lanaud C (1993) Genetic diversity in
cocoa revealed by cDNA probes. Theoretical and Applied Genetics,
88, 193 –198.
N’Goran JAK, Laurent V, Risterucci AM, Lanaud C (1994) Comparative genetic diversity studies of Theobroma cacao L. using
RFLP and RAPD markers. Heredity, 73, 589–597.
Rychlik W (1992) OLIGO 4.06, Primer Analysis Software. National
Biosciences Inc. Publishers, Plymouth, USA.
8n00
no
808
1999
10primer
Graphicraft
o supplier
issuenotes
no. ID
Limited,
number
Hong
of article
Kong
Polymorphic microsatellite DNA markers
in the ant Gnamptogenys striatula
T. G I R A U D , * R . B L AT R I X , * M . S O L I G N A C †
and P. J A I S S O N *
*Laboratoire d’éthologie expérimentale et comparée, Université de Villetaneuse,
avenue J. B. Clément, 93 430 Villetaneuse, France, †Laboratoire Populations,
Génétique et Evolution, CNRS, 91198 Gif-sur-Yvette Cedex, France
Keywords: ants, Gnamptogenys striatula, microsatellites
Received 18 June 1999; revision accepted 28 July 1999
Correspondence: T. Giraud. Fax: +33 1 30 83 31 95; E-mail:
[email protected]
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141–2152
Gnamptogenys striatula (Hymenoptera: Formicidae: Ponerinae)
is distributed throughout Central and South America, where
it can be found in open habitats and in humid forests (Lattke
1995). There have been few studies on social structure in this
genus (Prat 1994; Gobin et al. 1998). Colonies of G. striatula
are functionally polygynous (several mated queens reproduce simultaneously), which is uncommon in the subfamily
Ponerinae. Workers mate under some conditions and give
workers (R. Blatrix and P. Jaisson, unpublished). It is necessary to understand the genetic relationships between individuals within a colony to interpret the social structure of
G. striatula and its evolution. Variable genetic markers are
needed to estimate the genetic diversity, structure of population, reproductive strategy, and relatedness between colony
members.
G. striatula DNA was isolated using the QIAamp DNA
Mini Kit (Qiagen) and was digested with Sau3A (Eurogentec).
A partial genomic library was constructed according to Estoup
et al. (1993). Restriction fragments were ligated into the pUC19
vector, and electrotransformed into Escherichia coli DH5-α
strains. Two-thousand clones of the resulting library were
screened with the following oligonucleotide probes: (TC)10
(TG)10 (CAC)5CA, CT(CCT)5, CT(ATCT)6 and (TGTA)4TG.
A total of 100 colonies were positive. Thirty-one positive
colonies were sequenced and all revealed microsatellite loci:
28 dinucleotide repeats, two trinucleotide repeats and one
tetranucleotide repeat.
A group of 24 microsatellites was selected to test the variablity of G. striatula populations from Brasil. PCR primers were
designed from nucleotide sequences flanking the microsatellites, using the computer program OLIGO™ (Macintosh
version 4.0, National Bioscience). Each locus was screened
for variation using a panel of 39 ants: 31 individuals were
sampled in 1999 from different colonies along a 50-km transect
and the eight remaining individuals were collected in the same
region in 1997. PCR amplifications were performed using a
Biometra thermal cycler, with 35 cycles (except for L8, for
which 50 cycles were necessary) of 94 °C for 30 s, 53/55/57/
60 °C (Table 1) for 30 s, and 72 °C for 30 s. Each reaction
(10 µL) contained 1 µL of 10× reaction buffer (50 mm KCl,
Repeat array
in cloned allele
L2
(GA)12A3(GA)3
L3
(CA)10
L4
(AG)14GG(AG)21
L6
(CA)6(TA)22
L7
(CA)2CG(CA)9
L8
(TG)35
L12
(TC)12
L16
(GA)35
L19
(AG)12
L20
(CT)18
Primer sequence (5′– 3′)
F: AGATACAGCGGTCGGTCAGG
R: CGTACGGATGCGATTTCAGC
F: AGCACATCCTTGTTTCCTCTTC
R: TTTCACGCGTGACTTGAACAAT
F: TGCCGTGACGACCCATACCA
R: CGCAAAGCAGAGGAAGAGAA
F: AAGCTTACGACCGAGAAGGA
R: CGACGTGCGCTAACTCTTGG
F: TAGAGCACATCCTTGTTTCCTC
R: TCTTCTTCGAGTGATTTTCA
F: CGTTAACGCGTGTGTGAGG
R: GGGGATAAGAGTGTGAGATGAG
F: AATCCCTTTTCCTCTTTGTTCT
R: TTCTACTTACGCAGACCATA
F: GCTCGTTCGGACAGGATGC
R: TTCCCTCCGTCCATCTCCTG
F: ATTGCCGGGGGAGACATTAT
R: TCCTTACCCCTTTGCCACTC
F: ACATGCGCGAGGGATACATC
R: GAAGGAGAGGGCTCATTCAA
GenBank
Accession no.
Annealing
temp. (°C)
MgCl2
(mm)
Size of PCR
product (bp)
No. of
alleles
AF146882
60
1.5
150
3
AF170285
60
1.5
163
2
AF170286
55
1.5
230
3
35
46
Cb, Et, Er, Pa, Po
AF170287
55
1.5
237
2
32
51
Cb, Et
AF170288
60
1.5
173
2
AF170289
53
2
234
3
13
37
Er, Pa, Cn
AF170290
57
1.25
146
2
32
47
Cb
AF170291
60
2
250
5
48
52
Cb, Et, Er
AF170292
60
1.5
130
4
55
58
Cb, Et, Er, Pa, Po, Cn
AF170293
60
2
220
3
16
15
Cb, Et, Er, Pa, Po, Cn
HO (%)
6
HE (%)
Amplification in
other species*
18
Cb, Et, Er, Pa, Cn
Cb, Er, Pa, Po, Cn
Cb, Er, Cn
*Et, Ectatomma tuberculatum; Er, E. ruidum; Tb, Tetramorium bicarinatum; Cb, Cerapachys biroi; Po, Pachycondyla obscuricornis; Pa, P. apicalis; Ci, Cataglyphis iberica; Cn, Cataglyphis niger.
MEC802.fm Page 2144 Saturday, December 18, 1999 2:05 PM
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141– 2152
Locus
2144 P R I M E R N O T E S
Table 1 Characteristics of microsatellite loci in the ant Gnamptogenys striatula, including repeat array, primer sequences, GenBank Accession no. of the complete cloned sequence,
conditions for amplification, and polymorphism data. The observed (HO) and expected heterozygozity (HE) were calculated for the 31 individuals sampled in 1999, for the loci where
more than one allele was detected in these 31 ants. Successful cross-species amplification is indicated by species abbreviations*
MEC802.fm Page 2145 Saturday, December 18, 1999 2:05 PM
P R I M E R N O T E S 2145
0.1% Triton X-100, 10 mm Tris-HCl, pH 9.0), 75 µm of dCTP,
dGTP, dTTP, 6 µm of dATP, 0.02 µL of 33P-dATP, 0.2 µg/µL
BSA, 1.25/1.5/2 mm MgCl2 (Table 1), 2.5 pmol of each primer,
0.25 U of Taq DNA polymerase (Boehringer Mannheim),
and approximately 10 ng of sample DNA. PCR products
were analysed in 6% polyacrylamide gels and visualized
by autoradiography. Alleles were scored by length in base
pairs.
Only 10 of the 24 microsatellite loci tested in G. striatula
were polymorphic in the 39 individuals tested (Table 1). The
number of alleles per locus was rather small (2– 5), with a
mean of 2.5. The number of alleles seemed to be independent
of the length of the repeat array. There seems to be less polymorphism in G. striatula than in other insect species, perhaps
due to a recent bottleneck or a small effective population
size (Ne). The observed (HO) and expected heterozygosity
(HE) were calculated taking into account only the 31 individuals sampled in 1999 (Table 1). HO was lower than HE
for almost all loci, which may reflect population subdivision,
inbreeding, or the presence of null alleles. The microsatellites
identified in this work should permit study of fine-scale
genetic variation and relatedness in G. striatula.
We examined the ability of primers to amplify appropriately sized products in several other species of ant (35 cycles
of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s; 2 mm
MgCl2). No PCR products were obtained in Anomma nigricans
and Eciton burchelli, but amplification was successful in
several other species. The loci for which a clear amplification
was obtained are indicated in Table 1. The polymorphism in
these species was not tested, but the markers could potentially be useful in several genera of ants. This is especially
valuable as all primers that have previously been published
for ant species have been cloned only in distant genera.
Acknowledgements
We thank Yves Brygoo and Pathologie Végétale group (INRA,
France) for allowing part of the work to be done in their laboratory, Dominique Vautrin for technical assistance, Jacques Delabie
for field and taxonomy expertise, Cyril Astruc for testing the
primers on some ant species, Emmanuelle Baudry for many
invaluable contributions, and Owen Parkes for correcting the
English text. This research was partly funded by the Cellule des
relations internationales de l’Université Paris 13.
References
Estoup A, Solignac M, Harry M, Cornuet J-M (1993) Characterization of (GT)n and (CT)n microsatellites in two insect
species: Apis mellifera and Bombus terrestris. Nucleic Acids Research,
21, 1427–1431.
Gobin B, Peeters C, Billen J (1998) Colony reproduction and
arboreal life in the ponerine ant Gnamptogenys menadensis
(Hymenoptera: Formicidae). Netherlands Journal of Zoology, 48,
53 – 63.
Lattke JE (1995) Revision of the ant genus Gnamptogenys in the
new world (Hymenoptera: Formicidae). Journal of Hymenoptera
Research, 4, 137–193.
Pratt SC (1994) Ecology and behaviour of Gnamptogenys horni
(Formicidae: Ponerinae). Insectes Sociaux, 41, 255–262.
8if raphicraft
no
761
primer
12G
known
supplier
notes
ID
Limited,
number
Hong
of article
Kong
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141–2152
Microsatellites from a teleost, orange
roughy (Hoplostethus atlanticus), and
their potential for determining
population structure
C A T H E R I N E S . O K E , * Y. C H I N G C R O Z I E R , *
R O S S H . C R O Z I E R * and R O B E R T D . WA R D †
*Department of Genetics, La Trobe University, Bundoora, Victoria 3083,
Australia, †CSIRO Marine Research, GPO Box 1538, Hobart, Tasmania 7001,
Australia
Keywords: fisheries population structure, Hoplostethus atlanticus,
microsatellites, orange roughy
Received 21 May 1999; revision accepted 19 June 1999
Correspondence: C. Oke. Fax:+ 61 3-94792480; E-mail:
[email protected]
Orange roughy, Hoplostethus atlanticus, supports commercially important fisheries across its circumglobal distribution
(Kaiola et al. 1993). Within these fisheries, population structure is uncertain, hindering effective management. Genetic
and nongenetic studies have been conducted in Australian
waters. The nongenetic studies [parasites (Lester et al. 1988),
otolith microchemistry (Edmonds et al. 1991) and morphology
(Elliott & Ward 1992) ] have generally suggested multiple
stocks, whereas the genetic studies [allozyme (Elliott & Ward
1992) and mitochondrial DNA (Smolenski et al. 1993) ] have
generally suggested a single Australian stock. The two types
of studies could be reconciled if sufficient gene flow renders
populations homogenous, with most individuals self-recruiting
to natal areas.
Microsatellites offer higher resolving power for fisheries
population genetic studies than previous molecular markers
(Wright & Bentzen 1994). In some fish [northwest Atlantic
cod (Gadus morhua) (Ruzzante et al. 1996) and Arctic charr
(Salvelinus alpinus) (Brunner et al. 1998) ], microsatellites have
revealed population structure not apparent from earlier
allozyme or mitochondrial DNA (mtDNA) analysis. The
10 microsatellite loci characterized here may help to clarify
the currently uncertain situation regarding orange roughy
stock composition.
Locus polymorphism was tested using 87– 458 fish (Table 1)
combined from 1 to 9 separate collections from across their
global range. Six populations were taken from Australia
(1997–1998), two from the North Atlantic (1991, 1998) and
one from Namibia (1998). All samples were collected for the
authors except those from the North Atlantic in 1991, kindly
supplied by Elliott et al. (1994), and stored at either –20 °C
(in ethanol) or –70°C.
Genomic DNA was cut with Sau3A and RsaI and sizeselected fragments (500 – 800 bp) were cloned into BamHI and
Hinc2-digested pUC19 purified with GENECLEAN (BIO101
Inc.). Membrane lifts [Hybond N+(Amersham) ] of the resulting
library were prehybridized in 0.5% sodium dodecyl sulphate
(SDS), 6× sodium chloride/citrate (SSC), 5× Denhardt’s
solution and then hybridized with di-(GT)10 or tetra-(GTGA)8
nucleotide repeat probes (5′ end-labelled with [γ33P]-ATP
using T4 polynucleotide kinase) in 0.1% SDS, 6× SSC, 5×
MEC802.fm Page 2146 Saturday, December 18, 1999 2:05 PM
2146 P R I M E R N O T E S
Table 1 Microsatellite loci in the teleost Orange roughy (Hoplostethus atlanticus Collett). GenBank Accession nos are given under their
appropriate locus
Locus
N (n)
Repeat motif
No. of
alleles
HO*
HE*
Flanking primer sequences (5′–3′)
Size (bp)
Hat2a
AFI46636
Hat3
AFI46637
Hat78b
AFI46638
Hat9a
AFI46639
Hat49
AFI46640
Hat41
AFI46641
Hat4
AFI46642
Hat7
AFI46643
Hat45
AFI46644
Hat19
AFI46645
150 (6)
(GCTC)3(ACTC)4
24
0.807
0.879
166 –244
458 (9)
(GT)12
24
0.721
0.770
174 (2)
(CT)3(GT)10
16
0.85
0.862
183 (2)
(CA)15
18
0.87
0.89
87 (2)
(CA)4(15 bp) (CA)24
16
0.910
0.764
96 (1)
(GT)4(TTT)(GT)5
14
0.743
0.864
177 (2)
(GT)12
14
0.847
0.767
137 (2)
(CT)14 (CA)27
24
0.910
0.93
92 (3)
(GT)29
24
0.978
0.867
192 (2)
(GT)8 (4 bp) (GT)10
16
0.73
0.70
F: GTGTGCAATTTCCTTACCTAC
R: GCAATTTACAGTTGTGCAATTTG
F: GATCCAGAGAAACTGAAAATCTT
R: ACTACAAATACTCCATTCTGATG
F-CCACTATCAGGGTTTTTATCG
R: GCGTGGTAGAGATATGGCAT
F: CAAGCCTGGACAATGTATCT
R: AACACAAACTCTCTAATTCAC
F: GACTGTGAACTCCGACCTC
R: TATGACCATGATTACGCCAAG
F: GTCAGAACGTCATGGCAGG
R: GCCTGTTGATAGTCTTCCTC
F: GCTTAATGGATAATGAGTGGAC
R: TAGGGATGTTATAGTGGTTCTT
F: GTGACTTTGGGGTTGAGGG
R: GCCTTGTAACTCATTCCGCT
F: CTCCTTATCTGCTGCTTTATG
R: CACTACCACTCAACCTCAAC
F: GCTACAATAAAACCTGACTGG
R: CTACCTGGGACAATGGACTT
116 –164
61–149
126 –164
207– 400
156 –184
166 –220
207–267
120 –168
108 –140
*Mean heterozygosity across all individuals calculated according to Nei (1987). N is the total number of individuals screened; n is the
number of populations combined to screen.
Denhardt’s solution. The membranes were washed at room
temperature in 400 mL of 6× SSC twice for 6 min, then at 55 °C
in 600 mL of 6× SSC for 4 min, then at room temperature in
2× SSC for 4 min, before autoradiography. Twenty-four clones
were cycle-sequenced and run on a Perkin-Elmer ABI 377. Ten
primer pairs were designed using Oligo 4.0 (National Biosciences Inc.). No clones with the GTGA repeat were found.
For polymerase chain reaction (PCR) amplification, DNA was
extracted by two separate methods, cetyltri methylammoniumbromide (CTAB) or a modified Chelex extraction method
(Fitzsimmons et al. 1997). For the latter, 800 µL of 5% Chelex
and 5 µL of 14 mg/mL proteinase K were added to 5 mm3 of
crushed muscle tissue and left overnight at 57 °C.
PCR was carried out without oil in a 25-µL reaction made
up of 11.3 –15.3 µL of dH2O, 2.5 µL Taq 10× buffer without
MgCl2 (500 mm KCI, 100 mm Tris-HCl (pH 9.0 at 25 °C), 1.0%
Triton X-100) (Promega), 3 µL of 25 mm MgCl2, 1 µL of 10 mm
dNTP mix, 1 µL of 5 µm reverse primer, 1 µL of 5 µm fluorescent labelled (Hex, Tet or Fam) forward primer, 0.2 µL of Taq
polymerase (5units/mL, Promega) and 1 µL (CTAB DNA) or
5 µL (Chelex) of DNA. PCR was performed in a PerkinElmer 9700 thermocycler with the profile: initial denaturing
for 3 min at 94 °C then 10 cycles of 30 s denaturing at 94 °C;
30 s annealing at 45 °C: 30 s extension at 72 °C followed by 35
cycles of 30 s denaturing at 94 °C: 30 s annealing at 50 °C: 30 s
extension at 72 °C. The size of amplification products was
determined using TAMRA400 internal size standard and the
Genescan 2.0.1™ (Perkin-Elmer/ABI) software.
The loci were highly polymorphic with between 14 and
24 alleles (Table 1). Observed heterozygosities for the pooled
populations ranged from 0.721 (Hat3) to 0.978 (Hat45).
The populations were analysed separately using genepop
(Raymond & Rousset 1995) and the genotypic and genic
differentiation tests and FST values calculated indicated
population differentiation. These preliminary results suggest
structuring between populations which, if confirmed, will
have significance for stock management.
All primers amplified individuals from three other Hoplostethus species (H. lateus, H. intermedius and H. gigas) and
two other fish from the Trachichthyidae family, Darwins roughy
(Gephyroberyx darwinii) and sandpaper fish (Paratachichthys
sp.) (N = 6 per species). There was an indication of variation
within and amongst the three genera.
Acknowledgements
We thank the Australian Fisheries Research and Development
Corporation and the Australian Research Council for financial
support, Peter Grewe for help with automated sequencing,
the Marine and Freshwater Research Institute, Victoria and
Dennis White for help in obtaining the Australian samples, Peter
Smith for help in obtaining the Namibian collections, and Pascal
Lorance of IFREMER France for help in obtaining the 1998 North
Atlantic samples.
References
Brunner PC, Douglas MR, Bernatchez L (1998) Microsatellite and
mitochondrial DNA assessment of population structure and
stocking effects in arctic charr Salvelinus alpinus (teleostei,
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141– 2152
MEC802.fm Page 2147 Saturday, December 18, 1999 2:05 PM
P R I M E R N O T E S 2147
salmonidae) from central alpine lakes Source. Molecular Ecology,
7, 209–223.
Edmonds JS, Caputi N, Morita M (1991) Stock discrimination by
trace element analysis of otoliths of orange roughy (Hoplostethus atlanticus) a deep water marine teleost. Australian
Journal of Marine and Freshwater Research., 42, 383–389.
Elliott NG, Smolenski AJ, Ward RD (1994) Allozyme and mitochondrial DNA variation in orange roughy, Hoplostethus
atlanticus (Teleostei: Trachichthyidae): little differentiation
between Australian and North Atlantic populations. Marine
Biology, 119, 621– 627.
Elliott NG, Ward RD (1992) Enzyme variation in orange roughy,
Hoplostethus atlanticus (Teleostei: Trachichthyidae), from southern Australian and New Zealand waters. Australian Journal of
Marine and Freshwater Research, 43, 1561–1571.
Fitzsimmons NN, Limpus CJ, Norman JA, Goldizen AR, Miller
JD, Moritz C (1997) Philopatry of male marine turtles inferred
from mitochondrial DNA markers. Proceedings of the National
Academy of Sciences of the USA, 94, 8912–8917.
Kaiola PJ, Williams MJ, Stewart PC, Reichelt RE, McNee A,
Grieve C (1993) Australian Fisheries Resources. Bureau of Resource
Sciences, Department of Primary Industries and Energy and
The Fisheries Research and Development Corporation,
Canberra, Australia.
Lester RJG, Sewell KB, Barnes A, Evans K (1988) Stock discrimination of orange roughy, Hoplostethus atlanticus, by parasite analysis. Marine Biology, 99, 137–143.
Nei M (1987) Molecular Evolutionary Genetics. Columbia University Press, New York.
Raymond M, Rousset F (1995) GENEPOP (Version 1.2) — population genetics software for exact tests and ecumenicism. Journal
of Heredity, 86, 248 –249.
Ruzzante DE, Taggart CT, Cook D (1996) A nuclear DNA basis
for shelf- and bank-scale population structure in northwest
Atlantic cod (Gadus morhua): Labrador to Georges Bank. Molecular
Ecology, 7, 1663 –1680.
Smolenski AJ, Ovenden JR, White RWG (1993) Evidence of stock
separation in southern hemisphere orange roughy (Hoplostethus
atlanticus) from restriction-enzyme analysis of mitochondrial
DNA. Marine Biology, 116, 219 – 230.
Wright JM, Bentzen P (1994) Microsatellites: genetic markers for
the future. Reviews in Fish Biology and Fisheries, 4, 384–388.
8n00
no
785
1999
10primer
Graphicraft
o supplier
issuenotes
no. ID
Limited,
number
Hong
of article
Kong
Polymerase chain reaction (PCR)
primers for the amplification of
five nuclear introns in vertebrates
V. L . F R I E S E N , * B . C . C O N G D O N , * †
M . G . K I D D * ‡ and T. P. B I RT *
*Department of Biology, Queen’s University, Kingston, Ontario K7L 3N6,
Canada
Keywords: intron, nuclear genes, PCR, SSCP, vertebrate
Received 1 April 1999; revision received 27 June 1999; accepted 2 July 1999
Correspondence: V. L. Friesen. Fax: + 01-613-533–6617;
E-mail: [email protected]
Present addresses: †School of Tropical Biology, James Cook University,
Cairns, Queensland 4870, Australia; ‡The Alder Institute, PO Box 774,
Station C, St. John’s, Newfoundland A1C 5L4, Canada.
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141–2152
Advancements in evolutionary genetics, as well as the conservation of biodiversity, increasingly require direct analyses
of sequence variation in nuclear DNA. Recent studies indicate that nuclear introns have variabilities useful for both
phylogenetics and population genetics (reviewed in Friesen
2000); however, use of introns is currently limited by a paucity
of polymerase chain reaction (PCR) primers that have been
demonstrated to have broad taxonomic utility (although
several primers with less general or uncertain utilities have
been published; reviewed in Friesen 2000). We have designed
30 general PCR primers for nuclear introns for vertebrates.
Genes for which sequences were available for a variety of
vertebrates and for which sizes and locations of introns were
known for at least one species were extracted from GenBank
and aligned by eye. Primers were designed from sequences
of avian DNA to anneal to conserved sites within exons and
to amplify introns 100 –500 bp in size (the size optimal for
analysis of single-stranded conformational polymorphisms
[SSCPs]; Hayashi 1991) as well as approximately 50 bp of
flanking exon (so that gene homology can be confirmed by
sequencing; Palumbi & Baker 1994). When possible, primers
were designed to enable amplification and sequencing at
high temperatures if required either to improve specificity or
to overcome strong secondary structure. Sequence variation
in 121 marbled murrelets (Brachyramphus marmoratus) was
analysed, and cross-species reactivity of primers was tested
using the protocols for amplification, analysis of SSCPs and
sequencing described in Friesen et al. (1997).
All but 30 of 1200 loci examined were rejected because:
(i) they lacked introns; (ii) sizes or locations of introns were
not reported for any species; (iii) exon sequences were not
available for any birds; (iv) loci occurred in large multigene
families; (v) loci had pseudogenes; (vi) sequences could not
be aligned unambiguously; and/or (vii) conserved priming
sites could not be identified. Primer sequences, PCR protocols
and results of test amplifications for four loci are reported
in Friesen et al. (1997); primers have now been tested for
five more loci (Fig. 1):
(1) Lactate dehydrogenase (LDH) catalyses the interconversion of NADH/pyruvate to NAD+/lactate, and serves as
a lens protein (ε-crystallin) in the vertebrate eye; it is a member of a small multigene family. Primers were designed from
sequences that are conserved across species for LDH-B but
that are variable across analogues, so that only one locus
should amplify.
(2) Myelin proteolipid protein (MPP) creates a hydrophilic
layer on the outside of myelin sheaths, thus enabling close
juxtaposition of neighbouring membranes (Schliess & Stoffel
1991).
(3) Ornithine decarboxylase (OD) catalyses the conversion
of ornithine to putrescine, which functions in the control of
cell growth, development and division (Yao et al. 1995).
(4) Ribosomal protein 40 (RP40) functions primarily in ribosome formation and regulation of ribosome activity, but also
serves as a precursor for a membrane-associated laminin
receptor. The chicken has one gene; mammals have multiple
copies, most of which are probably pseudogenes (Clausse
et al. 1996).
Tropomyosin (TROP) is a myofibrillar protein involved in
MEC802.fm Page 2148 Saturday, December 18, 1999 2:05 PM
2148 P R I M E R N O T E S
Fig. 1 Sequences of PCR primers (for
representative birds) and corresponding
priming sites (for other species) for five
nuclear introns for various vertebrates.
Numbers represent GenBank accession
numbers. Chicken, Gallus gallus; cow, Bos
taurus; dog, Canis familiaris; duck, Anas
platyrhynchus; frog, Xenopus laevis; human,
Homo sapiens; killifish, Fundulus heteroclitus; mouse, Mus musculus; pig, Sus scrofa;
quail, Coturnix coturnix; rabbit, Oryctolagus
cuniculus; rat, Rattus norvegicus; zebrafish,
Danio rerio.
Table 1 Primer locations, results of test amplifications, observed (HO) and expected (HE) heterozygosities and numbers of alleles among
121 marbled murrelets for five nuclear introns
Results of test amplifications*
Locus
Primer locations
murrelet
murre
rhea
finch
snake
rabbit
frog
redfish
HO
HE
No. of alleles
LDH-B
MPP
OD
RP40
TROP
exons 3 & 4
exons 4 & 5
exons 6 & 8
exons 5 & 6
exons 5 & 6
480
390
730
410
1360
525
400
625
450
275
Y
Y
Y
Y
Y
700
400
650
400
450
~
~
400
~
800
275
~
x
150
210
x
~
x
~
~
x
~
x
~
450
0.32
0.40
0.65
0.73
0.69
0.33
0.42
0.65
0.78
0.69
7
4
8
8
7
*rhea, Rhea spp.; murre, Uria aalge; finch, Poephila acuticaudata; snake, Sistrurus catensus; rabbit, Sylvilagus floridanus; frog, Rana pipens;
redfish, Sebastes marinus. Numbers denote approximate sizes of amplification products (including primers); ‘x’ denotes no amplification;
‘~’ denotes multiple bands or smears on agarose gels.
‘Y’ indicates amplifications for rheas produced clean products, but sizes are not available.
regulating contraction and relaxation of muscle fibres; it is
part of a multigene family, and consists of α and β subunits
(Cummins & Perry 1973).
Sequence analyses indicated that amplification products
for murrelets probably represented the target loci: of 29
nucleotide differences between exon sequences of murrelets
and the reference bird, only four involved amino acid differences. Among murrelets, heterozygosities were ~10× higher
than in a complementary study of allozymes (mean for 29
presumptive loci = 0.026, SE = 0.011; Friesen et al. 1997; Table 1).
Substitutions did not differ from expectations of the neutral
theory: most substitutions within exons were silent; substitutions were distributed randomly within introns; transitions
outnumbered transversions; and tests for selection were not
significant (V. L. Friesen & B. C. Congdon unpublished).
Primers yielded a PCR product in all species of birds and
mammals that were tested (Table 1); in other vertebrates,
primers often yielded either multiple bands or a smear, but
should yield a clean band with either refinement of PCR
protocols or use of primers with sequences more specific for
these taxa (Fig. 1).
References
Clausse N, Jackers P, Jares P et al. (1996) Identification of the
active gene coding for the metastasis-associated 37LRP/p40
multifunctional protein. DNA and Cell Biology, 15, 1009–
1023.
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141– 2152
MEC802.fm Page 2149 Saturday, December 18, 1999 2:05 PM
P R I M E R N O T E S 2149
Cummins P, Perry SV (1973) The subunits and biological activity
of polymorphic forms of tropomyosin. Biochemistry Journal, 133,
765 –777.
Friesen VL (2000) Introns. In: Molecular Methods in Ecology (ed.
Baker AJ) Blackwell Science, Oxford, in press.
Friesen VL, Congdon BC, Walsh HE, Birt TP (1997) Intron variation
in marbled murrelets detected using analyses of single-stranded
conformational polymorphisms. Molecular Ecology, 6, 1047–
1058.
Hayashi K (1991) PCR-SSCP: a simple and sensitive method for
detection of mutations in the genomic DNA. PCR Methods and
Applications, 1, 34 – 38.
Palumbi SR, Baker CS (1994) Contrasting population structure
from nuclear intron sequences and mtDNA of humpback
whales. Molecular Biology and Evolution, 11, 426–435.
Schliess F, Stoffel W (1991) Evolution of the myelin integral
membrane proteins of the central nervous system. Biological
Chemistry Hoppe-Seyler, 372, 865 – 874.
Yao J, Zadworny D, Kühnlein U, Hayes JF (1995) Molecular
cloning of a bovine ornithine decarboxylase cDNA and its use
in the detection of restriction fragment length polymorphisms
in Holsteins. Genome, 38, 325 – 331.
8n00
no
809
1999
10primer
Graphicraft
o supplier
issuenotes
no. ID
Limited,
number
Hong
of article
Kong
Isolation and characterization of
microsatellite loci in the freshwater
gastropod, Biomphalaria glabrata, an
intermediate host for Schistosoma
mansoni
C AT H E R I N E S . J O N E S , * A N N E E .
L O C K Y E R , * D AV I D R O L L I N S O N , †
S T U A RT B . P I E RT N E Y * and
LESLIE R. NOBLE*
*Zoology Department, Aberdeen University, Tillydrone Avenue, AB24 2TZ, UK,
†Zoology Department, Natural History Museum, Cromwell Road, London SW7
5BD, UK
Keywords: Biomphalaria glabrata, freshwater snails, genetic markers,
microsatellites
Received 18 June 1999; revision accepted 31 July 1999
Correspondence: C. S. Jones. Fax: +44 (0) 1224 272396; E-mail:
[email protected]
Planorbid snails of the genus Biomphalaria are important
intermediate hosts of medically significant schistosomes.
Attempts to control freshwater snails should be an integral
part of schistosomiasis control programmes but chemical
and biological methods tend to be labour intensive and
costly and require long-term commitment to be successful.
However, susceptibility to the parasite has a strong genetic
component, offering the potential for investigation into host–
parasite interactions at the molecular level, perhaps leading to novel control approaches. Hence, the identification,
mapping and ultimately the molecular characterization of
genes which influence parasite compatibility in intermediate
hosts is fundamental to the control of parasitic diseases, as
well as being of intrinsic interest to evolutionary biologists
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141–2152
and epidemiologists studying coevolution and population
dynamics of host–parasite systems. Our hunt for resistance
genes in Biomphalaria glabrata (Rollinson et al. 1998), with the
aim of advancing current understanding of the evolutionary
processes driving parasite resistance and host speciation,
has prompted a search for molecular markers to anchor preliminary linkage maps. Microsatellite loci are highly polymorphic, single-locus, and codominant and have been advocated
as excellent markers for linkage analysis of quantitative trait
loci (Lymbery 1996).
Here, we report the isolation and characterization of the
first microsatellite loci from the tropical freshwater snail B.
glabrata, which acts as an intermediate host for Schistosoma
mansoni in South America and the Caribbean. This species
is a facultatively self-fertile hermaphrodite, and therefore a
necessary prerequisite of linkage analysis is to confirm that
progeny in F2 mapping populations are the product of outcrossing. Hence there is a need for accurate, unambiguous
genetic markers for the assessment of parentage and estimates
of map distances. These highly polymorphic loci may also
be used to study the population structure and mating system
of this hermaphroditic species.
Total genomic DNA was extracted using a modified
phenol-chloroform extraction procedure following Vernon et al.
(1995) using foot tissue from ethanol-preserved specimens
from two snail strains, resistant 1778 (Belo Horizonte, Brazil)
and susceptible 1742 (Puerto Rico). Size-selected (Rassmann
et al. 1991) partial genomic libraries (300– 800 bp) were constructed by ligating Sau3AI-digested DNA into either λ-Zap
phage (Strategene) or dephosphorylated pUC18 vector
digested with BamHI (Pharmacia). These libraries were
probed with α32P-labelled AG, AC (10 000 transformants
from the first library gave 15 positives; 3400 recombinant colonies from the second library gave 26 positives), TAA, TAAA,
GAAA and CAAA (two positives from the second library
only) oligonucleotide repeats at high stringency. All positives
were sequenced using an ABI 377 automated sequencer
(cycle sequenced using the Big Dye dye-terminator kit according to the manufacturer’s protocols) and primers were designed,
using the software program OLIGO™ Macintosh version 4.1
(National Biosciences Inc., USA), from the unique sequence
flanking the microsatellite repeats found.
Initially, the amplification efficacy of 20 primer pairs from
both microsatellite screening methods was tested against
individuals which constituted the original clone and a few
randomly chosen individuals from the resistant and susceptible strains. Amplified samples were run on 2% 1× TBE
agarose gels at 80 V and stained with ethidium bromide.
Polymerase chain reactions (PCRs) were performed on a
Hybaid PCR Express thermal cycler using the ‘touchdown’
protocol (Don et al. 1991). PCR reaction mixes contained
10 ng of template DNA, 1.5 –2.5 mm MgCl2, 0.2 mm of each
nucleotide, 5 pmoles of each primer, 0.2 units of Taq polymerase (Bioline), and 1× NH4 buffer (16 mm (NH4)2SO4,
67 mm Tris-HCL, pH 8.8, 0.01% Tween-20), in a final reaction
volume of 10 µL.
Nine primer pairs produced bright resolvable products
in all samples and each of these usable primers were then
tested on a further 80 snails from the two strains. In addition,
MEC802.fm Page 2150 Saturday, December 18, 1999 2:05 PM
2150 P R I M E R N O T E S
Table 1 Characterization of six Biomphalaria glabrata microsatellite loci. The repeat structure, primer sequences, allele size range, and
annealing temperatures are given for each locus. Additionally, the number of alleles and the per cent observed heterozygosity (%HO) is
presented for each of the two snail strains, resistant (R) and susceptible (S). The alleles were observed from approximately 40 individuals
from each of two strains (total n = 80)
No. of alleles
Locus*
Repeat
Primer sequences (5′– 3′)
Size range
(bp)†
Bgµ8
(TG)7TT(TG)10
Bgµ10
(CA)11
Bgµ15
(GA)14(G)11
Bgµ16
(TC)24(TATC)6
µBg1
(TC)20
µBg2
(GT)20
F: GCACGAATGTTTGTTGAC
R: CCTATTGATTGAAGTGTTTCC
F: AAACACCCACTCACTCTCC
R: GTTCAATAAGGTCAGGCAAG
F: AGGTTTGTATGTCTTGCTG
R: GGTTCACTCAGATACATCC
F: CTGTTATTCATTATTTCATAGAGC
R: GGGGATCTAACACATCAG
F: TTAATTCTACTGGACTCACATGG
R: CTGCCAATGTTTACATGCTG
F: AGTCTGCTCCAGATTCATTACG
R: GCTTATTTTCACCTCTGAATGC
131–107
(131)
107–93
(95)
182–162
(178)
138–124
(138)
200–160
(186)
280–248
(254)
%HO
T (°C)
both
R
S
both
53
3
1
2
2.5
0
5
55
3
2
2
4.2
3
5
50
3
2
2
20
25
52
3
2
2
57
6
2
5
52
50
54
58
5
2
3
52
32
73
22
8.7
R
2.5
S
16
*Bgµ, locus nomenclature for pUC18/BamHI vector microsatellite isolation method; µBg, locus nomenclature for λ-Zap phagemid vector
microsatellite isolation method.
†Cloned insert size in parentheses; GenBank Accession nos are AF157698–AF157701 and AF157703–AF157704, respectively, of the
sequenced clones from which the primers were designed.
we checked a cross between the two strains (both parents,
10 F1 and 10 F2) to assess inheritance of markers. These amplified products were resolved either on 4% 1× TBE, MetaPhor
(FMC) agarose gels, run at 80 V on long gels (20 × 30 cm)
for 8 h and ethidium bromide stained or 8% denaturing
polyacrylamide gels stained with a Silver Sequence kit
(Promega). Product sizes were determined by comparison
with an M13mp8 DNA sequence standard produced by
cycle sequencing from the Silver Sequence kit on polyacrylamide gels or to a 20-bp ladder (Advanced Biotechnologies)
and to known products on agarose gels. Promega’s protocol
recommends the use of 6% gels as higher polyacrylamide
concentrations cause the gel to crack and become unscoreable.
However, superior resolution (differences of 2 bp) was
obtained with 8% gels in which cracking was circumvented
by the addition of a final step of soaking the stained gel in
3% glycerol solution for 1 h prior to air drying.
Three loci were fixed for alternative alleles in the resistant
and susceptible strains and are not described further. Results
of optimization and screening of six polymorphic loci in the
two B. glabrata strains are given in Table 1. Analysis of the
banding patterns within the family crosses indicate that all
loci segregate according to Mendelian expectations, with no
evidence of null alleles. The number of alleles at each locus
ranged from 3 to 6 and observed heterozygosity ranged from
2.5% at locus Bgµ8 to 52% for locus µBg2, with the susceptible strain exhibiting the highest heterozygosities across
loci. These primers should prove suitable for linkage analyses
utilizing crosses of inbred laboratory strains and in elucidating
levels of population structuring in wild-caught samples.
Furthermore, we show that presumably orthologous
microsatellite loci can be successfully amplified in related
species of Biomphalaria (Table 2). Although sample sizes for the
neotropical species tested were small (n = 3) the African
species B. pfeifferi proved more polymorphic than the Neotropical species B. straminea, B. occidentalis and B. tenagophila,
with the latter amplifying with the least number of loci. The
results ranged from four out of six variable loci amplified
from B. pfeifferi to only three yielding scoreable PCR products in B. tenagophila, none of which was polymorphic. The
proportion of amplifiable loci in each species is consistent with the phylogeny suggested by Woodruff & Mulvey
(1997). Based on 20 allozyme loci, they deduced that South
American B. glabrata had closer affinities to African than to
other neotropical species, with B. tenagophila being most distantly related. Further, their work suggests that the African
species are younger than their neotropical congeners, with
the B. pfeifferi–protoglabrata lineage conservatively estimated
to have evolved in Africa from neotropical founders 2.3– 4.5
million years ago as a result of earlier transatlantic dispersal
from the Americas. Consequently, the markers described
here for B. glabrata may be more useful for examining population structure of other African than neotropical Biomphalaria
species.
Acknowledgements
This work was supported by the Wellcome Trust (project grant
042687/Z/94/Z to L.R.N. and a Biodiversity Fellowship to
C.S.J.), BBSRC (Advanced Fellowship to C.S.J., grant number 1/
AF09056) and NERC (S.B.P.). We would also like to thank Alison
Perry, Sarah Hughes and Mike Anderson for technical assistance
and Dr Cecelia Pereira de Souza (Centro de Pesquisas ‘René
Rachou’, Belo Horizonte, Brazil) for providing the snails.
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141– 2152
MEC802.fm Page 2151 Saturday, December 18, 1999 2:05 PM
P R I M E R N O T E S 2151
Table 2 Amplifications across species within the genus Biomphalaria
Locus
Species
N
µBg1
µBg2
Bgµ8
Bgµ10
Bgµ15
Bgµ16
B. pfeifferi
13
*p
(4)
*p
(2)
*
*
*p
(2)
*
*
*
—
*
*p
(5)
*
*p
(3)
*
*
—
—
—
*
—
—
*
*
*
B. straminea
3
B. occidentalis
B. tenagophila
3
3
*, One or two clear bands; *p, polymorphic, where polymorphism is detected the minimum number of alleles is given in parentheses;
—, multiple bands, smear or no product; N, sample size. Phylogenetic distance from B. glabrata moving down the first column in the
table.
References
Don RH, Cox PT, Wainright BT, Baker K, Mattick JS (1991)
‘Touchdown’ PCR to circumvent spurious priming during
gene amplification. Nucleic Acids Research, 19, 4008.
Lymbery AJ (1996) Finding genetic markers for complex phenotypic traits in parasites. International Journal of Parasitology, 26,
7–17.
Rassmann K, Schlotterer C, Tautz D (1991) Isolation of simple
sequence loci for use in polymerase chain reaction-based DNA
fingerprinting. Electrophoresis, 12, 113–118.
Rollinson D, Stothard JR, Jones CS et al. (1998) Molecular characterization of intermediate snail hosts and the search for
resistance genes. Memorias Do Instituto Oswaldo Cruz, 93, 111–116.
Vernon JG, Jones CS, Noble LR (1995) Random amplified polymorphic DNA (RAPD) markers reveal cross-fertilisation in
Biomphalaria glabrata from wild populations. Journal of Molluscan
Studies, 61, 455 – 465.
Woodruff DS, Mulvey M (1997) Neotropical schistosomiasis:
African affinities of the host snail Biomphalaria glabrata
(Gastropoda: Planorbidae). Biological Journal of the Linnean Society,
60, 505 – 516.
8n00
no
803
1999
10primer
Graphicraft
o supplier
issuenotes
no. ID
Limited,
number
Hong
of article
Kong
Isolation and characterization of
microsatellite loci in the European plaice,
Pleuronectes platessa L. (Teleostei:
Pleuronectidae)
P. C . WAT T S , * R . D . M . N A S H , †
S . G . G E O R G E ‡ and S . J . K E M P *
*Laboratory 1.03, Donnan Laboratories, Crown Street, School of Biological
Sciences, University of Liverpool, Liverpool, L69 7ZD, UK, †Port Erin Marine
Laboratory, School of Biological Sciences, University of Liverpool, Port Erin, Isle
of Man, IM9 6JA, ‡Institute of Aquaculture, University of Stirling, Stirling,
FK9 4LA, UK
Keywords: European plaice, glutathione S-transferase, microsatellite,
Pleuronectes platessa
Received 19 May 1999; revision accepted 13 July 1999
Correspondence: P. C. Watts. Fax: + 44-(0)-151-794-3655; E-mail:
[email protected]
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141–2152
European plaice, Pleuronectes platessa (L.), is a commercially
important flatfish that inhabits shelf waters of the Northeastern
Atlantic. It is particularly common in European seas, where
it is a major constituent of the demersal fisheries. Plaice
annually migrate and have specific spawning grounds within
regional seas, although the amount of interbreeding among
fish from different areas is not known. Morphometric
and biological characteristics suggest that this species comprises
several stocks. Knowledge of the movements of different
stocks and their breeding habits is essential for the management of exploited fish species. Molecular markers offer a
method of determining migration and stock structure of
marine fish (Carvalho & Pitcher 1995). Due to their high levels
of polymorphism, microsatellites are especially likely to
prove useful for stock discrimination, but no microsatellite
loci have yet been described for P. platessa.
DNA was isolated from the muscle tissue of a single plaice
and approximately 10 µg was digested with SauIIIA. The 400–
900 bp size fraction was recovered and cloned into dephosphorylated pUC18 (Boehringer Mannheim) digested with BamHI.
Ligation products were subsequently transformed into competent cells (Stratagene). Approximately 500 recombinant clones
were fixed to Hybond-N membranes (Amersham) and screened
for microsatellites using a (CA)10 probe. Hybridizations were
undertaken using a nonradioactive digoxigenin (Boehringer
Mannheim) protocol (Estoup & Turgeon 1996). Nineteen
positive clones were isolated, of which 12 were sequenced
using an ABI 377 sequencer. Primers were designed on the basis
of sequences flanking the repeat regions using a computer
programme (S. J. Kemp, unpublished results) for six loci, of
which four amplified consistently and showed products
within the expected size range (Table 1).
Several dinucleotide repeat regions are also present in a
contiguous sequence of approximately 14 kb, which contains
three glutathione S-transferase genes in P. platessa (Leaver
et al. 1997) (GenBank Accession no. X95199). Primers were
designed for five of these microsatellite loci, of which three
proved to be polymorphic (Table 1).
DNA was extracted from the muscle tissue of 14 individuals caught from Port Erin Bay, Isle of Man using conventional methods (Sambrook et al. 1989) and resuspended
in TE (pH 8.0). Primers were labelled through incubation
MEC802.fm Page 2152 Saturday, December 18, 1999 2:05 PM
2152 P R I M E R N O T E S
Table 1 Characterization and estimates of variability at seven microsatellite loci for 14 plaice collected from Port Erin Bay, Isle of Man.
Accession numbers of the cloned sequences deposited in GenBank are: LIST1001–AF149831; LIST1002–AF149830; LIST1003 –AF149829;
LIST1004 –AF149828. The base locations of the primer and microsatellite sequences within the glutathione S-transferase gene cluster
(GenBank X95199) are: Pplgst1–2769..2925; Pplgst2–3109..3217; Pplgst4–6623..6800
Locus
Primer sequences (5′(r)3′)
Repeat array
Ta
LIST1001
AATCCAAAAGCAGGGGTCC
GGTTGTAGTTATACTCAGGC
CTTTTCATCACCTGTTCCG
CTATTCTCTCAATGCCTGG
AGAGCTATTGTGGTTCCACC
CATGTCCTGAGATTCACTGC
GATTAACTATGGGCAAGTGC
TCTAGAGGATCCCTTTCCC
TCAGTTTAAGTCTCAGGGCC
CACGTTTAGAGTGTTGGTGC
CAGCGCAAACAGACACATGG
AGACGATCACATCAGCCAGC
AACTCAAACTCTGGGAGG
AAAACGGTCACATCAGCC
(AC)9(CA)
50
(CA)11(A)2(CA)6(C)CA
LIST1002
LIST1003
LIST1004
Pplgst1
Pplgst2
Pplgst4
bp
NA
HO
HE
MgCl2
87–97
6
0.58
0.60
1.5
50
187–189
2
0.14
0.14
1.5
(CTT)(TTT)(CTT)(TCT)(CTT)4(CTG)
50
156–175
4
0.25
0.27
1.5
(CA)(TA)(CA)82(A)4
49.5
113–165
8
0.82
0.38
2.0
(AC)3(TC)(AG)(AC)6
50
155–157
2
0.33
0.40
1.5
(AC)6
50
124–132
3
0.39
0.46
1.5
(CA)(A)(CA)2(A)2(CA)14
45
178–196
8
0.81
0.71
1.5
Ta, primer annealing temperature (°C); bp, size range of alleles; NA, number of alleles; HO, observed heterozygosity;
HE, expected heterozygosity; MgCl2, MgCl2 concentration for PCR (mm).
with PNK and [γ 33P]-ATP for 45–55 min at 37 °C. PCR amplification was undertaken in a 5-µL volume using a PTC-10096V MJ thermal cycler (MJ Research Inc.) and Reddy-Load
PCR Mix (Advanced Biotechnologies). Each reaction contained 20 – 50 ng of template DNA, 75 mm Tris-HCl, 20 mm
(NH4)2SO4, 0.01% (v/v) Tween 20, 0.2 mm of each dNTP,
1.5 –2.0 mm MgCl2, 0.66 pmol of each primer and 0.125 units
of Taq polymerase (Advanced Biotechnologies). PCR conditions were: (i) an initial denaturation for 1 min at 95 °C;
(ii) six cycles of denaturation for 30 s at 95 °C, 30 s at the
specified annealing temperature (Table 1) and extension for
45 s at 72 °C; (iii) a subsequent 26 cycles of 30 s denaturation
at 92 °C, 30 s of primer annealing and 55 s at 72 °C; and
(iv) a final extension at 72 °C of 30 min.
Microsatellite variability was determined by electrophoresis
on a 6% denaturing polyacrylamide gel (Severn Biotech Ltd).
Vertical gels were run at 90 W for 3.5 h, fixed, dried for 45 min
and then exposed to a phosphor screen (Molecular Dynamics
Inc.) overnight; the phosphor screens were scanned using
a Storm imaging system (Molecular Dynamics Inc.). All
alleles were run alongside an M13 sequencing ladder.
Similar to those of other coldwater marine teleosts, many
of the microsatellite arrays identified here comprise imperfect repeats (Table 1); the reasons for this phenomenon are
still unclear (but see Brooker et al. 1994). The number of
alleles varied between two and eight and the observed heterozygosities ranged between 0.14 and 0.82 (Table 1). Given that
the fish used were taken from a relatively limited part of this
species’ geographical range, these microsatellite loci will
probably prove very useful for studying gene flow and stock
dynamics. The glutathione S-transferases from which these
sequences are derived are thought to be primarily involved
in the detoxification of oxidation products of essential polyunsaturated fatty acids (Leaver & George 1998). However,
this superfamily of enzymes also detoxifies drugs, pollutants
and pesticides. The microsatellite loci described here may
therefore act as valuable genetic markers with which to investigate tolerance to pollution in plaice. It must be remembered
that the Pplgst microsatellites described here are linked
and thus their independence cannot be assumed when used
for population studies.
Acknowledgements
We are grateful to Dr Mark Hughes and Faye Barker for their
input and Dr Harry Noyes for sequencing. The University of
Liverpool supported this work through RDF Grant no. 2531.
References
Brooker AL, Cook D, Bentzen P, Wright JM, Doyle RW (1994)
Organisation of microsatellites differs between mammals
and cold-water teleost fishes. Canadian Journal of Fisheries and
Aquatic Sciences, 51, 1959–1966.
Carvalho G, Pitcher TJ (1995) Molecular Genetics in Fisheries.
Chapman & Hall, London.
Estoup A, Turgeon J (1996) Microsatellite markers: isolation
with non-radioactive probes and amplification. http://
www.inapg.inra.fr/dsa/microsat/microsat.htm.
Leaver MJ, George SG (1998) A piscine glutathione S-transferase
which effectively conjugates the end-products of lipid peroxidation. Marine Environmental Research, 46, 71–74.
Leaver MJ, Wright J, George SG (1997) Structure and expression
of a cluster of glutathione S-transferase genes from a marine
fish, the plaice (Pleuronectes platessa). Biochemical Journal, 321,
405–412.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning:
a Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory
Press, New York.
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 2141– 2152