Development and characterization of 11 microsatellite loci in
a historically introduced carnivoran, the common genet
(Genetta genetta)
P. G A U B E R T ,*‡ I . D E L C E R R O ,*§ F. PA L O M A R E S * and J . A . G O D O Y †
*Departamento de Biología de la Conservación, †Departamento de Ecología Integrativa, Estación Biológica de Doñana, CSIC, Pabellón
del Perú, Avda María Luisa s/n, 41013 Sevilla, Spain, ‡UR IRD 131 — UMS MNHN 403, Département Milieux et Peuplements
Aquatiques, Muséum National d’Histoire Naturelle, 43 rue Cuvier, 75005 Paris, France, §Genética de la Conservación Animal, IRTA,
Ctra de Cabrils, s/n 08348 Cabrils, Barcelona, Spain
Abstract
Microsatellite markers were developed to assess population structure and patterns of translocation in the introduced European common genet (Genetta genetta). Primer pairs were
designed for 60 microsatellite sequences enriched for CA, GA, CATC and TAGA repeat
motifs. Eleven loci that proved to be polymorphic were genotyped in 33 individuals from
southwestern France. The number of alleles per locus and observed heterozygosities varied
from three to seven and from 0.2121 to 0.7576, respectively. One locus (B103) showed significant departure from Hardy–Weinberg equilibrium, probably due to the presence of null
alleles. Tests of linkage disequilibrium did not detect significant associations among loci.
Keywords: Carnivora, Europe, Genetta genetta, introduced species, microsatellite, Viverridae
Received 13 January 2008; revision accepted 7 April 2008
Genets (Genetta spp.) are nocturnal, solitary, medium-sized
predators representing the most speciose carnivoran genus
in sub-Saharan Africa (Gaubert et al. 2005). One species, the
common genet Genetta genetta (Linnaeus, 1756), also occurs
naturally in Maghreb and in the Arabian Peninsula. It was
probably introduced from North Africa into southwestern
Europe during historical times (Delibes & Gaubert in press),
representing one rare example of successful, historical
translocation of a wild African mammal into Europe
(Gaubert et al. 2008). Thus, the study of the genetic structure
and historical demography among its populations may
shed light on the population dynamics and short-term
evolution at disequilibrium of invading meso-predators.
We report herein the 11 first microsatellite markers
developed for G. genetta, and assess their level of
polymorphism within a French population.
Microsatellite isolation was performed by Genetic Identification Services (http://www.genetic-id-services.com)
following the magnetic bead-based enrichment procedure
of Jones et al. (2000, 2002). Genomic DNA was extracted
Correspondence: Philippe Gaubert, Fax: (33)1 40 79 37 71;
E-mail: gaubert@mnhn.fr
from a muscle sample of a single individual, and was
partially restricted with a cocktail of seven blunt-end cutting enzymes (RsaI, HaeIII, Bsr B1, PvuII, StuI, ScaI, and
EcoRV). Libraries were prepared in parallel using BiotinCA(15), Biotin-GA(15), Biotin-CATC(8), and Biotin-TAGA(8)
(Integrated DNA Technologies) as capture molecules
(libraries A, B, C and D, respectively). A total of 100 positive
plasmid clones containing inserts 350–700-bp long were
sequenced, of which 84 contained a microsatellite (CA, 24/26;
GA, 26/28; TAGA, 21/25; CATC, 13/16).
Polymerase chain reaction (PCR} primers were designed
for 60 microsatellite-containing clones using DesignerPCR
version 1.03 (Research Genetics), and PCR was optimized
for 26 microsatellites from all four libraries, selecting primers
to span different product size ranges. Amplification products
were initially labelled fluorescently through an M13–5′-tailed
forward primer approach (Boutin-Ganache et al. 2001).
These markers were first evaluated for amplification in
three tissue samples using ‘touchdown-PCR’. Twenty-four
markers rendering clean and efficient amplification of the
expected product were evaluated for polymorphism in a
set of 11 samples of diverse origin (five from North Africa,
four from Spain, one from France and one from Italy). At
Table 1 Characterization of 11 microsatellite loci in Genetta genetta among 33 individuals from southwestern France. The letter preceding
each locus name refers to the library from which it was selected (see text)
Locus Repeated motif
GenBank
Accession no. Primer sequences (5′–3′)
A5
(GT)16
EU362975
A104 (CA)23
EU362976
A108 (CA)16(TGCACACGCACGCG) EU362977
(CA)12
A110 (AC)22
EU362978
A112 (GT)21
EU362979
B103* (GA)26
EU362980
B104 (AG)20
EU362981
B105 (GA)18
EU362982
C101 (ATGG)12
EU362983
D4
(TATC)15
EU362984
D111
(TAGA)14
EU362985
F: GAACTCGGGGCTTAGATGTC
R: CTGGAAAGATGAGGGGACTT
F: TGAAAGAATTGCTTGGTATGG
R: GCATGGTTGGTGAACATTC
F: TGCATTACAATCACTCACTCTC
R: TAGGTGGAAATCAATCTGTTG
F: TCGTGCTGACGTGTTTAGC
R: TTTGCCTTCCACAAAGAGG
F: CCAACTGCCTCTGTGACTC
R: CCAAAACCTATCCGAGAATG
F: CTTTCTTGCTGTGTCCTCACA
R: GGGTATAGGGCCTTTAAGGAG
F: ATCTGCTACTGGCAAGTCAAC
R: GCCTGTTTCAGTTTCTGTGTC
F: CGTGTATGTGTGTGGTGTGTG
R: CCCCTACCTTCTTCATCCAAC
F: TCCCACAGAAGGAACAGTC
R: GCTTGTCCCATCAGAGTGT
F: TTGGAGAGGATTTCACTGAC
R: TAGGCTTAGGAGATTTAGCAAG
F: TGCTTTTTCTTTAATCCCTCTC
R: TATCCTCAGCAGTCCTCAGAG
Ta MgCl2 Size range
Na HO
Label (°C) (mm) (bp)
HE
FIS
FAM 54
2
127–131
3
0.6061 0.6318 0.056
VIC
53
1.5
117–133
4
0.6970 0.5914 –0.164
NED 54
1.5
127–137
5
0.3333 0.5376 0.393
NED 54
2
256–274
5
0.4242 0.4766 0.125
PET
54
2
289–311
4
0.5455 0.5083 –0.058
VIC
60
1
178–190
4
0.2121 0.6331 0.673
VIC
54
1.5
247–261
5
0.5455 0.5106 –0.053
PET
54
2
169–173
2
0.3030 0.2571 –0.164
FAM 54
2
264–276
4
0.3030 0.4187 0.290
PET
2
209–249
7
0.6970 0.7856 0.128
1.8
185–201
5
0.7576 0.7273 –0.026
54
NED 54
F, forward; R, reverse. Label, fluorescent dye used. Ta, annealing temperature. Na, number of alleles. HO, observed heterozygosity. HE,
expected heterozygosity. FIS, inbreeding coefficient.
*Locus showing departure from Hardy–Weinberg equilibrium (P < 0.05).
this stage, we were able to obtain a first estimate of the
numbers of alleles and allelic size range for each marker.
Twelve markers that showed easily scorable amplification
products and high variability were grouped in four
nonoverlapping sets that were distributed among the
four possible fluorescent labels, so that they could be
simultaneously analysed in a single run of capillary
electrophoresis.
Assessment of polymorphism within 33 individuals
from southwestern France was conducted using 5′-fluorescently labelled forward primers and 5′-GTTTCTT-tailed
reverse primers (Brownstein et al. 1996). Amplification
reactions were performed in 20 μL final volumes consisting
of: reaction Buffer 1× (Bioline), 1–2 mm MgCl2 (see Table 1),
0.25 mm dNTPs, 0.25 μm primers, 0.1 mg/mL BSA,
0.025 U/μL Taq DNA polymerase, and c. 50 ng of template
DNA. Temperature cycling was performed on MJ Research
PTC-100 thermocyclers using the following conditions:
initial denaturation of 94 °C for 2 min, followed by 40
cycles of 92 °C (30 s), 53–60 °C (see Table 1) (30 s), and
72 °C (30 s), and terminated with a final extension at 72 °C
for 5 min. Products were analysed on an ABI 3100 DNA
sequencer and alleles were scored with GeneMapper 4.0
(Applied Biosystems).
The genetic diversity of the microsatellite loci was
quantified by the number of alleles per locus, observed
heterozygosity (HO), and expected heterozygosity (HE) using
Genetix version 4.05 (Belkhir et al. 1996–2004). Departures
from Hardy–Weinberg equilibrium and linkage disequilibrium for each pair of loci were calculated using permutation
tests with fstat version 2.9.3. (Goudet 1995). P values were
adjusted for multiple test of significance using the sequential
Bonferroni correction at the 5% nominal level (Rice
1989). One locus proved monomorphic for southwestern
France — but was polymorphic at a larger geographical
scale — and is thus not further discussed here [A113:
(GT)5(ATAT)(GT)4(GA)(GT)18); F: 5′-TAAAGGGGTGTGTGTGTATATG-3′ — R: 5′-CCCTATAACACTCTGGCTATTG-3′;
annealing temperature: 54 °C; MgCl2: 2 mm; GenBank
Accession no. EU362986].
We were able to detect three to seven alleles per locus
(average = 4.36). Observed and expected heterozygosities
varied from 0.2121 to 0.7576 and from 0.2571 to 0.7856,
respectively (Table 1). One locus (B103) showed significant
departure from Hardy–Weinberg equilibrium (P = 0.0008)
probably due to presence of null alleles. Tests of linkage
disequilibrium revealed no significant associations among
all pairs of loci.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
We have identified a set of 11 microsatellites that will be
useful for studying population structure, gene flow (natural
or following translocations) and historical demography
among populations of G. genetta introduced into Europe.
An additional locus (A113) will be useful in broader-scale
examinations across the entire Mediterranean Basin. This is
an important step towards our understanding of the translocation patterns at the origin of the European populations
of common genets (Gaubert et al. in press).
Acknowledgements
We thank Mark Todd and Ken Jones at Genetic Identification
Services (Chatsworth, CA) for building the microsatellite library
and for fruitful comments on the manuscript. We are grateful to
Ana Piriz and Laura Soriano for laboratory work assistance at the
Laboratorio de Ecologia Molecular. Pascal and Christine Fournier
(Groupe de Recherche et d’Etude pour la Gestion de l’Environnement, Villandraut, France) supplied samples from southwestern
France (other field collectors are thanked in Gaubert et al. in press).
An anonymous reviewer provided useful comments on the early
version of the manuscript. This work was funded by the European
Commission 6th PCRDT ‘EPISARS’ (FP6-2003-SSP-2-SARS;
no. 51163).
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