Isolation and characterization of 11 tetranucleotide
microsatellite loci in the Egyptian mongoose
(Herpestes ichneumon)
M Ó N I C A R O D R I G U E S ,*§ C A R L O S A . F E R N A N D E S ,*†§ F R A N C I S C O PA L O M A R E S ,‡ I S A B E L R .
A M O R I M ,¶¥ M I C H A E L W. B R U F O R D † and M A R G A R I D A S A N T O S - R E I S *
*Centro de Biologia Ambiental, Universidade de Lisboa, Campo Grande Edifício C2 5° Piso, 1749-016 Lisboa, Portugal,
†Cardiff University, Museum Avenue, Cardiff CF10 3US, UK, ‡Departamento de Biología de la Conservación, Estación Biológica de
Doñana-CSIC, Avenida de Maria Luisa s/n, Pabellón del Perú, E-41013 Seville, Spain, ¶Departamento de Ciências Agrárias-CITAA,
Universidade dos Açores, 9700-851 Angra do Heroísmo, Portugal, ¥Grupo Lobo, 1749-016 Lisboa, Portugal
Correspondence: Carlos A. Fernandes, Fax: 00351-217500028; E-mail: CaFernandes@fc.ul.pt
§Mónica Rodrigues and Carlos A. hernandes contributed equally to this work.
Abstract
We report the isolation of 11 polymorphic tetranucleotide microsatellite loci in the Egyptian
mongoose (Herpestes ichneumon). In a sample of 27 individuals, we observed between 4
and 7 alleles per locus and their observed and expected heterozygosities ranged from 0.37
to 0.85 and from 0.44 to 0.79, respectively. All genotypic frequencies conformed to Hardy–
Weinberg equilibrium expectations and there were no instances of linkage disequilibrium
detected between pairs of loci.
Keywords: Herpestes ichneumon, Herpestidae, microsatellites, mongoose, tetranucleotide
Received 6 October 2008; accepted 26 January 2009
The Egyptian mongoose, Herpestes ichneumon (Linnaeus,
1758), is a mammalian carnivore of the family Herpestidae.
Although essentially African, ranging widely across the
continent with the exception of extreme deserts and humid
forests, it has spread from northeast Egypt into the Mediterranean Middle East, up to southern Turkey (Kingdon
1997; Palomares in press). The species is also found in the
southwest of the Iberian Peninsula (Borralho et al. 1996;
Palomares in press), where it is generally considered
the result of introduction(s) by Arabs from North Africa
(Riquelme-Cantal et al. 2008). The introduction of nonnative species has long been a topic of interest in evolutionary research and an issue of concern in biodiversity
conservation (Ficetola et al. 2008; Suarez & Tsutsui 2008).
Most invasive species show a substantial reduction of
genetic diversity in introduced populations due to
demographic bottlenecks associated with the founding
event(s), and this loss is particularly marked at discrete
molecular markers (Dlugosch & Parker 2008). Because the
mitochondrial DNA variation of the Egyptian mongoose in
the Iberian Peninsula seems to be rather low (Fernandes
et al. 2008; C. Fernandes, unpublished), only highly variable
markers such as microsatellite loci might be powerful
enough to allow a more detailed reconstruction of the
invasion history (e.g. Thulin et al. 2006). Moreover, they open
the way for population genetics studies within and between
introduced and native populations (Colautti et al. 2005).
To identify polymorphic microsatellites for genetic
investigations of H. ichneumon, we constructed an enriched
genomic DNA library following the protocol of Glenn &
Schable (2005) with minor modifications. Genomic DNA
for library construction was extracted from an ear punch
of an individual captured at the CBA Field Station in
Grândola, Baixo Alentejo (southern Portugal) and stored in
a saturated NaCl solution (6 m) containing 25% dimethyl
sulfoxide. DNA was extracted using the Puregene Tissue
Kit (Gentra Systems) and approximately 2 μg of the genomic
DNA were digested with RsaI (Promega) and ligated to
double-stranded SuperSNX24 linkers (forward: 5′-GTTTAAGGCCTAGCTAGCAGAATC-3′; reverse: 5′-pGATTCTGCTAGCTAGGCCTTAAACAAAA-3′). Fragments
were size-separated on a 1% agarose/TAE gel and the ones
in a range of 500–1500 base pairs were excised and purified
with the GFX PCR and Gel Band Purification kit (Amersham). To ensure ligation was successful, we carried out a
polymerase chain reaction (PCR), using SuperSNX24
Forward as primer, on the linker ligation as described
by Glenn & Schable (2005). For enrichment, the products of
this PCR, and not the original linker-ligated DNA, were
hybridized with 3′-end biotinylated tetranucleotide
probes (AAAG)6, (AGAT)6, and (GATA)6. We chose to isolate
tetranucleotide microsatellite loci because they can be
scored less ambiguously and are less likely to suffer from
slippage-generated scoring errors than dinucleotide
repeats. Successfully hybridized fragments were captured
on streptavidin-coated magnetic beads, Dynabeads M-280
(Invitrogen), washed, recovered from the beads, purified,
and eluted as in Glenn & Schable (2005). To increase the
amount of enriched DNA, PCR amplification, using
SuperSNX24 Forward as primer, of the eluted fragments
was performed and the resulting PCR products were used
for a second round of enrichment. The PCR products from
this second enrichment were ligated into pCR 2.1-TOPO
vector (Invitrogen) and used to transform One Shot TOP10
chemically competent Escherichia coli cells (Invitrogen). The
cells were cultured on selective agar media, with ampicillin,
X-Gal, and IPTG, for blue/white colony selection using the
β-galactosidase gene. Recombinant colonies (white) were
screened for microsatellite containing-inserts using a threeprimer PCR protocol as in Gardner et al. (1999). The PCR
amplifications were performed with two vector primers
(M13 forward (–20): 5′-GTAAAACGACGGCCAG-3′; M13
reverse: 5′-CAGGAAACAGCTATGAC-3′) plus the respective
probe used in the enrichment. Clones giving two (or more)
bands, after 2% agarose electrophoresis, were considered
likely to contain a microsatellite and were amplified with
the two vector primers. Products of this PCR were purified
using the GFX PCR and Gel Band Purification kit, and
sequenced at Macrogen Inc.Sequences were edited and
assembled using Sequencher 4.7 (Gene Codes).
A total of 90 positive clones were sequenced and 69
contained microsatellite repeats. Primers for PCR were
designed, from the flanking sequences of unique microsatellites, to amplify the repetitive element of 20 loci. Eighteen
Table 1 Microsatellite loci in Herpestes ichneumon including locus name, primer sequences, repeat in the cloned allele, number of alleles (Na),
allele size range (base pairs), expected heterozygosity (HE), observed heterozygosity (HO), and polymorphic information content (PIC).
Cloned sequences have been deposited with GenBank under Accession nos FJ357430–FJ357441
Locus ID
Primer sequences (5′–3′)
Repeat
Na
Size range
HE
HO
PIC
Hic 1.95
F: CGGGTAAATGCATATAGC
R: GCGTTCCTTTTACAGCAT
F: AAGGTCTCATCCAGGACT
R: CGCTCTTTGATCAACTGA
F: AAACCTGCTTGGGATTCG
R: ACTGTCTCGGTGTTAGTC
F: CCGTTTAGAATACTGCCT
R: CCTATTTACACTCAACTG
F: GGCTTCTTCTTTCATAGCT
R: GGGTAAAGAAAATGTGAC
F: TTAAGCCCCAATCCTGAC
R: TTTGGATTCCAGATAGGC
F: GACACACTTGTAATCCTG
R: CCGGGTTAAGTTTAAGCA
F: CGCCATACTTACTCTAGT
R: CGACTGATTATGGCGCTA
F: GAAATTGAGGCTTCCATG
R: AGAGTGTGGAAACAGGAC
F: TTGGAATCAGGCAAGTCT
R: GCATCTCCTATATACCAG
F: TCCTCATCTCTCAAGTCT
R: AGAACAGTCTTCCTACCG
(TAGA)11
4
141–153
0.63
0.70
0.57
(TATC)10
5
213–229
0.68
0.52
0.62
(TAGA)17
7
174–198
0.79
0.85
0.75
(TCTA)12
4
187–215
0.73
0.56
0.66
(TATC)11(AAAC) (TATC)2
7
159–183
0.79
0.82
0.74
(CTAT)12
4
163–175
0.70
0.63
0.63
(TAGA)13
5
192–220
0.65
0.63
0.60
(TAGA)12
4
216–232
0.53
0.37
0.47
(TCTA)14
6
249–269
0.73
0.63
0.68
(TCTA)10
4
248–260
0.44
0.41
0.39
(CTAT)7
4
141–173
0.64
0.48
0.56
Hic 2.30
Hic 2.52
Hic 3.16
Hic 3.19
Hic 3.20
Hic 3.22
Hic 4.27
Hic 4.30
Hic 4.47
Hic 4.59
of these were perfect repeats, because detectable homoplasy appears to be more common in compound and/or
interrupted repeats (Adams et al. 2004). To the 5′-end of
each forward primer, we added a universal M13 tag
(5′-AGGGTTTTCCCAGTCACGACGTT-3′) for use in the
universal dye-labelling method described by BoutinGanache et al. (2001). We optimized PCR conditions and
tested for variability with 27 samples from southern
Portugal. Amplification products were labelled with 6-FAM,
VIC, or NED M13 tag oligonucleotides. PCRs of 10 μL
contained 1× PCR Buffer (Invitrogen), 3 mm MgCl2, 0.2 mm
of each dNTP (Bioline), 0.5 μm of each primer plus 0.5 μm
of labelled M13 tag oligonucleotide, 0.5 U of Taq DNA
Polymerase (Invitrogen), 0.5 μg/μL BSA (New England
Biolabs) and 2 μL of DNA extract. The reactions were run
in a 2720 Thermal Cycler (Applied Biosystems) and cycling
conditions consisted of an initial denaturation at 94 °C for
3 min, followed by five cycles of 30 s at 94 °C, 30 s at the
reverse primer ’s annealing temperature (Ta) + 10 °C, 30 s
at 72 °C, 10 cycles of 30 s at 94 °C, 30 s at Ta + 5 °C, 30 s at
72 °C, and 22 cycles of 30 s at 94 °C, 30 s at Ta, 30 s at 72 °C.
The final extension was 20 min at 72 °C. Fragment lengths
of PCR products were determined with an ABI PRISM 310
Genetic Analyser using the GeneScan 500 LIZ size standard
(Applied Biosystems), and analysed using GeneMapper
3.7 (Applied Biosystems).
Of the 20 primer pairs examined, 13 had easily recognizable phenotypes and 11 of these were polymorphic in the
samples analysed here (Table 1). GenePop 4.0 (Rousset 2008)
was used to calculate heterozygosities, number of alleles,
allele size ranges, as well as to carry out tests of Hardy–
Weinberg equilibrium (HWE) and linkage disequilibrium.
The presence of null alleles, large allele dropout, and
stuttering were tested using Micro-Checker 2.2.3 (Van
Oosterhout et al. 2004). The polymorphic information
content and the probability of identity were estimated
using Cervus 3.0 (Kalinowski et al. 2007). We identified
between 4 and 7 alleles per locus and the observed heterozygosities ranged from 0.37 to 0.85. After Bonferroni
correction for multiple comparisons (α = 0.05), no significant
deviation from HWE was detected in each locus and no
linkage disequilibrium was identified between any sets of
loci. No null alleles, large allele dropouts, or stuttering
were detected at the 99% confidence level. The probability
of identity using all 11 loci was 2.01 × 10–9. Given that the
individuals we screened were of an isolate potentially
derived from a single founding event in historical times, it is
feasible that the loci will show more variation in other areas
of the (native) species range. These novel polymorphic microsatellites will therefore be useful for population genetic
and phylogeographical studies of the Egyptian mongoose.
Acknowledgements
We thank BRISA for their invaluable contributions to the collection
of carnivore biological samples maintained at the CBA. We
acknowledge financial support from the FCT (MCTES, Portugal),
through the research projects POCTI/2000/BSE/407888 and
POCI/BIA-BDE/61491/2004 (POCI 2010 program), and the
postdoctoral fellowship awarded to Carlos Fernandes (BPD/
20317/2004).
References
Adams RI, Brown KM, Hamilton MB (2004) The impact of microsatellite electromorph size homoplasy on multilocus population
structure estimates in a tropical tree (Corythophora alta) and an
anadromous fish (Morone saxatilis). Molecular Ecology, 13, 2579 –
2588.
Borralho R, Rego F, Palomares F, Hora A (1996) The distribution
of the Egyptian mongoose Herpestes ichneumon (L.) in Portugal.
Mammal Review, 26, 1–8.
Boutin-Ganache I, Raposo M, Raymond M, Deschepper CF (2001)
M13-tailed primers improve the readability and usability of
microsatellite analysis performed with two different allelesizing methods. BioTechniques, 31, 24–28.
Colautti RI, Manca M, Viljanen M et al. (2005) Invasion genetics of
the Eurasian spiny waterflea: evidence for bottlenecks and
gene flow using microsatellites. Molecular Ecology, 14, 1869 –
1879.
Dlugosch KM, Parker IM (2008) Founding events in species
invasions: genetic variation, adaptive evolution, and the role of
multiple introductions. Molecular Ecology, 17, 431– 449.
Fernandes CA, Ginja C, Pereira I et al. (2008) Species-specific
mitochondrial DNA markers for identification of non-invasive
samples from sympatric carnivores in the Iberian Peninsula.
Conservation Genetics, 9, 681– 690.
Ficetola GF, Bonin A, Miaud C (2008) Population genetics reveals
origin and number of founders in a biological invasion. Molecular
Ecology, 17, 773 –782.
Gardner MG, Cooper SJB, Bull CM, Grant WN (1999) Isolation of
microsatellite loci from a social lizard, Egernia stokesii, using a
modified enrichment procedure. Journal of Heredity, 90, 301–304.
Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci.
Methods in Enzymology, 395, 202–222.
Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the
computer program Cervus accommodates genotyping error
increases success in paternity assignment. Molecular Ecology, 16,
1099 –1106.
Kingdon J (1997) The Kingdon Field Guide to African Mammals.
Academic Press, London.
Palomares F (in press) Herpestes ichneumon. In: The Mammals
of Africa (eds Kingdon J, Butynski T, Happold D). Academic
Press, Amsterdam, The Netherlands.
Riquelme-Cantal JA, Simón-Vallejo MD, Palmqvist P, CortésSánchez M (2008) The oldest mongoose of Europe. Journal of
Archaeological Science, 35, 2471–2473.
Rousset F (2008) GenePop’007: a complete re-implementation of
the GenePop software for Windows and Linux. Molecular
Ecology Resources, 8, 103 –106.
Suarez AV, Tsutsui ND (2008) The evolutionary consequences of
biological invasions. Molecular Ecology, 17, 351–360.
Thulin C-G, Simberloff D, Barun A et al. (2006) Genetic divergence
in the small Indian mongoose (Herpestes aeropunctatus), a
widely distributed invasive species. Molecular Ecology, 15, 3947–
3956.
Van Oosterhout CV, Hutchinson WF, Wills DPM, Shipley P (2004)
Micro-Checker: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes, 4,
535 –538.
doi: 10.1111/j.1755-0998.2009.02624.x
© 2009 Blackwell Publishing Ltd
Isolation and characterization of 11 polymorphic
microsatellite loci in the millipede Antichiropus variabilis
Attems (Diplopoda: Polydesmida: Paradoxosomatidae)
J A N I N E M . W O J C I E S Z E K and L E I G H W. S I M M O N S
Centre for Evolutionary Biology, School of Animal Biology (M092), University of Western Australia, 35 Stirling Highway,
Crawley, WA 6009, Australia
Abstract
Eleven polymorphic microsatellite loci were isolated from the Western Australian millipede
Antichiropus variabilis. The number of alleles observed ranged from 2 to 12, with observed
heterozygosities ranging from 0.20 to 0.80. All loci were in Hardy–Weinberg equilibrium,
and no pairs of loci were in linkage disequilibrium. Many of the loci amplified successfully
in eight other Antichiropus species.
Keywords: Diplopoda, microsatellite, millipede, sexual selection, sperm competition
Received 8 December 2008; accepted 27 January 2009
Correspondence: Janine M. Wojcieszek, Fax: +61 86488 1029; E-mail: wojcij01@student.uwa.edu.au