Indian Academy of Sciences
Isolation and characterization of microsatellite loci in the Neotropical
ﬁsh Astyanax altiparanae (Teleostei: Characiformes)
and cross-species ampliﬁcation
ROSÂNGELA LOPES ZAGANINI
, DIOGO TERUO HASHIMOTO
, LUIZ HENRIQUE GARCIA PEREIRA
, FERNANDO FERNANDES MENDONÇA
, FAUSTO FORESTI
and FÁBIO PORTO-FORESTI
Departamento de Ciências Biológicas, Faculdade de Ciências, Universidade Estadual Paulista,
Júlio de Mesquita Filho, 17033-360, Bauru, SP, Brazil
Departamento de Morfologia, Instituto de Biociências, Universidade Estadual Paulista, Júlio de Mesquita Filho,
18618-000, Botucatu, SP, Brazil
[Zaganini R. L., Hashimoto D. T., Pereira L. H. G., Oliveira C., Mendonça F. F., Foresti F. and Porto-Foresti F. 2012 Isolation and characteri-
zation of microsatellite loci in the Neotropical ﬁsh Astyanax altiparanae (Teleostei: Characiformes) and cross-species ampliﬁcation. J. Genet.
91, e24–e27. Online only: http://www.ias.ac.in/jgenet/OnlineResources/91/e24.pdf]
We isolated and characterized 11 polymorphic microsatellite
loci from the Neotropical ﬁsh Astyanax altiparanae, consid-
ered of economic interest, whose stocks have been seriously
endangered by the introduction of predatory ﬁshes. The anal-
yses in a population of 33 specimens detected a large number
of alleles (ranging from 4 to 11) and high levels of heterozy-
gosity (0.64–0.88) at these loci, indicating their usefulness in
population genetic studies. Cross-species ampliﬁcation was
successful only in species of Astyanax, 43% of which were
The Characiformes constitute one of the dominant and
more diverse orders among tropical ﬁshes, with more than
1800 species, among which the family Characidae is the
most diverse, with species spread throughout the Neotropical
region. However, the interrelationships among the Characi-
dae are poorly known (Reis et al. 2003) and remain under
discussion (Javonillo et al. 2010; Mirande 2010).
The genus Astyanax (Characiformes, Characidae) com-
prises 163 described species (Froese and Paulay 2010), and
its systematics are very complex and several studies have
currently shown that the genus needs to be more thoroughly
characterized. A. altiparanae, encountered along the south
and southeast Brazilian rivers, was formerly included in the
complex ‘A. bimaculatus’ (Garutti and Britski 2000), which
is widely distributed in South America. A. altiparanae is
of great economic interest, also being utilized as bait in
For correspondence. E-mail: firstname.lastname@example.org.
sport ﬁshing and in aquaculture programmes (Garutti and
Britski 2000; Porto-Foresti et al. 2010). However, the stocks
of this species are seriously endangered by introduced preda-
tory ﬁshes, such as tucunaré (Cichla spp.) and corvina (Pla-
gioscion squamossissimus) (Agostinho et al. 2007).
Many molecular markers have been frequently used for
the Astyanax species (Prioli et al. 2002; Leuzzi et al. 2004;
Peres et al. 2005). However, there are no microsatellite data
available for this group. These markers can be valuable tools
to investigate genetic variability, with applications to conser-
vation and population genetics (Oliveira et al. 2006). Their
use in stock characterization of A. altiparanae may have
practical implications for ﬁsheries, ﬁsh farming, and con-
servation biology. We describe the isolation and character-
ization of 11 novel microsatellite loci from A. altiparanae,
and their cross-ampliﬁcation for potential utility in studies of
Materials and methods
A microsatellite-enriched genomic library was obtained for
A. altiparanae following the protocol described by Billotte
et al.(1999). Genomic DNA was extracted using the com-
mercial Wizard Genomic DNA Puriﬁcation kit (Promega,
São Paulo, Brazil). The total DNA was digested with RsaI
and enriched in (AC)
repeats. Enriched frag-
ments were ampliﬁed by polymerase chain reaction (PCR)
and then linked into a pGEM vector (Promega) and trans-
formed into competent XL1-blue Escherichia coli cells. Pos-
itive colonies were tested by PCR to conﬁrm the presence
Keywords. molecular markers; ﬁsh conservation; aquaculture; polymorphic DNA.
Journal of Genetics Vol. 91, Online Resources e24
Isolation of microsatellites in Astyanax altiparanae
of inserts. Selected recombinant colonies were sequenced
using the primers T7 (5
) and SP6 (5
) and the
BigDye Terminator kit (Applied Biosystems, São Paulo,
Brazil), and electrophoresed on an ABI Prism 377 automated
sequencer (Applied Biosystems, Foster City, USA). Flank-
ing primers were designed with Primer3 software (Rozen and
Results and discussion
We isolated and sequenced a total of 48 positive colonies,
resulting in 25 good quality ﬂanking sequences. The selected
sequences were used to characterize a sample of 33
A. altiparanae specimens, collected in the Batalha river
W), Brazil, and tested in ﬁve
individuals of the species Salminus brasiliensis, Brycon
amazonicus, Brycon hilarii, A. fasciatus, A. bockmanni,
A. paranae, A. abramis, A. schubarti, A. ribeirae and A.
jacuhiensis. PCR was performed in 20 μL reaction volume
containing approximately 10.9 μLH
O miliQ, 2.75 μLPCR
50 mM, 1.5 μLdNTP1.25mM
(Invitrogen, São Paulo, Brazil), 1 μL of each primer
10 μM, 0.1 μL Taq DNA polymerase (Invitrogen) 5U/μL
and 1.5 μL of genomic DNA. The conditions for ampliﬁ-
cation were 5 min at 95
C, 30 s at the annealing temperature (see table 1),
C, and a ﬁnal extension time of 5 min at 72
The ampliﬁcation products were separated in 6% denaturing
polyacrylamide gel and visualized by silver nitrate staining,
photographed, and analysed using the Kodak Digital Science
program Eastman Kodak Company, Rochester, USA. Allele
scoring was done using the 10-bp DNA Ladder (Invitrogen,
São Paulo, Brazil) as size standard.
Among the 25 tested primer pairs, 11 loci were highly
polymorphic (GenBank accession numbers JQ246359 to
JQ246369). The allele number varied from 4 (Asty12) to
11 (Asty21) by locus; the value of expected heterozygos-
ity varied from 0.64 (Asty12) to 0.88 (Asty13), and three
loci showed deviation from the Hardy–Weinberg equilibrium
(HWE) (P < 0.01) (table 1). They were calculated using
GENALEX v6.1 software (Peakall and Smouse 2007). Pair-
wise tests for linkage disequilibrium among loci were calcu-
lated using GENEPOP 3.3 package (Raymond and Rousset
1995), and were nonsigniﬁcant. Micro-Checker (Van Ooster-
hout et al. 2004) was used to verify possible causes of HWE
departures, and the analysis showed no evidence of stutter-
ing, allelic dropout, or null alleles as a possible cause of
Cross-species ampliﬁcation was investigated in 10 addi-
tional species of the same family (table 2). All 11 primers
analysed revealed a high level (89%) of cross-ampliﬁcation
in species of Astyanax, 43% of which were polymorphic.
On the other hand, with noncongeneric species (Salminus
brasiliensis, Brycon amazonicus and B. hilarii), the cross-
ampliﬁcation did not show positive results. Barbará et al.
(2007) revealed that the transferability for ﬁsh species can
be around 70% in congeners, lowering to 60% among
Table 1. Description of microsatellite loci and primer sequences in Astyanax altiparanae.
Primer sequence (5
) Repeat motif Lenght (bp) T
C) nA H
Asty 4 GGTCACTGGAGGACAGATGTT (AC)
200 53 31 5 0.852 0.754 −0,129
Asty 11 TAAATCTATAAAGTCACCAT (AC)
151 50 32 9 0.630 0.837 0,248
Asty 12 AGACACAATCAGCCGCCGAAATG (GT)
163 58 25 4 0.742 0.638 −0,162
Asty 13 AAATGGGTGCAAGCAACG (GT)
160 58 25 10 0.563 0.877 0,359
Asty 15 CAACTTTTACTTAAAACCTGC (AC)
212 50 33 6 0.500 0.773 0,353
Asty 16 AAAGTAAAGGGCATCTGTGGAGAA (AC)
165 52 32 8 0.769 0.860 0,106
Asty 21 TTTATGGGGACCGTGAGATGTGC (CA)
150 57 24 11 0.500 0.852 0,413
Asty 23 TCAATGGAACCTATGGACAAC (CA)
160 52 30 6 0.600 0.743 0,193
Asty 24 AGACCAAACACTAGGGCTCAG (GT)
139 52 32 7 0.677 0.800 0,153
Asty 26 CCCATTGATCCTGCCTCTAA (GT)
190 58 25 8 0.654 0.856 0,236
Asty 27 GCATTGTTCAGGTTGGGTCT (GT)
150 58 25 9 0.667 0.809 0,176
Intrapopulational analysis: T
, annealing temperature; n, number of individuals; A, allele number; H
, observed heterozygosity; H
expected heterozygosity; F
, endogamy index.
Loci that displayed signiﬁcant deviations from Hardy–Weinberg equilibrium (P < 0.01).
Journal of Genetics Vol. 91, Online Resources e25
Rosângela Lopes Zaganini et al.
Table 2. Cross-ampliﬁcation of the 11 polymorphic loci in seven species of Astyanax and three others species of Characidae.
Species 4 11121315162123242627
Astyanax paranae PMPMPMPMPMM
A. bockmanni –PP–PMPPPPM
A. fasciatus –PPPPPPPPPM
A. jacuhiensis PPPPPPPPPPP
A. abramis MPMP –MMP P PM
A. schubarti MP P – – MMP MMM
A. ribeirae MPPP–MPPPPM
S. brasiliensis M– M – MMMMMM –
B. amazonicus M– MMMMMMMMM
B. hilarii M– MMMMMMMMM
P, polymorphic; M, monomorphic; –, no ampliﬁcation.
genera of the same family, which showed that the success of
transferability of microsatellite loci were directly linked to
phylogenetic relationship between the groups tested.
Astyanax spp. represent an excellent model group for
studies of evolutionary mechanisms (Langecker et al. 1991;
Jeffery 2001) because they are naturally distributed into
structured groups (Garutti and Britski 2000), favouring
vicariant processes responsible for the great diversity of
Neotropical ﬁshes (Castro 1999). Additionally, several
Astyanax spp. are distributed in species complexes, such as
bimaculatus, fasciatus and scabripinnis (Moreira-Filho and
Bertolo 1991; Fernandes and Martins-Santos 2004; Artoni
et al. 2006), which are abundant in rivers and other aquatic
habitats throughout the Neotropical region (Reis et al. 2003).
Consequently, the results obtained here reinforce the applica-
bility of the microsatellites for parentage population genetic
and studies in this heterogeneous group of ﬁshes.
This work was supported by grants from Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional
de Desenvolvimento Cientíﬁco e Tecnológico (CNPq) and Coorde-
nação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
We are grateful to Laboratório de Análise Genética e Molecu-
lar, CBMEG – UNICAMP, for help with microsatellite library
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Received 28 October 2011, in ﬁnal revised form 30 December 2011; accepted 4 January 2012
Published on the Web: 28 March 2012
Journal of Genetics Vol. 91, Online Resources