c ? Indian Academy of Sciences
Isolation and characterization of microsatellite loci in the Neotropical
fish Astyanax altiparanae (Teleostei: Characiformes)
and cross-species amplification
ROSÂNGELA LOPES ZAGANINI1, DIOGO TERUO HASHIMOTO1, LUIZ HENRIQUE GARCIA PEREIRA2,
CLAUDIO OLIVEIRA2, FERNANDO FERNANDES MENDONÇA2, FAUSTO FORESTI2
and FÁBIO PORTO-FORESTI1∗
1Departamento de Ciências Biológicas, Faculdade de Ciências, Universidade Estadual Paulista,
Júlio de Mesquita Filho, 17033-360, Bauru, SP, Brazil
2Departamento 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)n and (AG)n 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
Isolation of microsatellites in Astyanax altiparanae
of inserts. Selected recombinant colonies were sequenced
using the primers T7 (5?-TAATACGACTCACTATAGGG-
3?) and SP6 (5?-ATTTAGGTGACACTATAGAA-3?) 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 weredesigned with Primer3software(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
(22◦6?40.92??S, 49◦16?5.81??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 μL H2O miliQ, 2.75 μL PCR
buffer 10×, 1.25 μL MgCl250 mM, 1.5 μL dNTP 1.25 mM
(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 followed by 35 cycles of 30 s
at 95◦C, 30 s at the annealing temperature (see table 1),
5 s at 72◦C, and a final extension time of 5 min at 72◦C.
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?-3?)Repeat motif Lenght (bp)
151 50329 0.630 0.837
16358 254 0.742 0.638
160 58 25100.563 0.877
Asty 15 (AC)17– (CT)6
212 50 336 0.500 0.7730,353∗
15057 2411 0.500 0.8520,413∗
190 58258 0.654 0.8560,236
150 58259 0.6670.809 0,176
Intrapopulational analysis: Ta, annealing temperature; n, number of individuals; A, allele number; Ho, observed heterozygosity; He,
expected heterozygosity; Fis, endogamy index.∗Loci that displayed significant deviations from Hardy–Weinberg equilibrium (P < 0.01).
Journal of Genetics Vol. 91, Online Resources
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.
Primer - Asty
16 Species4 111213 15 21232426 27
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
Agostinho A. A., Gomes L. C. and Pelicice F. M. 2007 Ecolo-
gia e manejo de recursos pesqueiros em reservatórios do Brasil.
EDUEM, Maringá, Brazil.
Artoni R. F., Shibatta O. A., Gross M. C., Schneider C. H., Almeida
M. C., Vicari M. R. and Bertollo L. A. C. 2006 Astyanax aff.
fasciatus Cuvier, 1819 (Teleostei; Characidae): evidences of a
species complex in the upper rio Tibagi Basin (Paraná, Brazil).
Neotrop. Ichthyol. 4, 197–202.
Barbará T., Palma-Silva C., Paggi G. M., Bered F., Fay M. F. and
Lexer C. 2007 Cross-species transfer of nuclear microsatellite
markers: potential and limitations. Mol. Ecol. 16, 3759–3767.
Billotte N., Lagoda P. J. L., Risterucci A. M. and Baurens F. C.
1999 Microsatellite-enriched libraries: applied methodology for
the development of SSR markers in tropical crops. Fruits 54,
Castro R. M. C. 1999 Evolução da ictiofauna de riachos sul-
americanos: Padrões gerais e possíveis processos causais. In
Ecologia de Peixes de Riachos (ed. E. P. Caramashi, R. Mazzoni
and P. R. Peres-Neto), pp. 139–155. PPGE-UFRJ, Rio de Janeiro,
Fernandes C. A. and Martins-Santos I. C. 2004 Cytogenetic stud-
ies in two populations of Astyanax altiparanae (Pisces, Characi-
formes). Hereditas 141, 328–332.
Froese R. and Paulay D. 2010 FishBase. World Wide Web electron-
ics publication, http://www.fishbase.org/. (Acessed 03 November
Garutti V. and Britski H. A. 2000 Descrição de uma nova espécie de
Astyanax (Telostei, Characidae) da bacia do Alto Paraná e con-
siderações sobre as demais espécies do gênero na bacia. Comun.
Mus. Ciênc. Ser. Zool. PUCRS 13, 65–88.
Javonillo R., Malabarba L. R., Weitzman S. H. and Burns J. R. 2010
Relationships among major lineages of characid fishes (Teleostei:
Mol. Phyl. Evol. 54, 498–511.
Jeffery W. R. 2001 Cavefish as a model system in evolutionary
developmental biology. Dev. Biol. 231, 1–12.
Langecker T. G., Wilkens H. and Junge P. 1991 Introgressive
hybridization in the Pachon cave population of Astyanax fascia-
tus. Ichthyol. Explor. Freshw. 2, 209–212.
Leuzzi M. S. P., Almeida F. S., Orsi M. L. and Sodré
L. M. K. 2004 Analysis by RAPD of the genetic structure of
Astyanax altiparanae (Pisces, Characiformes) in reservoirs on
the Paranapanema River, Brazil. Genet. Mol. Biol. 27, 355–
Mirande J. M. 2010 Phylogeny of the family Characidae (Teleostei:
Characiformes): from characters to taxonomy. Neotrop. Ichthyol.
Moreira-Filho O. and Bertollo L. A. 1991 Astyanax scabripinnis
(Pisces: Characidae), a species complex. Rev. Bras. Genet. 14,
Oliveira E. J., Pádua J. G., Zucchi M. I., Vencovsky R. and Vieira
M. L. C. 2006 Origin, evolution and genome distribution of
microsatellites. Genet. Mol. Biol. 2, 294–307.
Peakall R. and Smouse P. E. 2007 GENALEX 6.1: genetic anal-
ysis in Excel. Population genetic software for teaching and
research. Australian National University, Canberra, Australia,
Journal of Genetics Vol. 91, Online Resources
Isolation of microsatellites in Astyanax altiparanae Download full-text
Peres M. D., Vasconcelos M. S. and Renesto E. 2005 Genetic
variability in Astyanax altiparanae Garutti and Britski, 2000
(Teleostei, Characidae) from the Upper Paraná River basin,
Brazil. Genet. Mol. Biol. 28, 717–724.
altiparanae). In Espécies nativas para piscicultura no Brasil
(ed. B. Baldisserotto and L. C. Gomes), pp. 101–115. Editora
UFSM, Santa Maria.
Prioli S. M. A. P., Prioli A. J., Júlio Jr H. F., Pavanelli C. S.,
Oliveira A. V., Carrer H. et al. 2002 Identification of Astyanax
altiparanae (Teleostei, Characidae) in the Iguaçu River, Brazil,
based on mitochondrial DNA and RAPD markers. Genet. Mol.
Biol. 25, 421–430.
Raymond M. and Rousset F. 1995 Genepop (version 1.2): popula-
tion genetics software for exact tests and ecumenicism. J. Her.
Reis R. E., Kullander S. O. and Ferraris C. J. 2003 Check list of the
freshwater fishes of South and Central America. Edipucrs, Porto
Rosen S. and Skaletsky H. 2000 Primer3 on the WWW for gen-
ods and protocols: methods in molecular biology (ed. S. Misener
and S. A. Krawetz), pp. 365–386. Humana Press, Totowa, New
Van Oosterhout C., Hutchinson W. F. , Wills D. P. M. and Shipley
typing errors in microsatellite data. Mol. Ecol. Notes 4, 535–538.
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