Molecular Ecology Notes (2007) 7, 3234

doi: 10.1111/j.1471-8286.2006.01484.x

PRIMER NOTE
Blackwell Publishing Ltd

Isolation and characterization of eight microsatellite loci

in Leporinus macrocephalus (Characiformes: Anostomidae)
and cross-species amplification
K A R I N A A . M O R E L L I , E L O I S A R E V A L D A V E S , C L A U D I O O L I V E I R A and F A U S T O F O R E S T I
Departamento de Morfologia, Instituto de Biocincias, Universidade Estadual Paulista, CEP 18608-000, Botucatu, So Paulo, Brazil

Abstract
Leporinus macrocephalus is a species endemic to the Paraguay River basin and an important
fishery resource, as well as a valuable species in aquaculture programs. A total of eight
polymorphic microsatellite loci were isolated and characterized. A population survey was
conducted involving 45 specimens whereby a large number of alleles (range 517 among
loci), a highly observed (0.16670.6129) and an expected (0.69670.9448) heterozygosity was
detected, indicating its usefulness in population genetics studies. Cross-species amplification was successful in eight Characiformes species.
Keywords: aquaculture, cross-amplification, evolution, fish, microsatellite isolation, repetitive
sequences, Leporinus macrocephalus, microsatellite
Received 12 April 2006; revision accepted 3 June 2006

The Anostomidae family (Characiformes), with 12 genera

thus far recognized (Nelson 1994), is an important freshwater fish group distributed throughout the Neotropical
region (Gry 1977). Leporinus, the most diversified genera
of Anostomidae, is distributed from Central to South
America. The species Leporinus macrocephalus is an endemic
species found on the Paraguay River basin and is an
important fishery resource and a valuable species in
aquaculture programs. Several wild populations have been
suffering a drastic reduction despite its great economic
importance. Genetic characterization of populations from
this region may hold practical implications for biodiversity
conservation. Microsatellite loci have recently been used for
many biological applications, including conservation genetics.
To further genetic population analysis of natural and hatchery
populations, we designed microsatellite primers for L.
macrocephalus and tested their application in related species.
Microsatellite loci were isolated from a partially
enriched library, following the protocols of Hamilton et al.
(1999). Total genomic DNA was extracted from the liver
using phenolchloroform protocol described by Sambrook
& Russel (2001). The DNA was digested with DraI and SspI
restriction enzymes. After electrophoresis, 400 800-bp
Correspondence: Karina A. Morelli, Fax: 55 14 38116264; E-mail:
kmorelli@ibb.unesp.br

fragments were recovered from 2% agarose low-melting

gel and purified with phenolchloroform. These fragments
were ligated in double stranded SNX linker (SNXr
5-P-GCTTCTGCTAGCAAGCCTTAGA-3 and SNX
5-CTAAGGCCTTGCTAGCAGAAGC-3). The selection
of segments with repetitive motifs was performed using
biotinylated (AC)18 and (AG)18 oligonucleotides and
magnetic beads covered with streptavidin.
The selected fragments were amplified by polymerase
chain reaction (PCR) using the SNX adaptors. Amplified
fragments were ligated into a pGEM-T vector (pGEM-T
Vector System, Promega) and inserted into competent
Escherichia coli cells (Subcloning Efficiency DH5 Competent Cells, Invitrogen). Positive colonies were sequenced
on an ABI PRISM 377 automated DNA sequencer using
BigDye Terminator version 3.1 Cycle Sequencing Ready
Reaction kit (Applied Biosystems).
Sixty-four clones were sequenced and 10 flanking
primers pairs were designed using the primer 3 software
(Rozen & Skaletsky 1998). The analysis was conducted
with a sample of 45 fishes collected in the Taquari River,
Mato Grosso do Sul, Brazil. PCRs were carried out in 25-L
volume with the following components: 10 ng of template
DNA, 0,2 m of each primer, 0.2 U of Taq DNA polymerase, 0.16 m of dNTP, 1 PCR buffer. The conditions for
amplifications were 94 C for 2 min, followed by 30 cycles
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Journal compilation 2006 Blackwell Publishing Ltd

P R I M E R N O T E 33
Table 1 Characteristics of eight polymorphic microsatellite loci in Leporinus macrocephalus
Locus/GenBank
Accession no.
Lmac 02
DQ469271
Lmac 03
DQ469272
Lmac 04
DQ469273
Lmac 05
DQ469274
Lmac 06
DQ469275
Lmac 07
DQ469276
Lmac 08
DQ469277
Lmac 09
DQ469278

Repeat motif

Primer sequence (5 3)

(TG)28(AG)10

F: ACTTCCCTCCCTAATCTGTG
R: AGGGTGTAAGATGTATGAAG
F: TGTATGTGGTGTGGCTGAATG
R: AAACAAAGTAGAAGTGAACG
F: CTGTCACTCTGTCAACTTTT
R: TAAGGGAAAGCGGATACGGG
F: CGTGTGCTTCTGTTTGTGTG
R: GGCTGAAGTATGAGAGGTAAG
F: CTCTACTTCACTTTTACAGCAG
R: CCCGAGCCGCGTCACACTTC
F: GTGGGAACATTTGGGATTAT
R: CAGAAGAGAGGGCGAGAGGG
F: CCATTTCTGTCTTACCTTTG
R: CTTCGGGAGTTGACAGCACC
F: TAAACAAACGGTGGGCAGTG
R: TATCTTCACTAATCAAACTCC

(AC)28
(CT)6(TT)(CT)13
(CT)14
(CA)17
(TC)17
(TC)23
(AG)4(GG)(AG)19

Ta
(C)

Allele size*
(range)

Na

HO

HE

HWE

57

256 (212264)

28

17

0.6071

0.9448

**

56

258 (214252)

31

15

0.6129

0.9207

**

64

265 (248272)

36

0.1667

0.7398

**

64

231 (212256)

30

14

0.5714

0.8779

**

58

123 (112134)

41

11

0.4000

0.8608

**

58

147 (140150)

35

0.2941

0.6967

**

55

167 (148168)

35

0.5294

0.8578

**

56

177 (164180)

34

0.5588

0.7669

**

Ta, annealing temperature; N, sample size; Na, number of alleles; HO, observed heterozygosity; HE, expected heterozygosity; HWE,
HardyWeinberg equilibrium; **P < 0.05. *Allele size refers to the base-pair size of the cloned allele, and the range of the observed alleles
(based on the population analysis) in parentheses.

Table 2 Cross-amplification data of eight additional Characiformes species using the primers developed for Leporinus macrocephalus. N,
sample size; P, polymorphic; M, monomorphic; no amplification
Locus
Species

Lmac02

Lmac03

Lmac04

Lmac05

Lmac06

Lmac07

Lmac08

Lmac09

Leporinus elongatus
Leporinus obtusidens
Leporinus friderici
Schizodon nasutus
Caenotropus labyrinthicus
Prochilodus lineatus
Cyphocharax modesta
Steindachnerina insculpta

5
5
5
5
5
5
5
5

P
P

P
P
P
M
P
M
P
P

P
P
M

M
P

P
P
P

P
P
P
M
P
P

P
P
P
M
P
P

P
P
P
M
P

P
P
P
P
P
P

of 30 s at 94 C, 30 s at annealing temperature (Table 1),

30 s at 72 C, and a final extension time of 72 C for 2 min.
PCR products were separated on a 6% polyacrylamide gel
electrophoresis and visualized using a silver-staining
protocol. The allele sizes were calculated by comparison
with the band mobilities of a known DNA size standard
(10 bp DNA ladder, Invitrogen).
popgene version 3.1 software (Ref 2) was used to calculate
expected heterozygosity (considering the Levenes correction) and genepop version 3.4 (Ref 1) was used to access
HardyWeinberg equilibrium (HWE) and genotypic
linkage disequilibrium. Eight highly polymorphic loci
were successfully amplified. The number of alleles varied
from five to 17 and the values of both the observed and the
expected heterozygosities ranged from 0.1667 to 0.6129
2006 The Authors
Journal compilation 2006 Blackwell Publishing Ltd

and from 0.6967 to 0.9448, respectively (Table 1). All loci

showed a significant departure from HardyWeinberg
equilibrium (P < 0.05), due to the heterozygote deficiency.
These deviations, which will be further investigated, may
have resulted from genetic drift, Wahlund effect, mating
systems or null alleles. There was no significant linkage
disequilibrium for all pairs of loci, suggesting independent
inheritance.
Cross-species amplification in eight related taxa was
investigated (Table 2). A high level of cross-amplification
was observed in the genus Leporinus. The amplification
product of the Lmac04 and Lmac05 for Leporinus elongatus
and Leporinus obtusidens, was sequenced and confirmed
that the repetitive loci are conserved between related
species. These results suggest that some of the markers

34 P R I M E R N O T E
here developed may be applied to further genetic diversity
studies in wild and cultivated population of L. macrocephalus and related species, once the cross-transferability
of microsatellites was successful.

Acknowledgements
We thank Fbio Porto-Foresti, Priscila Gusmo-Pompiani and
Fernando Pompiani for their help with fish collection. Financial
support for this study was provided by CNPq and FAPESP.

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2006 The Authors

Journal compilation 2006 Blackwell Publishing Ltd