Identification and characterization of polymorphic microsatellite loci in the blue shark Prionace glauca, and cross-amplification in other shark species.

Abstract

Two to 14 alleles were found to be segregating per locus (mean 5·2), with observed and expected heterozygosities ranging from 0·08 to 0·78 and 0·08 to 0·94, respectively. Cross-amplification of six of these microsatellite loci indicated that they are also polymorphic in three species of Carcharhiniformes and two species of Lamniformes. The newly developed primers reported here constitute a useful tool for genetic population analyses on Prionace glauca and, potentially, other related species.

Full text

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Journal of Fish Biology (2012) 80, 2643–2646
doi:10.1111/j.1095-8649.2012.03291.x, available online at wileyonlinelibrary.com
Identification and characterization of polymorphic
microsatellite loci in the blue shark Prionace glauca,
and cross-amplification in other shark species
F. F. Mendonc ¸a*†, L. H. F. Ussami*, D. T. Hashimoto‡,
L. H. G. Pereira*, F. Porto-Foresti‡, C. Oliveira*, O. B. F. Gadig§
and F. Foresti*
*Laborat´ orio de Biologia e Gen´ etica de Peixes, Departamento de Morfologia, Instituto de
Biociˆ encias de Botucatu, Universidade Estadual Paulista, CEP 18618-000, S˜ ao Paulo, Brazil,
‡Laborat´ orio de Gen´ etica de Peixes, Departamento de Biologia, Faculdade de Ciˆ encias,
Universidade Estadual Paulista, CEP 17033-360, S˜ ao Paulo, Brazil and §Laborat´ orio de
Pesquisa em Elasmobrˆ anquios, Campus Experimental do Litoral Paulista, Universidade
Estadual Paulista, CEP 11330-900, S˜ ao Paulo, Brazil
(Received 7 January 2011, Accepted 27 February 2012)
Two to 14 alleles were found to be segregating per locus (mean 5·2), with observed and expected
heterozygosities ranging from 0·08 to 0·78 and 0·08 to 0·94, respectively. Cross-amplification of six
of these microsatellite loci indicated that they are also polymorphic in three species of Carcharhini-
formes and two species of Lamniformes. The newly developed primers reported here constitute
a useful tool for genetic population analyses on Prionace glauca and, potentially, other related
species.
Journal of Fish Biology © 2012 The Fisheries Society of the British Isles
© 2012 The Authors
Key words: conservation; elasmobranch; genetic variation; marine ecology; polymorphism; popula-
tion structure.
The increasing fishing pressure on sharks worldwide and of their generally low recov-
ery potential has heightened concerns about the vulnerability of shark populations
to overfishing. Blue sharks Prionace glauca (L. 1758) are the most heavily fished
sharks in the world with annual catches (mainly through by-catch) estimated between
10 and 20 million individuals. Despite the potential effect on populations worldwide,
monitoring data are inadequate to assess the scale of any population decline (Stevens,
2009). Globally, P. glauca is listed as near threatened by the International Union for
the Conservation of Nature (IUCN; Stevens, 2009).
The number of studies about the genetic structure of shark and ray popula-
tions is low, contrasting with the increasing number of elasmobranches captured.
†Author to whom correspondence should be addressed. Tel.: +55 14 3811 6264; email: fernandofm@
ibb.unesp.br
2643
© 2012 The Authors
Journal of Fish Biology © 2012 The Fisheries Society of the British Isles

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F. F. MENDONC ¸ A ET AL.
Many elasmobranch species are continually added to the IUCN endangered species
list or are considered under risk of extinction. These conditions coupled with a
lack of knowledge necessary for the adequate management of the exploited species,
raises considerable concerns on the sustainability of these resources (Mendonc ¸a
et al., 2009).
The development of polymorphic nuclear microsatellite loci for future investiga-
tions involving the assessment of levels of genetic diversity and population genetic
structure of P. glauca is presented. A total of 40 individuals were sampled in landings
by commercial fisheries of southern Brazil at latitudes 30◦5?–33◦08?S. To develop
and characterize nuclear microsatellite loci was employed the enrichment protocol
described by Glenn & Schable (2005). Genomic DNA (gDNA) was extracted from
one individual using the phenol–chloroform protocol, according to Sambrook & Rus-
sel (2001). The gDNA was digested with the restriction enzyme RsaI (New England
Biolabs Inc., www.neb.com; standard protocols). Following restriction digest, Super-
SNX24 linkers were ligated on to the ends of gDNA fragments. SNX linkers serve
as priming sites for polymerase chain reaction (PCR). Five biotinylated tetranu-
cleotide probes (AAAT, AACT, AAGT, ACAT, AGAT; all probes had eight repeats)
were hybridized linker-ligated restriction fragments, and the resultant probe-gDNA
complex was added to steptavidin-coated magnetic beads following the protocol sug-
gested by the manufacturer (Dynabeads M-280 Invitrogen; www.invitrogen.com).
Enriched fragments, eluted in water, were then used as template in a recovery PCR,
and PCR fragments were cloned using the TOPO TA Cloning kit (Invitrogen) fol-
lowing the manufacturer’s protocol. Ninety-six bacterial colonies were amplified
using universal Rsa21 primer (5?CTCTTGCTTACGCGTGGACTA 3?), and PCR
products were cleaned using MultiScreen-PCR filter plates following the manufac-
turer’s protocol (Millipore; www.millipore.com). DNA sequencing was performed
using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems;
www.appliedbiosystems.com) with sequencing reactions run on an ABI PRISM 3730
DNA Analyser (Applied Biosystems).
Primer pairs were designed for a total of 15 putative microsatellite loci using
PRIMER3 software (Rozen & Skaletsky, 1998). Tests for polymorphism were carried
out in a sample comprised of 40 P. glauca. The PCRs were performed in a 10 μl
reaction volume containing c. 10 ng of DNA, 1× PCR buffer, 0·3 μM of each
primer, 1·5 mM MgCl2and 0·25 U Pht Taq polymerase (Invitrogen). The conditions
for amplification were 2 min at 95◦C followed by 30 cycles of 30 s at 95◦C, 30 s
at the annealing temperature (see Table I) and 30 s at 72◦C with a final extension
time of 10 min at 72◦C.
Among the 15 tested nuclear microsatellite loci, eight loci were found to be poly-
morphic. The number of alleles at each polymorphic locus, their size range and
observed and expected heterozygosities were calculated using POPGENE version
1.31 (Yeh & Boyle, 1997). Tests for linkage disequilibrium were performed using
GENEPOP (Raymond & Rousset, 1995). The eight polymorphic loci displayed two
to 14 alleles per locus (mean across loci was 5·2) and the expected heterozygos-
ity (He) ranged from 0·08 to 0·78, while the observed heterozigosity (Ho) ranged
from 0·08 to 0·94 (Table I). Only one of eight polymorphic loci (TB02) revealed
a deviation from the Hardy–Weinberg Equilibrium (P < 0·05). These deviations
might have resulted either from null alleles, non-random sampling, mating systems
or inbreeding (Hoarau et al., 2002).
© 2012 The Authors
Journal of Fish Biology © 2012 The Fisheries Society of the British Isles, Journal of Fish Biology 2012, 80, 2643–2646

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MICROSATELLITE MARKERS FOR PRIONACE GLAUCA
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Table I. Summary statistics for microsatellite loci amplified from Prionace glauca sampled off southern Brazil
Locus
Repeat motif
Primer (5?-3?)
Ta(◦C)
Size range
(bp)
A Ho He
HWE
P-value
Other
species
Accession
number
TB01
(GA)9
F: TTGGTTGGGAAGAAGACTA
50
150–162
4 0·44 0·39
>0·05
Rp, Rl, Sl
GQ845157
R: GGAAATATGACCTGTAACCA
TB02*
(GT)14
F: TGCATTAAATCACCACAACTC
50
218–247 14 0·75 0·54
<0·01
Rp, Rl, Sl
GQ845158
R: GCCTAGGCTTAAAACTCCA
TB04
(CT)8
F: ATGATCAAACTTAAACTGGGTGTC
50
131–138
2 0·65 0·70
>0·05
Rp, Rl, Sl, Pk
GQ845159
R: AGAAAGCTGGCATCTGGTAGAAA
TB5
(CA)7
F: GGCAGCCGTTTTCATCAAG
56
167–198
3 0·55 0·52
>0·05
Rp, Sl, Pk, Io
GQ845160
R: GGGGTACGGGTCCACAC
TB07
(GT)5(GA)5
F: AGATAAATGGAGAGCTCACGACTG
53
170–213
9 0·67 0·70
>0·05
—
GQ845162
R: TTATCATTTCACATTTGCCATCC
TB08
(TG)16AGGAA F: TGTTGGATAGCAGCAGAC
51
191–202
2 0·78 0·94
>0·05
—
GQ845163
(GA)2CA(GA)5 R: ATCACCATAAGCTCAGC
TB13
(TG)6
F: ATGGGGTCAGAAGCAGAGAA
50
155–179
4 0·40 0·43
>0·05
Rp, Rl, Pk
GQ845166
R: ACTTATCCACCCCACCATC
TB15 (CA)5TACG(CA)4F: ATTTTAGGATGGCAGGTG
51
310–319
3 0·08 0·08
>0·05
Rp, Rl, Sl, Pk, Io GQ845168
R: TGTGTCTGCATATGTGTTTATT
Ta, annealing temperature; A, number of alleles; Ho, observed heterozygosity; He, expected heterozygosity;
*loci that displayed deviations from the Hardy–Weinberg Equilibrium (HWE). In ‘other species’ are the polymorphic nuclear microsatellite loci: Rp, Rhizoprionodon
porosus; Rl, Rhizoprionodon lalandii; Sl, Sphyrna lewini; Pk, Pseudocarcharias kamoharai; Io, Isurus oxyrinchus.
© 2012 The Authors
Journal of Fish Biology © 2012 The Fisheries Society of the British Isles, Journal of Fish Biology 2012, 80, 2643–2646

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F. F. MENDONC ¸ A ET AL.
The eight polymorphic microsatellite loci developed for P. glauca were tested
for cross-amplified in six individuals of each of five species, Sphyrna lewini (Grif-
fith & Smith 1834): five polymorphic loci, Rhizoprionodon porosus (Poey 1861):
six polymorphic loci and Rhizoprionodon lalandii (M¨ uller & Henle 1839): five
polymorphic loci (family Carcharhinidae), Isurus oxyrinchus Rafinesque 1810: two
polymorphic loci and Pseudocarcharias kamoharai (Matsubara 1936: four polymor-
phic loci) (family Lamnidae). These results indicate that the markers developed in
this study can potentially also be used to examine the population genetic structure,
levels of genetic diversity and mating systems of other shark species and, thus, can
contribute substantially to support conservation programmes.
Microsatellite enrichment was carried out in the Laborat´ orio de An´ alise Gen´ etica e Molec-
ular, CBMEG–UNICAMP. The authors render thanks to A. Pereira de Souza and F. Cidade.
They would also like to thank Fundac ¸˜ ao de Amparo ` a Pesquisa do Estado de S˜ ao Paulo
(FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnol´ ogico (CNPq), for
their financial support.
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© 2012 The Authors
Journal of Fish Biology © 2012 The Fisheries Society of the British Isles, Journal of Fish Biology 2012, 80, 2643–2646