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Development and characterization of polymorphic microsatellite markers from the
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Geoffroy's side-necked turtle (Phrynops geoffroanus)
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Priscilla M. S. Villela1*, Ana Luiza B. Longo1, Erika C. Jorge2, Ricardo A. Brassaloti1,
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Aline R. Martins3, Thiago S. Marques4, Luciano M. Verdade4, Antonio Figueira5, Luiz L.
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Coutinho1
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Universidade de São Paulo, Piracicaba, Estado de São Paulo 13418-900, Brazil.
Departamento de Zootecnia, Escola Superior de Agricultura Luiz de Queiroz,
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MG, 31270-901, Brazil.
Departamento de Morfologia, Universidade Federal de Minas Gerais, Belo Horizonte,
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Universidade de São Paulo, Piracicaba, Estado de São Paulo 13418-900, Brazil.
Departamento de Ciências Biológicas, Escola Superior de Agricultura Luiz de Queiroz,
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Universidade de São Paulo, Piracicaba, Estado de São Paulo 13416-000, Brazil.
Laboratório de Ecologia Isotópica, Centro de Energia Nuclear na Agricultura,
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Universidade de São Paulo, Piracicaba, Estado de São Paulo 13418-900, Brazil.
Laboratório de Melhoramento de Plantas, Centro de Energia Nuclear na Agricultura,
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*Corresponding author: Priscilla M. S. Villela. Fax: +55 19 34294215. E-mail:
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[email protected]
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Abstract
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Phrynops geoffroanus is a freshwater turtle species, with a wide distribution in South
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America, living in many types of habitats, including polluted urban rivers. However, the
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lack of knowledge hinders broader approaches to various ecological and evolutionary
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aspects of this species. In this study eight polymorphic microsatellite markers were isolated
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and characterized in 48 individuals from two natural populations. In the Piracicaba river
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population, the number of alleles for the eight loci ranged from 4 to 15, whereas the
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observed and expected heterozygosities per locus varied from 0.29 to 0.87 and from 0.31 to
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0.90, respectively. In Piracicamirim stream population, the number of alleles ranged from 4
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to 13, and the observed and expected heterozygosities per locus varied from 0.46 to 0.75
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and from 0.53 to 0.88, respectively. These microsatellites provide efficient genetic markers
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for population structure, species relationships and phylogeography studies.
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Keywords: genetic diversity, microsatellites, polymorphism, population genetics,
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freshwater turtles
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Geoffroy's side-necked turtle, Phrynops geoffroanus, is widely distributed in South
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America, occurring from Venezuela and Colombia to southern Brazil, Paraguay, northern
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Argentina and Uruguay (Pritchard & Trebbau 1984; Ernst & Barbour 1989). This
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occurrence is common in many urban rivers (Souza & Abe 2000; 2001; Souza 2005; Piña
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et al. 2009), but little is known about the species biology under natural conditions (Medem
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1960). The species taxonomy still remains controversial, with some authors maintaining
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this taxon as super-species, and others, arguing that it is a polytypic species with subspecies
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(Rueda-Almonacid et al. 2007).
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Microsatellite markers are widely used in population genetics studies. The highly
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polymorphic, co-dominant nature of these markers, make them ideal for a diverse range of
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applications (Allendorf & Luikart 2007, Frankham et al. 2008). Clarification of taxonomic
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and evolutionary relationships, evaluation of demographic and behavioral mechanisms of
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populations, such as migratory routes, mating systems, and survivorship, along with
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pedigree studies and wildlife forensics, are just a few areas where these molecular markers
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are employed (Frankham et al. 2008; Allendorf & Luikart 2007).
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Microsatellite markers originally developed for related species, such Podocnemis expansa
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(Sites et al. 1999; Valenzuela 2000) and Emydura sp. (Farley 2004) have not been
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successfully transferred to P. geoffroanus (unpublished results). For this reason, in this
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study, we report the development and characterization of eight specific microsatellite
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markers for P. geoffroanus.
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In this study we collected samples from animals from two water bodies of southeastern
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Brazil: Piracicaba river (22°41'5.70"S; 47°33'46.59"W) and one of its tributaries, the
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Piracicamirim stream (22°42'52.18"S; 47°37'38,95"W). The Piracicaba river has
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approximately 370 km, from its source on ‘Serra da Mantiqueira’ in the state of Minas 3
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Gerais to its confluence with the Tietê river in the state of São Paulo, being in average 50 m
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wide at the study site. Land use around Piracicaba river has changed over time, affecting its
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water quality (Del Grande et al. 2003). The current major pollution source in this area is
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direct domestic sewage (Ferraz et al. 1996) and heavy metals have been detected (Ferraz et
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al. 1996; Favaro et al. 2004, Meche et al. 2009, Piña et al. 2009). The Piracicamirim stream
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is one of the most important tributaries of the Piracicaba river, with a total length of 24.6
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km mostly across agricultural and urban landscapes, being in average 6 m wide at the study
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site (Teixeira & Duarte 2008).
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A total of 48 individuals of P. geoffroanus were sampled, 24 in the Piracicaba river and 24
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in the Piracicamirim stream. Blood was collected from the external jugular vein (Rogers &
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Booth, 2004). After collection, blood was stored in lysis buffer: 100 mM Tris-HCl, pH 8.0;
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100 mM EDTA, pH 8.0; 10 mM NaCl; 0.5% SDS (w/v), as described in Hoelzel (1992).
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Blood samples were digested with proteinase K to a final concentration of 0.5 mg ml-1,
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proteins precipitated with 1.2M NaCl and total DNA was precipitated with ethanol (Hoezel
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1992; Olerup and Zetterquist, 1992).
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Genomic DNA (5 µg) extracted from an individual P. geoffroanus from Piracicaba river
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population was used to construct one genomic library enriched for microsatellites, based on
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Billotte et al. (1999) protocol. DNA was digested overnight at 37°C with 60 U RsaI
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(Fermentas, Burlington, Canada) and 400 mM spermidine, in a total volume of 100 µL.
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Rsa21 and Rsa25 (0.2 µM of each) adapters were ligated to the digested DNA ends (1 µg)
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at 20°C for 2 h, using 5 U T4 DNA ligase (Promega®, Madison, Wisconsin, USA) in a final
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volume of 200 µL. Biotinylated oligonucleotide [(CT)8 and (GT)8] were used as probes for
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library enrichment for microssatelites, as described by Kijas et al. (1994). Amplified
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fragments were cloned in pGEM-T Easy Vector (Promega®) at room temperature for 2 h, 4
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using 50 ng vector, 3 µL of amplification product, and 3 U T4 DNA ligase. Ligation
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product was used to transform electrocompetent Escherichia coli (DH10B) and plated onto
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selective media (Luria – Bertani with 100 mg L-1 ampicillin). Positive colonies (288 clones)
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were transferred to 96-wells plates, containing the same selective media, with 8% glycerol.
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After growth for 24 h at 37°C, plates were stored at -80°C. Plasmids from two 96-wells
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plates were purified using alkaline lyses followed by filtration with a Millipore (MAGV
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N22; EMD Millipore®, Billerica, Massachusetts, USA) filter and sequenced using an ABI
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PRISM Terminator Cycle sequencing kit and an ABI 3100 Automated Sequencer (Applied
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Biosystems®, CA).
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Sixty-six per cent of the cloned inserts contained microsatellite sequences: from 94 inserts
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identified after sequencing, 63 had a microsatellite, and 20 were suitable for primer design;
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the remaining sequences were not exploited because repetitive sequences were either too
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short or too close to one of the sequence ends.
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Primer pairs complementary to sequences flanking the repeat element were designed using
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the Primer3 software (Rozen and Skaletsky, 1998) for 20 selected loci, with one primer in
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each pair labeled with a fluorescent dye, either HEX or FAM. From these, 10 successfully
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amplified P. geoffroanus DNA samples. Amplification conditions were optimized using a
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thermal gradient of annealing temperatures (55–65°C). Microsatellite loci were amplified
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under the following conditions: 94°C for 2 min, followed by 35 cycles of 94°C for 45 sec,
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the appropriate annealing temperature (Ta, Table 1) for 45 sec, and amplification at 72°C
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for 1 min. Samples were amplified in a 15 µL final volume of 1X PCR buffer (20 mM Tris-
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HCl, pH 8.4; 50 mM KCl), 1.0 mM MgCl2, 0.2 mM each dNTP, 0.2 mM of each primer,
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0.02 U ml-1 Taq DNA polymerase (Invitrogen®), and 100 ng of DNA. GeneScanTM 500
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RoxTM Size Standard (Applied Biosystems®) was included as internal marker for size 5
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standard. PCR products were separated by gel electrophoresis using an ABI PRISM®
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3130xl Genetic Analyzer (Applied Biosystems®). Fragment sizes were determined using
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Gene Mapper TM v4.0 software, and confirmed manually (Applied Biosystems®).
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The number of alleles per locus, the allele size range, the observed (HO) and expected (HE)
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heterozygosities, deviation from Hardy-Weinberg equilibrium (HWE) and linkage
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disequilibrium (LD) were estimated using GENEPOP 4.0 (Raymond and Rousset, 1995).
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To correct for multiple comparisons, a sequential Bonferroni correction for both LD and
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HWE tests was used. The presence of null alleles and scoring errors was estimated using
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MICRO-CHECKER 2.2.3 (Van Oosterhout et al. 2004).
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From the 10 loci successfully amplified in P. geoffroanus, two of them (loci Phg09 and
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Phg11) did not revealed polymorphism in the two populations studied. The remaining
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markers were highly polymorphic and presented a mean Polymorphism Information
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Content (PIC) from the remaining eight loci greater than 0.7 (Table 1).
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In the Piracicaba river population, the number of alleles for the eight loci ranged from 4 to
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15, whereas the HO and HE varied from 0.29 to 0.87 and from 0.31 to 0.90, respectively
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(Table 2). In Piracicamirim stream population, the number of alleles ranged from 4 to 13,
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and the HO and HE varied from 0.46 to 0.75 and from 0.53 to 0.88, respectively (Table2).
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One locus (Phg8) in the Piracicaba population and three (Phg4, Phg6, Phg7) loci in the
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Piracicamirim population appear to deviate from HWE (before a Bonferroni’s correction).
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Based on telemetry data (unpublished data) from about 10 individuals from each population
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we notice that the home range for most animals do not reach more than 1 km for stream
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population and 3 km for river population, and during the monitoring time we never
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observed migration for animals captured and marked on each habitat. So based on these
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observations we believed that most part of the heterozygosity deficits observed are likely 6
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the result of the breeding system of this species, as it can exercise substantial inbreeding in
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isolated breeding sites. After Bonferroni correction (P
