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Cross-amplification of microsatellites from the Atherinopsidae Odontesthes perugiae and Odontesthes argentinensis to Chilean silversides of the genus Odontesthes and Basilichthys
Amplificación cruzada de microsatélites de los Atherinopsidae Odontesthes perugiae y Odontesthes argentinensis en pejerreyes chilenos del género Odontesthes y Basilichthys
Pablo Muñoz1,2, Claudio Quezada-Romegialli1,2, Irma Vila1 & David Véliz1,2*
1Departamento de Ciencias Ecológicas and 2Instituto de Ecología y Biodiversidad (IEB) Universidad de Chile, Casilla 653, Ñuñoa, Santiago, Chile *E-mail: dveliz@uchile.cl
ABSTRACT
]]> We tested the amplification potential of 8 microsatellites initially described for Odontesthes perugiae and O. argentinensis in 3 species of Odontesthes and 4 species of Basilichthys. Most of the microsatellites amplified and showed polymorphism; thus they will be useful in genetic conservation plans for these species.RESUMEN
Se prueba el potencial de amplificación de 8 microsatélites descritos inicialmente para Odontesthes perugiae y O. argentinensis en 3 especies de Odontesthes y 4 especies de Basilichthys. La mayoría de los partidores microsatélites amplificaron correctamente y presentan polimorfismo por lo que serán de utilidad en planes de conservación genética de estas especies.
Biodiversity protection measures should consider both the conservation of the habitat and the genetic variability of the species involved, in order to maintain effective population sizes and the evolutionary potential of the species (Reed & Frankham 2003; Reed 2004).
Among the molecular techniques frequently used in conservation studies, microsatellites have been particularly important. These molecular markers are tandem repeats of simple sequences (usually composed of about -0 - 50 repetitions of - - 6 base pairs) which are abundant and randomly distributed in the genome of the majority of eukaryotes. The variation in the number of repetitions produces considerable polymorphism (Hearne et al. -992). These genetic markers have been described as evolutionarily neutral with Mendelian inheritance (Jarne & Lagoda -996); thus they provide many advantages in the study of populations (Selkoe & Toonen 2006).
Although there are published protocols for locating these tandem repeats (e.g. Zane et al. 2002), specialized laboratory services are required to obtain the clones that allow development of microsatellite loci. Given the high cost of this process, some investigators in Chile are already using primers described for related species with good results (e.g. Canales-Aguirre et al. 20-0).
Most of aquatic species with conservation problems in Chile are those which inhabit rivers and estuaries (Habit et al. 2006). For many native species, life cycles, migrations, spatial patterns, age and population structure, among other aspects, are unknown, thus studies of these aspects are important in order to establish effective conservation plans. One of the groups of threatened species is that of the silversides of the genera Odontesthes and Basilichthys, which includes marine Odontesthes regia (Humboldt, -821); freshwater Odontesthes mauleanum (Steindachner, -896); estuarine Odontesthes brevianalis (Günther, 1880), and freshwater Basilichthys semotilus (Cope, -874), Basilichthys microlepidotus (Jenyns, -841) and Basilichthys australis (Eigenmann, -928) species.
Taking into account the need to undertake genetic conservation at the species level, and the scarce population information available for these species (e.g. Quezada-Romegialli et al. 20-0), this study tests the utility of microsatellite primers described for Atlantic species of the genus Odontesthes in species of the genera Basilichthys and Odontesthes species from rivers, estuaries and coasts of Chile.
Specimens of Odontesthes regia (from Iquique and Puerto Montt), O. mauleanum (Valdivia estuary), O. brevianalis (Conchalí wetland), Basilichthys semotilus (Lima, Perú), B. aff. semotilus (Loa River), B. microlepidotus (Choapa River) and B. australis (Maule River) were obtained from Chile and Perú (Table 1). Total genomic DNA was extracted from ethanol-preserved fin clips using the salt extraction method (Aljanabi & Martinez -997); purified DNA was stored at -20° C in 50 |iL of water until analysis.
]]> All the microsatellite primers (N = -2) described for Odontesthes perugiae (Odont09 and Odont 38) and Odontesthes argentinensis (Odont02, Odont07, Odont08, Odont--, Odont-6, Odont23, Odont25, Odont27, Odont29 and Odont39) by Beheregaray & Sunnucks (2000) were tested in a first step. The PCR reaction mixture had a final volume of -0 pL, including - .3 μL buffer PCR -0X (50 mM KCl, -0 mM tris-HCl, pH 8), 0.5 μL MgCl2 50 mM, 0.5 μL of the forward and of the reverse primers 50 ngμL, 2.4 μL dNTPs 2.5 mM, 4.68 μL H2O2, 0.-2 μL Taq polymerase. To this mixture we added -.5 μL of DNA 50 ngμL. The amplification protocol used a PCR touchdown method as described by Beheregaray & Sunnucks (2000), with the modifications indicated in the above paragraph.The PCR products obtained were visualized on -.5% agarose gels and stained with ethidium bromide for positive amplification. We selected 8 pairs of microsatellite primers which gave us the best amplification (Odont02, Odont07, Odont08, Odont09, Odont23, Odont27, Odont38 www.macrogen.com) for fragment analysis and allelic sizes were scored against the size standard LIZ-500 (Applied Biosystems) using PeakScanner software (Applied Biosystems).
Most of the primers amplified the microsatellite loci in all species under the conditions of our modified protocol (Table 1); the results showed a clean amplification and characteristic microsatellite peaks. Only two polymorphic microsatellite loci were found in Basilichthys aff. semotilus from the Loa River, while five were polymorphic in Odontesthes mauleanum. Comparing Chilean and Argentinean Odontesthes, four out of eight microsatellites had a shared range of allele size (Odont07, Odont08, Odont09 and Odont23); a similar pattern was observed when Odontesthes species were compared with the genus Basilichthys. Furthermore, microsatellites showed common alleles in almost all species studied here (e.g. allele -23 for Odon08; allele 1-9 for Odon02). In order to test Hardy-Weinberg Equilibrium (HWE), two sites containing 30 specimens of B. microlepidotus each were analyzed (Table 2). The test found not evidences of Hardy Weinberg disequilibrium for all loci, except the microsatellite Odont23 in samples from Choapa River. With the individuals sampled, only two loci (Odont27 and Odont38) could be compared among Argentinean Odontesthes and Chilean Basilichthys. For these loci, Basilichthys showed a lower number of alleles than Argentinean Odontesthes, as expected for microsatellites amplified from a related genus (see Barbará et al. 2007).
In conclusion, the microsatellite primers described by Beheregaray & Sunnucks (2000) can be used to amplify polymorphic loci in species of Odontesthes and Basilichthys from Chile with very clear allele peaks in the electropherograms.
Considering that watersheds in which these species are distributed are those with the greatest alterations in their aquatic systems (Vila et al. -999; Dyer 2000; Habit et al. 2006; Quezada-Romegialli et al. 2009), these variable microsatellites will be useful in future genetic conservation plans, allowing the monitoring of changes in allele frequencies over time, and complementing studies of genetic migration within and between watersheds, spatial structure of the populations and paternity analysis of Basilichthys and Odonthestes in Chile.
ACKNOWLEDGEMENTS
Thanks to R Gauci, M Espinoza, L Eaton, C Jara, R Quispe and H Ortega. Financial support: Fondecyt 1100341, 11060496, PFB-23, ICM P05-002 to DV; Doctoral CONICYT Grant to CQR.
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Recibido: 14.02.11
Aceptado: 05.05.11
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