Description of novel microsatellite loci in the Neotropical fish Prochilodus argenteus and cross-amplification in P. costatus and P. lineatus

Abstract

Prochilodus is one of the most important fish resources of South America, in addition to the important role it plays in nutrient cycling of Neotropical rivers. In the present study, we describe the isolation and characterization of nine novel microsatellite loci in Prochilodus argenteus. The number of alleles per polymorphic locus varied from 5 (Par76) to 21 (Par85), revealing a total of 116 alleles. The values of observed and expected heterozygosities ranged from 0.629 (Par69) to 0.926 (Par85 and Par86) and from 0.643 (Par66) to 0.931 (Par80), respectively. Furthermore, the ability of these and other previously described microsatellite markers to amplify orthologous loci was tested in two related species, Prochilodus costatus and Prochilodus lineatus. These loci will be useful for studies of population genetic structure in this group of fishes, and in aiding future genetic mapping studies of P. argenteus.Key words: cross-species amplification, enrichment libraries, microsatellite, Prochilodus. Family Prochilodontidae constitutes one of the most important fish resources of South America (Bayley and Petrere, 1989), in addition to its important role in nutrient cycling in Neotropical rivers (Flecker, 1996). Prochilodus comprises 49 nominal species of which only 13 species are valid (Castro and Vari, 2003). Among the species found in the São Francisco River basin, Prochilodus argenteus, popularly known as curimatã-pacu, forms a bulk of the subsistence fishery of the region, although numbers harvested have drastically declined in the last years (Sato and Godinho, 2004).Microsatellites are polymorphic DNA sequences containing short tandemly arranged repetitions (Tautz, 1989), distributed throughout the genome (Litt and Luty, 1989), and found in all prokaryotic and eukaryotic genomes studied until now (Zane et al., 2002). Due to their high variability, these genetic markers have been widely used in genetic mapping (Knapik et al., 1998;Shimoda et al., 1999;Coimbra et al., 2003) and population structure studies Primmer et al., 2006). However, one of the great impediments for the wider use of microsatellites is the need to isolate and characterize these markers via cloning and sequencing of genomic libraries for each species of interest (Angers and Bernatchez, 1997). Nevertheless, once the flanking sequences of microsatellite markers are known, a large number of individuals may be rapidly genotyped.Although the Neotropical ichthyofauna is the world's most diversified (Lowe-McConnel,1969;1987), microsatellite primers have been published only for Piaractus mesopotamicus (Calcagnotto et al., 2001), Astyanax fasciatus (Strecker, 2003), Arapaima gigas (Farias et al., 2003), Brycon opalinus (Barroso et al., 2003), Eigenmannia sp. (Moysés et al., 2005), Pseudoplatystoma corruscans (Revaldaves et al., 2005), Brycon hilarii (Sanches and Galetti, 2006) and Prochilodus costatus (Carvalho-Costa et al., 2006). The isolation and characterization of microsatellite loci has also been performed in P. argenteus, and thirteen loci ...

Cross-amplification of heterologous microsatellite markers in Piracanjuba

“…Similar to genus Brycon, it has been shown that different species of Prochilodus share transferability of microsatellite primers (BARBOSA et al, 2008). However, current analysis has shown for the first time that cross-amplification was possible between the genera through Par80 (P. argenteus).…”

The roles of marginal lagoons in the maintenance of genetic diversity in the Brazilian migratory fishes Prochilodus argenteus and P. costatus

“…Genomic DNA was extracted using a saline solution with proteinase K described by Aljanabi & Martinez (1997). Amplifications were carried out using six microsatellite loci developed by Barbosa et al (2008) for Prochilodus argenteus: Par66, Par69, Par71, Par76, Par83 and Par85; the same loci were used for P. costatus, exception the locus Par80 was used instead of Par76 (monomorphic for this species). The reactions were performed in a total volume of 12.5 µl with 7.3 µl of ultrapure H 2 O, 1.25 µl of Taq DNA buffer (10X), 0.45 µl of MgCl 2 (50 mM), 0.4 µl of dNTP (2 mM), 0.5 µl of each primer (10 µM), 0.1 µl of Platinum Taq DNA Polymerase enzyme (Invitrogen; www.invitrogen.com) (5 U/µl) and 2.0 µl of genomic DNA (10-50 ng).…”

Genetic population structure of two migratory freshwater fish species (Brycon orthotaenia and Prochilodus argenteus) from the Sao Francisco River in Brazil and its significance for conservation

“…Genetic diversity was analyzed using ten specific microsatellite loci: Par12, Par14, Par15, Par21, Par35 (Barbosa et al, 2006), Par66, Par69, Par80, Par82 and Par85 (Barbosa et al, 2008) (Table 1). PCRs were performed using the method described by Schuelke (2000) and were carried out in a final volume of 10 µL, containing 100 ng of DNA, 200 μM of dNTPs, 1 x PCR buffer (20 mM Tris-HCl, pH 8.4 and 50 mM KCl; LGC Biotecnologia), 4 pmol of each reverse and 6-FAM or NED M13 (-21) primers as well as 1 pmol of the forward primer, 1.5 mM MgCl 2 and 1 U of Taq DNA Polymerase (LGC Biotecnologia).…”