Construction of dams, overfishing and the introduction of hybrids from aquaculture have placed populations of the catfishes Pseudoplatystoma reticulatum and Pseudoplatystoma corruscans at continuous risks of genetic extinction. These taxa have not been assessed for the IUCN, and the development of multiallelic markers, as microsatellites, may provide important genetic data for management and conservations plans.

Genomic DNA was extracted from fin clips following the “Wizard Genomic DNA Purification Kit” (Promega) protocols. The DNA from three individuals of P. reticulatum was pooled on a Roche 454 GS FLX sequencer using the Titanium platform “Genome sequencer 20 System”. Primers were designed using Primer3 software (Rozen and Skaletsky 2000). We studied 22 individuals of P. reticulatum from the Cuiaba River, Paraguay basin, Brazil. Cross species amplification were conducted on 12 specimens of P. corruscans from four populations of Paraguay and Upper Parana basins, Brazil.

Multiplex PCRs were performed in a final volume of 10 μl containing 100 μM of each dNTP, 1.5 mM MgCl2, 1× Taq buffer, 0.5 units Taq polymerase (Quiagen Master mix), 0.4 μM reverse primer, 0.4 μM fluorescently-labeled forward primer (Applied Biosystems), 30 ng of genomic DNA; and performed in a thermal cycler Veriti™ (Applied Byosystems) for 15 min. at 95 °C followed by 30 cycles of 30 s at 94 °C, 1 min at 58 °C, 1 min at 72 °C and a final extension of 60 °C for 30 min. Each multiplex PCR was loaded on an automated DNA sequencer ABI prism_3730 (Applied Biosystems). Allele sizes were scored using Gene Mapper 3.7 (Applied Biosystems).

Genetic diversity estimations using Cervus 3.0 (Marshall et al. 1998) revealed a mean of 7.8 alleles per locus and an average expected heterozygosities (He) of 0.728 for P. reticulatum (Table 1). Genepop 3.4 (Raymond and Rousset 1995) demonstrated significant deviations from Hardy–Weinberg Equilibrium (HWE) (P < 0.05) for Prt3, 37 and 39. Microchecker 2.2.3 (Van Oosterhout et al. 2004) suggested the presence of null alleles for Prt3 and Prt37, reflected by a homozygote excess. P. corruscans presented 14 polymorphic microsatellites (medium He of 0.710 for polymorphic loci) (Table 1) and Prt4, 37 and 39 were not in HWE, what can be explained by Wahlund effect. Moreover, six loci presented non-overlapping allelic sizes between species and can be future tested as diagnostic markers. These results indicated that the microsatellites here developed exhibited sufficient allelic variation to be successfully applied in genetic studies of these species, including population genetics and hybridization studies.

Table 1 Primer sequences and characterization of 16 microsatellites in P. reticulatum and P. corruscans
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