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The new-generation 454 GS-FLX Titanium pyrosequencing  was used to isolate microsatellite markers for the Brazilian Guanabara frog, Euparkerella  brasiliensis, an Atlantic forest endemic species. Three multiplex  polymerase chain reaction sets were optimized for genotyping of 11 polymorphic  (di- and tetranucleotide) microsatellite markers. Genetic diversity was  assessed in 21 individuals from a population (Reserva Ecológica de Guapiaçu,  REGUA) located in the central region of the Rio de Janeiro State, in Brazil.  The mean number of alleles per locus ranged from 3 to 12. Observed and expected  heterozygosities ranged from 0.095 to 0.905 and from 0.094 to 0.904,  respectively. After using the Bonferroni correction for multiple tests, there  was no evidence of linkage disequilibrium between pairs of loci but deviations  for Hardy-Weinberg equilibrium were found in 4 loci. We found no evidence for  allele dropouts or stuttering, but we detected the presence of null alleles at  loci Eb10 and Eb36. These markers will be useful for analyses of fine-scale  population structure and determination of relative effects of habitat loss and  fragmentation on population genetic variability within species.

genetics and molecular research

Methodology Development of microsatellite markers for the Neotropical endemic Brazilian Guanabara frog, Euparkerella brasiliensis, through 454 shotgun pyrosequencing