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- Highly polymorphic microsatellite loci for the Parsley frog (Pelodytes punctatus): characterization and testing for cross-species amplificationPublication . Van De Vliet, M. S.; Diekmann, O. E.; Serrão, Ester; Beja, PedroA microsatellite library was developed using genomic DNA of the Parsley frog, Pelodytes punctatus, an amphibian species which inhabits Mediterranean temporary pond systems. Number of alleles and heterozygosity ranged from 10 to 25 and from 0.66 to 0.90, respectively. Crossspecies amplification was successful for 13 of the 15 developed loci for the related species, Pelodytes ibericus. The high levels of polymorphism revealed by these loci will be extremely useful for characterizing the population genetic diversity and structure and to estimate levels of dispersal and gene flow in the species P. punctatus and P. ibericus.
- Isolation of highly polymorphic microsatellite loci for a species with a large genome size: Sharp-ribbed salamander (Pleurodeles waltl)Publication . Van De Vliet, M. S.; Diekmann, O. E.; Serrão, Ester; Beja, PedroFourteen highly polymorphic microsatellite markers were developed and characterized for the sharp-ribbed salamander, Pleurodeles waltl. Isolating microsatellites with more than 12 single repeat type units was only successful for a tetranucleotide repeat (ATAG). Compared to microsatellite libraries constructed simultaneously for two anuran amphibian species, a greater number of primer pairs designed for P. waltl had to be discarded, due to consistent amplification problems. Low amplification success rate for P. waltl may be due to its larger genome size. Consequently, to avoid nonspecific binding and to increase amplification success, polymerase chain reaction programmes with touchdown cycles were used. For 14 microsatellite markers, amplification was successful and consistent with number of alleles and expected heterozygosity ranging from seven to 22 and from 0.79 to 0.94, respectively. All 14 microsatellite markers will be extremely useful for metapopulation studies of this unique amphibian species.
- Development and characterization of highly polymorphic microsatellite loci for the Western Spadefoot toad, Pelobates cultripesPublication . Van De Vliet, M. S.; Diekmann, O. E.; Serrão, Ester; Beja, PedroNine highly polymorphic microsatellite markers were isolated and characterized for the Western Spadefoot, Pelobates cultripes. Remarkably, for this amphibian species high numbers of microsatellites were found as part of larger repeat containing regions, making primer design difficult. For nine loci, primers were designed successfully and genotyping of individuals was reliable and consistent. Number of alleles and heterozygosity for these loci ranged from 9 to 34 and from 0.72 to 0.94, respectively. The high levels of polymorphism revealed by our developed loci should provide insight into population genetic structure and levels of dispersal for this typical Mediterranean temporary pond-breeding amphibian.