Possibility of utilization of molecular methods for study of population genetics of noble crayfish Astacus astacus

The noble crayfish (Astacus astacus) is one of two native species of crayfish living in this country who are all over our country strictly protecte, because they are identified as critically endangered species. In Europe it is one of five native species of crayfish, reported by IUCN as an endangered species that needs protection management. Its population was dramatically reduced due to crayfish plagues, which carry non-native crayfish species from North America, who were introduced in the past in Europe and later to the Czech Republic. In the past, have been isolated and described microsatellite markers for crayfish (Koiv et al., 2008a). As part of this work was tested using the eight microsatellite markers on samples obtained from the czech population of crayfish. Testing was performed on 53 samples from six populations of crayfish. Test samples were subjected to isolation of DNA from tissues of the third walking legs using DNA Lego Kit. Then test isolate DNA electrophoresis on agarose gel. Testing the temperature cycle of PCR amplification based on the original publication Koiv et al., (2008a), followed by PCR cycles were adjusted according to the quality of PCR products obtained in our laboratory. Primer annealing temperature 60°C was chosen as the best for six tested loci i.e. Aas 3666, Aas 3115, Aas 790, Aas 1198, Aas 3950 and Aas 766, for two other loci Aas 2489 and Aas 3040 was chosen 55°C annealing temperature. The resulting PCR products were tested on agarose gel and subsequently fragment analysis on an automatic sequencer Beckman Coulter CEQ8000 determining the lenght of PCR products in multiplex consisting of several loci. In the individual loci were in our 53 samples found from 1 to 13 alleles ? one of the loci was monomorphic in all samples analyzed. The moravian population in the Boskovice tank showed the greatest variability, the average number of alleles per locus 3.86, then the north bohemian population from Jaroměř 3.43 alleles per locus. Zelenohorská population of 3 alleles at a locus and Světlohorská population of 2.57 alleles per locus. The lowest average number of alleles per locus had a population from Landštejn 2.43. The aim of this bachelor thesis was to develop a literature search of methods use in molecular biology studies of crayfish and also validate the use of eight microsatellite markers have described for the noble crayfish. In laboratory conditions were succesfully optimized using seven microsatellite markers from the eight described. These microsatellite markers should be used for population studies, or for determining parentage (paternity) in breeding experiments. The verification of the applicability of microsatellite markers have been evaluate and some fundamental characteristics of the population, usability testing and optimization of microsatellite markers, however, was done only on a very small number of samples, because these characteristics have only a very limited explanatory power.