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The goal of this study was to measure the utility of 10 pairs of starter sequences described scientifically to amplify microsatellites in the genome of canines and measure the usefulness of the commercial set StockMarks® for Dogs Canine Genotyping Kit from Applied Biosystems company in individual identification. An additional goal is to check the feasibility of using the set to control the origin of species from Canidae familly. The tests were conducted on animals from three species: Red Fox (Vulpes vulpes), Arctic Fox (Alopex lagopus), and Raccoon Dog (Nyctereutes procyonoides), from which blood was drawn into sterile vacuum test-tubes with the anticoagulant EDTA from the saphena vein. DNA was isolated from the blood using QIAamp DNA Blood Mini Kit, a set for isolating genetic material. The obtained products PCR were exposed to electrophoretic separation in the genetic analyzer ABI Prism 3100 Avant, then analyzed using the computer program Gene Mapper TM v. 3.5. The frequency of alleles was measured for all microsatellite loci. All calculations were performed using the statistical packet SAS - module SAS/GeneticsTM 9.1.3. The analysis of the microsatellite sequences (FHC 2010, FHC 2054, FHC 2079, PEZ1, PEZ20, PEZ12, PEZ3, PEZ5, PEZ6, PEZ8) in the population of the Arctic Fox, Red fox, and Raccoon Dog, indicates a high degree of polymorphism and high usefulness in the majority of tested microsatellite loci to control the origin of species and identification of specimen in fox and raccoon dog farms. Moreover, it has been confirmed that in many cases clear differences between the length of specific alleles indicates a potential use in identifying species.
The aim of the study was to assess the biodiversity of farmed fur animals from the Canidae family (common fox, polar fox, and raccoon dog) using nuclear and mitochondrial markers. The study involved 434 animals. The biological material included whole peripheral blood or skin tissue. The isolated genetic material was subjected to qualitative and quantitative analyses. Mitochondrial DNA (mtDNA) gene fragments (COX1, COX2, CYTB) and nuclear DNA (nDNA) gene fragments (MSTN1, MSTN2, MSTN3, IGF1, GHR) were amplified with the PCR (polymerase chain reaction) technique. The amplicons obtained were sequenced or subjected to PCR-RFLP (restriction fragment length polymorphism) reaction, and bioinformatics analyses were performed. The interspecific analysis of the nDNA sequences revealed a total of 25 polymorphisms. On the other hand, the interspecific analysis of the mtDNA gene fragments identified 277 polymorphisms. The COX1 gene fragment exhibited the greatest variability. It was shown that the frequency of polymorphisms within the mitochondrial genome was almost 20-fold higher than that in the nuclear genome of the raccoon dog. It was found that the genetic distances revealed by the analysis of the mitochondrial gene fragments were similar to the results obtained by the nDNA analysis. The genetic distance between the raccoon and common fox was the greatest. The smallest phylogenetic distance was revealed between the two fox species. The study results indicate mitochondrial and nuclear genes may be alternatively used for determining the phylogenetic relationships between fur animals from the Canidae family.
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