Research On Kunming Dog's Genetic Polymorphisms And Multiplex PCR Of Fifteen Microsatellite Loci

As a class of highly polymorphic DNA marker, microsatellite DNA, named as short tandem repeats(STR), is widely applied to genetics and forensic science such as making physical map, guiding the breeding of animal and plant, paternity test and so on. Being an excellent purebred dog, Kunming dog which was bred by Chinese has been bred into three steady inherited breeds wolf cyan, grass yellow and black back Kuning dog. At the first part of this dissertation, fifteen highly polymorphic canine STR loci (PEZ1, PEZ2, PEZ3, PEZ5, PEZ6, PEZ8, PEZ12, PEZ15, PEZ20, FHC2010, FHC2054, FHC2079, FHC2087Ub, FHCL2132, FHC2611) were chosen and the primers were designed by ourselves. By PCR (Polymerase Chain Reaction) and non-denatured PAGE (Polyacrylamide Gel Electrophoresis) and silver-staining, we analyzed the genotype of 74 Kunming dogs at 15 STR loci and computed the statistical data in population genetics and forensic science. For the fifteen canine STR loci, it shows that the average Heterozygosity (H), the average Ploymorphism Information Content (PIC), the Cumulative probability of paternity exclusion (CPE) and the Total Power of Discrimination (TDP) is 0.638, 0.604, 0.9996 and 0.999999999997 respectively. Twelve loci correspond to Hardy-Weinberg equilibrium except PEZ15, FHC2079 and FHC2087Ub. The evolutionary relationships among three kinds of Kunming dog and German shepherd were estimated by genetic distance Ds and Da-With two methods Neighbour-Joining (NJ) and unweighted pair group method with arithmetic (UPGMA), we constructed evolution tree among them. According to the pairwise genetic distances, wolf cyan Kunming dog and grass yellow Kunming dog had shorter diverse evolutionary histories and black back Kunming dog and German shepherd had shorter diverse evolutionary histories.In the first part of this dissertation, we adopted singleplex PCR which was time-consuming and expended largely with high cost and heavy work, especially when there were a lot of samples to be tested. In contrast with singleplex PCR, multiplex PCR consisted of two or more loci, is more used for the merits that it is low-cost and timesaving with a little sample DNA and plentiful information. At the second part of this dissertation, we substituted FHC2132 for FHCL2132 and redesigned the primers for PEZ6, PEZ15 and FHC2054 after investigating the range of alleles of 15 loci severally. We have five multiplex systems as follow: I (PEZ2, FHC2079, PEZ8), II...