The Genetic Diversity Of 12 STR Loci In Four Dog Breeds And The Application In Individual Discrimination

The purpose of this study was:1) to analyze genetic diversity of the four dog breeds and provide a theoretical basis for breeds conservation and breeding.2) to empolder the domestic canine identification kit by the application of fluorescent multiplex amplification technology, establish convenient, fast, standard dog DNA detection methods, lay the groundwork for the establishment of Chinese working dog DNA database;3) enrich the capacity of Chinese working dog DNA database, providing technical support for criminal investigation and ensure the benefit of the victims.The first part experimental animals were 244 working dogs, including 72 Labrador Retrievers,49 English Springer Spaniels,52 German Shepherd dogs and 71 Tibetan Mastiffs; the second part test samples were a few hair follicle and blood samples from a medium-sized dog who attacking human, as well as salivary stain on the cloth. The main content of this experiment included the following two aspects:1. Study on genetic diversity of four dog breeds population by 12 STRWe constructed the fluorescently labeled multiplex ampliction system, including 12 STR loci (FHC2010, FHC2054, FHC2087, FHC2132, FHC2137, FHC2302, FHC2328, FHC2412, FHC2611, PEZ2, PEZ20 and PEZ3).To investigate the variability of 12 canine microsatellite loci in four dog breeds,Polymorphism information content (PIC), index of genetic diversity (H), expected heterozygosity (h(e)), observed heterozygosity (h(0)),effective number of allele (ne), genetic distance, cluster analysis and F-statistic were evaluated.And the forensic data of 12 STR loci were also analyzed, the result showed that: (1) the 12 fluorescent-labeled STR loci multiplex system had been successfully constructed, good amplification product,good intracolor and intercolor balance, no extra peak and no overlapping, the PHR(peak height ratio) concentrated on 1500-2000RFU.(2) 211 alleles were detected in 12 STR loci, gene frequency distribution was between 0.007 and 0.590, the average number of alleles for each STR was 18.58, indicating that all loci in four dog breeds were high polymorphic;.(3) All STR loci were highly polymorphic(PIC>0.5), the average PIC of Labrador Retriever, English Springer Spaniel, German Shepherd dog and Tibetan mastiff. was 0.763,0.768,0.728 and 0.847,the average heterozygosity of 4 dog breeds was0.844,0.802, 0.720 and 0.851.(4) The total population inbreeding coefficient (Fit) of 4 dog breeds was 0.0680, the within group inbreeding coefficient (Fis) was -0.0054, inter-group genetic differentiation coefficient (Fst) was 0.073, indicating 7.3% of genetic variation was from the inter-group, while 92.7% of genetic variation was caused by the differences between individuals within group; the average gene flow (Nm) was 3.1739.(5) The genetic distance between German shepherd dog and Labrador retriver was farthest (0.9171), while the genetic identity between them was minimum (0.3997); the genetic distance between Springer Spaniel and Tibetan Mastiff was nearest (0.3289), while the genetic identity between them was highest (0.7197). Cluster analysis results showed that the German Shepherd dog was outside-group, the phylogenetic tree showed that the German Shepherd dog clustered as a single group, Labrador together clustered with the Tibetan mastiff as a group and then clustered with the Springer Spaniel, the clustering results was generally consistent with their breeding history.(6)Forensic analysis showed that:the average heterozygosity was 0.811, the average PIC was 0.777.And the Cumulative matching probability (TPm), the the Total Power of Discrimination (TDP) and Cumulative probability of paternity exclusion (CPE) in the four Dog breeds were as follows:Labrador Retriever,3.56×10-13, 0.9999999999996 and 0.99999999565 respectively; Springer Spaniel,1.83×10-13,0.9999999999998 and 0.99999803150 respectively; German Shepherd Dog,1.33 ×10-11,0.9999999999867 and 0.99981801953 respectively; Tibetan mastiff,3.29×10-16,1.00000000000 and 0.99999995452 respectively.2. The individual identification of suspected biting dog by the application of 12 fluorescent-labeled STR multiplex amplificationAfter we received biological specimens, we took DNA testing in the 12 fluorescent-labeled STR loci multiplex system (FHC2010, FHC2054, FHC2087, FHC2132, FHC2137, FHC2302, FHC2328, FHC2412, FHC2611, PEZ2, PEZ20 and PEZ3). The biological specimens included salivary stains on the pants,,the attacking's dog hair follicle samples, as well as suspected dog's blood sample. By DNA genotype matching,,we found that the DNA genotype of Suspects' blood sample was completely consistent with hair follicle samples and salivary stains.And the probability of match(PM) was 4.2×10-19, likelihood ratio (LR) is 2.38×1018. According to these data,we could confirm that the Suspected dog was just the attacking dog,providing a theoretical basis to protect interests of the victim.