Allele Frequencies and the Probability of Identity for 18 STR Loci Generated via the Canine GenotypesTM Panel 2.1 Kit in Golden Retriever, Miniature Dachshund, and Shiba Inu Groups for Forensic Applications

1. Introduction

According to the Japan Pet Food Association (JPFA), approximately 7 million dogs were bred and raised in Japan in 2022 [1]. According to the report, 9.7% of Japanese households had one or more purebred dogs. The human–dog (or human–companion animal) bond is strong in Japan. Dogs are very attached to human society. Forensically relevant cases that involve dogs include dog bite accidents and the identification of affected or illegally dumped animals. The estimate for dog bites is more than 40,000 cases in the last 10 years in Japan [2]. According to the JPFA report, approximately 6,500 dogs were either killed or separated from their owners due to the Great East Japan Earthquake that struck on March 11, 2011 [3]. The total number of dog abandonments reported in the newspapers exceeds 100 dogs [4].

The utility of mitochondrial DNA (mtDNA) [5,6,7,8,9,10,11,12,13,14,15,16] and/or short tandem repeat (STR) polymorphisms in forensic analysis has been extended in recent years [17,18,19,20,21,22,23,24,25]. In Japan, very little research on forensic genetics has focused on STR typing in dogs. Recently, the Canine genotype^TM panel 2.1 Kit (Thermo Fisher Scientific, Waltham, MA, USA) can be used to determine 18 STR loci and sex, and it is commercially available and can be purchased in Japan. Then, we first show the identity data for 18 primarily autosomal STR loci tetranucleotide markers, and we use the kit on 150 purebred dogs: 50 non-related Golden Retrievers, 50 Miniature Dachshunds, and 50 Shiba Inus.

2. Materials and Methods

The material used comprised DNA stored in our laboratory, which was previously extracted from blood using the QIAamp DNA Blood Mini Kit (QIAGEN, Germantown, MD, USA). The blood samples were obtained from the Department of Veterinary Clinical Pathology at Nippon Veterinary Life Science University (NVLU) in Musashino, Tokyo; they were originally collected at the Veterinary Medical Teaching Hospital at NVLU with the written consent of each owner.

We selected two purebred breeds that are relatively popular in Japan and that have been analyzed before using the Canine Genotypes Panel 1.1 Kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, we used dog DNA from non-related 50 Golden Retrievers, 50 Miniature Dachshunds, and 50 Shiba Inus. DNA concentrations were measured with Nano Drop (Thermo Fisher Scientific).

The amplification of 18 STR loci (PEZ02, PEZ17, FH2017, FH2309, PEZ05, FH2001, FH2328, FH2004, FH2361, PEZ21, FH2054, FH3377, FH2107, FH2088, vWF.X, FH2010, PEZ16, and FH3313) and one sex-determinant locus (ZFX and ZFY) was performed using the Canine Genotypes Panel 2.1 Kit (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions (Table 1). Capillary electrophoresis was conducted using a 3730xl DNA Analyzer (Applied Biosystems, Waltham, MA, USA) in accordance with the manufacturer’s instructions.

To carry out fragment analysis, Peak Scanner Fragment Analysis 2 (Thermo Fisher Scientific) was used. Moreover, statistical analysis was performed using Cervus (http://www.fieldgenetics.com/pages/home.jsp, accessed on　1 October 2023). Cervus was used to determine expected heterozygosity (He), observed heterozygosity (Ho), p-value (P) for the Hardy–Weinberg equilibrium exact test using a Markov chain, the power of the discriminant (PD), the power of exclusion (PE), polymorphic information content (PIC), and matching probability (MP). The accumulated power of discrimination (APD), which is a comprehensive PD, was calculated from the PD of 18 loci. Similarly, APE was also calculated using the PE of 18 loci.

3. Results and Discussion

The allele frequencies of the 18 canine STR loci in 50 Golden Retrievers, 50 Miniature Dachshunds, and 50 Shiba Inu groups are shown in Table 1. A total of 130 alleles were observed with respect to 18 STR loci in the purebred Golden Retriever group, with allele frequency distributions from 4 to 16; 126 alleles were observed in the purebred Miniature Dachshund group, with frequency distributions from 3 to 15; 131 alleles were observed in the Shiba Inu group, with allele frequency distributions from 3 to 17. The average number of alleles per locus was 7.2 in the Golden Retriever group, 7.0 in the Miniature Dachshunds group, and 7.3 in the Shiba Inu group. In the purebred group, FH3313 exhibited the largest allele numbers, with a total of 16 in the Golden Retriever group, 15 in the Miniature Dachshund group, and 17 in the Shiba Inu group. The lowest number of alleles was observed in the following: PEZ02, PEZ05, PEZ21, and vWF.X in the Golden Retriever group; FH2010 in the Miniature Dachshund group; and FH2017 in the Shiba Inu group.

Table 2 also shows that the forensic application values of 18 STR loci in the Golden Retriever, Miniature Dachshund, and Shiba Inu groups were explored by calculating forensic parameters, including the power of the discriminant (PD), power of exclusion (PE), polymorphic information content (PIC), matching probability (MP), and accumulated power of discrimination (APD). In the Golden Retriever group, the He value ranged from 0.471 to 0.882 with an average of 0.677. The Ho value ranged from 0.340 to 0.780 with an average of 0.588. PIC values ranged from 0.428 to 0.860 with an average of 0.626. The PD, PE, and RMP values ranged from 0.677 to 0.972, 0.258 to 0.748, and 0.028 to 0.323, respectively. APD, which was calculated via the PD of 18 STR loci was 1–3.257 × 10⁻¹⁶. In other words, Random Match probability (RMP) was 3.257 × 10⁻¹⁶ for 18 STR loci in Golden Retrievers groups. In the Miniature Dachshund group, the He values ranged from 0.481 to 0.891 with an average of 0.727. The Ho values ranged from 0.380 to 0.860 with an average of 0.652. PIC values ranged from 0.409 to 0.872 with an average of 0.675. The PD, PE, and MP values ranged from 0.658 to 0.976, 0.232 to 0.767, and 0.024 to 0.342, respectively. APD, which was calculated via the PD of 18 loci, was 1–3.933 × 10⁻¹⁸. RMP was 3.933 × 10⁻¹⁸ for 18 STR loci in Miniature Dachshunds groups. In the Shiba Inu group, He values ranged from 0.500 to 0.865 with an average of 0.732. Ho values ranged from 0.354 to 0.820 with an average of 0.604. PIC values ranged from 0.382 to 0.840 with an average of 0.682. The PD, PE, and MP values ranged from 0.632 to 0.964, 0.196 to 0.717, and 0.036 to 0.368, respectively. APD, which was calculated via the PD of 18 loci, was 1–2.107 × 10⁻¹⁸. RMP was 2.107 × 10⁻¹⁸for 18 STR loci in the Shiba Inus groups.

We investigated the genetic polymorphisms of 18 STR loci using the Canine Genotypes Panel 2.1 Kit in three purebred Golden Retriever, Miniature Dachshund, and Shiba Inu groups. The results of the genotype showed that this kit would be informative for the identity testing of canines in Japan. We also previously applied the Canine Genotypes Panel 1.1 Kit on 40 Golden Retrievers and 40 Miniature Dachshunds, and APD was estimated to be 1–1.4337 × 10 ⁻¹⁴ (G.R.) , 1–1.61466 × 10⁻¹⁵ (M.D.) [27]. Both kits performed equally well with respect to obtaining genotyping results for the identity testing of canines. Moreover, the interruption of the peak's size was easier to distinguish in Panel 2.1, which was based on tetranucleotides, than in Panel 1.1, which was based on dinucleotide structures, in our laboratory. Our findings correspond with a previous paper by Kanthaswamy et.al [24].

Fang et al. have reported that the 19 STR loci have forensic application values in Shiba Inu groups via the commercial PBG Canine Genotype STR system (Beijing Protect Baby Technology, BeijinChina) [26]. In this study, we also showed population data with high genetic polymorphisms relative to the Shiba Inu breed in Japan, which was determined using the Canine Genotypes Panel 2.1 Kit. The Shiba Inu is an ancient breed of Japanese origin. The STR analysis is also informative for pedigree management.

4. Conclusions

In this study, 18 STR loci exhibited high genetic polymorphism in Golden Retrievers, Miniature Dachshunds, and Shiba Inus using the Canine Genotypes Panel 2.1 Kit. This is the first time Japanese canine population data were reported using this kit. The results of the genotype showed that the Panel 2.1 Kit could be informative for the identity testing of canines in Japan.