Forensic Application Evaluation of a Novel Canine STR System in Pembroke Welsh Corgi and Shiba Inu Groups

Aim To evaluate the forensic application values of 19 autosomal short tandem repeat (STR) loci in canines. Methods The 19 STR loci in two canine groups (Pembroke Welsh Corgis, n = 200; Shiba Inus, n = 175) were analysed by the capillary electrophoresis platform. The allele frequencies and forensic parameters were calculated, and the genetic relationships between these two canine groups and a previously reported Labrador group were analysed. Results These two canine groups conformed to the Hardy-Weinberg equilibrium at all STRs except for locus VGL3438 in the Pembroke Welsh Corgi group, and there was no linkage disequilibrium among pairwise loci at the 19 STRs. All STRs were polymorphic in the Pembroke Welsh Corgi and Shiba Inu groups, of which the locus C38 had the highest polymorphism. And it was found that the genetic relationship between the Pembroke Welsh Corgi and Labrador groups were closer in the three canine groups (Pembroke Welsh Corgi, Shiba Inu and Labrador). Conclusion The 19 STR loci had high genetic polymorphisms and forensic application values in Pembroke Welsh Corgi and Shiba Inu groups.


Introduction
In recent years, it has been found that the study of nonhuman DNA genetic polymorphism has great significance for case investigation in the forensic practice, especially for canine which is closely related to human. Canine is the most frequently kept pet in the world today. Meanwhile, caninerelated cases are increasing rapidly, such as cruelty to animal, attack on people or animal, involvement in crime scene, property damage, and the identification of lost pet. In 1999, Schneider et al. used mitochondrial DNA as case evidence that were extracted from canine hair [1]. In 2002, Padar et al. analysed short tandem repeats (STR) loci to detect a case involving a Hungarian canine which attacked a child to death [2]. In 2004, Wang et al. successfully extracted the canine DNA from the victim's mixed stain and genotyped by using the canine STR multiplex amplification technology in a vicious rape case involving domestic canine, and identified that the mixed stain contained the canine sperm. In 2016, Barrientos et al. reported a robbery and homicide case in which highly degraded DNA was extracted from canine stool samples [3]. In 2017, a fatal attack case was solved with canine genetic markers by Ciampolini et al. [4].
The analyses of DNA genetic polymorphisms are helpful for the identifications and pedigree controls of canine individuals. In 2011, the International Society for Forensic Genetics (ISFG) clarified that the non-human DNA analysis was similar to human DNA testing [5]. STRs are widely existing tandem repeat sequences with fragment length polymorphisms in the biological genome. It has been widely used in forensic identification and paternity testing. The STR analysis is not only low-cost and easy to operate in the capillary electrophoresis platform but also a widely popularized and highly utilized method in primary forensic DNA laboratories.
The molecular genetic markers in canine DNA also need to be evaluated in different groups like the genetic markers in human DNA. However, there are still relatively few studies on genetic polymorphisms and forensic application values of molecular genetic markers in different canine groups. Pembroke Welsh Corgis, the long-bodied and short-legged canines with erect ear and fox-like head, are the small canines originated from Wales of England. Shiba Inu is a kind of canine which has ancient origin from Japan. In this study, 200 purebred Pembroke Welsh Corgis and 175 purebred Shiba Inus were used as research objects to evaluate the genetic polymorphisms of 19 STR loci. It can not only assist in breeding pure Pembroke Welsh Corgis and Shiba Inus but also provide scientific evidences for the case investigations involving Pembroke Welsh Corgis or Shiba Inus in forensic caseworks.

Materials and Methods
2.1. Sampling and DNA Extraction. Saliva samples were taken from 200 purebred healthy Pembroke Welsh Corgis and 175 purebred healthy Shiba Inus based on the recommendations of the ISFG on non-human DNA analysis and the ethics committees of the Xi'an Jiaotong University Health Science and Southern Medical University. All canines were registered with the National General Kennel Club. Informed consents had been obtained from the owners of the canines before the study began. Genomic DNA was extracted by using the Chelex-100 method [6]. . The 19 autosomal STR loci (PEZ02,  PEZ20, FH2010, FH2054, FH2001, vWF.X, FH2088,  PEZ21, PEZ17, FH2328, FH2361, VGL2136, VGL3235,  PEZ01, VGL3438, FH2004, C38, FH2611, and FH2137) of the commercial PBG Canine Genotype STR system (Beijing Protect Baby Gene Technology, China) were amplified in a

Data Analyses.
The Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were tested by the Arlequin software v3.5 [7] and the SHEsis online software [8], respectively. The allele frequencies and forensic parameters of STRs were calculated by the online software STRAF [9]. The inbreeding coefficient (F IS ) was evaluated by genepop software v4 [10]. The principal component analysis (PCA) was performed by the R software v3.6.2 (https:// cran.r-project.org/bin/windows/base/old/3.6.2/). The phylogenetic tree was conducted by MEGA X software [11]. The genetic structure was analysed by the STRUCTURE software v 2.3.4 [12], and the optimal K value was calculated by the online software Structure Harvester [13].  C38  FH2001  FH2004  FH2010  FH2054  FH2088  FH2137  FH2328  FH2361  FH2611  PEZ01  PEZ02  PEZ17  PEZ20  PEZ21  VGL2136  VGL3235  VGL3438 vWF.X vWF.X  Linkage correlation coefficient could be used to not only test the existence of LD but also evaluate the strength of LD. The correlation coefficient between the two loci was reflected by r 2 values in this study. Figures 1(a) and 1(b) showed the r 2 values of the linkage disequilibrium tests, in which 0 represented the value less than 0.01, 1 represented the value was from 0.01 to 0.02, and 2 represented the value was from 0.02 to 0.03. The results showed that all r 2 values were less than 0.02, indicating that there was no linkage disequilibrium at the 19 STR loci in these two canine groups.
The F IS was calculated to evaluate the level of inbreeding in these two canine groups. The F IS value was 0.0619 in the Pembroke Welsh Corgi group, while the F IS of the Shiba Inu group was 0.0865. Figure 2, the forensic application values of 19 STR loci in two canine groups were explored by calculating forensic parameters including matching probability (MP), polymorphic information content (PIC), observed heterozygosity (Ho), expected heterozygosity (He), power of discrimination (PD), and power of exclusion (PE).

Forensic Parameters. As shown in
In the corgi group, the Ho values of 19 STR loci ranged from 0.3000 to 0.8250, and the He values were from 0.2965 to 0.8426. The heterozygosity values (Ho and He) of loci PEZ01 and FH2004 were less than 0.5, and there were 7 loci (C38, VGL2136, FH2328, FH2137, PEZ02, FH2611, and FH2001) which were greater than 0. 7 Figure 3, the differences of allele numbers at 19 STRs were compared among these two studied canine groups and a Labrador group (n = 214) [14]. The allele numbers of locus FH2137 were the largest in three canine groups, while the loci FH2010, FH2088, PEZ01, PEZ21, and vWF.X were relatively small.

Interpopulation Genetic Analyses. As shown in
The PCA and genetic structure analyses were performed to reveal the genetic structure of three canine groups. As shown in Figure 4(a), the clustering diagram of PCA divided the Pembroke Welsh Corgis, Shiba Inus, and Labradors into three clusters, and the small parts of three clusters overlapped with each other: Shiba Inus fell on the upper left of the axis, Labradors fell on the upper right, and Pembroke Welsh Corgis located on the lower. The numbers of assumed populations (K) were set to 2-7 in the structure analysis, and the optimal K value was determined to be 3 after calculations. As shown in Figure 4(c), the three canine groups had different ancestral components when K = 3. In Figure 4(b), a phylogenetic tree was conducted to understand the genetic relationships among these groups, and the results showed that the genetic relationship between the Pembroke Welsh Corgi group and the Labrador group was closer in all three groups.

Discussion
In this study, we investigated the genetic polymorphisms of 19 STR loci in 200 Pembroke Welsh Corgis and 175 Shiba Inus. Canine samples were collected, stored, and conducted in the manner that were similar with corresponding human forensic DNA analysis process, and the allele frequencies and forensic genetic parameters of the 19 loci in these two canine groups were evaluated, respectively, according to the recommendations of ISFG on non-human DNA analysis in forensic cases [5].
The present results showed that there was no linkage disequilibrium among the 19 STR loci, and only one locus  BioMed Research International (VGL3438) deviated from Hardy-Weinberg equilibrium in the corgi group, which may be the reason for the sampling.
In the canine breeding process, inbreeding is often the most common way to pursue the purity of the breed. And canine pedigrees are usually not as well documented as those of humans, so it is difficult to establish the canine family tree. Therefore, the disturbance of the inbreeding still could not be completely avoided, although we tried to track breeding to avoid collecting the biological related samples in the sampling process, and exclude possible genetic related samples after comparing the genotyping results. How to circumvent this problem is still the difficulty of non-human DNA analysis. In addition, F IS values were calculated to evaluate the level of inbreeding in this study, and the results showed the inbreeding levels of these two studied canine groups were low (0.0619 in the Pembroke Welsh Corgi group and 0.0865 in the Shiba Inu group), respectively. In addition, the locus C38 with the highest heterozygosity, PIC, and PD values was high genetic diversities in the Pembroke Welsh corgi and Shiba Inu groups, which may have high potential in the applications of individual identifications of canines. The Pembroke Welsh Corgi, Shiba Inu, and Labrador groups had different genetic structures and a little genetic communications based on the results of PCA and structure analyses. The phylogenetic tree revealed that genetic relationship between the Pembroke Welsh Corgi group and Labrador group was closer in three canine groups. The Pembroke Welsh Corgi is an ancient breed of canine which originated from Wales, England. The modern Labrador is a king of breed that has been systematically bred and improved after being introduced into Britain in the 19th century, although they originally came from Newfoundland. The Shiba Inu is an ancient breed of Japanese origin. This may be the reason that the genetic relationship between the Pembroke Welsh Corgis and Labrador groups was closer than the genetic relationships between these two groups and the Shiba Inu group.

Conclusion
In this study, we found that the 19 STR loci have high genetic polymorphisms and forensic application values in the Pembroke Welsh Corgi and Shiba Inu groups. By genetic analysis among the two studied groups and the previously reported canine group, the Pembroke Welsh Corgi, Shiba Inu, and Labrador groups had different genetic backgrounds and structures, and the genetic relationship between the Pembroke Welsh Corgi and Labrador groups was relatively closer in three kinds of canine groups.

Data Availability
The data underlying the findings of the study can be obtained by contacting the corresponding authors.