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

Authors: Yating Fang; Jinlong Yang; Yajun Deng; Bofeng Zhu
Journal: BioMed Research International
 (2021)

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## Abstract

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.


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## Body

## 1. Introduction

In recent years, it has been found that the study of non-human 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, canine-related 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.


## 2. 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].


### 2.2. Amplification

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 25μl system, which included 1 μl DNA template, 5 μl PCR buffer, 5 μl Primer Mix, and 14 μl sterile water. The amplification was performed on a GeneAmp® 9700 PCR thermocycler. The reaction conditions were as follows: initial denaturation at 95°C for 2 min, followed by 29 cycles at 94°C for 5 s and at 60°C for 1 min, and final incubation at 60°C for 10 min. And then, amplified products were held at 10°C.


### 2.3. Genotyping

After amplification, 1μl of the product or 1 μl of ladder were combined with 9 μl Hi-Di formamide and 0.3 μl internal size standard. The 19 STR genotypes were conducted by capillary electrophoresis on Applied Biosystems™ 3500xL genetic analyser with default instrument settings. Subsequently, the raw data and the allelic ladder were analysed by GeneMapper ID-X software. All sizes were calculated by using internal size standards on the 70, 80, 100, 125, 150, 175, 200, 233, 266, 300, 333, 366, 400, 445, 490, and 500 bp.


### 2.4. 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 (FIS) 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].


## 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].


## 2.2. Amplification

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 25μl system, which included 1 μl DNA template, 5 μl PCR buffer, 5 μl Primer Mix, and 14 μl sterile water. The amplification was performed on a GeneAmp® 9700 PCR thermocycler. The reaction conditions were as follows: initial denaturation at 95°C for 2 min, followed by 29 cycles at 94°C for 5 s and at 60°C for 1 min, and final incubation at 60°C for 10 min. And then, amplified products were held at 10°C.


## 2.3. Genotyping

After amplification, 1μl of the product or 1 μl of ladder were combined with 9 μl Hi-Di formamide and 0.3 μl internal size standard. The 19 STR genotypes were conducted by capillary electrophoresis on Applied Biosystems™ 3500xL genetic analyser with default instrument settings. Subsequently, the raw data and the allelic ladder were analysed by GeneMapper ID-X software. All sizes were calculated by using internal size standards on the 70, 80, 100, 125, 150, 175, 200, 233, 266, 300, 333, 366, 400, 445, 490, and 500 bp.


## 2.4. 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 (FIS) 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].


## 3. Results

### 3.1. Allele Frequency Distributions

A total of 165 alleles were observed at 19 STR loci in 200 purebred Pembroke Welsh Corgis with the allele frequency distributions from 0.0025 to 0.8325 (Table1), and 180 alleles were in the Shiba Inu group with the allele frequency distributions from 0.0029 to 0.6371 (Table 2). In both the two groups, the allele distributions of locus C38 were the most extensive with a total of 18 alleles in the Shiba Inu group and 19 in the Corgi group (numbers of allelic repeat units were ranged from 10 to 37.1). In the 19 loci, locus FH2137 had the largest allele numbers with a total of 19 and 20 alleles in the Shiba Inu and the Pembroke Welsh Corgi groups, respectively. PEZ21 and vWF.X were the lowest number of alleles in the Pembroke Welsh Corgi group (only a total of 4), so were FH2010 and vWF.X in the Shiba Inu group.Table 1
Allele frequencies for 19 short tandem repeat loci in the Pembroke Welsh Corgi group (n=200).
Alleles
C38
Alleles
FH2004
Alleles
FH2137
Alleles
FH2361
Alleles
PEZ02
Alleles
VGL2136
14.2
0.1025
12
0.0250
17.3
0.0025
14
0.0250
10
0.0250
8
0.2875
15
0.0050
13
0.0575
18.3
0.0125
14.1
0.0025
11
0.0650
9
0.2125
15.2
0.0400
14
0.0400
19
0.0050
15
0.4750
13
0.0200
10
0.0975
16
0.1100
15
0.7650
19.3
0.0600
16
0.3425
14
0.1425
11
0.0100
16.2
0.0150
16
0.0300
20
0.0250
17
0.0900
15
0.3450
12
0.0800
17.2
0.3200
29
0.0200
20.2
0.0025
17.1
0.0025
16
0.3825
13
0.0925
18.2
0.0825
31
0.0300
20.3
0.3000
18
0.0250
17
0.0025
14
0.1825
19.2
0.1400
32
0.0150
21
0.0025
19
0.0350
18
0.0175
15
0.0375
20.2
0.0125
33
0.0100
21.3
0.1950
23
0.0025
Alleles
PEZ17
Alleles
VGL3235
21.2
0.0325
34
0.0075
22
0.0300
Alleles
FH2611
13
0.0025
12
0.0075
31.1
0.0025
Alleles
FH2010
22.3
0.1950
17
0.0025
14
0.4000
13
0.0200
33.1
0.0050
9
0.0075
23
0.0825
17.2
0.0025
15
0.1250
14
0.3800
34.1
0.0200
10
0.2875
23.2
0.0025
18
0.0475
16
0.3500
15
0.0750
35
0.0125
11
0.2300
23.3
0.0325
18.2
0.0300
17
0.1150
16
0.4775
35.1
0.0600
12
0.4725
24
0.0325
19
0.0325
18
0.0075
17
0.0100
36
0.0050
13
0.0025
24.3
0.0025
19.2
0.3625
Alleles
PEZ20
18
0.0100
36.1
0.0275
Alleles
FH2054
25
0.0025
20
0.0100
12
0.1600
19
0.0200
37
0.0050
16
0.0250
26
0.0075
20.2
0.0625
13
0.3750
Alleles
VGL3438
37.1
0.0025
17
0.2850
27
0.0025
21
0.0025
14
0.2250
12
0.2250
Alleles
FH2001
18
0.2575
28
0.0050
21.2
0.3250
15
0.1975
13
0.0050
8
0.0500
19
0.3475
Alleles
FH2328
22
0.0050
16
0.0425
14
0.0825
9
0.0725
20
0.0100
13
0.1225
22.2
0.0325
Alleles
PEZ21
15
0.0200
10
0.1125
21
0.0150
15
0.1775
23.2
0.0250
8
0.4925
16
0.1325
11
0.3850
23
0.0600
16
0.0025
24.2
0.0300
9
0.0150
17
0.3575
12
0.1075
Alleles
FH2088
17
0.0100
25.2
0.0300
10
0.2175
18
0.0125
13
0.2400
14
0.4250
18
0.3675
Alleles
PEZ01
11
0.2750
19
0.0525
14
0.0300
15
0.0175
19
0.0250
10
0.0050
Alleles
vWF.X
20
0.1025
14.2
0.0025
16
0.0975
20
0.0950
11
0.0175
9
0.6000
21
0.0100
17
0.3725
21
0.1950
12
0.0850
10
0.2900
18
0.0875
22
0.0050
13
0.8325
11
0.0025
14
0.0600
12
0.1075Table 2
Allele frequencies for 19 short tandem repeat loci in the Shiba Inu group (n=175).
Alleles
C38
Alleles
FH2004
Alleles
FH2137
Alleles
FH2361
Alleles
PEZ02
Alleles
VGL2136
10
0.0029
11
0.0029
17
0.0029
14
0.1229
12
0.0086
8
0.0229
11
0.0114
12
0.1029
17.3
0.0600
15
0.2429
13
0.4400
9
0.2343
14.1
0.0171
13
0.1771
18
0.0057
15.2
0.0343
14
0.0857
10
0.0029
15.2
0.1143
14
0.5429
18.3
0.0457
16
0.1714
15
0.2686
12
0.0029
16
0.0029
15
0.0571
19
0.0543
16.2
0.0029
16
0.1143
13
0.0514
16.2
0.0771
29
0.0057
19.1
0.0286
17
0.0829
17
0.0057
14
0.4229
17.2
0.0914
30
0.0943
19.3
0.0029
17.2
0.0029
18
0.0771
15
0.0086
18.2
0.4086
31
0.0086
20
0.0143
18
0.2057
Alleles
PEZ17
16
0.0343
19.2
0.0257
32
0.0057
20.1
0.0143
19
0.0686
14
0.1514
17
0.2171
20.2
0.0200
33
0.0029
20.3
0.0029
19.1
0.0086
15
0.2629
18
0.0029
26.1
0.0029
Alleles
FH2010
21
0.0143
19.2
0.0057
16
0.2343
Alleles
VGL3235
27.1
0.0029
9
0.1143
21.1
0.5743
20
0.0057
17
0.3057
12
0.2800
29.1
0.0229
10
0.2371
22
0.0114
20.1
0.0314
18
0.0343
13
0.2600
30.1
0.0800
11
0.0943
22.1
0.0686
20.2
0.0114
19
0.0029
14
0.1143
31.1
0.0914
12
0.5543
22.2
0.0029
21.1
0.0029
20
0.0086
15
0.0086
32.1
0.0257
Alleles
FH2054
23
0.0714
Alleles
FH2611
Alleles
PEZ20
16
0.0257
35.1
0.0029
17
0.0514
23.2
0.0057
18.2
0.0086
11
0.1514
17
0.1743
Alleles
FH2001
18
0.2000
24
0.0171
19.2
0.0657
12
0.0200
18
0.0543
6
0.0171
19
0.3514
24.2
0.0029
19.3
0.0229
13
0.5857
19
0.0771
8
0.1486
20
0.1714
Alleles
FH2328
20
0.0029
14
0.1714
20
0.0057
9
0.0114
21
0.0943
13
0.0114
20.2
0.1943
15
0.0571
Alleles
VGL3438
10
0.0171
22
0.1171
14
0.0029
21
0.0029
16
0.0114
11
0.1800
11
0.3143
23
0.0114
15
0.1543
21.2
0.3029
19
0.0029
12
0.3143
12
0.0314
24
0.0029
16
0.1657
22
0.0143
Alleles
PEZ21
13
0.1771
13
0.1086
Alleles
FH2088
17
0.0600
22.2
0.3000
9
0.0343
14
0.0200
13.2
0.3486
12
0.0029
17.1
0.0029
23
0.0229
10
0.0886
15
0.2257
14
0.0029
14
0.2314
18
0.0343
23.2
0.0314
11
0.6000
16
0.0657
Alleles
PEZ01
15
0.3771
18.1
0.2629
24
0.0057
12
0.2714
17
0.0086
9
0.0029
16
0.2543
19
0.1000
24.2
0.0029
13
0.0057
18
0.0057
11
0.6371
17
0.1286
19.1
0.0143
25
0.0200
Alleles
vWF.X
19
0.0029
12
0.0143
18
0.0057
20
0.0057
27
0.0029
9
0.1743
13
0.2400
21
0.1514
10
0.3286
14
0.1000
22
0.0343
11
0.3400
15
0.0057
12
0.1571


### 3.2. HWE, LD, andFIS Tests

In HWE tests, the correction level was adjusted to 0.0026 (0.05/19) after the Bonferroni correction. The estimatedP values of all 19 loci were greater than 0.0026, suggesting that the two canine groups in this study were consistent with HWE except for locus VGL3438 in the Pembroke Welsh Corgi group.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 byr2 values in this study. Figures 1(a) and 1(b) showed the r2 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 r2 values were less than 0.02, indicating that there was no linkage disequilibrium at the 19 STR loci in these two canine groups.Figure 1
Linkage disequilibrium tests were conducted between pairwise STR loci in the studied canine groups. (a) Pembroke Welsh Corgi group (n=200). (b) Shiba Inu group (n=175). Linkage correlation coefficient (r2) values magnified 100 times were easy to be displayed in the small box.
(a)
(b)TheFIS was calculated to evaluate the level of inbreeding in these two canine groups. The FIS value was 0.0619 in the Pembroke Welsh Corgi group, while the FIS of the Shiba Inu group was 0.0865.


### 3.3. Forensic Parameters

As shown in Figure2, 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).Figure 2
Forensic parameters for 19 short tandem repeat loci in the Pembroke Welsh Corgi group (n=200) and the Shiba Inu group (n=175).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. All PIC values were greater than 0.25, and 16 loci were greater than 0.5. The MP values ranged from 0.0450 to 0.5101, and the combined random match probability (CMP) was3.04×10−17. The PD and PE values ranges from 0.4899 to 0.9551 and 0.0635 to 0.6462, respectively. The combined power of discrimination (CPD) and the combined power of exclusion (CPE) were 0.9999999999999999696 and 0.999889, respectively.In the Shiba Inu group, the He values were a range from 0.5277 to 0.8428 with the average of 0.7166. The Ho values of all loci were greater than 0.5 (except for PEZ01 and PEZ21), so were the PIC values. The MP, PD, and PE values were in the range from 0.0475 to 0.2837, 0.7163 to 0.9525, and 0.1443 to 0.5471, respectively. The CMP, CPD, and CPE were8.98×10−19, 0.999999999999999999102, and 0.999911, respectively.


### 3.4. Interpopulation Genetic Analyses

As shown in Figure3, 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.Figure 3
Comparison of the allele numbers between the studied two canine groups and the Labrador group based on 19 loci.The PCA and genetic structure analyses were performed to reveal the genetic structure of three canine groups. As shown in Figure4(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.Figure 4
The genetic structures and genetic relationships of the Pembroke Welsh Corgi, Shiba Inu, and Labrador groups. (a) A clustering diagram of principal component analysis. (b) A phylogenetic tree. (c) A bar plot of structure analysis when the number of assumed populations (K) was 3.
(a)
(b)
(c)


## 3.1. Allele Frequency Distributions

A total of 165 alleles were observed at 19 STR loci in 200 purebred Pembroke Welsh Corgis with the allele frequency distributions from 0.0025 to 0.8325 (Table1), and 180 alleles were in the Shiba Inu group with the allele frequency distributions from 0.0029 to 0.6371 (Table 2). In both the two groups, the allele distributions of locus C38 were the most extensive with a total of 18 alleles in the Shiba Inu group and 19 in the Corgi group (numbers of allelic repeat units were ranged from 10 to 37.1). In the 19 loci, locus FH2137 had the largest allele numbers with a total of 19 and 20 alleles in the Shiba Inu and the Pembroke Welsh Corgi groups, respectively. PEZ21 and vWF.X were the lowest number of alleles in the Pembroke Welsh Corgi group (only a total of 4), so were FH2010 and vWF.X in the Shiba Inu group.Table 1
Allele frequencies for 19 short tandem repeat loci in the Pembroke Welsh Corgi group (n=200).
Alleles
C38
Alleles
FH2004
Alleles
FH2137
Alleles
FH2361
Alleles
PEZ02
Alleles
VGL2136
14.2
0.1025
12
0.0250
17.3
0.0025
14
0.0250
10
0.0250
8
0.2875
15
0.0050
13
0.0575
18.3
0.0125
14.1
0.0025
11
0.0650
9
0.2125
15.2
0.0400
14
0.0400
19
0.0050
15
0.4750
13
0.0200
10
0.0975
16
0.1100
15
0.7650
19.3
0.0600
16
0.3425
14
0.1425
11
0.0100
16.2
0.0150
16
0.0300
20
0.0250
17
0.0900
15
0.3450
12
0.0800
17.2
0.3200
29
0.0200
20.2
0.0025
17.1
0.0025
16
0.3825
13
0.0925
18.2
0.0825
31
0.0300
20.3
0.3000
18
0.0250
17
0.0025
14
0.1825
19.2
0.1400
32
0.0150
21
0.0025
19
0.0350
18
0.0175
15
0.0375
20.2
0.0125
33
0.0100
21.3
0.1950
23
0.0025
Alleles
PEZ17
Alleles
VGL3235
21.2
0.0325
34
0.0075
22
0.0300
Alleles
FH2611
13
0.0025
12
0.0075
31.1
0.0025
Alleles
FH2010
22.3
0.1950
17
0.0025
14
0.4000
13
0.0200
33.1
0.0050
9
0.0075
23
0.0825
17.2
0.0025
15
0.1250
14
0.3800
34.1
0.0200
10
0.2875
23.2
0.0025
18
0.0475
16
0.3500
15
0.0750
35
0.0125
11
0.2300
23.3
0.0325
18.2
0.0300
17
0.1150
16
0.4775
35.1
0.0600
12
0.4725
24
0.0325
19
0.0325
18
0.0075
17
0.0100
36
0.0050
13
0.0025
24.3
0.0025
19.2
0.3625
Alleles
PEZ20
18
0.0100
36.1
0.0275
Alleles
FH2054
25
0.0025
20
0.0100
12
0.1600
19
0.0200
37
0.0050
16
0.0250
26
0.0075
20.2
0.0625
13
0.3750
Alleles
VGL3438
37.1
0.0025
17
0.2850
27
0.0025
21
0.0025
14
0.2250
12
0.2250
Alleles
FH2001
18
0.2575
28
0.0050
21.2
0.3250
15
0.1975
13
0.0050
8
0.0500
19
0.3475
Alleles
FH2328
22
0.0050
16
0.0425
14
0.0825
9
0.0725
20
0.0100
13
0.1225
22.2
0.0325
Alleles
PEZ21
15
0.0200
10
0.1125
21
0.0150
15
0.1775
23.2
0.0250
8
0.4925
16
0.1325
11
0.3850
23
0.0600
16
0.0025
24.2
0.0300
9
0.0150
17
0.3575
12
0.1075
Alleles
FH2088
17
0.0100
25.2
0.0300
10
0.2175
18
0.0125
13
0.2400
14
0.4250
18
0.3675
Alleles
PEZ01
11
0.2750
19
0.0525
14
0.0300
15
0.0175
19
0.0250
10
0.0050
Alleles
vWF.X
20
0.1025
14.2
0.0025
16
0.0975
20
0.0950
11
0.0175
9
0.6000
21
0.0100
17
0.3725
21
0.1950
12
0.0850
10
0.2900
18
0.0875
22
0.0050
13
0.8325
11
0.0025
14
0.0600
12
0.1075Table 2
Allele frequencies for 19 short tandem repeat loci in the Shiba Inu group (n=175).
Alleles
C38
Alleles
FH2004
Alleles
FH2137
Alleles
FH2361
Alleles
PEZ02
Alleles
VGL2136
10
0.0029
11
0.0029
17
0.0029
14
0.1229
12
0.0086
8
0.0229
11
0.0114
12
0.1029
17.3
0.0600
15
0.2429
13
0.4400
9
0.2343
14.1
0.0171
13
0.1771
18
0.0057
15.2
0.0343
14
0.0857
10
0.0029
15.2
0.1143
14
0.5429
18.3
0.0457
16
0.1714
15
0.2686
12
0.0029
16
0.0029
15
0.0571
19
0.0543
16.2
0.0029
16
0.1143
13
0.0514
16.2
0.0771
29
0.0057
19.1
0.0286
17
0.0829
17
0.0057
14
0.4229
17.2
0.0914
30
0.0943
19.3
0.0029
17.2
0.0029
18
0.0771
15
0.0086
18.2
0.4086
31
0.0086
20
0.0143
18
0.2057
Alleles
PEZ17
16
0.0343
19.2
0.0257
32
0.0057
20.1
0.0143
19
0.0686
14
0.1514
17
0.2171
20.2
0.0200
33
0.0029
20.3
0.0029
19.1
0.0086
15
0.2629
18
0.0029
26.1
0.0029
Alleles
FH2010
21
0.0143
19.2
0.0057
16
0.2343
Alleles
VGL3235
27.1
0.0029
9
0.1143
21.1
0.5743
20
0.0057
17
0.3057
12
0.2800
29.1
0.0229
10
0.2371
22
0.0114
20.1
0.0314
18
0.0343
13
0.2600
30.1
0.0800
11
0.0943
22.1
0.0686
20.2
0.0114
19
0.0029
14
0.1143
31.1
0.0914
12
0.5543
22.2
0.0029
21.1
0.0029
20
0.0086
15
0.0086
32.1
0.0257
Alleles
FH2054
23
0.0714
Alleles
FH2611
Alleles
PEZ20
16
0.0257
35.1
0.0029
17
0.0514
23.2
0.0057
18.2
0.0086
11
0.1514
17
0.1743
Alleles
FH2001
18
0.2000
24
0.0171
19.2
0.0657
12
0.0200
18
0.0543
6
0.0171
19
0.3514
24.2
0.0029
19.3
0.0229
13
0.5857
19
0.0771
8
0.1486
20
0.1714
Alleles
FH2328
20
0.0029
14
0.1714
20
0.0057
9
0.0114
21
0.0943
13
0.0114
20.2
0.1943
15
0.0571
Alleles
VGL3438
10
0.0171
22
0.1171
14
0.0029
21
0.0029
16
0.0114
11
0.1800
11
0.3143
23
0.0114
15
0.1543
21.2
0.3029
19
0.0029
12
0.3143
12
0.0314
24
0.0029
16
0.1657
22
0.0143
Alleles
PEZ21
13
0.1771
13
0.1086
Alleles
FH2088
17
0.0600
22.2
0.3000
9
0.0343
14
0.0200
13.2
0.3486
12
0.0029
17.1
0.0029
23
0.0229
10
0.0886
15
0.2257
14
0.0029
14
0.2314
18
0.0343
23.2
0.0314
11
0.6000
16
0.0657
Alleles
PEZ01
15
0.3771
18.1
0.2629
24
0.0057
12
0.2714
17
0.0086
9
0.0029
16
0.2543
19
0.1000
24.2
0.0029
13
0.0057
18
0.0057
11
0.6371
17
0.1286
19.1
0.0143
25
0.0200
Alleles
vWF.X
19
0.0029
12
0.0143
18
0.0057
20
0.0057
27
0.0029
9
0.1743
13
0.2400
21
0.1514
10
0.3286
14
0.1000
22
0.0343
11
0.3400
15
0.0057
12
0.1571


## 3.2. HWE, LD, andFIS Tests

In HWE tests, the correction level was adjusted to 0.0026 (0.05/19) after the Bonferroni correction. The estimatedP values of all 19 loci were greater than 0.0026, suggesting that the two canine groups in this study were consistent with HWE except for locus VGL3438 in the Pembroke Welsh Corgi group.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 byr2 values in this study. Figures 1(a) and 1(b) showed the r2 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 r2 values were less than 0.02, indicating that there was no linkage disequilibrium at the 19 STR loci in these two canine groups.Figure 1
Linkage disequilibrium tests were conducted between pairwise STR loci in the studied canine groups. (a) Pembroke Welsh Corgi group (n=200). (b) Shiba Inu group (n=175). Linkage correlation coefficient (r2) values magnified 100 times were easy to be displayed in the small box.
(a)
(b)TheFIS was calculated to evaluate the level of inbreeding in these two canine groups. The FIS value was 0.0619 in the Pembroke Welsh Corgi group, while the FIS of the Shiba Inu group was 0.0865.


## 3.3. Forensic Parameters

As shown in Figure2, 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).Figure 2
Forensic parameters for 19 short tandem repeat loci in the Pembroke Welsh Corgi group (n=200) and the Shiba Inu group (n=175).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. All PIC values were greater than 0.25, and 16 loci were greater than 0.5. The MP values ranged from 0.0450 to 0.5101, and the combined random match probability (CMP) was3.04×10−17. The PD and PE values ranges from 0.4899 to 0.9551 and 0.0635 to 0.6462, respectively. The combined power of discrimination (CPD) and the combined power of exclusion (CPE) were 0.9999999999999999696 and 0.999889, respectively.In the Shiba Inu group, the He values were a range from 0.5277 to 0.8428 with the average of 0.7166. The Ho values of all loci were greater than 0.5 (except for PEZ01 and PEZ21), so were the PIC values. The MP, PD, and PE values were in the range from 0.0475 to 0.2837, 0.7163 to 0.9525, and 0.1443 to 0.5471, respectively. The CMP, CPD, and CPE were8.98×10−19, 0.999999999999999999102, and 0.999911, respectively.


## 3.4. Interpopulation Genetic Analyses

As shown in Figure3, 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.Figure 3
Comparison of the allele numbers between the studied two canine groups and the Labrador group based on 19 loci.The PCA and genetic structure analyses were performed to reveal the genetic structure of three canine groups. As shown in Figure4(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.Figure 4
The genetic structures and genetic relationships of the Pembroke Welsh Corgi, Shiba Inu, and Labrador groups. (a) A clustering diagram of principal component analysis. (b) A phylogenetic tree. (c) A bar plot of structure analysis when the number of assumed populations (K) was 3.
(a)
(b)
(c)


## 4. 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 (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,FIS 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.


## 5. 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.



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*Source: 5355109-2021-11-25.xml*