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. 2020 Oct 30:8:e10215.
doi: 10.7717/peerj.10215. eCollection 2020.

Genetic variations and dog breed identification using inter-simple sequence repeat markers coupled with high resolution melting analysis

Wannapimol Kriangwanich  1 , Korakot Nganvongpanit  1   2 , Kittisak Buddhachat  2   3 , Puntita Siengdee  2   4 , Siriwadee Chomdej  2   5 , Siriluck Ponsuksili  4 , Chatchote Thitaram  6
Affiliations

Genetic variations and dog breed identification using inter-simple sequence repeat markers coupled with high resolution melting analysis

Wannapimol Kriangwanich et al. PeerJ. .

Abstract

The identification of differing physical characteristics of dogs is an uncomplicated and straightforward way to categorize dog breeds. However, many dog owners and veterinarians still struggle to distinguish between pure breed and mixed variations in certain breeds of dogs. Presently, the absence of the tools and methods needed to confirm a pure breed dog is a significant problem since the only method available to validate pure or mongrel breeds is the official pedigree system. Inter-simple sequence repeat markers have been successfully used to assess genetic variations and differentiations. Notably, inter-simple sequence repeat markers coupled with high resolution melting analysis were effectively used for the breed identification of 43 breeds of dogs (total 463 dogs). The 10 primers chosen for analysis resulted in a range of 31-78.6% of breed discrimination when using one primer, while a combination of two primers was able to successfully discriminate between all of the 43 dog breeds (100%). Shannon's index information (I = 2.586 ± 0.034) and expected heterozygosity (H e = 0.908 ± 0.003) indicated a high level of genetic diversity among breeds. The fixation index (F st ) revealed a value of 10.4%, demonstrating that there was a high level of genetic subdivision between populations. This study showed that inter-simple sequence repeat marker analysis was effective in demonstrating high genetic diversity among varying breeds of dogs, while a combination of Inter-simple sequence repeat marker analysis and high resolution melting analysis could provide an optional technique for researchers to effectively identify breeds through genetic variations.

Keywords: Breeds; Canine; Classification; Genetic variation; Melting temperature.

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Conflict of interest statement

Korakot Nganvongpanit is an Academic Editor for PeerJ.

Figures

Figure 1
Figure 1. Percent polymorphic bands of 36 breeds of dogs classified by FCI groups.
Each color represents each group. The highest percent of polymorphic bands was 93% in the Shih Tzu breed and the lowest was 28.04% in the Alaskan Malamute. *Westies = West Highland White Terrier, Alaskan = Alanskan Malamute and CKCs = Cavalier King Charles Spaniel.
Figure 2
Figure 2. Clustering assignment of 22 dog breeds based on data of 10 ISSR makers. STRUCTURE was used to determine the admixture of each dog.
Each breed is represented by 4-47 animals at K values (number of genetic clusters) from 2 to the most suitable K at 13. Each vertical line represents an individual dog. A clear indication of two subgroups was obtained at all K values were at 2 and four subgroups had a K value of 4. The figure shows representative runs at K = 2, K = 4, K = 6 and K = 13 and labels 13 distinctive breeds as 1 = Chihuahua, 2 = Golden Retriever, 3 = Thai Bangkaew, 4 = Siberian Husky, 5 = Jack Russell Terrier, 6 = Cocker Spaniel, 7 = Pomeranian, 8 = Beagle, 9 = Yorkshire Terrier, 10 = Poodle, 11 = Pug, 12 = French Bulldog, 13 = Shih Tzu.
Figure 3
Figure 3. Phylogenetic dendrogram of 33 different dog breeds separated into three main clusters based on genetic distance which is presented in three different colors.
Figure 4
Figure 4. Heatmap showing percentages of breed discrimination from each breed and combination of Inter-simple sequence repeat (ISSR) markers coupled with High Resolution melting analysis (HRM) for identification of dog breeds.
Figure 5
Figure 5. HRM derivative melting curve from 43 different breeds of dogs based on data of UBC823 and dog groups classified by FCI nomenclature.
The numbers shown in brackets represent the FCI nomenclature group. German Shepherd (A), Border Collie (B), Welsh Corgi (C), Miniature Pinscher (D), Schnauzer (E), Bulldog (F), Rottweiler (G), Doberman Pinscher (H), Dogo Argentino (I), Fila Brasileiro (J), Great Dane (K), Yorkshire Terrier (L), Jack Russell Terrier (M), Bull Terrier (N), West Highland White Terrier (O), Dachshund (P), Pomeranian (Q), Siberian Husky (R), Thai Ridgeback (S), Samoyed (T), Spitz (U), Chow Chow (V), Thai Bangkaew (W), Alaskan Malamute (X), Shiba Inu (Y), Akita Inu (Z), Beagle (AA), Dalmatian (BB), Golden Retriever (CC), Labrador Retriever (DD), Cocker Spaniel (EE), Chihuahua (FF), Shih Tzu (GG), French Bulldog (HH), Poodle (II), Pug (JJ), Bichon Frise (KK), Boston Terrier (LL), Pekingese (MM), Cavalier King Charles Spaniel (NN), Papillon (OO), American Bully (PP) and American Pit Bull Terrier (QQ).
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Grants and funding

Financial support for this study was provided by the Royal Jubilee Ph.D. Program, Thailand Research Fund (TRF), Faculty of Veterinary Medicine, Chiang Mai University, Thailand (R000022228). Additional support was obtained from Chiang Mai University through the research administration office which provided necessary budget via the Excellence Center in Veterinary Bioscience (ECVB), Chiang Mai University, Thailand. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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