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Genetic distance between three breeds of dogs based on selected microsatellite sequences

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The aim of this study was to evaluate genetic diversity and genetic distance between three breeds of dogs: Bernese Mountain Dog (BMD), Chihuahua (Ch) and Caucasian Shepherd Dog (CSD), based on microsatellite sequences commonly used to confirm/exclude dogs’ parentage. The study included 60 dogs (20 dogs per breed) from Czech kennels. One dog was randomly selected from a single litter while the litters were unrelated down to the second generation. A commonly used panel of microsatellite markers (PEZ1, FHC2054, FHC2010, PEZ05, PEZ20, PEZ12, PEZ3, PEZ6, PEZ8 and FHC2079) was applied in the study. Loci of each microsatellite revealed different polymorphism levels with an average of 5.1 alleles per locus (from 2 to 9 across breeds). PEZ3 and PEZ12 were the most informative markers (7.0 and 6.7 alleles, respectively). Specific alleles were detected within each breed. Loci PEZ5 and PEZ20 showed the lowest average number of alleles (3.7). Number of observed genotypes ranged from 3 to 14, but only few genotypes were common to all breeds. Average heterozygosity ranged from 0.49 in BMD to 0.72 in Ch. Within CSD, solely the PEZ20 locus was not highly polymorphic (PIC<0.5), while in BMD only half of the microsatellites were highly polymorphic (PIC>0.5). The lowest genetic distance was found between CSD and Ch, whilst relatively large genetic distance was estimated between Ch and BMD, as well as between the CSD and BMD, which may support the hypothesis of these breeds’ separation at an early stage of domestication.
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Animal Science Papers and Reports vol. 34 (2016) no. 1, 95-102
Institute of Genetics and Animal Breeding, Jastrzębiec, Poland
Genetic distance between three breeds of dogs
based on selected microsatellite sequences
Katarzyna1,*, Andrzej Filistowicz1, Marzena1,
Piotr2, Irena3, Anna E.1
1 Wroclaw University of Environmental and Life Sciences, Institute of Animal Breeding,
Chełmońskiego 38c, 51-630 Wrocław, Poland
2 Leszno State Vocational College, Institute of Polytechnic and Agriculture,
64-100 Leszno, Mickiewicza 5, Poland
3 Mendel University in Brno, Department of Morphology, Physiology and Animal Genetics,
61-300 Brno, Zemědělská 1, Czech Republic
(Accepted November 3, 2015)
  

   

     
           
Loci  
locus 
Loci

              
     locus   
            

                

: 
genetic distance
*Corresponding author: e-mail: katarzyna.czyz@up.wroc.pl
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All dog (Canis lupus f. familiaris) breeds have the same ancestor, which is the
grey wolf (Canis lupus). After the initial domestication of the wolf, selection started
to improve functional traits and consequently to create different breeds and groups of
breeds. However, only at the turn of the 19th century dog breeding became systematized
by formation of breed associations promoting specic types (breeds) of dogs and
organizing dog shows to nominate the perfect representatives of the breeds [Parker
2012]. Selection which was focused on desirable traits, while restricting natural
selection, caused that dog breeds were becoming increasingly isolated from each
other, and thus their gene pools became eventually more and more limited [Parker
2012] within a population.
An analysis of microsatellite sequences short tandem repeats (STR) can be
used for genetic distance determination. Populations having many similar alleles
(microsatellite sequences) are considered genetically close, which means that they
share a proximate ancestor.
Out of the indicators of genetic variation within a breed, heterozygosity (H) and
effective number of alleles at a locus (EA) are usually employed to describe a population.
They demonstrate the degree of diversity based upon analyzed polymorphic loci
within a breed. High heterozygosity and high effective number of alleles inform about
a greater genetic diversity of a breed. In addition to those indicators, polymorphic
information content (PIC) is widely used as a measure of genetic polymorphism
within a locus used as a marker in the linkage analysis [Botstein et al. 1980].
The dog breeds selected for the present study are characterized by diverse origin
resulting in morphological and behavioral traits. The BMD was used to herd cattle,
and because of its size and strength, was also used as a draught dog. Its origin is not
known in full. Some researchers suggest that this breed originates from the Tibetan
dog, which came to Europe from Asia with the migrating men, while others, relying
on archaeological excavations, propose an exclusively European ancestry of this breed
[Räber 1965].
Furthermore, Wayne and Von Holdt [2012] placed BMD in the group of
mastiffs, along with Rottweiler, Newfoundland, Boxer, Bulldog, and other large breeds,
for which the common ancestor was a big dog of the contemporary mastiff type.
The origin of Chihuahua is not clear, either. Probably, its direct ancestor was
a dwarf dog, Techichi, bred by the Toltec people that had lived on the territory of
present Mexico [Van Asch et al. 2014]. The literature data also suggest Chinese roots
of that breed, along with a common to the Chinese Crested Dog progenitor, which was
brought to South America by merchants [Von Holdt et al. 2010, Wayne and Von Holdt
2012]. According to Van Asch et al. [2014], the Mexican Chihuahua breed comes
directly from the Mexican pre-Columbian dogs. Only the origin of CSD is certain. It is
an old breed of herding dogs from Caucasus area (Georgia, Armenia, and Azerbaijan)
used to guard ocks of sheep and belongings. For centuries, these dogs had lived in
a complete isolation; rst specimens were brought to Europe only in 1969, and the
breed was registered by the FCI in 1984 [Kopaliani et al. 2014].
K. Czyż et al. al.
97
The aim of the study was to conrm common or different parentage of the
examined dog breeds which are spread all over the world (Chihuahua, Caucasian
Shepherd, Bernese Mountain Dog), and for this purpose the genetic distance between
three breeds was determined.
Material and methods
The DNA was sampled from saliva. Samples of 20 individuals from each of the
three breeds: Chihuahua, Caucasian Shepherd Dog and Bernese Mountain Dog were
selected from a large collection of biological material of various breeds of dogs kept
in the Czech Republic. Dogs came from breeders from across the country, and the
criterion for selection was belonging to a breed and to a different family (unrelated for
two generations) within a breed.
DNA isolation from buccal epithelial cells employed the conventional phenol/
chloroform method [Sambrook and Russell 2001]. Concentration of the extracted
DNA was determined spectrophotometrically (BioPhotometer, Eppendorf). The stock
DNA was stored at -20°C.
Ten microsatellite loci (PEZ01, FHC2054, FHC2010, PEZ05, PEZ20, PEZ12,
PEZ03, PEZ06, PEZ08 and FHC2079) were genotyped by the StockMarks®Dogs
Genotyping kit (Applied Biosystems, USA), under conditions recommended by the
manufacturer.
Frequencies of alleles and genotypes at the loci of 10 microsatellite sequences in
each of the three breeds were calculated. To further analyze the genetic variation, the
effective number of alleles (EA), the expected heterozygosity (H) and the polymorphic
information content (PIC) indicators were computed. The above statistics were calculated
for each locus separately for each breed or for all the loci within each breed. To determine
the genetic distance between the breeds the Nei formula was used [Nei 1972].

There was no monomorphic loci in any of the three breeds. Observed, however,
were alleles specic to a single breed, while a case when an allele was present in two
breeds and was missing in the remaining breed was not recorded. The number of alleles
ranged from 2 to 9. PEZ3 and PEZ12 were found to be the most informative markers,
because they showed the highest number of alleles (average number of alleles: 7.0
and 6.7, respectively). The lowest average number of alleles (3.7) was detected at the
PEZ5 and PEZ20 loci (Tab. 1).
The number of genotypes ranged from 3 to 14 (Tab. 2). Although a large number
of genotypes was found in all breeds, not many genotypes were common: FHC2010
– 4, FHC2054 – 3, FHC2079 – 2, PEZ1 – 2, PEZ5 – 1, PEZ6 – 4, PEZ8 – 4, PEZ12
1, PEZ20 3. The PEZ3 locus showed no common genotype. Locus FHC2054,
which had the highest number of genotypes in two breeds, had only three genotypes
common to all the breeds.
Genetic distance between three breeds of dogs
98
The H values ranged from 0.49 in BMD to 0.72 in Ch (Tab. 3).
The highest values of EA, H and PIC were recorded for loci PEZ12 and FHC2054
in Ch, for PEZ3 and FHC2054 in CSD and for PEZ8 in BMD (Tab. 3). All the
microsatellites in Ch were highly polymorphic (PIC and H > 0.5) and in CSD only
locus PEZ20 was not highly polymorphic (PIC < 0.5). In turn, in BMD merely half
of the microsatellites were highly polymorphic (FHC2054, PEZ1, PEZ6, PEZ8 and
PEZ12). The lowest values of EA, H and PIC indices were obtained for Ch and
CSD for locus PEZ20 and for BMD for locus PEZ5. Thus, the lowest variability
was observed at loci PEZ20 and PEZ5. The highest polymorphism was found at loci
PEZ3, PEZ8 and PEZ12, in every breed.
Genetic distances between the three breeds, based on 10 microsatellite loci, are
presented in Table 4. Genetic distance between the Ch and CSD breeds was the closest.
A relatively large distance was estimated between Ch and BMD, as well as between
CSD and BMD.
Ten popular microsatellite sequences were used in this study. The same or similar
microsatellite sequences have been studied by several authors [e.g. De Nise et al.
2004, Cho 2005, Pribánová et al. 2009, Dimitrijevic et al. 2013] in populations of
K. Czyż et al.
Table 1. Expected size of alleles and number of alleles at each locus in three dog breeds
Dog breed
Locus
Expected
size (bp)
Chihuahua
Caucasian
Shepherd Dog
Bernese
Mountain Dog
Average
for 3 breeds
FHC2010
92-136
4
4
4.3
FHC2054
140-183
6
4
5.7
FHC2079
210-260
4
3
3.7
PEZ1
97-121
4
4
5.0
PEZ3
170-201
7
5
7.0
PEZ5
250-320
4
2
3.7
PEZ6
95-154
6
4
5.7
PEZ8
164-214
5
5
5.7
PEZ12
222-260
7
6
6.7
PEZ20
263-299
4
3
3.7
Mean
5.1
4.0
5.1
Table 2. Highest and lowest number of genotypes in chosen loci in three dog breeds
Dog breed
Number of
genotypes
Locus
Chihuahua
Caucasian
Shepherd Dog
Bernese
Mountain Dog
FHC2010
7
Lowest
PEZ5
4
3
FHC2054, PEZ1, PEZ12
13
FHC2054
14
Highest
PEZ8
8
Average
all 10 loci
10.6
8.5
5.4
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dogs of various breeds. These sequences are commonly used to conrm or exclude
parentage [Halverson and Edwards 2000].
Our results showing a high polymorphism at loci FHC2054, PEZ1, PEZ12 and
PEZ3 are consistent with results obtained by De Nise et al. [2004]. The authors
recognized these loci as the richest in information basing on a research conducted
on 9561 dogs of 108 breeds. De Nise et al. [2004] indicated locus PEZ3 as the
most informative, due to its highest average number of alleles (6.7) per breed. Also
in our study, this sequence had the highest number of variants. Our results are also
in accordance with a research on the Yugoslav Shepherd Dog breed (Šarplanina)
[Dimitrijevic et al. 2013]. Locus PEZ3 had the highest number of alleles in that breed,
but it was characterized by lower values of the basic diversity indicators. Similarly
low H and PIC values were found for locus PEZ3 in the present study, but only for
BMD. The least informative loci, in the Yugoslav Shepherd Dog, were FHC2010 and
PEZ5, which is coherent with our results for all three breeds and with several previous
studies [De Nise et al. 2004, Pribánová et al. 2009, Dimitrijevic et al. 2013].
The lowest variability, as described by the EA, H and PIC indices, was observed
in the BMD breed, and the highest in the Ch breed. CSD breed took an intermediate
position, however its statistics were closer to those of Ch. The representatives of the
BMD breed were characterized by the lowest average number of alleles at a locus
and the lowest H value (0.49), which points to a small variability within the breed,
caused probably by an intensive selection based only upon phenotypic evaluation.
Genetic distance between three breeds of dogs
Table 3. Effective number of alleles (EA), heterozygosity (H) and polymorphic information
content (PIC) in three dog breeds
Chihuahua
Caucasian Shepherd Dog
Bernese Mountain Dog
Locus
EA
H
PIC
EA
H
PIC
EA
H
PIC
FHC2010
3.33
0.70
0.65
2.56
0.61
0.55
1.69
0.41
0.38
FHC2054
5.33
0.81
0.79
4.49
0.78
0.74
2.61
0.62
0.55
FHC2079
2.64
0.62
0.55
2.77
0.64
0.57
1.36
0.27
0.25
PEZ1
4.49
0.78
0.75
3.56
0.72
0.67
2.37
0.58
0.51
PEZ3
4.02
0.75
0.72
5.33
0.81
0.79
1.87
0.47
0.42
PEZ5
3.00
0.67
0.63
2.84
0.65
0.59
1.30
0.23
0.20
PEZ6
3.57
0.72
0.68
4.30
0.77
0.73
2.40
0.58
0.53
PEZ8
4.12
0.76
0.72
3.20
0.69
0.64
3.42
0.71
0.66
PEZ12
5.44
0.82
0.79
3.29
0.70
0.66
2.23
0.55
0.51
PEZ20
2.62
0.62
0.57
2.37
0.58
0.49
1.89
0.47
0.38
Mean
3.86
0.72
0.68
3.47
0.69
0.64
2.11
0.49
0.44
Table 4. Genetic distance between the Chihuahua, Caucasian Shepherd Dog and
Bernese Mountain Dog breeds
Breed
Caucasian Shepherd Dog
Bernese Mountain Dog
Chihuahua
0.33
0.50
Caucasian Shepherd Dog
0.44
100
Similar results were obtained by Cho [2005], Irion et al. [2003] and Kopaliani et al.
[2014]. Research conducted by Cho [2005], in which comparable microsatellite loci
in six dog breeds in Korea were used, conrm high values of expected heterozygosity
and PIC in the Ch breed. The author found the highest H and PIC values in the native
Jindo breed (0.796 and 0.755, respectively) and in the Ch breed (0.713 and 0.665,
respectively), while in Poongsan and German Shepherd breeds these values were the
lowest (Poongsan – 0.550 and 0.502; German Shepherd – 0.567 and 0.538). Irion et al.
[2003] obtained varying H indicators for 28 dog breeds, including terriers (from 0.387
for Bull Terrier up to 0.758 for Jack Russell Terrier), and moderate H value (0.543) for
BMD (23rd position in 28 analyzed breeds). Kopaliani et al. [2014] estimated lower
expected heterozygosity in wolves (0.78) comparing to CSD (0.83), whereas values of
the observed heterozygosity were similar (0.74 and 0.73, respectively).
H values in this study were comparable with values estimated for other breeds:
0.46 – German Shepherd, 0.63 – Kunming Wolfdog, 0.63 – Labrador Retriever, 0.66
– English Springer Spaniel, 0.75 – Belgian Shepherd, 0.76 – Tibetan Mastiff [Ye et al.
2009] and 0.43 – Yugoslav Shepherd Dog [Dimitrijevic et al. 2013].
Pribánová et al. [2009] analyzed genetic variability of 6 dachshund breeds and
showed that the average observed heterozygosity ranged from 0.58 to 0.70. Similar
values for expected (0.62-0.79) and observed (0.70-0.72) heterozygosity were found
in four Turkish breeds [Altunok et al. 2005].
The PIC value can provide a better information piece on the degree of variability, as
it depends on both, polymorphism and frequency of alleles, contrary to heterozygosity.
The higher the value of PIC, the more information can be obtained from a marker
[Ciampolini et al. 2011]. In our study, the highest PIC values characterized Ch and
the lowest BMD. Similar results were obtained by Ye et al. [2009] for several breeds:
German Shepherd – 0.49, Kunming Wolfdog – 0.62, Labrador Retriever – 0.69, English
Springer Spaniel – 0.7, Belgian Shepherd – 0.72 and Tibetan Mastiff – 0.74. A similarly
high PIC value (0.66) was also found for the Yugoslav Shepherd Dog by Dmitrijevic
et al. [2013].
Our estimates of genetic distance were consistent with those obtained by Cho
[2005]. The author used similar microsatellite loci (FHC2010, FHC2054, FHC2079,
PEZ1, PEZ6, PEZ8, PEZ10, PEZ11, PEZ12, PEZ15 and PEZ17) to determine
genetic distance between ve breeds in Korea (Chihuahua, Jindo, Miryang, Poongsan
and German Shepherd). The highest genetic distance was found between German
Shepherd and Miryang breeds (1.244), and the lowest between Chihuahua and Jindo
breeds (0.154). Ye et al. [2009] carried out a research on six breeds (Tibetan Mastiff,
Kunming Wolfdog, German Shepherd, Belgian Shepherd, Labrador Retriever and
English Springer Spaniel), using the same 10 microsatellites as in our study, but
estimated much lower values of the genetic distances. The closest breeds were
Labrador Retriever and English Springer Spaniel (0.099) and the most distant were
German Shepherd and Labrador Retriever (0.254).
A relatively high genetic distance was estimated between Ch and the BMD, as
K. Czyż et al.
101
well as between CSD and BMD, which may suggest that phylogenetically Ch and
CSD breeds have been separated from BMD at an early stage of domestication. A
at topology of phylogenetic tree created by Parker et al. [2012] largely conrms a
common founder of dog breeds and an extensive gene ow between breeds with very
different phenotypes, even before the advent of kennel associations and restrictions on
breeding due to determination of the breed standards.
The Ch and CSD breeds were characterized by the lowest genetic distance and
also by the highest heterozygosity, which is in accordance with the research reported
by Leroy et al. [2009].
Von Holdt et al. [2010] analyzed the shared proportion of microsatellite haplotypes
characteristic for four populations of the gray wolf (Middle East wolf, European wolf,
Chinese wolf and North American wolf) in genotypes of dogs of 80 breeds. The
contribution of haplotypes of the Middle East and Chinese wolves in Ch and BMD
genotypes were similar (35% and 20%, respectively), while these breeds differed with
regard to the proportion of haplotypes of the European and North American wolves
(Ch – 30% and 15%, respectively; BMD – 35% and 10%). The authors did not analyze
CSD, but according to Kopaliani et al. [2014] this breed has approximately 68% of
haplotypes characteristic to East Asian wolf. Hence, the greater genetic similarity
between Ch and CSD breeds and a larger genetic distance between the both breeds
and BMD may be explained.
The presence of the same alleles in 10 microsatellite loci observed in all three dog
breeds is an evidence of their belonging to one species. However, in each breed alleles
not found in the other two breeds have been detected, which differentiates them.
Data on microsatellite polymorphism of dogs may be useful in studies concerning
the segregation of microsatellite sequences associated with quantitative traits or genetic
diseases. Because inbreeding occurs often in purebred dogs, genetic markers should
be as much polymorphic as possible, which would increase chances of detection of
markers associated with genetic diseases. The lowest EA, H and PIC values obtained
for loci PEZ5 and PEZ20 indicate a low degree of variability and a low suitability of
these microsatellite sequences to examine the origin and genome mapping of dogs.
Genetic diversity among the studied breeds may reect genetic isolation, and thus
the differences in gene ow between them in the past. The large variation of these
breeds of dogs may also result from selection by man. The large values of genetic
distance between BMD versus Ch and CSD breeds support the hypothesis of their
separation at an early stage of domestication.
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... That, along with the historical variations in population sizes, have resulted in the considerable inbreeding and loss of genetic variability in certain purebred dog populations [5][6][7][8], as well as in the increased incidence of inherited diseases [9][10][11]. Although genetic make-up of numerous dog breeds has been studied to date [4,[11][12][13][14][15][16][17][18][19][20][21][22][23], the knowledge regarding genetic structure of non-cosmopolitan dog breeds and local canine populations is still grossly lacking [21,24]. ...
... It is genetically well distinguished from other livestock guard dogs from the Western Balkans, Karst Shepherd and Tornjak [25]. We report levels of genetic diversity in YSD which are in the range of those reported by Dimitrijević [2008] and Ceh and Dovc [25], and are comparable and in some cases even higher than those found in numerous dog breeds studied to date by means of nuclear microsatellites [4,[11][12][13][14][15][16][17][18][19][20][21][22][23]. Thus, despite the decline of the YSD population after both 20 th century World Wars, this breed remains genetically highly diverse. ...
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