Nghiên cứu đa dạng di truyền quần thể chó phú quốc dựa trên trình tự HV1 thuộc vùng CR trên hệ gen ty thể tt tiếng anh

MINISTRY OF EDUCATION

MINISTRY OF AGRICULTURE

AND TRAINING

AND RURAL DEVELOPMENT

VIETNAM ACADEMY OF AGRICULTURAL SCIENCES


THAI KE QUAN

EVALUATION OF GENETIC DIVERSITY OF PHU
QUOC RIDGEBACK DOGS BASED ON
MITOCHONDRIAL DNA HYPERVARIABLE-1 REGION
Speciality: Biotechnology
Code: 9420201

SUMMARY OF DOCTORAL DISSERTATION

HO CHI MINH CITY - 2019


This dissertation has been completed in Ho Chi Minh city
Supervisors:
1. Assoc. Prof. Dr. Tran Hoang Dung
2. Dr. Chung Anh Dung
Reviewer 1:...................................................................................
Reviewer 2: ..................................................................................
Reviewer 3:...................................................................................

The dissertation will be presented to the Institute-level Dissertation
Defense Committee at the Institute of Agricultural Science for
Southern Vietnam on ......, 2019
This dissertation can be found at:
1. National library of Vietnam
2. Library of Vietnam academy of agricultural sciences
3. Library of Institute of Agricultural sciences for
Southern Vietnam.


1
PREAMBLE
1. Introduction
Phu Quoc ridgeback dog, a valuable dog breed originating in
Phu Quoc Island (Kien Giang province, Vietnam), is one of the three
dog breeds in the world harboring a ridge of hair running along the
back in the opposite direction from the rest of the coat. Currently,
Phu Quoc ridgeback dog has not yet been officially identified as a
dog breed. It is said that the Phu Quoc ridgeback dog originated
from the Thai ridgeback dog because of the same characteristic of
the ridge. However, no scientific evidence has been provided to
prove this hypothesis.
Mitochondrial DNA (mtDNA), especially the control region
(CR), has a high mutation rate, leading to genetic differences
between individuals. The project "Evaluation of genetic diversity of
Phu

Quoc

ridgeback

dogs

based

on

mitochondrial

DNA

hypervariable-1 region" assesses the genetic diversity in the CR of
the Phu Quoc ridgeback dog population to understand the genetic
relationships between Phu Quoc ridgeback dog with other dog
breeds in the world, thereby serving as a basis for tracking the origin
of this special dog breed.
2. Aim of the study
Tracking the origin of Phu Quoc ridgeback dog based on
information about the mitochondrial DNA hypervariable-1 region
variation.
3. The new contributions and implications of the study
- For the first time in the world, a web-based tool has been
developed for haplotyping the mtDNA HV1 of dog.


2
- The genetic diversity of mtDNA HV1 of Phu Quoc
ridgeback dog was evaluated successfully.
- A hypothesis about the origin of Phu Quoc ridgeback dog
was given based on information collected from this study.

4. The meaning of the study
4.1. Scientific meaning
Examined and identified the genetic diversity of mtDNA
HV1 of Phu Quoc ridgeback dog, this study has provided scientific
information supporting the tracking of the origin of Phu Quoc
ridgeback dog. A simple protocol for DNA extraction from dog hairs
was also established, which therefore can be applied to other studies
on genetic of dog. The web-based tool for quick haplotyping of
mtDNA HV1 has been available for public using via world wide
web.
4.2. Practical meaning
This study has provided initial information and scientific data
for tracking the origin of Phu Quoc ridgeback dog. The study has
also provided the information on genetic diversity of Phu Quoc
ridgeback dog, which can be a premise for the genetic resources
conservation of this valuable Vietnamese dog breed .
5. Subject and scope of the study
5.1. Subject
This dissertation has carried out the study on 582 bp
fragment in the mtDNA HV1 of dogs. Totally, 200 individuals (100
Vietnamese village dogs and 100 Phu Quoc ridgeback dogs) have
been examined in this study .


3
5.2. Scope
This study focuses on the 582 bp fragments in the mtDNA
control region of 100 Vietnamese village dogs and 100 Phu Quoc
ridgeback dogs. Other dog breeds, which is used in determining the
genetic relationships with Phu Quoc ridgeback dog, are used based

on the availability of data and the information about the migration
route of dog. Not all dog breeds on the world are included in this
study.


4

CHAPTER 1. LITERATURE REVIEW
1.1. Phu Quoc ridgeback dog
1.1.1. Introduction to Phu Quoc ridgeback dog
Ridgeback dog living on Phu Quoc island in Kien Giang
province is a dog breed characterized by many valuable
characteristics. There are currently three dog breeds on the world
harboring the ridgeback trait. Of those, the Rhodesian ridgeback dog
in South Africa and the Thai ridgeback dog are recognized by the
Federation Cynologique Internationale (FCI), while Phu Quoc
ridgeback dog of Vietnam has not been approved. Many historical
evidences have been reported to prove the special origin of Phu Quoc
ridgeback dog, but the persuasion of these data is not high. So far, the
origin and the genetic relationship of this dog breed with others has
not been thoroughly studied.
1.1.2. Taxonomy of the Phu Quoc ridgeback dog
1.1.3. Morphological and behavioral characteristics of the Phu
Quoc ridgeback dog
1.1.4. Studies on the origin and genetics of Phu Quoc ridgeback
dog
So far, there has been no study on the origin and genetics of
Phu Quoc ridgeback dog outside of Vietnam. Tran Hoang Dung et al.
(2017) initially analysed DNA and proved the genetic relationship
between the Phu Quoc ridgeback dog harbouring the E4 haplotype

with the Pungsang dog in North Korea.


5
1.2. Inheritance of the ridgeback trait
The ridge trait in ridgeback dog is caused by the autosomal
dominant allele (R) on chromosome 18. Besides, dogs harbouring the
homozygous dominant alleles (RR) have a higher risk of getting
Dermoid sinus (DS), which negatively affect the dog’s health (Table
1.1).
Table 1.1: Correlation between genotype and ridge, DS
Genotype

Ridge

DS

RR

yes

high risk

Rr

yes

low risk

rr

none

none

1.3. Mitochondrial DNA of dog
1.3.1. Structure of mitochondrial DNA of dog
1.3.2. Structure and inheritance of mtDNA control region
The mtDNA control region of dog consists of three small
regions, in which, the hypervariable region 1 (HV1) and
hypervariable region 2 (HV2) are highly polymorphic. The genetic
diversity of HV1 is usually evaluated to study the origin of dog
breeds.

Figure 1.4: Position and structure of mtDNA control region
1.4. Studies on the origin, phylogeny and genetic diversity of dogs


6
1.4.1. Studies using archaeological remains
1.4.2. Studies using modern genetic methods
1.4.2.1. Molecular markers
Many different target DNAs have been used in genetic
research in dogs such as microsatellite, Y chromosomes, COI, CytB
... Especially, many studies in the world have focused on the
polymorphism of 582 bp fragment on mtDNA to learn about the
origin and history of many different dog breeds.
1.4.2.2. Studies on the origin of domestic dogs
On the phylogenetic tree based on the 582 bp fragments of
HV1, dogs’ and wolves’ haplotypes are grouped into six distinct

haplogroups A, B, C, D, E, F. There are totally 72.34% of dogs
worldwide belonging to haplogroup A; 97.40% of dogs belongs to
haplogroup A, B, C. The distribution of haplogroups D, E, F are
more geographic specificity which can be found only in particular
regions. These haplogroups are rare and believed to be the result of
postdomestication wolf-dog bybridization. Based on the distribution
of haplotypes, Wang et al (2016) unraveled the place of origin and
the migration route of dog on earth.

Figure 1.8: The place of origin and the migration route of domestic
dog


7
1.4.2.3. Studies on the genetic diversity of dog breeds
The genetic diversity of different dog breeds are varied,
implicating the place and date of origin of dog breeds. The haplotype
diversitys of dog breeds living in isolated regions are low, such as
Portuguese Castro Laboreiro dog (0.1) while the haplotype diversity
of Shiba dog in Japan, which was bred from three bloodlines from
three different regions in Japan, is quite high (0.8161).
1.5. DNA databank GenBank and the mtDNA HV1 data
1.5.1. GenBank
1.5.2. mtDNA HV1 data on GenBank
mtDNA HV1 data is stored vastly on GenBank. As in many
previous publications, there is a lot of inaccurate or inconsistent
information that needs to be corrected and reconciled.
1.6. Perception on the study on the genetic diversity of the Phu
Quoc ridgeback dog
- The 582 bp fragments of mtDNA HV1 will show the genetic

diversity of the Phu Quoc ridgeback dog population.
- A reliable and accurate dataset on worldwide dog haplotypes should
be developed for the comparison and studying of the genetic diversity
of the Phu Quoc ridgeback dog.
- The genetic diversity of the Phu Quoc ridgeback dog, along with
the genetic relationship with other dog breeds, will be the basis for
tracking the origin of this dog breed.


8

CHAPTER 2. CONTENT AND RESEARCH METHOD
2.1. Research contents
- Content 1: Building the database and the web-based tool for
haplotyping the 582 bp fragment of mtDNA HV1 of dog.
- Content 2: Identifying the nucleotide sequence of 582 bp fragment
of mtDNA HV1 of 100 Phu Quoc ridgeback dogs and 100
Vietnamese village dogs.
- Content 3: Evaluating the genetic diversity of Phu Quoc ridgeback
dog based on the 582 bp fragments of mtDNA HV1.
- Content 4: Assuming the origin of Phu Quoc ridgeback dog.
2.2. Research methods
2.2.1. Building the database and the web-based tool for
haplotyping the 582 bp fragment of mtDNA HV1 of dog
2.2.1.1. Building the database of 582 bp fragment of mtDNA HV1
Database of 582 bp fragments of mtDNA HV1 is designed
using the relational model of data, consisting of 6 tables with logical
connections. Each table in the database contains distinct data about
the haplotype, nucleotide sequence, taxonomy data… Nucleotide
sequences collected from Genbank using an in-house developed

computer program. Information of sequences are extracted from
GenBank records and stored in the database.
2.2.1.2. Nucleotide numbering and mutation illustration
2.2.1.3. Haplotyping and data reconcilation
Assigned haplotypes and the haplotype of a sequence would
be identified by following rules:


9
1. The sequence which was firstly used to report a haplotype
would be the standard sequence for that haplotype.
2. If two assigned haplotypes have the same mutation profile,
the later (newer) will be eliminated.
3. If a sequence was annotated as an assigned haplotype but
it is not identical to the standard sequence of that haplotype, the
annotation is considered as wrong and its haplotype is defined
according to its mutation profile
2.2.1.4. Web-based tool for haplotyping 582 bp fragment of mtDNA
HV1
The web-based haplotyping tool is developed in Perl
programming language and HyperText Markup Language (HTML).
The tool will analyse the query sequence, recognize the polymorphic
site to identify the haplogroup and/or haplotype of the input
sequence.
2.2.1.5. Construction of the phylogenetic tree
Phylogenetric trees for the 582 bp sequences of mtDNA HV1
was inferred using PAUP*, with Neighbor-Joining method and 2000
repetitions bootstrapping.
2.2.2. Nucleotide dentification of the 582 bp fragment of mtDNA
HV1 of Vietnamese village dog and Phu Quoc ridgeback dog

2.2.2.1. Equipment applied in the laboratory
2.2.2.2. Sampling
Hair roots were collected from 100 Phu Quoc ridgeback dogs
and 100 Vietnamese village dogs living in Phu Quoc island, Rach
Gia (Kien Giang province), Ho Chi Minh city and nearby provinces.


10
2.2.2.3. DNA extraction from dog hairs
DNA from hair roots are extractred using the protocol which
is established in this project. Proteinase K is used for digestion of
protein, while phenol:chloroform is used to separate DNA from
others. After precipitation in absolute ethanol, DNA will be
resuspended in water and stored at -30oC.
2.2.2.4. Checking the DNA purity using spectrometer
2.2.2.5. Amplification of mtDNA HV1 by PCR
The mtDNA HV1 region is amplified by PCR with the
primers pair 15412F-16625R, as suggested by Gundry et al. (2007).
2.2.2.6. Sequencing and sequence editing
Nucleotide sequences are sequenced using Sanger method.
Two strands of each nucleotide sequence are sequenced separately
for checking the consistency.
2.2.3. Evaluation of the genetic diversity of Phu Quoc ridgeback
dogs based on the 582 bp fragment of mtDNA HV1
Data from this study was statistically analysed using R.
Genetic diversity measures, AMOVA and genetic distances among
dog populations were estimated as average nucleotide differences
using Arlequin 3.5.
2.2.3.1. Haplotype diversity
Haplotype

diversity

is

defined

as

the

probability that two randomly chosen haplotypes are different in
population.
2.2.3.2. Nucleotide diversity
Nucleotide diversity is defined as the probability that two
randomly chosen nucleotide at one random site are different.


11

2.2.3.3. Medium-spanning network of haplotypes
Medium-spanning network of haplotypes, calculated by
Arlequin

3.5,

illustrates

relationships

among

haplotypes

in

population. In cases of haplotypes that differs from nearsest
haplotype more than one nucleotide, the intermediate reported
haplotype, if available, will be inserted into the network at the
corresponding position.
2.2.3.4. Estimating the genetic distance between two populations
Genetic distance between two populations is defined as the
average number of nucleotide differences between two populations.
2.2.3.5. Analysis of molecular variance (AMOVA)
Analysis of Molecular Variance (AMOVA) is a method of
estimating population differentiation at different level: among groups
within population, among different demes with groups and among
haplotypes within demes. The significance of difference is evaluated
by the F value of Fisher test.
2.2.4. Assuming the origin of Phu Quoc ridgeback dog
The origin of Phu Quoc ridgeback dog is assumed based on
the scientific data collected from this study and previously reported
data related to dog all over the world.


12

CHAPTER 3. RESULT AND DISCUSSION
3.1. Building the database of 582 bp fragments of mtDNA HV1
Using BLAST against the GenBank nucleotide database,

5567 nucleotide sequences originating from individuals of Canis
lupus species with high similarity to the reference mtDNA HV1
sequence (GenBank accession: U96639.2) were collected and stored
in the in-house developed database. Of these, 1921 entries are the 582
bp partial sequences which are not sufficient for haplotyping; 414
entries were correctly haplotyped and annotated with haplotype
information using A-F system, 3232 entries were annotated with
wrong haplotype identification or with the haplotyping information
using other systems or did not contain any information about
haplotype. The lack of haplotype information and inconsistencies
derived from the GenBank were modified or corrected in our
database.
Web-based tool for rapid haplotyping of the 582 bp fragment
of mtDNA HV1 (namely Haplotype identifier) was developed in Perl
programming language, and freely available via the Internet at
. Testing on 50 random sequences in the
database showed that Haplotype identifier was working correctly,
resulting in a more appropriate and faster haplotyping than the same
work done with phylogenetic trees. The advantages of haplotypings
with the Haplotype identifier compared to those with phylogenetic
tree can be summarized in the following table (Table 3.5).


13

Table 3.5: Comparison between haplotyping with Haplotype identifer
and with Phylogenetic tree
Haplotype identifier
Sequence

called

identical

with corresponding

reported

exactly

Phylogenetic tree

the could not

haplotype

haplotype
Sequence
identical

not called

exactly

with haplogroup

the called

exactly

the

haplogroup

reported
haplotype
Running time

fast

(about

second/sequence)

1 long, depend on the
method used (ranging
from 10 minutes to 8
hours)

Specialized

no

yes

software
Experience

The experience on User must be familiar
haplotyping is

required

not with

methods

of

inferring Phylogenetic
tree


14

3.2. Sequencing the mtDNA HV1 of Phu Quoc ridgeback dogs
and Vietnamese village dogs
3.2.1. Establishing the protocol for DNA extraction from dog
hairs
After examining the conditions of sample incubation
(temperature, time), using common chemicals in laboratory of
molecular biology, a protocol for DNA extractrion from dog hairs
was established. It can be summarized as follows (Figure 3.5).

Figure 3.5: Protocol for DNA extraction from dog hairs
Using this extraction process, total DNA (including nuclear
DNA and mtDNA) from a source of about 40 dog hairs can be
obtained. The extracted DNA may be used in further studies.
3.2.2. DNA extraction from dog hairs
Two hundred samples of dog hairs (100 Phu Quoc ridgeback
dogs and 100 Vietnamese village dogs) were used to extract DNA

using the established protocol. The quality of obtained DNAs were
good, which can be used in subsequent PCR.


15

3.2.3. Amplification of mtDNA HV1
The extracted DNA was used as materials for the
amplification of mtDNA HV1, and the PCR products in turn were
used in nucleotide sequencing. The purity of PCR products was
checked through electrophoresis using 1% agarose gel. The signals of
the DNA bands are bright, clear, located between the 1000 bp band
and 1500 bp band of 1Kb DNA ladder, matching the expected size of
DNA products (about 1200 bp).
3.2.4. Sequence editing
PCR products were sequenced using 15412F and 16114R
primers. The chromatogram files representing DNA sequencing
results were read by FinchTV software. For each sample, two
nucleotide sequences primed by 15412F and 16114R primers were
compared to verify and result in a final sequence. Sequencing result
of 200 DNA sequences in this study are clear, especially in the region
of 582 bp.
3.2.5. Haplotyping of Phu Quoc ridgeback dogs’ and Vietnamese
village dogs’ sequences
All 200 examined sequences were submitted into the
Haplotype identifier for quick haplotyping. Most of sequences belong
to 3 common haplogroups (A, B, and C). 197 out of 200 sequences
are identified harbouring haplotypes which are previously reported.
Besides, three new haplotypes - An1 (haplogroup A), Cn1, Cn2
(haplogroup C) - have never been reported so far. In Phu Quoc

ridgeback dogs, the proportion of haplogroup E’s haplotypes ís


16
notably high while these haplotypes are rare in the world’s dog
population.
3.3. Genetic diversity of mtDNA HV1 in Phu Quoc ridgeback
dogs
3.3.1. Nucleotide diversity
In the Phu Quoc ridgeback dogs, 100 examined sequences
were classified into 19 groups based on the identity of nucleotide
sequences, corresponding to 19 reported haplotypes. Through the
length of 582 bp fragment of Phu Quoc ridgeback dog, there are
totally 33 polymorphic sites. Of these, 31 sites are nucleotide
substitutions and 2 sites are observed with insertion or deletion
(indel). Most of polymorphic sites are with transition (30/31 sites),
only 2 sites (the nucleotide 15479 and 15639) are with transversion
(G/C substitution; T/A,G substitution). Nucleotide at position 15639
can be observed with both transversion and transition. In Vietnamese
village dogs, a total of 34 polymorphic sites were recorded. Of these,
32 sites are nucleotide substitutions and 2 sites are with indels. All
nucleotide substitutions are transitions, except for the site 15639, as
seen in the Phu Quoc ridgeback dogs, that can be observed with both
transversion and transition. Compared to other dog breeds in the
world, the nucleotide diversity of Phu Quoc ridgeback dogs and
Vietnamese village dogs are quite high (0.014588 ± 0.007534 and
0.014035 ± 0.007270; respectively).
3.3.2. Haplotype diversity
One hundred 582 bp sequences of Phu Quoc ridgeback dogs
were sequenced and identified as 19 different haplotypes, including a

new unpublished haplotype. All 18 remaining haplotypes were


17
previously seen in the world dog population. 77% of sequences
harbour haplotypes in common haplogroups A, B, C, none of
sequences harbours haplotype in rare haplogroup D and F. Notably,
23% of sequences harbour haplotypes in the rare haplogroup E which
only accounts for a very low proportion in the world (along with
haplogroup D and F accounts for less than 3%). With 19 haplotypes
detected among 100 examined samples, the haplotype diversity of
Phu Quoc ridgeback dog is up to 0.9042 ± 0.0127, meaning that the
probability to take two different haplotype in the samples is about
90%. Compared with some other dog breeds in the world, the
haplotype diversity of Phu Quoc ridgeback dog is lower than that of
Thai dogs. However, the haplotype diversity is still high, similar to
those of Pungsang dogs (Korea), Vietnamese village dogs and much
higher than the Portuguese sheepdogs, German shepherd dogs,
Tibetan Mastiff dogs, Kangal dogs (Turkey), or Shiba dogs (Japan)
and Jindo dogs (Korea).
3.3.3. Genetic relationship between Phu Quoc ridgeback dogs
and other dog breeds
3.3.3.1. Genetic relationship between Phu Quoc ridgeback dogs
and other dog breeds
AMOVA analysis of mtDNA HV1 data shows that although
the differences among examined dog breeds are small, they are
statistically significant (Table 3.11).


18

Table 3.11: AMOVA analysis of some dog breeds in the world
Source of
variation
Among

Sum of
squares

Variation

Percentage of
variation

F value

P value

groups of dog

512.086

0.55663

13

0.13001

0.01369

111.69

0.348993

8.15

0.09369

0

2538.619

3.37582

78.85

0.21152

0

3162.395

4.28145

100

population
Among

dog

populations
Within
populations
Total

It is notable that Thai dog is not too genetically close to the
Vietnamese village dog (Figure 3.13). The genetic distance between
the Phu Quoc ridgeback dog and Thai dog is 2.27668, nearly 7 times
higher than that between the Phu Quoc ridgeback dog and the
Vietnamese village dog (0.33899). This is the basis for asserting Phu
Quoc ridgeback dog originating from Vietnamese village dogs, not
from Thai dogs as previously believed.


19

Figure 3.13: Genetic distance among some world dog breeds
3.3.3.2. Genetic relationship between Phu Quoc ridgeback dogs and
Vietnamese village dogs
AMOVA analysis showed a small and unsignificant
difference between Phu Quoc ridgeback dog and Vietnamese village
dog (Table 3.12).
Table 3.12: AMOVA analysis of dog populations in Vietnam
Source of
variation
Between two
breeds
Among
populations

Within
population
Total

Sum of
squares

Variation

Percentage
of variation

F value

P value

21.120

0.19002

4.38

0.04378

0.10655

6.923

-0.04919

-1.13

-0.01185

0.84360

818.857

4.19927

96.76

0.03245

0.06647

451.278

4.31726

100%

According to the calculation of Savolainen et al. (2009), 1
unit of genetic distance between two populations (or mutation rate) in
the mtDNA HV1 of dogs is 1 substitution per about 40000 years.


20
Thus, considering the entire examined dog populations in this study,
with the genetic distance of 0.33899, Phu Quoc ridgeback dog and

Vietnamese village dog differentiated from a common ancestor about
6780 years ago.
3.3.3.3. Genetic relationship between Phu Quoc ridgeback dogs and
Vietnamese village dogs living in Kien Giang province
The similarity of the haplotype compositions of Phu Quoc
ridgeback dogs and Vietnamese village dogs living in Kien Giang,
especially the common haplotypes with high ratio in the population,
indicates the phenomenon of gene flow from Vietnamese mainland to
Phu Quoc island (Figure 3.14).

Hình 3.14: The similarity of haplotypes between dogs living on the
mainland and on the Phu Quoc island
AMOVA analysis of mtDNA HV1 data also showed no
significant difference between two groups of dogs living on the
mainland and on the island in Kien Giang province (Table 3.13).


21
Table 3.13: AMOVA analysis of dog populations living in Kien
Giang province
Source of
variation
Between
two groups
Among
populations
Within
population
Total

Sum of
squares

Variation

Percentage
of
variation

F value

P value

10.204

0.14484

3.38

0.03383

0.34018

2.970

-0.03645

-0.85

-0.00881

0.52590

438.104

4.17242

97.47

0.02532

0.13392

451.278

4.31726

100%

Notably, the genetic distance between Vietnamese village
dogs living on the mainland of Kien Giang and Phu Quoc ridgeback
dogs living on the Phu Quoc island (0.36176) is similar to that
between Vietnamese village dogs and Phu Quoc ridgeback dog in
general (0.33899). In a meanwhile, the genetic distance between
Vietnamese village dogs and Phu Quoc ridgeback dogs living on the
Phu Quoc island is much smaller (0.07154). Based on the mutation
rate in this mtDNA HV1 region, the time point of differentiation of
these two populations is about 1430 year before (0.07154 x 40000/2).
3.4. Assuming the origin of Phu Quoc ridgeback dog
Based on the analyses of this study, along with previous

studies on the migration route of dogs on earth, and the origin of the
Australian dingo, as well as the genetic characteristics of the ridge
trait, the origin of Phu Quoc ridgeback dog can be hypothesized as
follows:
The domestic dog originated in the Vietnam - China border
and migrated to different lands, resulting in the diversity and wide
distribution of current dogs. Beside a group of dogs moving west to


22
Europe, and a group of dogs moved north, at this place of
domestication, a group of dogs moved south, which are the ancestors
of current Vietnamese dogs. During the migration to the south, the
mating between the male dog and female wolf harbouring haplotype
E has added this gene into the dog's gene pool. About 6780 years
before, a group of Vietnamese village dogs separated from
populations, forming a new deme and migrating towards the land
currently known as Kien Giang today. Until about 1430 years before,
the human migration process from the mainland (Kien Giang
province) brought a group of Vietnamese village dogs carrying
haplotypes A, B, C and E to Phu Quoc island. These dogs did not
have dorsal hair ridge, they carried two alleles r/r on chromosome 18.
Under the condition of geological isolation and the limitation of
nutrient sources, allele r was mutated into allele R in dogs harbouring
different haplotypes. The advantage of haplotype E harbouring
individuals in the habitat, together with inbreeding due to
geographical isolation, has contributed to raising the frequency of
haplotype E and allele R in the population of Phu Quoc ridgeback
dogs.

23

CONCLUSION – RECOMMENDATION
I. Conclusion
1. From 5567 sequences stored in GenBank, a database and a
tool for mtDNA HV1 haplotyping were developed.
2. A protocol for DNA extraction from dog hairs was
established. 582 bp fragments of 100 Phu Quoc ridgeback dogs and
100 Vietnamese village dogs were sequenced, analysed and edited. In
the population of Phu Quoc ridgeback dogs, 19 haplotypes belonged
to 4 different haplogroups were detected. Of these, 23 dogs harbours
rare haplotype E. In the population of Vietnamese village dogs, 24
haplotypes belonged to 4 different haplogroups were detected. Of
these, 4 dogs harbours rare haplotype E.
3. The genetic diversity of Phu Quoc ridgeback dogs is high,
with the haplotype diversity of 0.9042 ± 0.0127 and the nucleotide
diversity of 0.014588 ± 0.007534. The genetic diversity of Phu Quoc
ridgeback dogs is higher than that of many other dog breeds on the
world.
Phu Quoc ridgeback dog is genetically close to the
Vietnamese village dog, with the genetic distance of 0.33899, while
the genetic distance between Phu Quoc ridgeback dog and Thai dog
is quite high (2.27668).
4. The above scientific data initially asserted that Phu Quoc
ridgeback dog originated from Vietnamese village dog and did not
originate from Thai dogs as previously stated. The natural conditions