Nghiên cứu mối liên quan giữa kiểu gen và kiểu hình đặc trưng trong bảo tồn chó phú quốc TT TIENG ANH

MINISTRY OF EDUCATION
AND TRAINING

MINISTRY OF AGRICULTURE
AND RURAL DEVELOPMENT

VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
---------

QUAN QUOC DANG

RESEARCH OF THE RELATIONSHIP BETWEEN GENOTYPE
AND PHENOTYPE IN PHU QUOC DOG CONSERVATION

Major: Biotechnology
Major code: 94 202 01

SUMMARY OF THE DOCTORAL THESIS IN AGRICULTURE

HO CHI MINH CITY – 2021


Thesis was completed at: Ho Chi Minh City
Supervisors:
1. Dr. Chung Anh Dung
2. Assoc. Prof. Dr. Tran Hoang Dung
First Reviewer : .........................................................................
Second Reviewer : .........................................................................
Third Reviewer : .........................................................................

The thesis will be defended in front of the Institute-level Thesis Judging

Committee in ..........................................................................................
Month ... Day ... 2021
The thesis can be found at:
1. National Library
2. Vietnam Academy of Agricultural Sciences Library


1
INTRODUCTION
1. Urgency of study
The Phu Quoc Dog is a unique type of dog of Phu Quoc island, Vietnam.
The distinguishing feature of them to differ from other dogs is the dorsal ridges.
Phu Quoc dog is one of three dogs breeds with ridges in the world, although not
all individuals in this spicies have dorsal ridges.
Phu Quoc dogs have many different colors coat: black, brown, yellow,
brindle, gray and other colors. But the most popular colors are black and yellow
which have 60%. Straight hair style have over 98%. Another feature that is very
typical for Phu Quoc dogs that noticed is the dorsal ridge, called ridgeback. The
ridgeback is very diverse and symmetrical in the dorsal, the most common forms
can be swords, arrows, saddles, violins and others.
However, at present, there are only two breeds of ridgeback dog in the
world, the African Rhodesian and the Thai Ridgeback recognized by the
Fédération Cynologique Internationale (FCI), and Phu Quoc dog is mentioned
belong with and considered to be descended from the Thai Ridgeback.
Nowadays, there is non-scientific for phenotype and genotype basis evidence as
a proof for recognition this breed from two others.
In addition, most customers only base on the type of ridgeback to decide
to choose a Phu Quoc dog breed, while not all Phu Quoc dogs have back ridges.
Therefore, the process of breeding to meet the needs of trading leads to the
degeneration of this breed.

The research about the structural and morphological features of the Phu
Quoc dog in order to develop a set of morphological standards which serve for
identification this breed is very important and necessary, not only for buyers but
also for the breeders and preservers on the Phu Quoc dog.
2. Aim of study
Develop a set of criteria for identifying Phu Quoc dogs based on
morphological dimensions, determine the relationship between genotype and


2
specific dorsal ridgeback phenotype to serve for selection, breeding and
conservation Phu Quoc dog.
3. Scientific and practical significance of study
Provide data on the basic dimensions of Phu Quoc dogs, contributing to
standardizing the selection of Phu Quoc dogs through morphological assessment
(compared to current evaluation and selection by emotion).
Provide scientific data to determine genotypes related to the characteristic
ridgeback trait of Phu Quoc dogs, contributing to species conservation in the
breeding process.
4. Object and scope of study
Dogs were born in Phu Quoc island and dogs at Phu Quoc breeders in
other regions but have clear origins, both parents from Phu Quoc island.
This study investigated some morphological parameters of 175 Phu Quoc
dogs (96 males, 79 females) in Phu Quoc island and Ho Chi Minh City and
surrounding areas, where Phu Quoc dog breeding farms are concentrated.
Among of them, 32 individuals had non-ridgeback and 143 individuals had
ridgeback.
Analysis of the R gene region for the ridgeback in 15 Phu Quoc dogs with
and without ridgeback (both parents have ridgeback) on Phu Quoc island,
compared with 03 domestic dogs in Ho Chi Minh City.

5. New contributions of study
- Phu Quoc dog morphological standard has been developed first time in
Vietnam, helping to quickly and accurately identify this breed.
- The first published to determine the relationship between genotype and
ridgeback phenotype in the Phu Quoc dog in the world.
6. Dissertation layout
Thesis consists 112 pages (excluding the Appendix), including the
following parts: Introduction (4 pages), Chapter 1: Literature Review and
Scientific Evidence (39 pages), Chapter 2: Materials and Research Methods (13
pages), Chapter 3: Research Results and Discussion (46 pages), Conclusion and


3
Recommendations (1 page), References (9 pages), used 8 Vietnamese and 112
English Published Papers. Thesis has 25 Tables, 49 Figures and 9 Appendices, 4
Published Papers.


4
Chaper 1 - LITERATURE REVIEW AND SCIENTIFIC EVIDENCE
The thesis has consulted and summarized Vietnamese and English
published papers, with relevant contents including: 1. Classification and
characteristics of Vietnamese Phu Quoc dogs, 2. Origin and morphology of Phu
Quoc dogs, 3. Ridgeback trait in Phu Quoc dogs, 4. Genetic studies and ancient
documents on Phu Quoc dogs, 5. Necessity of research on morphology and
genotype of ridgeback trait on Vietnamese Phu Quoc dogs.

1.1. Classification and characteristics of Vietnamese Phu Quoc dogs
1.1.1. Classification
In the biological classification system, Phu Quoc dogs have the following

classification position:
Gender: Animals (Animalia).
Sub-Gender: Multicellular animals (Metazoa).
Class: Chordata
Subphylum: Vertebrates (Vertebrata).
Class: Mammal (Mammalia).
Order: Carnivores (Carnivora).
Family: Dogs (Canidae).
Sub-Family: Dogs (Caniae).
Breed: Dog (Canis Linnaeus, 1758).
Species: Dog (Canis lupus Linnaeus, 1758).
1.1.2. Characteristics
The Phu Quoc Dog is a type of unique dog of Phu Quoc island in
Vietnam. The distinguishing feature of dog breed is a dorsal ridgeback on most
of individuals in population This dog breed is one of the three dog breeds with
has dorsal ridgeback in the world. Phu Quoc dogs are precious with many
outstanding characteristics that other dog breeds do not have such as:
intelligence, agility, ability to hunt and keep good house. They are medium
sized, easy to raise, suitable for rural areas and rivers. The main characteristics
of the Phu Quoc dog are different from other breeds: Small dog head suitable for


5
long skull, small and standing ears, black muzzle, brown eyes. The body is slim
and the chest is enlarged, the belly is slim, especially for male dogs. The dog's
four legs are strong, the muscles are prominent, straightened when standing, and
the feet are webbed. Tail with short hair, often in a curved position with a
curvature of ẵ to ẳ of a circle.

1.2. Origin and morphology of Phu Quoc dogs

Phu Quoc dog is one of 3 dog breeds with ridgeback in the world. The
other two types of ridgeback dogs are the African Rhodesian Ridgeback and the
Thai Ridgeback. However, at present, there are only two breeds of ridgeback
dog in the world, the African Rhodesian and the Thai Ridgeback recognized by
the Fédération Cynologique Internationale (FCI), the Phu Quoc dog is
mentioned belong with and considered to be descended from the Thai
Ridgeback.
1.3. Ridgeback trait in Phu Quoc dogs
The ridgeback trait is a mutation affecting skin and hair follicle
development during infancy in dogs, both of them originate in the ectodermal
neural tube.
The ridgeback trait in South African Ridgeback and the Thai Ridgeback is
caused by a mutation on chromosome 18 in which a 133,400 nucleotide of DNA
has been duplicated. The ridgeback trait consists of a set of genes called R
(Ridge) with a normal phenotype r (non-ridgeback). The ridgeback trait is
completely dominant.
1.4. . Genetic studies and ancient documents on Phu Quoc dogs
1.4.1. Current domestic dog genetic studies and Copy Number Variation
quantification methods (CNV)
Waldo et al. proposed a method for homozygous or heterozygous
genotyping of the African Ridgeback phenotype based on the quantification of
CNVs of the R gene region by Ct quantification through real-time-PCR. The
results show that, it is possible to determine the ratio between the regions


6
established on the 133kb area containing the gene group for the ridge trait in the
African and Thai Ridgebacks..
1.4.2. Phenotype ancient documents on Phu Quoc dogs
With the ancient documents left by the French, the descriptions and

learning about the Phu Quoc dog are quite similar. The similarities show that the
Phu Quoc dog is a wild hunting dog with many great personalities. and closely
resembles a wolf. The most special was the dorsal ridgeback and was passed on
to the next generations, a trait that was unlike any other dog breed they knew at
that time.
In the old literature, the measurements were mainly based on very few
individuals collected. The measurements are therefore not representative of the
general population of the former and current Vietnamese Phu Quoc dog
populations. In addition to some characteristics of temperament, habits, and
hunting behavior, phenotypic characteristics are necessary for standardization to
have a more complete view of domestic dogs.
1.4.3. Recent studies on the morphology and genotype of Phu Quoc dogs in
Vietnam
Currently in Vietnam, Phu Quoc dogs are very interested. In 2000, Dr.
Nguyen Huu Chiem, Msc. Nguyen Van Bien together with staff from Faculty of
Agriculture, Can Tho University and Department of Science and Technology of
Kien Giang province carried out the project "Investigation, research and
conservation of animal genes: Phu Quoc Dog, Kien Giang Province". However,
the topic of the Can Tho University team is almost reused at the basic level of
investigation, not really going into the assessment of genetic resources to have a
specific conservation direction. In 2009, Hoang Tuan Thanh and his colleagues
from the Institute of Animal Husbandry studied the growth and reproduction
ability of Phu Quoc dogs raised in Ho Chi Minh City.
Since 2012, Tran Hoang Dung and his colleagues, Department of
Genomics & Bioinformatics, Nguyen Tat Thanh University have conducted the
project "Research on mitochondrial genome sequencing of Phu Quoc dogs to


7
evaluate genetic diversity of Phu Quoc dog breeds in Ho Chi Minh City”

produced many outstanding results. Research results show that Phu Quoc dogs
appear haplotype E, which is a very rare form in the world. In 2016, Thai Ke
Quan and his colleagues studied the area analysis when using the D-loop area to
analyze the genetic characteristics of Phu Quoc dogs living on Phu Quoc Island,
This breed dog detected carrying halotype E1 with a very high frequency of
more than 15% in the world.
1.5. Necessity of research on morphology and genotype of ridgeback trait
on Vietnamese Phu Quoc dogs
Currently, the problem of recognizing the Phu Quoc dog breed is very
difficult because the Phu Quoc Ridgeback and the Thai Ridgeback have similar
appearance, live in two relatively close geographical areas and at the same time
the Thai Kennel Association successfully registered Thai Ridgeback standard
with Fédération Cynologique Internationale (FCI) in 2003.
The phenotypic criteria of Phu Quoc dogs are mainly based on ancient
documents from E. Oustalet and Henri de Bylandt based on only a few
individuals brought from Vietnam to France and those criteria are not
representative of the Phu Quoc dog population in Vietnam.
During the development of the Phu Quoc dog herd, the period 2007
witnessed a decrease in mortality caused by epidermoid cysts closely related to
the trait of ridgeback, which is an outbreak of the disease. Due to the inbreeding
process to create individuals according to the needs of buyers, then at the same
time, homozygous genotypes will appear to increase the spread and disease
expression, which will degrade the Phu Quoc dog population.
Currently, in Vietnam, there aren’t methods to evaluate and test genotypes
of ridgeback trait in Phu Quoc dogs with the aim of determining homozygous
and heterozygous genotypes in crossbred parent dogs in order to reduce the
incidence of ridgeback. Spread rate of dorsal ridgeback phenotype with
homozygous dominant genotype in the population. Also evaluate the role of the
alleles R and r in the development of the captive Phu Quoc dog population.

8
Chapter 2 - MATERIALS AND RESEARCH METHODS
2.1. Materials
This study surveyed 175 Phu Quoc dogs, including 96 males, 79 females
originating in Phu Quoc island district and in Ho Chi Minh City and surrounding
areas, where Phu Quoc farms and breeding centers are located. Of which 32
individuals without ridgeback and 143 individuals with ridgeback, 32
individuals with ridgeback were collected at breeding facilities in Phu Quoc and
143 individuals with and without ridgeback were obtained at natural and seminatural breeding establishments originating in Ho Chi Minh City and
surrounding areas. The surveyed individuals must be at least 18 months old,
sexually mature and have relatively stable body sizes and have both parents are
Phu Quoc dogs.
Blood samples were collected from a total of 18 dogs, including 03
control dogs originating in Ho Chi Minh City and 15 individuals of Phu Quoc
dogs (with and without ridgeback were collected from Phu Quoc and Ho Chi
Minh City).
2.2. Research Section
Section 1: Research and evaluate the specific body characteristics of Phu Quoc
dogs.
Section 2: Research and establish morphological indexes to select Phu Quoc
dogs based on morphological characteristics
Section 3: Study on the relationship between genotype and ridgeback phenotype
in Phu Quoc dogs.
Section 4: Study on improving conservation models (insitu-in vivo and exsitu-in
vivo) of Phu Quoc dogs based on a set of morphological criteria.


9
2.3. Research Methods

Section 1: Research and evaluate the specific body characteristics of Phu Quoc
dogs.
Technique of measuring large animal samples, applying current
measurement techniques when studying in domestic dog breed around the
world.
Quantitative characteristics: (1) Body height at withers (BHW), (2) Body
length (BL), (3) Tail length (TL), (4) Muzzle length (ML), (5) ) Ear length (EL),
(6) Chest circumference (ChC), (7) Waist circumference (WC) , (8) Weight
(BW)
Qualitative criteria such as coat color, eye color, swimming membrane,
ridgeback shape were estimated to distribute the traits as a percentage of the
survey sample, evaluating the data from high to low.

Figure 0.1 Quantitative characteristics of Phu Quoc dogs.

Section 2: Research and establish morphological indexes to select Phu Quoc
dogs based on morphological characteristics
Body index of Phu Quoc dog was selected, evaluated and adjusted based
on previous publications on other dog breeds such as Italian Cane Corso or


10
Spanish dog. Evaluate the correlation between body height at withers and body
length, chest circumference and waist circumference.
Set the morphological indexes according to the formulas:
Body Index = body length x 100/ chest circumference (BI = BL x 100/ChC)
Proportionality Index = body height at withers x 100/ body length (PI = BHW x
100/BL)
Section 3: Study on the relationship between genotype and ridgeback phenotype
in Phu Quoc dogs

The method of determining the homozygous or heterozygous genotype of
the African Ridgeback phenotype by real-time PCR proposed by Waldo et al.,
when performed on Thai and African Ridgebacks.
Relative Number Copy calculated according to Livak's formula modified
by Pfaffl: RCN = 2∆Ct(C/Tg-T/Tg)/2∆Ct(C/Ref-T/Ref)
Table 0.1 Primer pairs were used to quantify Ct by RT-PCR

Forward

Reverse

Set 1

TGCCGCTCAGATGATCAAC

TCTGCTTTTCTCTGCTCCC3

Set 2

ATTGGCAGTGTCCGTGTGAG AAGCCCCGCAGACAATGAAC

Set 3

GCATCCACCTAAGCAATCTG CCCTATTCTCTTCCACCCATC

Set 4

GCTTCTGCTTTGATACCCTTC GTTCTGCAACAGCATCTCC

β-actin

CATGGATGCCGCAGGATT

GTTCCGCTGCCCAGAGG

Section 4: Study on improving conservation models (insitu-in vivo and exsitu-in
vivo) of Phu Quoc dogs based on a set of morphological characteristic
Developing a set of information conventions on Phu Quoc dogs,
consulting on semi-conservative farming models. Phu Quoc dogs were recruited
into the farm based on the phenotypic criteria advised to the owners.
Establish a guide on how to identify homozygous and heterozygous
genotypes of back vortex trait through population genetic relationships, keeping
individuals with appropriate genetic resources to preserve Phu Quoc dogs.


11
Chapter 3 - RESEARCH RESULTS AND DISCUSSION
3.1. Research and evaluate the specific body characteristics of Phu Quoc
dogs
From the survey results on basic body measurements of Phu Quoc dogs, it
is possible to determine the characteristic parameters of these morphological
characteristics as follows: body weight: 19,5-19,6kg; body length: 50,2-50,6cm;
body height at withers: 45,3-45,9cm; muzzle length: 10,2-10,3cm; chest
circumference: 55,3-55,9cm; waist circumference: 45,1-45,6cm; ear length:
9,8cm; tail length: 28,1-28,9cm; cranial length: 20,3-20,4cm; cranial wide
20,3cm.
Table 0.1 Basic characteristics observed in Phu Quoc dogs (n=175, 𝑋̅ ± σ)

No

Body characteristics

Sex
Male

Female

1

Body weight (BW, kg)

19,6 ± 2,3

19,5 ± 2,2

2

Body length (BL, cm)

50,6 ± 6,4

50,2 ± 6,5

3

Body height to shouder (BHW, cm)

45,9 ± 6,3

45,3 ± 6,2

4

Muzzle length (ML, cm)

10,3 ± 0,6

10,2 ± 0,5

5

Chest circumference (ChC, cm)

55,9 ± 8,0

5,3 ± 7,9

6

Waist circumference (WC, cm)

45,6 ± 6,9

45,1 ± 6,8

7

Ear lenght (EL, cm)

9,8 ± 0,8

9,8 ± 0,8

8

Tail length (TL, cm)

28,9 ± 3,0

28,1 ± 2,3

9

Cranial length (CrL, cm)

20,4 ± 1,2

20,3 ± 1,0

10

Cranial wide (CrW, cm)

20,3 ± 1,2

20,3 ± 1,0

Phu Quoc dog features:
- Coat color: black or yellow coat color, brindle color ratio is quite small; The
ridgeback shape is mostly sword or violin;

- Eyes: black and brown.
- Ears: pointed and erect


12
- Tongue: has black spots occupying about 40% of the tongue area
- Legs: have 1-2 claws on both front and hind legs and have swimming
membranes.
- Tail: curved up 2/4 to 3/4 circle.
The size measurements have partially quantified the external morphology
of the surveyed Phu Quoc dogs and are representative of the population.
However, each individual may have a different overall size, so to standardize the
size based on an index may not be accurate, but must be based on the average
ratio between the basic measurements (body length , body height at withers, ,
cranial length/wide ...) will more accurately reflect the characteristic physical
structure of that species.
3.2. Research and establish morphological criteria to select Phu Quoc dogs
based on characteristic morphological characteristics

(Morphological

Indexes)
In the world, dog breed identification has long been applied in clinical
veterinary medicine, including in administrative procedures for breed
certification and the information required from owners about dog breeds, in
treatment programs or health assessment, certification of disease in domestic
dogs. The process of identifying the Phu Quoc dog breed in Vietnam is currently
quite vague and unclear, leading to the appearance of mixed breeds from many
different sources. The most difficult part in the identification process is that it is
not possible to identify the parents of the hybrid, then the process of determining

the breed is mainly based on the basic phenotypic manifestations. Evolutionary
genetic studies have shown a very small association between the known
genotype and the associated behavioral phenotype. Thus, the identification of
dog breeds provides information that helps owners think about different
directions of breeding from the original breed.


13
3.2.1. Determine and normalize the ratio of cranial to muzzle length
Observational size and initial statistics in this study show that Phu Quoc
dogs belong to the average head group with almost equal length and width of the
cranial with a ratio nearly 1:1; while the ratio of muzzle length to cranial length
is close to 1:2.
The individuals in Phu Quoc dog population observed has vary size, but
the ratio between body sizes is always stable. Therefore, determining the size
ratios obtained from section 1 is an important to build and standardize the
specific size ratios for the representative sample of observed Phu Quoc dogs.
Table 0.2 Summary of proportions and shapes normalizing of basic characteristics

Characteristics

Size, Color and Shape
̅ ± σ , CI 95%, unit: cm)
(X

Muzzle length (ML, cm)

10,3±0,6

Cranial length (CrL, cm)

20,4±1,1

Cranial wide (CrW, cm)

20,3± 1,

Cranial length / Cranial wide

1:1

Muzzle length / Cranial length

1:2

Correlation function of muzzle length / ML=0,5CrL + 0,6, R2=0,875729
cranial length with R coefficient
Ear length and shape

9-11cm, ears are straight forward.

3.2.2. Determine and standardize the proportions of the body, chest, waist;
body height to shouder/body length ratio
The body height at withers (BHW)/body length (BL) ratio and chest
circumference (ChC)/ waist circumference (WC) are common indicators used in
the calculation and construction of specific sizes for domestic dog.


14
Table 0.3 Ratio of body height at withers/body length and chest circumference/waist

circumference of Phu Quoc dog samples (n=175, 𝑋̅ ± σ, CI 95%)

Characteristics

Size

Function

Body length (BL)

50,6 ± 6,4 BL = 0,9xBHW + 8,4 R2=0,80

Body length at withers (BHW)

45,9 ± 6,3

Chest circumference (ChC)

55,9±8,0 ChC = 1,1xWC + 5,5 R2=0,88

Waist circumference (WC)

45,5±6,9

3.2.3. Determination of morphological indexes in Phu Quoc dogs
a) Body Index (BI)
The analysis results show that the average body index of Phu Quoc dogs
is 92,44±1,72 in male and 92,06±1,91 in female; The results of statistical
analysis comparing the t-test with different variances showed that there was no
difference in body index between male and female Phu Quoc dogs.

b) Proportionality Index (PI)
The analysis results show that the average body ratio coefficient is
90,86±0,57 in male and 90,59±0,61 in female; The results of statistical analysis
comparing t-tests with different variances showed that there was no difference in
body coefficient between male and female Phu Quoc dogs.
Bảng 0.4 Summary Body Index and Proportionality Index of Phu Quoc dogs (n=175)

Medium

Standard error

t - estimate

t - compare P value

-0,15015

1,9743

0,88

-0,33021

1,9741

0,74

Body Index
Male

92,44

1,72

Female

92,06

1,91

Proportionality Index
Male

90,86

0,57

Female

90,59

0,61


15
3.3. Study on the relationship between genotype and ridgeback phenotype
in Phu Quoc dogs
3.3.1. Evaluating the primers effectiveness of set 1 to set 4 and β-actin gene

Well 1: ladder (100bp)

Well 2 - 16: primer from set 1
to set 4 and β-actin for three
domestic dogs (1, 2, 3)
Well 17: empty
(a) Checking primer sequence of CD1, CD2, CD3

Well 1: ladder (100bp)
Well 2 - 16: primer from set
1 to set 4 and β-actin for
three Phu Quoc dogs (4, 11,
12)
Well 17: empty
(b) Checking primer sequence of PQ4, PQ11, PQ12

Well 1: ladder (100bp)
Well 2 - 16: primer from set 1
to set 4 and β-actin for three
Phu Quoc dogs (5, 6, 13)
Well 17: empty

(c) Checking primer sequence PQ5, PQ6, PQ13


16

Well 1: ladder (100bp)
Well 2 - 16: primer from set 1
to set 4 and β-actin for three
Phu Quoc dogs (7, 8, 9)
Well 17: empty

(d) Checking primer sequence PQ7, PQ8, PQ9

Well 1: ladder (100bp)
Well 2 - 16: primer from set 1
to set 4 and β-actin for three
Phu Quoc dogs (10, 17, 18)
Well 17: empty

(e) Checking primer sequence PQ10, PQ17, PQ18

Well 1: ladder (100bp)
Well 2 - 16: primer from set 1
to set 4 and β-actin for three
Phu Quoc dogs (14, 15, 16)
Well 17: empty

(f) Checking primer sequence PQ14, PQ15, PQ16
Figure 0.1 Checking primer sequence the samples

All 18 dogs including domestic and Phu Quoc dogs are numbered from 1
to 3 for domestic dogs (symbol: control dog - CD), 4 to 18 for Phu Quoc dogs
(symbol: Phu Quoc dog - PQ ). Because ridgeback ridge trait is an autosomal
dominant mutation, it is not possible to distinguish the sex of the individual dogs


17
that are used as a model for evaluation. Each electrophoresis tank consists of 3
samples with a scale and an empty well final. Each individual runs on 5 pairs of
primers: including 4 sets of primers from 1 to 4 into the wells in order, the last

pair is the primer of β-actin.
3.3.2. Evaluation of the frequency of R and r genes in the observed sample
using Pfaffl's improved Livak method
Total 18 DNA samples were extracted from the collected dog blood
samples. In which, there are 3 domestic control dogs, 13 Phu Quoc dogs with
and 2 Phu Quoc dogs without ridgeback (Male=M, Female=F, with ridgeback:
w/r, without ridgeback; wt/r)
Table 0.3 Threshold and phenotype for genotype estimation (n=15)

Phenotype

Genotype

Frequency

No

Code

RCN

1

PQ4

1.422299

F, w/r

Rr

RR

5/15

33%

2

PQ5

1.331038

F, w/r

Rr

Rr

7/15

47%

3

PQ6

1.242135

M, w/r

Rr

rr

2/15

13%

4

PQ7

0.880758

M, wt/r

rr

NA

1/15

7%

5

PQ8

(âm tính)

M, w/r

NA

Difference

6

PQ9

2.370577

M, w/r

RR

RR

Rr

rr

7

PQ10 2.440037

M, w/r

RR

33%

47%

13%

8

PQ11 2.058692

M, w/r

RR

25%

50%

25%

9

PQ12 2.012307

F, w/r

RR

+7%

-3%

-12%

10

PQ13 1.626127

M, w/r

Rr

11

PQ14 1.240628

M, w/r

Rr

12

PQ15 1.349758

M, w/r

Rr

13

PQ16 1.792784

F, w/r

RR

14

PQ17 1.503264

M, w/r

Rr

15

PQ18 0.892569

M, wt/r

rr


18
According to the experimental and observational results from the process
of Waldo et al. on the African Ridgeback and combining phenotypes, it is
possible to initially determine the frequency of the ridgeback gene in Phu Quoc
dogs.
RCN 3

2,5
2
1,5
1
0,5
0
R/R

R/r

r/r

Kiểu gen

RCN 3
2,5
2
1,5
1
0,5
0
R/R

R/r
Kiểu gen

r/r

Hình 0.2 The relative copy number (RCN) of the observed individuals (top)
and the mean of them (bottom) of homozygous dominant (RR), heterozygous (Rr),

homozygous recessive (rr) genotype of R gene in Phu Quoc dogs. (n=15)

The ridgeback mutation of the African and Thai ridgeback is located in
the 133kb region on chromosome 18, based on the method of Waldo et al.,


19
identified in the Phu Quoc dog also has similar segments of the two breed dogs
studied before.
According to previous studies on the condition of the African Ridgeback,
it has been confirmed that the Phu Quoc dog has a similar genotype for this trait.
This study opens a new direction in conservation and breeding of Phu Quoc
dogs in order to limit the spread of DS by determining the homozygous or
heterozygous genetic resources of the parents' ridgeback trait. It is necessary to
combine phenotype and mitochondrial and phenotypic genomic studies to
identify specific biomarkers of this rare breed.
3.4. Study on improving conservation models (insitu-in vivo and exsitu-in
vivo) of Phu Quoc dogs based on a set of morphological characteristic
The results of research and standardization of phenotypic sizes and body
proportions and genotypes of the ridgeback trait show that the important role of
selection of leading dogs plays an important role and determines the
development of the herd during rearing, breeding and conservation. The
selection process should follow morphological characteristic and ratios, along
with homozygous or heterozygous allele testing on ridgeback trait.
3.4.1. Building distribution map on Phu Quoc island
Table 0.4 Number of Phu Quoc dogs in breeding camps
No

Group Locate

1

1-20

Cửa Dương

15

2

1-20

Gành Dầu

3

20-30

4

>30

Total

Dogs

Male

Female

Captive

7

8

17

Captive

6

11

Dương Đông

22

Captive

10

12

Dương Tơ

32

Semi-wild

18

14

41

45

86

Feeding methods

In the semi-wild environment, dogs are allowed to roam freely in an area
that includes their respective species of flora and fauna, they tend to use the


20
plants as a remedy for intestinal and digestive ailments, this is a natural behavior
that needs to be preserved because it is not found in captive dogs.
3.4.2. Design and arrange farms according to the natural behavior and
optimal living conditions for Phu Quoc dogs on Phu Quoc Island
Phu Quoc dogs are genealogy determined to avoid inbreeding before took
into the mating area to pair up and conceive. During this process, the father and
mother dogs need to be tested for genetics simultaneously for diseases. The
principle in mating is to limit the homozygous dominant traits and try to
maintain a stable population with heterozygous individuals. With the research
results showing that the construction of crossbred scenarios is reasonable control
their genetics while also reassessing the effects of environment and nutrition on
culture.

Figure 0.3 Phu Quoc dog sanctuary

The results of the crosses obey the laws of inheritance of dominant traits
that are completely sex-unrelated. There are 09 combinations, which 05
combinations give 100% phenotype with ridgeback trait; 01 combination gives a
phenotypic ratio with/without ridgeback trait of 3:1; 02 combinations give a


21
phenotypic ratio with/without ridgeback trait of 1:1; 01 combination gives 100%
phenotypic ratio without ridgeback trait.
Table 0.5 Crossbred Model on Phu Quoc dogs
Ridgeback
trait

Father gene
RR
R100% RR

Mother gene

R

r

Rr

Rr

50% RR

100% Rr

(100% with

50% Rr

(100% with ridgeback)

ridgeback)

(100% with ridgeback)

R50% RR

25% RR

50% Rr

50% Rr

50% Rr

50% rr

(100% with

25% rr

(50% with ridgeback

ridgeback)

(75% with ridgeback

50% without ridgeback)

25% without ridgeback)
r100% Rr
r

50% Rr

100% rr

(100% with

50% rr

(100% without ridgeback)

ridgeback)

(50% with ridgeback
50% without ridgeback)

Normally, according to crossbred models, individuals with dominant traits
will be crossed with homozygous recessive individuals for genotyping, but this
method has the disadvantages of taking a long time and ethically. Animal
bioethics does not allow. With the results from the analysis by real-time PCR

method, the breeder can collect and send analysis samples of the individuals
after crossing with the ridgeback trait phenotype to determine the homozygous
dominant or heterozygous genotype as well as possible.
Previous studies have shown that it is necessary to limit the homozygous
dominant phenotype in the herd because of its association with fatal DS and
many health related diseases for individual. Breeders need to breed and retain
heterozygous individuals with ridgeback phenotype while minimizing the spread
and spread of DS, as well as other physical diseases.


22
3.4.3. Implant and attach electronic chip
Staff prepare anesthetics in syringes, cotton swabs and rubbing alcohol
and other tools. Anesthesia must be prepared, the anesthetic dose is 3cc
(Ketamine). Check electronic chip: Each chip has a unique code. This check
ensures that the chips are readable and in good working order.
Two days before chip implantation took, it is necessary to inform the
owner not to feed their dog one day before the chip is inserted to avoid the case
of weak dogs in shock, vomiting, food in their stomachs, and obstructing the
respiratory tract. Do not let the dog drink water and take bath for one day before
and after inserting the chip to avoid reacting with the drug.
To avoid confusion and cross-check the chip readings before and after
implantation simultaneously to check that the chip has been implanted in the
animal, it is imperative to hold the reader and double-check the readings on the
electronic board.
After chip implanting, it is necessary to immediately monitor the health of
the Phu Quoc ridgeback dogs. Strong ones can slowly return to normal after half
an hour. Dogs that show signs of difficulty breathing need veterinarian
intervention and immediate treatment.

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CONCLUSION AND RECOMMENDATIONS
Conlusions
Based on the the thesis research results were obtained, the following
conclusions can be reached:
1. Important typical Phu Quoc dogs phenotypic characteristics have been
identified: body weight 19.5-19.6kg; body length 50.2-50.6cm; body height to
withers 45.3-45.9cm; muzle length 10.2-10.3cm; chest circumference 55.355.9cm; waist circumference 45.1-45.6cm; ear length 9.8cm; tail length 28.128.9cm.
2. The first time developed a set of morphological standards to make out
Phu Quoc dogs which to serve selection and conservation, based on the
following measurements: Cranial index is 1:1 (length: width of cranial) and 1:2
(muzle length:cranial length); Body index (body length/chest circumference) is
92.06-92.44 and Proportionality Index (body height to withers/body length) is
90.59-90.86.
3. Affirming the relationship between the genotypes related to the
characteristic ridgeback phenotype of Phu Quoc dogs is similar to the
recognized other dogs in the world, the Thai Ridgeback and the African
Ridgeback. This phenotype is caused by a completely dominant gene located on
chromosome 18, denoted R (ridge), where R is the phenotype with ridgeback
and r is the phenotype without ridgeback.
4. The study proposed a method of using morphological criteria combined
with genotyping of ridgeback trait to select Phu Quoc dogs before being
introduced into conservation and breeding camps.
Recommendations
Continuing to conduct related studies between R genotype and nutrition
provided during rearing and breeding; as well as evaluate the impact of different
environmental conditions on the development and reproduction of Phu Quoc
dogs.