MINISTRY OF EDUCATION AND
TRAINING
MINISTRY OF AGRICULTURE AND RURAL
DEVELOPMENT
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
----------***----------
TRAN THI BINH NGUYEN
EVALUATING GENETIC RESOURCES AND ANALYZING MOLECULAR
MARKERS RELATED TO EGG YIELD TRAITS IN LIEN MINH
CHICKEN BREED
Specialization: Biotechnology
Code number: 9420201
SUMMARY OF AGRICULTURAL DOCTORAL THESIS
Hanoi, 2019
The dissertation has been completed at: Vietnam Academy of Agricultural Sciences
Supervisors:
1. Dr. Nguyen Thi Dieu Thuy
2. Dr. Nguyen Huu Duc
Reviewer 1:
Reviewer 2:
Reviewer 3:
The dissertation will be defended at the Dissertation committee at the
National level Vietnam Academy of Agricultural Sciences, on Date
month
year
The full text of the dissertation can be found in the following libraries:
- The National Library of Vietnam;
- The Library of Vietnam Academy of Agricultural Sciences.
DANH MỤC CÔNG TRÌNH CÔNG BỐ LIÊN QUAN ĐẾN LUẬN ÁN
1.
Tran Thi Binh Nguyen, Nguyen Huu Duc, Do Vo Anh Khoa, Nguyen Thi
Dieu Thuy (2018), “Effect of nucleotide polymorphism of candidate genes on
egg production traits in native Lien Minh chicken”, Livestock Research for
Rural Development 30(6).
2.
Tran Thi Binh Nguyen, Nguyen Huu Duc, Vu Duc Quy, Pham Thu Giang,
Nguyen Manh Linh, Dinh Thi Ngoc Thuy, Nguyen Thi Dieu Thuy (2018)
“Polymorphism in candidate genes of Lien Minh chicken”, Vietnam Journal of
Agricultural Sciences 1(2), tr. 174-182.
3.
Tran Thi Binh Nguyen, Nguyen Hung Cuong, Vu Thi Tien, Ta Thi Loan,
Do Vo Anh Khoa and Nguyen Thi Dieu Thuy (2018).,“Association of
neuropeptide gene polymorphism with egg production trait in Lien Minh
chicken”, National Biotechnology conference 2018, Hanoi 26/10/2018, pp.
1828-1833.
4.
Tran Thi Binh Nguyen, Nguyen Huu Duc and Nguyen Thi Dieu Thuy
(2018), ,“Association of prolactin gene polymorphism with egg production
trait in Lien Minh chicken”, Vietnam Journal of Biotechnology, 16(2), pp. 1-8.
5.
Tran Thi Binh Nguyen, Do Thi Lieu, Nguyen Huu Duc, Hoang Thi Yen, Vu
Cong Quy, Nguyen Hung Cuong and Nguyen Thi Dieu Thuy (2016),
,
“Reproductive characteristics and preliminary analysis on polymorphism of
candidate genes concerning with egg production traits of Lien Minh chicken
breed”, Journal of Animal Husbandry Sciences and Technics, 212, pp. 2-8
INTRODUCTION
1. The necessity of research
Lien Minh chicken is an indigenous breed with several favorable properties, such
as good meat quality and associated with the economic development of the people in
the Lien Minh village, Cat Hai, Hai Phong. Such breed comes in beautiful physical
appearance with its nice feather color, yellow skin and produces high-quality meat
with thinfat under the skin. The source of the Lien Minh chicken gene was assessed at
the level of dangerous threat (FAO, 2007), and has been listed on the conservation list
since 2008. The conservation and efficient use of Lien Minh chicken were developed
in 2013. Research results from more than 30 households studied in Tran Chau
commune, Cat Hai district, Hai Phong city the grazing revealed that, aAge of first egg
laying in Lien Minh chickens was quite late (197.5 days of age) and low egg yield 75.6
eggs/chicken/year (Doan BH et al, 2016). It is therefore essential to increase Lien
Minh chicken egg production in order to conserve, exploit, and develop such poultry
breeds.
Chicken egg production can be affected by a number of factors such as genetics,
nutrition, light, and veterinary care regimes. Among these factors, Genetic
polymorphism is currently studied in order to improve economic characteristics of
indigenous chickens. The polymorphisms of PRL (Prolactin), VIP (Vasoactive
Intestinal Peptide), VIPR1 (Vasoactive Intestinal Peptide Receptor 1), NPY
(Neuropeptide Y), GH (Growth hormone) and GHR (Growth hormone receptor) have
been defined to have significant association with fertility in some in some indigenous
chicken breeds around the world. Therefore, genetic polymorphism can be used as
DNA markers to improve egg production in indigenous chickens.
Additionally, little research has been carried out on the evaluation of the genetic
diversity based on the nucleotide sequence of D-loop mitochondrial genes in Lien
Minh chickens. The results of these studíe can be used to provide information on
genetic diversity, to support for registering the copyright of the Lien Minh chicken
breed. Furthermore, this work is a solid foundation for building the panorama of Lien
Minh chicken’s genetic diversity levels.
Therefore, "Evaluating genetic resources and analyzing molecular markers
related to egg yield traits in Lien chicken breed” was implemented to provide genetic
information of the D-loop region of mitochondrial genes and candidate genes
improving egg production as well as support conservation, exploitation and
development of Lien Minh chicken.
1
2. Research objectives
- Evaluate genetic diversity of Lien Minh chickens based on sequences of
mitochondrial DNA D-loop region
- Determine the genetic relationship and the origin of Lien Minh chickens.
- Identif genetic relationships between candidate genes and egg yield traits in
Lien Minh chickens.
3. Scientific and practical significance
* Scientific significance
- Provides the initial information about the sequence of mitochondrial D-loop
region in Lien Minh chickens. The first scientific publication to study the frequency
of allele / genotype occurrence at the polymorphisms of PRL, VIP, VIPR1, NPY, GH,
and GHR in Lien Minh chickens;
- results on candidate genetic polymorphism with egg yield traits are the
scientific database for the study of the application of molecular markers to support the
selection of Lien Minh chickens that have high egg yield. Provide information
molecular markers of indigenous chickens Lien Minh for breeders, reference for
researchers, students.
* Practical significance:
Provide scientific information to promote and develop the market for indigenous
chickens Lien Minh products. Provide scientific information to promote and develop
the market for indigenous chickens Lien Minh products. Provide information for the
Hai Phong Center for Science and Technology Application, companies and livestock
farms to select the line Lien Minh chickens with high egg production.
4. Object and scope of the study
- The thesis applied bioinformatics tools to assessing the genetic resource of Lien
Minh chickens: Analyzing the nucleotide sequence of mtDNA D-loop region of Lien
Minh chicken to assess the diversity of genetic resources.
- Determine genotypic and allelic frequency of at PRL, VIP, VIPR1, NPY, GH,
and GHR genes in Lien Minh chickens.
- Assess the relationship between polymorphisms of PRL, VIP, VIPR1, NPY, GH,
and GHR genes and egg production traits.
5. Subject of research and scope of research
Subjects of research: Lien Minh indigenous chickens in Tran Chau commune,
Cat Hai district, Hai Phong city.
Scope of research: The Thesis applied biotechnology and bioinformatics tools
to analyze the genetic diversity of Lien Minh chicken, and to support conservation,
2
exploitation and development of Lien Minh chickens. .
The Thesis focused on candidate genes related to egg production ability, so that
the target genes could be identified, Contributing to the selection of Lien Minhchicken
with high egg yield.
6. Structure of the thesis
The thesis comprises of 107 pages (apart from references and appendices),
including: Introduction (5 pages); chapter 1: Literature review (37 pages); chapter 2:
Materials contents and methods (8 pages); chapter 3: Results and discussion (50
pages); Conclusion and recommendations (2 pages). The paper uses 180 references, 33
of which are in Vietnamese and 147 others are in English. The thesis includes 32
tables, 44 figures, 4 appendices and 5 publications.
CHAPTER 1. LITERATURE OVERVIEW
The thesis has used to and analyzed 33 reference materials in Vietnamese and
147 materials in English, which relate to the following contents: The mitochondrion is
a double-membrane-bound organelle found in most eukaryotic organisms.
Mitochondrial genomes are about 16 kb in almost all animals Mitochondrial DNA is
the DNA located in mitochondria with characteristics: i) multiple number of copies; ii)
exist in a haploid form and have almost no recombination; iii) relatively fast mutation
rates and iv) mother line genetics in most species, so it is used as a tool in analysis
between evolutionary relationships and evaluation of genetic variation within and
between species (Harpending et al, 1998). The nucleotide sequence in D-loop region is
the fastest evolving region compared to that of mtDNa, Making it an appropriate
region in in analyzing population genetic diversity, especially internal changes. of the
same species.
To study the low reproductive rate in Lien Minh chickens, the loci associated
with reproductive traits (candidate genes) were selected derived from the knowledge
of reproductive physiology and reports as to whether these genes showed an
association with reproductive performance in commercial poultry lines. Prolactin is a
polypeptide hormone secreted by the anterior pituitary gland, which is involved in
many physiological pathways: osmotic regulation, luteolysis (regressive ovary) and the
maintenance of incubation behavior in hens. Studies have shown that the PRL is
present in the hypothalamus, the pituitary gland, the oviduct, and the egg, with the
highest levels found in the pituitary gland (Li et al, 2009a). In chickens, the hormone
prolactin is one of the hormones that plays an important role in egg production.
Prolactin concentrations increase sharply in plasma, may induce broody behavior
3
(Sockman et al, 2000), and results in reduced egg production (Reddy et al, 2002). A
mutation that occurs in the promoter region can affect the expression of the PRL gene,
so it may affect egg production. Prolactin secretion in birds is predominantly regulated
by releasing factors, one of which is a vasoactive intestinal peptide (Kagya-Agyemang
et al, 2012). Vasoactive intestinal peptide (VIP) is a prolactin-releasing factor in birds
(El Halawani et al, 1990; 1997). VIP increases PRL secretion from pituitary glands,
especially when the pituitary gland responsiveness is enhanced with estrogen pretreatment (Sharp et al, 2005). VIP binds with the vasoactive intestinal peptide receptor
to give rise to the secretion and release of PRL (El Halawani et al, 1990).
Neuropeptide Y (NPY) is an important neuromodulator, which is associated with
gonadal function in birds and mammals (Hilal et al, 1996). NPY may affect the egg
production rate through its role in controlling ovulation (Dunn et al, 2004). Genetic
encoding for the neuropeptide Y gene is associated with the age of having the first egg
and the number of eggs laid (Xu et al, 2011a, Xu et al, 2011b, Dunn et al, 2004, Li et
al, 2009b). The NPY gene might produce markers for the age of the onset of lay, and
through its role in the control of ovulation, influence the egg production rate.
Injections of NPY change the plasma concentration of the luteinizing hormone and
growth hormone (Pierroz et al, 1996), suggesting that it may play a significant role in
the secretion of these hormones (Wang et al, 2001). Growth hormone (GH), when
combined with the growth hormone receptor in the liver and when forming the GHGHR-IGFs signal pathway, affects chicken development (Lau et al, 2007). The
identified alleles of the GH gene of White leghorn have been linked to the egg
production phenotype (Kuhnlein et al, 1997). The growth hormone receptor (GHR)
controls the number of follicles in animals that are recruited to the rapid growth phase
(Roberts et al, 1994; Monget et al, 2002).
Lien Minh breed comes in beautiful physical appearance with its nice feather
color, yellow skin and produces high-quality meat with thin subcutaneous fat layer,
crispy and chewy skin. It also possesses long legs and neck silky fur. The rooster has a
good appearance, colorful feathers and it can weigh up to 5kg, and a hen has 3kg. The
market demand for Lien Minh chicken is very large, but in the small-scale farming
conditions and extensive farming methods, Lien Minh hamlet and surrounding areas
only meet a very small part of market demand in Cat Hai district and Hai Phong city,
typically in Cat Ba.
Therefore, the study evaluated the genetic diversity of Lien Minh chickens by
using the D-loop nucleotide sequence, at the same time using genes related to fertility
to find gene polymorphisms can improve egg yield in Lien Minh chickens.
4
CHAPTER 2. MATERIALS, CONTENTS AND METHODS
2.1. MATERIALS
2.1.1. Animal materials
2.1.1.1. Chicken population
Blood samples of Lien Minh chicken raised in households in Lien Minh village were
collected for genetic diversity analysis Lien Minh chickens were selected based on the
following criteria: Limiting blood ties (from different farming households in Lien Minh
village, Tran Chau commune, Cat Hai district, Hai Phong), roosters (n = 12 individuals) and
hens (n = 12 individuals), mature individuals with typical characteristics of the breed,
confirmed by breeders participating in the project "Exploitation and development of Lien
chicken Minh in Hai Phong "at Decision No. 2398 / QD-BKHCN dated August 31, 2012 of
the Minister of Science and Technology. In addition, two indigenous chicken breeds are
Dong Tao chicken - GDT (n = 18) and Chicken Many Finger - G9C (n = 6) were sampled
from the locality where they originated (Khoai Chau District, Hung Yen and Xuan Son
National Park, Phu Tho).
2.1.1.2. Sample collection (or Egg collection)
We collected Lien Minh chicken eggs at households in Lien Minh hamlet. Then
eggs were hatched, tended, vaccinated and monitored at Hai Phong City of Science
and Technology Application Center in an individual form from hatching (01 day old)
to 44 weeks of age. Lien Minh chickens received the same veterinary cared, nutrition,
environment and evaluated characteristics egg production traits.
1.1.1.3. Primer sequences used for amplification in thesis
Table 2.2. Primer sequences used for amplification of the D-loop region
Locus
Polymophism
mtDNA
D-loop
Primer sequences [5’ -3’]
F: AGGACTACGGCTTGAAAAGC
R: CATCTTGGCATCTTCAGTGCC
Ta
(oC)
PCR
size
([bp]
56
1321
References
Oka et al, 2007
Table 2.3. Primer used in candidate gene analysis
Locus
PRL
Polymophism
Primer sequences [5’ -3’]
C-2402T
(PRL5)
358/indel
24bp(PRL24)
F: AGAGGCAGCCCAGGCATTTTAC
R: CCTGGGTCTGGTTTGGAAATTG
F:TTTAATATTGGTGGGTGAAGAGACA
R: ATGCCACTGATCCTCGAAAACTC
F: AGAGGCAGCCCAGGCATTTTAC
R: CCTGGGTCTGGTTTGGAAATTG
F:GCTTGGACTGATGCGTACTT
R:GTATCACTGCAAATGCTCTG
F: GCTTGGACTGATGCGTACTT
R: GTATCACTGCAAATGCTCTG
C2161G
C+338T
VIP
G5138982T
5
Ta
(oC)
PCR
size
([bp]
References
56
439
Cui et al, 2006
54
154/130
Xu et al, 2011
56
439
Cui et al, 2006
55
520
Zhou et al, 2010
55
520
Xu et al, 2011a
C1715301T
(VIPR1/TaqI)
C1704887T
(VIPR1/ HhaI)
Indel 4 bp tại
3139135
VIPR1
NPY
C31394761T
GH
C2983T
GHRi2
A294-541G
GHRi5
C571T
F:CTCCTCAGGCAGACCATCATG
R:CTTGCACGTATCCTTGGGTAGC
F:CCCCGTTAAACTCAGCAGAC
R:CCCAAAGTCCCACAAGGTAA
F: TCTCAGAGCTCCAACGTATGA
R: ATATTTCTGTGCCTGAACAACA
F:CGTGGCTGCTTTGCTTCCTTTC
R:GGGGTACGAGGCAAGGACATG
F: CTAAAGGACCTGGAAGAAGGG
R: AACTTGTCGTAGGTGGGTCTG
F: GGCTCTCCATGGGTATTAGGA
R: GCTGGTGAACCA ATCTCGGTT
F: ACGAAAAGTGTTTCAGTGTTGA
R: TTTATCCCGTGTTCTCTTGACA
61
486
Xu et al, 2011
61
434
Xu et al, 2011
56
248
Xu et al, 2011a
58
324
Xu et al, 2011
62
1164
Makhsous et al,
2013
58
718
Li et al, 2008
60
750
Li et al, 2008
F: Forward primer; R: Reverse primer; T (a): Annealing temperature.
Components of the cut reaction (total volume of a 15 µl reaction): deionized
water (2,5 µl), 10X cutting buffer (1,5 µl), PCR product (10 µl), restriction enzyme (1
µl), the incubation time is 16 hours, the incubation temperature is corresponding to
each enzyme cut (Table 2.4).
Table 2.4. Information for polymorphisms and restriction enzyme (RE)
Polymophism
RE
GenBank accession
number
PCR-RFLP size [bp]
Ta [oC]
C-2402T (PRL5)
AluI
AB011438
439/304/160/144/81/54
37
-
AF288765
154/130
37
C2161G
Csp6I
AB011438
439/405/34
37
C+338T
HinfI
NC_006090
520/480/40
37
G5138982T
ApoI
NC_006090
520/486/34
37
C1715301T
TaqI
NC_006089
486/310/176
65
C1704887T
HhaI
NC_006089
434/253/181
37
Indel 4 bp tại 3139135
DraI
M87298
248(252) 167/81
37
C31394761T
KpnI
GI:35797382 9
GH
C-2983T
SacI
JN675393
1164/1020/ 570/450/144
37
GHR
C571T (intron 5)
NspI
NC_006127
750/550/200
37
GHR
A294-541G
(intron 2)
HinIII
NC_006127
718/428/290
258/170/290
37
Locus
PRL
VIP
VIPR1
NPY
– 358/ indel
24 bp (PRL24)
324/200/124
200/124
37
Manufacturer
Themo, America
Themo, America
Themo, America
Themo, America
Themo, America
Themo, America
Themo, America
Themo, America
Themo, America
Themo, America
Themo, America
Themo, America
2.1.2. The chemicals
Chemicals used in molecular biology research of Sigma (Germany), Merck
(Germany), and Thermo (USA).
2.1.3. Machinery used
PCR machine eppendorf AQ mastercycle 5332YN056927 (Germany), horizontal
electrophoresis machine FCIE-PLAS-HU1030012479 (UK), water bath (JSRJSWB22T-Korea), storage refrigerator 40C (Panasonic - Japan), gel scanner (UVP-M6
26V / P / N95-0458-02, USA), centrifuge (HETTICH-UNI version 320R, Germany),
egg pressure gauge, Nano Drop Spectrophotometer.
2.2. RESEARCH CONTENT
- Content 1: Assessing genetic diversity of Lien Minh chicken based on
mitochondrial DNA
- Content 2: Analyzing the relationship between genetic markers and egg
production traits in Lien Minh chicken
+ Experiment 1: Identifying indicators related to egg production traits in 90
Lien Minh hens;
+ Experiment 2:
Determining genotypic and allelic frequency in candidate genes (PRL, VIP,
VIPR1, NPY, GH and GHR) related to egg yield traits;
Identifing the relationship between candidate genes and egg yield traits in Lien
Minh chickens.
2.3.
METHODS
2.3.1. Blood sampling
The blood samples from Lien Minh chickens were taken with a 5 ml syringe
from the vein of the wing vein, then quickly transferred to a blood tube containing
EDTA-K anticoagulant. Blood tube was stored at 4ºC, and -20ºC in the lab.
2.3.2. DNA extraction
Genomic DNA was extracted by a standard procedure using Proteinase K
digestion followed by phenol-chloroform extraction and precipitation with ethanol
(Ausubel et al, 1995). A nanodrop spectrometer and agarose gel electrophoresis were
used to estimate DNA concentration and purity. DNA samples were stored at -20oC
until analysis
2.3.3. PCR amplification
The PCR primers were designed based on the chicken candidate gene sequences
using Primer 3.0 software. PCR was performed in a 25 µl reaction containing 1x PCR
Buffer, 1.5 mM MgCl2, 1.25 mM each dNTPs, 5 pM primer, 1U Taq-polymerase
(Fermentas), and 100 ng genomic DNA. In PCR amplification, an initial denaturation
at 94oC for three minutes followed by 35 cycles of denaturation at 94oC for 45
seconds, annealing for 45 seconds and extension at 72oC for 90 seconds, and an
additional extension of 72oC for seven minutes was set.
2.3.4. PCR purification and gene sequencing
Purification of DNA fragments for sequencing: The purification process was
used with the QIAquick PCR Purification Kit (QIAGEN). The purified product was
7
sequenced on the ABI-3100 Avant Gentic Analyzer from Macrogen Company
(Korea).
2.3.5. Restriction Fragment Length Polymorphism
Genetic length polymorphism analysis used PCR-RFLP (Restriction Fragment
Length Polymorphism (RFLP) technique with appropriate restriction fragment
enzymes. PCR products were then digested with restriction enzymes overnight. The
restriction fragments were separated on 2% agarose gel stained with ethidium bromide.
2.3.6. Evaluate indicators related to reproductive traits in chickens
Based on the description of Bui Huu Doan et al (2011), the indicators…to
produce eggs include age at first egg laying (days), weight of first egg (gram), energy
egg yield (total number of eggs/hens/20 weeks) egg weight (grams) and egg shape
index (large diameter size/small diameter D/R).
2.3.7. Body weight
The body weight of Lien Minh chickens was weighed every Friday at 19 o'clock
(start from 01 day to 22 weeks), when chickens go to barns, using a scale of 5 g
accuracy.
2.3.8. Statistical analyses
Nucleotide sequences were identified with the Basic Local Alignment Search
Tool (BLAST) (Altschul et al, 1990). Multiple nucleotide alignments were carried out
with BioEdit (Hall, 1999). The diversity parameters, including the nucleotide diversity
haplotypes D-loop of all sequences were estimated using DnaSP v.5 software (Librado
and Rozas, 2009). Neighbor Joining (NJ) phylogenetic tree was conducted using
MEGA version 6.1 (Tamura et al, 2007). Phylogenetic tree analysis was constructed
based on classification of Oka et al, (2007) with reference mtDNA sequences from the
GenBank database.
, The gene frequencies were calculated by counting method as: p= 2(AA) +
(AB)/2N and q= 2(BB) + (AB)/2N where p = the gene frequency of allele A, q = the
gene frequency of allele B and N = the total number of chickens tested. The HardyWeinberg Equilibrium (HWE) was estimated using the method of Rodriguez et al,
2009. The association between genotype and egg production was analyzed based on
General Linear Model of Minitab software version 16.0: Yij= µ + Gi + ξij (where Yij:
traits observed; μ: general mean, Gi: influence of genotype; ξij: random error).
8
Chapter III. RESULTS AND DISCUSSION
3.1. GENETIC DIVERSITY ANALYSIS IN INDIGENOUS LIEN MINH
CHICKEN
3.1.1. DNA extraction
In order to carry out an evaluation of the Lien Minh chicken genetic diversity based on
the nucleotide sequence of D-loop region of mitochondrial DNA, it is necessary to
obtain pure and intact genomic DNA.
Table 3.1. Results of genomic Spectrophotometer
DNA samples
OD260nm/280nm
Concentration (ng/µl)
GLM01 → GLM24
1,82 → 1,99
100,56 → 694,80
G9C01 → G9C06
1,81 → 1,90
257,50 → 428,96
GDT01 → GDT18
1,85 → 1,97
225,60 → 515,37
Table 3.1 shows that the OD260/280 ratio of DNA samples are in the range of 1.8 2.0, which proves the extracted DNA is purified, the DNA concentration is from
100.56 to 694.80 ng/µl. Therefore, the quality of genomic DNA samples is assurance
to subsequent experiments.
3.1.2. Amplification of the mitochondrial DNA D-loop region
As shown in Figure 3.2, DNA sample is a bright band and has a molecular expected
size of about 1300 bp (the electrophoresis band is located between two standard DNA
bands of 1000 bp and 1500 bp).
Figure 3.2. Eectrophoresis of PCR products on 1% agarose gel
Wells 1 → 9: PCR products of Lien Minh chickens, M: Ladder DNA1kb (Merck)
3.1.3. Nucleotide sequence in D-loop region
The result of the nucleotide sequence in D-loop region of Lien Minh chicken were
registered on GenBank with the codes: from KY172116 to KY172121 and from
9
MH425591 to MH425608.
3.1.4. Analysis of nucleotide polymorphism in D-loop region of Lien Minh
chickens and other native chickens with code on Genbank
3.1.4.1. Diversity analysis in D-loop region
DnaSP was used to analyze the nucleotide polymorphism in mtDNA D-loop region of
Lien Minh chicken . The analytical parameters include: Number of polymorphic sites
(S); number of haplotype (h); haplotype diversity (Hd); nucleotide diversity (Pi);
Tajima test (D). The analysis results of the nucleotide polymorphic parameters (1050
bp) in Lien Minh chickens, other native chickens in Vietnam were shown in Table 3.2.
Table 3.2: Polymorphic sites, haplotype and nucleotide diversity of chicken
breeds
Breeds
Lien Minh
Dong Tao
Nhieu Ngon
N
24
18
6
S
23
11
4
h
12
7
3
Hd
0.913
0.856
0.867
Pi
0.007
0.004
0.002
NC
4
1
1
D
0.187*
1.721*
0.562*
N: number of samples, S: number of polymorphic sites (excluding sites with gaps), h: Number of haplotypes, Hd:
Haplotype diversity, Pi: nucleotide diversity, NC: number of clades, D:Tajima’s test.
3.1.4.2. Polymorphic nucleotide sequence in D-loop region
a. Analysis of D-loop nucleotide polymorphisms between Lien Minh chickens and
native Vietnamese chicken breeds (455 bp)
The analysis results indicate, there were 21 nucleotide substitution sites and no
nucleotide insert/deletion polymorphism when compared to a native Vietnamese
chicken coded GU564373 (Dong Tao chicken). Figure 3.4 showed that among 21
nucleotide substitution sites there are 11 T→C substitutive positions, four A→G
substitutive positions, five substitutive positions C→T, but only one G→A substitutive
nucleotide sites. Especially, T→C substitution at position 241 appeared in all Lien
Minh chickens, in addition this substitution at position 206 appeared in 17 of 24
studied samples.
b. Analysis of D-loop nucleotide polymorphisms between Lien Minh chickens and
native breeds in the world (in Genbank)
The results showed that from 24 nucleotide sequences were identified by 23
nucleotide polymorphic positions between Lien Minh chicken and the branch E breed
with code AB268540 (Gallus gallus domesticus), including 22 nucleotide substitution
sites and a nucleotide insert site. Special nucleotide G insertion at position 860 appears
in all Lien Minh chickens. Besides, nucleotide substitution T→C appears in 10
positions (167, 199, 217, 243, 246, 256, 270, 315, 367, 888) and substitutions C→T
appears only in position. 225, 261, 310 and 446.
10
3.1.4.3. Nucleotide identity of D-loop region
a. Analysis nucleotide identity of D-loop region between Lien Minh chickens and
native Vietnamese chicken breeds (455 bp)
Mostly, genetic diversity of indigenous chicken was analyzed by using 455 bp Dloop region (Le Tien et al, 2009; Cuc et al, 2011). Therefore, 455 bp D-loop region
was used to compare the level of nucleotide diversity of D-loop between Lien Minh
chicken and other Vietnam native chickens.
The analytical results indicated that the nucleotide identity among Lien Minh
chickens reached 97.5% - 100%, in which 14 Lien Minh individuals of Group I have
the nucleotide identity of 100% (GLM 01, GLM02, GLM03, GLM06, GLM 07, GLM
08, GLM 09, GLM 11, GLM12, GLM13, GLM14, GLM15, GLM16 và GLM18) have
Nucleotide identity index that reaches. 100%. The above Lien Minh chickens have a
nucleotide identity to the Lien Minh chickens group II (GLM10 and GLM17) reaching
97.5%.
As demonstrated from the pairwise comparison that the Lien Minh chickens
shared high nucleotide identity with their native counterparts in Vietnam, ranging from
96.8 to 99.7%. Particularly, the percent nucleotide identity of group I Lien Minh
chicken (n=14) compared to branch B native chicken ranged from 99.3 to 99.7%.
Also, Lien Minh chickens in this group have a high nucleotide identity coefficient
when compared to Dong Tao and Multi-Finger chickens (99.1-99.7). On the contrary,
when compared to Vietnamese native chicken breeds in branches C, D, E, G, I, the
percent nucleotide identity of Lien Minh chickens in group I reaching only from 97,3
% to 97,9%.
Lien Minh chickens of group II (GLM10 and GLM17) shared a high percent
nucleotide identity compared to native chickens in branch B8 (99.1%), while the
nucleotide identity of Lien Minh chickens in group III (GLM04, GLM19, GLM23),
GLM24) reached 99,1% of nucleotide identity whenc compared with the Vietnamese
native chickens in D1 branch. Lien Minh chickens in group IV (GLM20) and group V
(GLM05) had high nucleotide identity with Vietnamese native chickens of branch C1
(99.5-99.7%). Lien Minh chickens of groups VI (GLM21) and VII (GLM22) had high
percent nucleotide identity to those of E1 branched chickens (99.3-99.5%).
b. Analysis of D-loop nucleotide identity between Lien Minh chickens and
domestic chickens in the world (1050 bp)
1050 nucleotides in the D-loop region of Lien minh chicken were used to
compare with the native chickens of the world (Oka et al, 2007). Result analysis
illustrated that the nucleotide identity coefficient among Lien Minh chickens reached
11
98.7% - 100%. In particular, Lien Minh chickens in groups I (GLM 01, GLM 07,
GLM 08, GLM 09, GLM 11), group II (GLM02, GLM18), group III (GLM12,
GLM13, GLM14, GLM15), group IV ( GLM06) and group V (GLM03, GLM16)
shared a fairly high nucleotide identity from 99.7% to 99.9%, Lien Minh chickens in
the same group have a nucleotide identity of 100%.
The nucleotide identity of Lien Minh chickens and domestic chickens according
to research results of Oka et al (2007) reached 98.2% - 100%. Lien Minh chickens of
groups I, II, III, IV and V shared nucleotide identity with chickens of branch E1, E2,
E3, E4 (Oka et al, 2007) reaching 99.6% - 100.0%. Lien Minh chickens GLM10,
GLM17, GLM21, GLM22 shared a high nucleotide identity with chicken in branch A
(99.6% - 100.0%),Lien Minh chickens of group VIII (GLM04, GLM19, GLM23,
GLM24) shared a high nucleotide identity with domestic chickens in branch C,
reaching 99.5%. The nucleotide identity between GLM05, GLM20 with branch D
chicken is 99.8-99.9%. On the other hand, the nucleotide identity is lower when
compared Lien Minh chicken breed to the domestic chicken in branches G, F, merely
ranged 98.2 to 99.2%.
3.1.4.4. Distribution Haplotype Lien Minh chickens
Using DnaSP software version 5, determine haplotype on 1050 bp and 455 bp
Dloop region. A total of 12 haplotypes in 24 Lien Minh chickens based on 1050 bp of
mtDNA D-loop sequence, with in are focused most on three haplotypes (1, 3 and 8).
Beside this, a total of 7 haplotypes in 24 Lien Minh chickens based on 455 bp of
mtDNA D-loop sequence, with in are focused on haplotype 1 (14 chickens).
3.1.4.5. The Phylogenetic analysis of the Lien Minh chicken is based on the Dloop nucleotide sequence
a. Phylogenetic analysis between Lien Minh chicken and native Vietnamese
chicken breeds (455 bp)
Phylogenetic tree was constructed based on 48 D-loop sequences (24 GLM
sequences, six G9C sequences and 18 GDT sequences) along with 11 reference
sequences that corresponded to the nine clades defined by Liu et al, (2006) and 37
sequences of indigenous Vietnamese chickens distributed seven branches (Cuc et al,
2011). Using 455 bp D-loop mitochondrial DNA to determine the genetic relationship
between Lien Minh chickens and native Vietnamese chicken breeds (Figure 3.10).
12
Figure 3.10. The phylogenetic tree in Lien Minh chicken with native Vietnamese
chickens
The phylogenetic tree was generated by the neighbor‐joining method using MEGA6.1 with bootstrap values for
interior clades after 1,000 replications. Different clades and haplotypes are indicated. GLM, GNN, and GDT
breeds are marked (circle●, triangular▲, and square■, respectively). Numbers in bracket are number of samples
that appeared at identical haplotype.
13
As can be seen from the Phylogenetic tree which was constructed based on Dloop, Lien Minh chickens were distributed on seven haplotypes of 5 clades (A, B, C,
D, E). In general, 58,3% of the Lien Minh chickens were presented in clade B that has
also known as the dominant clade of indigenous Vietnamese chickens and Southeast
Asia. In this study, no samples of Lien Minh, Dong Tao, Many Finger chicken breeds
were distributed in clades G, H, I and F. This result was consistent with the study of
Cuc et al (2011).
b. Phylogenetic analysis between Lien Minh, Dong Tao, Many Fingers chicken breeds
and the world native chicken breeds (455 bp)
Figure 3.11. Phylogenetic tree in Lien Minh chicken with native chickens in the world
The phylogenetic tree was generated by the neighbor‐joining method using MEGA6.1 with bootstrap values for
interior clades after 1,000 replications. Different clade and haplotype are indicated. GLM, GNN and GDT breeds
are marked (circle●, triangular▲ and square■, respectively). Numbers in bracket are number of samples
appeared at identical haplotype.
Phylogenetic tree was constructed based on 48 D-loop sequences (24 GLM
sequences, six G9C sequences and 18 GDT sequences) along with 11 reference
14
sequences that corresponded to the seven clades defined by Oka et al (2007). Using 1050
bp of D-loop region and using NJ method to build Phylogenetic tree for Lien Minh
chicken, Multi Finger chicken, Dong Tao chicken and domestic chicken in the world.
Results showed that, 24 GLM samples were observed in five clade A, B, C, D
and E. Specifically, 14 samples (58.3%) of Lien Minh chickens belonging to haplotype
1, 2, 3 and 4 were distributed in clade E. Previous studies have shown that chickens of
clade E were observed in indigenous chickens in Asia, China, Laos, Japan and India
(Liu et al, 2006, Oka et al, 2007, Cuc et al, 2011, Kawabe et al, 2014) and were not
observed in native African chickens (Miao et al, 2013).
3.2. ANALYSIS POLYMORPHISM IN CANDIDATE GENES RELATED
WITH EGG PRODUCTION TRAITS IN LIEN MINH CHICKEN
3.2.1. Track five egg production traits related to egg yield traits of 90 Lien Minh
chickens
Experiment was conducted on 90 hens which were kept in separate cages for the
period of egg laying (25-44 weeks old). All hens were fed with same diet and
veterinary indicators during the experiment aiming to evaluate the correlation between
candidate genes and egg yield traits in Lien Minh chickens. During the 20-week
spawning period, each Lien minh chicken individual was evaluated on a daily basis
against five egg production traits which are as follows age at first egg, first egg’s
weight, number of eggs, eggs’ weight, and egg’s shape index (D/d).
3.2.2. The frequencies of the alleles and genotypes of polymorphisms of PRL,
PRLR, VIP, VIPR1, NPY, GH and GHR; analyze the relationship with egg yield
traits in Lien Minh chickens
3.2.2.1. DNA extraction
3.2.2.2. The frequencies of the alleles and genotypes of polymorphisms of PRL and
its association with egg yield traits
a. PCR amplification of gen PRL
b. Polymorphic analysis of PRL gene by restriction enzyme
c. Identification of PRL polymorphism by nucleotide sequencing
d. Analysis of the frequencies of the alleles and genotypes of polymorphisms of PRL
Table 3.6 illustrates polymorphisms of PRL24, PRL/2161genes were not
significantly different from the distribution expected under the assumption of Hardy
Weinberg equilibrium (P>0.05). The PRL/2402 polymorphism did not follow the
Hardy-Weinberg equilibrium (P<0.05)
e. Association of the PRL polymorphisms on egg production traits in Lien Minh
chicken
15
Table 3.9. Association between SNP PRL24 and egg production traits in Lien Minh
chicken
Genotype (N)
ID (24)
DD (66)
185.33±8.21
187.12±8.25
45.29±5.77
43.14±4.73
47.57±3.11a
45.05±4.33b
49.13±3.27a
46.58±4.68b
42.16±4.57
39.95±4.98
1.28±0.03
1.28±0.03
Egg production traits
Age at first egg (days)
Number of eggs in 20 weeks (egg)
Mean eggs weight in 20 weeks (g)
Mean eggs weight in 36-44 weeks (g)
First egg’s weight (g)
Eggs’ shape index (D/d)
a,
P
0.365
0.075
0.011
0.016
0.062
0.632
or b, values with no common superscripts within a column for each site differ significantly (P<0.05)
Analysis of the relationship between polymorphism of PRL24 and egg yield traits
revealed that the average egg weight of chickens with genotype ID (47.57±3.11 g) was
greater than that of chickens with DD (45.05±4.33 g) ( P<0.05) (Table 3.9).
For polymorphism of PRL/C2402T, the average egg weight in Lien Minh chickens
with genotype CT (46.91±4.29 g) was higher than genotype TT (44.89±3.93 g)
(P<0,05) (Table 3.10).
Table 3.10. Association between SNP PRL/C2402T and egg production traits in Lien
Minh chicken
Egg production traits
Age at first egg (days)
Number of eggs in 20 weeks (egg)
Mean eggs weight in 20 weeks (g)
Mean eggs weight in 36-44 weeks (g)
First egg’s weight (g)
Eggs’ shape index (D/d)
.
a,
Genotype (N)
CT (37)
TT (53)
185.84±7.99
187.21±8.42
44.95±5.63
42.85±4.53
46.91±4.29a
44.89±3.93b
48.27±4.52
46.56±4.36
41.17±4.44
40.11±5.27
1.28±0.03
1.28±0.03
P
0.440
0.054
0.023
0.073
0.319
0.585
or b, values with no common superscripts within a column for each site differ significantly (P<0.05)
For polymorphism of PRL/C2161G, there was no statistically significant association
between the SNP PRL/C2161G and egg production traits (Table 11)
Table 3.11. Association between SNP PRL/C2161G and egg production traits in Lien
Minh chicken
Egg production traits
Age at first egg (days)
Number of eggs in 20 weeks (egg)
Mean eggs weight in 20 weeks (g)
Mean eggs weight in 36-44 weeks
First egg’s weight (g)
Eggs’ shape index (D/d)
.
Genotype (N)
CC (4)
187.00±2.94
48.50±3.70*
48.99±3.25
51.14±3.37
41.48±4.45
1.27±0.02
*
: Data were not subjected for statistical analysis.
16
CG (26)
186.42±8.55
44.00±4.98*
45.10±3.58
46.94±3.74
39.69±5.89
1.27±0.02
GG (60)
186.72±8.41
43.27±5.10*
45.77±4.41
47.14±4.76
40.85±4.55
1.29±0.03
P
0.985
0.221
0.206
0.565
0.107
f. Effect of haplotype PRL gene at positions: -385; 2402; and 2161 to egg yield traits
in Lien Minh chickens
The analysis of the combination of two polymorphisms PRL24 and PRL/2402
(Table 3.12) showed that the average egg weight of haplotype IDTT (48.17±1.83)
was higher than that of haplotype IDCT (47.20±3.68), DDCT (46.71±4.73) and
DDTT (44.22±3.91) (P<0.01).
Table 3.12. Effect of nucleotide polymorphism of PRL24 and PRL/2402 on egg
production traits in Lien Minh chicken
Haplotype (N)
Egg production
traits
DDCT (22)
DDTT (44)
IDCT (15)
IDTT (9)
AFE (days)
FEW (g)
EN(eggs)
MEW (g)
MEW 36-44(g)
ESI (D/d)
188.05±7.64
40.69±4.76
44.64±4.96
46.71±4.73ab
47.99±5.08
1.28±0.03*
186.66±8.59
39.59±5.10
42.39±4.48
44.22±3.91b
45.88±4.36
1.28±0.03*
182.60±7.61
41.88±3.96
45.40±6.64
47.20±3.68ab
48.68±3.66
1.28±0.03*
189.89±7.42
42.62±5.67
45.11±4.29
48.17±1.83a
49.87±2.51
1.28±0.03*
P
0.128
0.230
0.107
0.006
0.022
-
AFE: age at first egg, FEW: first egg’s weight, EN: number of eggs in 20 weeks, MEW: mean eggs weight in 20
weeks, MEW 36-44: mean eggs weight in 36-44 weeks and ESI: eggs’ shape index. a,b or ab, values with no
common superscripts within a column for each site differ significantly (P<0.05); *: Data were not subjected for
statistical analysis.
Analysis of the combination of two polymorphs PRL24 and PRL/2161 also showed
that chickens with haplotype IDCC showed average egg weight and egg yield, higher
than chickens with the remaining haplotypes.
Table 3.13. Effect of nucleotide polymorphism of PRL24 and PRL/2161 on egg
Haplotype
(N)
production traits in Lien Minh chicken
MEW 36MEW (g)
AFE (days)
EN(eggs)
44(g)
ESI (D/d)
DDCC (3)
DDCG (16)
DDGG (47)
IDCC (1)
IDCG (10)
IDGG (13)
187.33±3.51
185.00±8.81
187.83±8.26
186.00±0.00*
188.70±8.03
182.69±7.99
52.34±2.89
45.70±3.96
46.52±4.81
47.52±0.00*
48.91±2.40
49.41±3.98
47.67±4.04
42.69±3.98
43.00±4.93
51.00±0.00*
46.10±5.88
44.23±5.78
49.41±3.85
43.83±3.77
45.18±4.41
47.75±0.00*
47.14±2.09
47.88±3.87
1.27±0.02
1.28±0.02
1.29±0.03
1.29±0.00*
1.27±0.02
1.29±0.04
P
0.391
0.038
0.167
0.041
0.372
AFE: age at first egg, FEW: first egg’s weight, EN: number of eggs in 20 weeks, MEW: mean eggs weight in 20
weeks, MEW 36-44: mean eggs weight in 36-44 weeks and ESI: eggs’ shape index (N): Number of chickens
Particularly, the average egg weight of chicken with haplotype DDCC (49.41±3.85)
was higher than that of chicken with haplotype DDCG (43.83±3.77) with 5.58 grams
(P <0.05). In addition, chickens with haplotype DDCC showed high egg yield of 47.76
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