Khóa luận tốt nghiệp Công nghệ sinh học: Investigation of prolactin gene polymorphism in the tb crossbred layer duck by PCR-RFLP technique

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESINVESTIGATION OF PROLACTIN GENE POLYMORPHISM IN THE TB CROSSBRED LAYER DUCK BY PCR-RFL

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

INVESTIGATION OF PROLACTIN GENE POLYMORPHISM

IN THE TB CROSSBRED LAYER DUCK BY PCR-RFLP

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

GRADUATIONTHESIS

INVESTIGATION OF PROLACTIN GENE POLYMORPHISM

IN THE TB CROSSBRED LAYER DUCK BY PCR-RFLP

TECHNIQUE

Instructor Student

NGUYEN NGOC TAN, Ph.D DO CHI HIEU

Thu Duc City, 08/2023

First and foremost, I would like to express my special thanks to the teachers of

the Faculty of Biological Sciences and Research Institute for Biotechnology andEnvironment, Nong Lam University — Ho Chi Minh City for their respectful greetings,

best wishes, and deep thanks I received attention, teaching, thoughtful guidance,

favorable conditions, and a research environment during the research andimplementation of the thesis

I would like to thank my instructor, Nguyen Ngoc Tan, Ph.D., for providing mewith the opportunity to conduct this research However, I must admit that I have felt

disappointed with his instructions, and I believe that the teaching style may not have

been entirely suitable for me I found it difficult to voice my concerns, as I felt that theteacher did not actively listen or show much interest in the progress of my research

Moreover, I must admit that my instructor did not review this thesis, and I completed it

by myself

I want to express my sincerest thanks to my family, who have always been a

source of encouragement and concern throughout the journey of preparing thisgraduation thesis Their unwavering support has motivated my continuous efforts to

strive and excel on my intellectual path

Furthermore, I would like to acknowledge the support of VIGOVA PoultryResearch and Development Center for providing genetic resources and necessaryinformation that enabled the smooth progress of this study

Lastly, I extend my sincere appreciation to my seniors, classmates, and everyone

in the research room for sharing and exchanging knowledge and experiences to help meimplement this project

AFFIRMATION AND COMMITMENT

Name : DO CHI HIEU ID: 19126249 Class: DH19SHC

Phone : 0888528341 Email: 19126249@st.hcmuaf.edu.vn

My major is Biotechnology at Nong Lam University-Ho Chi Minh City This is

my graduation thesis, which was conducted by myself All the data and information arecompletely honest and objective I take full responsibility in front of the committee forthese commitments

Thu Duc City, July 31% 2023

Student’s signature

Do Chi Hieu

ii

The Prolactin (PRL) gene, encoding the PRL hormone, plays a crucial role in

regulating various reproductive processes in animals, including egg production andbroodiness in poultry Studying the genetic variations and associations of the PRL gene

can provide valuable insights into enhancing breeding strategies and optimizing

reproductive performance in livestock species Additionally, the early assessment ofreproductive traits, especially in terms of productivity, carries great significance

Therefore, this study aimed to investigate the polymorphism at the PRL/PstI locus ofthe PRL gene in the second generation of TB crossbred ducks by the PCR-RFLPtechnique and the association of its polymorphism with reproductive traits A total of

167 blood samples were collected for DNA extraction and target fragment amplification

The target gene fragment in the exon 5 region was successfully amplified with a size of

536 bp The amplified PCR products were subsequently treated for digestion using thePstl restriction enzyme The results revealed that polymorphism at the PRL/Pstl locus

was detected with two alleles, C and T, and the allele frequencies were 0.808 and 0.192,respectively Three genotypes were observed; the genotype frequencies were 0.641,

0.335, and 0.024 for CC, CT, and TT in the total population The PIC (Polymorphic

Information Content) and He (expected heterozygosity) were 0.262 and 0.310,respectively In conclusion, polymorphism at PRL/PstI is identified, and the CC

genotype is dominant The distribution of genotypes in the population fits the

Hardy-Weinberg equilibrium Further analysis showed a significant association between thegenotypes and egg production Specifically, the CC genotypes exhibited higher eggyields up to 38 weeks of age compared to the CT genotype (corresponding to 99.25 +

9.82 eggs versus 95.55 + 7.25 eggs; P<0.05), following a regression line of

y=3.39x+89.02, with a determination coefficient of 0.039

Keywords: TB crossbred duck, Prolactin gene, Prolactin Hormone, exon 5, PCR-RFLP,

genetic polymorphism, reproductive traits

ill

điểm sinh sản Tổng cộng 167 mẫu máu được thu nhận dé ly trích DNA và khuếch đại

đoạn gen mục tiêu Doan gen mục tiêu trong vùng exon 5 đã được khuếch đại thành công với kích thước là 536 bp Sản pham PCR khuếch đại sau đó được tiến hành phân

cắt bởi enzym giới han PsíI Kết quả cho thay tính đa hình tại locus PRL/Pstl được phát

hiện với hai allele C và T Tần suất xuất hiện cua allele này là 0,808 và 0,192 Ba kiểu gen đã được quan sát Tần suất kiểu gen tương ứng là 0,641; 0,335 và 0,024 cho CC, CT

và TT trong quan thé Chi số PIC (hàm lượng thông tin đa hình) va He (di hợp tử mong đợi) lần lượt là 0,262 và 0,310 Tổng kết, locus PRL/Pstl đã phát hiện tính đa hình va kiểu gen CC chiếm ưu thế Phân bố kiểu gen trong quan thé phù hợp với cân bằng Hardy-

Weinberg Phân tích tiếp theo cho thấy mối quan hệ ý nghĩa giữa các kiểu gen và năng suất trứng Cụ thé, kiều gen CC cho thấy hiệu suất trứng cao hơn đến 38 tuần tuôi so với

kiểu gen CT (tương ứng với 99,25 + 9,82 trứng so với 95,55 + 7,25 trứng; P <0,05), theo

đường hồi quy y = 3,39x + §9,02, với hệ số xác định là 0,039.

Từ khóa: Vit lai TB, Prolactin gene, Hormone Prolactin, exon 5, PCR-RFLP, đa hìnhgen, tinh trang sinh san

iv

TABLE OF CONTENTS

Page

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CHAPTER 3 MATERIALS AND METHODS 0 cccceccecceceeeeseeseeseeseeseeseeseeseeseenens 22

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LIST OF ABBREVIATIONS

Abbreviations Description

AFLP : Amplified Fragment Length Polymorphism

ANOVA : Analysis of variance

Bp : Base pair

df : Degree of freedom

DNA : Deoxyribonucleic acid

EDTA : Ethylenediaminetetraacetic acid

Ha : The alternative hypothesis

PIC : Polymorphic Information Content

PRL - Prolactin gene

PRL : Prolactin hormone

QTL : Quantitative Trait Loci

RAPD : Random Amplified Length Polymorphism

REases : Restriction Enzyme

RFLP : Restriction Fragment Length Polymorphism

rpm : Round per minute

SD : Standard Deviation

SNP : Single Nucleotide Polymorphism

Vii

: Chi-square

Vili

LIST OF TABLES

Table 2.1 Summary of Genotypic and Allelic Frequencies at the PRL/PstI Locus 20

Table 3.1 Expected RFLP-digested PCR product sizes and genotype conventions 27Table 4.1 Information on the L536 primer pair - 5-5525 52 +52 <+£+s£+sc+sccee 31Table 4.2 Polymorphic analysis of the PRL/Pstl locus by PstI restriction enzyme 34

Table 4.3 Effect of genotypes on some reproductive fTa1{S - -+s++++ 36

ix

LIST OF FIGURES

Page

Figure 2.1 Crossbreeding scheme điagram 2-52 22+ 22+ +22 £+zE++vEezerreezerrreerere 4

Figure 2.2 Illustrations of portions of the prolactin gene in ducks 6Figuré 2.3 IIlustration of RFLP analysis.e.cccivsssiavssseavesveee oreseervessisessnmmnsensensesaesenses 14Figure 2.4 Illustration of PCR-RFLP anaÌyS1s - 55552 +52£+<<+2c+sc+scesrree 15Figure 4.1 Representative electrophoresis of the extracted DNA on 1% agarose gel.30

Figure 4.2 Representative electrophoresis of the PCR products of PRL in exon 5 on

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Figure 4.3 Representative electrophoresis PCR-RFLP pattern of the PRL gene

digested with Pst] on 2,5% agarose Ø€ÌL - LG HH TH ng hệt 33Figure 4.4 Genotypic effect of the PRZ/PstI locus on egg yield (EY) - 38

CHAPTER 1 INTRODUCTION

1.1 Problem statement

Along with the wet rice civilization, duck farming has been a long-standingtraditional business and played an important role in the agricultural economy in Vietnam.Since 1961, our country has consistently ranked second globally in terms of duckpopulations In 2020, Vietnam continued to hold the second position with a population

of over 86.5 million ducks, representing 7% of the global total (Jalaludeen et al., 2022)

Duck farming contributes remarkably to agricultural productivity by providing a

considerable source of duck meat and eggs Duck meat is highly sought-after for its

nutritional value and flavor (Sinanoglou et al., 2011) In 2019, Vietnam ranked fourth

globally in terms of duck meat production Duck eggs are also highly regarded for theirsuperior nutritional profile compared to chicken eggs, as well as their affordable price

They are commonly used in the production of salted eggs, thousand-year eggs (pidan),and balut In Vietnam, ducks are primarily raised for the production of table eggs, with

a large domestic market for various types of duck eggs as well as an export market forduck eggs (Jalaludeen et al., 2022) To meet market demand, The TC crossbred duck,

renowned for its highest egg yield in the world and suitability for Vietnamese raising

conditions, was bred and officially recognized as a breed in 2011 (Tran Thanh et al.,2018) As one of the countries heavily affected by climate change, adverse impacts on

agricultural activities are increasing day by day (Anh et al., 2023) Because of the limitedadaptability of duck breeds to climate change, there is a pressing need to breed and select

duck breeds that can ensure both high economic efficiency in production and adaptation

to climate change The TB crossbred layer duck, resulting from a crossbreeding program

between male TC ducks and female Bien ducks, is considered for its potential in adapting

to climate change This crossbred duck was specifically developed to achieve high egg

productivity and salt intrusion tolerance, making it a suitable choice for duck farming inregions affected by climate change and saltwater intrusion

The main method in selective breeding has been used for a long time to improve

livestock by selecting animals with superior traits In general, traditional selectionmethods have limitations in evaluating reproductive traits due to their late expression in

the animal's life Therefore, the early assessment of reproductive traits, particularly interms of productivity, carries great significance The term "Smart Breeding" is

occasionally used to refer to breeding strategies supported by marker-assisted selection(Al-Samarai and Al-Kazaz, 2015) Most RFLP markers are co-dominant, thus allowing

for the detection of both homozygous and heterozygous individuals The utilization ofPCR-RFLP for supporting livestock breeding offers several advantages, such as

affordability, robustness, and simplicity in both the execution and interpretation ofresults (Salisu et al., 2018)

The Prolactin (PRL) gene stands out as a significant candidate due to its impact

on brooding behavior, which can ultimately influence egg production by regulating

PRL hormone levels Despite the PRL gene has garnered attention in many studies,there is still limited information available on the polymorphism of the PRZ/PstI locusand its relationship with productive and reproductive traits in ducks (Ghanem et al.,2017) In my thesis, The PCR-RFLP technique was utilized to investigate the

polymorphism of the PRL/PstI locus of the PRL gene and its association with specificreproductive traits in the second generation of TB crossbred ducks The findings have

practical implications for improving duck breeding programs, specifically in increasingegg production and ensuring the sustainability and resilience of the industry, which

faces ongoing challenges posed by climate change

1.2 Objectives

To evaluate the prolactin gene polymorphism on the exon 5 and the association

of its polymorphism with some reproductive traits of TB crossbred ducks

1.3 Contents

To accomplish the objectives, this research conducted 03 contents

Content 1: Amplification of the target gene on the exon 5 region

Content 2: Polymorphic analysis of the PRL/Pstl by PstI restriction enzyme

Content 3: Effect of the genotype at PRL/PstI locus on some reproductive traits of TBcrossbred ducks

CHAPTER 2 LITERATURE REVIEW

2.1 Duck breed in research: an overview

In this study, I focused on studying the second generation of TB crossbred layer

ducks This section provides an overview of this duck breed

In parallel with the wet rice civilization for thousands of years in our country, theintegrated rice-duck farming system has been developed With that advantage, it is notsurprising that Vietnam has the second-largest duck population globally In rice-producing countries, there is a synergistic relationship between duck productivity and

rice cultivation Free-range ducks play a natural predator role by controlling insects and

snails while also consuming leftover grains after harvest As natural predators and

contributors of organic fertilizer, ducks contribute to higher crop yields (Ismoyowati andSumarmono, 2019) Vietnam has a wealth of genetic resources for duck breeds, from

indigenous duck breeds that are valued for their unique traits and high adaptability tolocal environments to exotic duck breeds that have also gained popularity in Vietnamdue to their fast growth, excellent meat quality, high egg-laying capacity, and

commercial potential However, Vietnam is one of the most countries heavily affected

by climate change These environmental changes pose a significant threat to agricultural

activities, including duck farming, which is known to be sensitive to climate variations

The effects of climate change, particularly saltwater intrusion, have been increasingsteadily over the years, with the salinity intrusion disaster of 2020 acknowledged as the

worst in recent memory for provinces in the Mekong Delta (Tran Ngoc Lam et al., 2022;Park et al., 2022) It is essential to develop salt-tolerant duck breeds with highproductivity and quality for economic efficiency and climate change adaptation

The Bien 15 duck breed, also known as Dai Xuyen, has the ability to adapt tovarious water environments, including fresh, brackish, and saline water It is the first

duck breed in Vietnam to possess the adaptation trait to a marine environment, which is

attributed to its salt gland This salt gland is an exocrine gland that functions toconcentrate salt and facilitate the excretion of ions from the body When raised indifferent water environments with varying salinity levels, the Bien 15 ducks maintainedhigh survival rates The maturity age was 21—22 weeks, with body weights at first laying

ranging from 2,591.12—2,648.31 g/duck The laying rate was between 67.51% and68.31% (Le Thi Mai et al., 2019).

The TC duck breed is a crossbred duck crossed by Vietnamese scientists by

combining the genetic traits of imported duck breeds (Triet Giang, China ducks) and the

well-known egg-laying duck breed in Vietnam (Co duck) This is a duck breed that is

evaluated to have a remarkably high egg production, a larger egg volume than Triet

Giang duck eggs, and an earlier laying age than Co ducks TC ducks have egg

productivity of 280.65—282.68 /duck/52 weeks; egg weight is 68—70 g/egg; egg embryorate is 96.63%; and hatching rate per total incubated eggs is 88.63% (Vu Hoang, 2019)

Based on the salt tolerance capability of the Bien ducks and leveraging the existing productivity advantages of the TC ducks, the process of reciprocal crossing

pre-between TC and Bien ducks was performed to obtain offspring Among the offspring,

the TB crossbred ducks were selected to conduct this research The TB crossbred duck

is considered to have great potential for high egg productivity and good salt tolerance.The breeding and development of this duck breed aimed to address the specific farming

conditions in Vietnam, particularly in response to the challenges posed by climate changeand saltwater intrusion

ơ TC breed / 2 Bien breed

^S

ká

TB crossbred

Figure 2.1 Crossbreeding scheme diagram

The TB crossbred duck generations have reached the second generation The study

on the polymorphism at the PRL/PstI locus was conducted in the first generation of TBcrossbred ducks, but it has not been performed in this generation yet The secondgeneration of TB crossbred ducks is a crucial stage in the breeding program, where thestability of desired characteristics observed in the first generation will be evaluated todetermine the consistency of traits passed on to subsequent generations If the traits

persist and demonstrate consistency in the second generation, it indicates the potential

of using the marker at the PRL/PstI locus to enhance egg productivity

2.2 Candidate genes are associated with egg-laying performance

The rate of egg production is a major economic determinant of the livelihoods of

poultry farmers The ovary is a significant part of the female reproductive system and ishighly important for egg formation Due to the cruciality of this organ, many studieshave been conducted to identify candidate genes associated with egg production in ducksthrough transcriptome analysis, Quantitative Trait Loci (QTL) mapping, and the

Genome-Wide Association Study (GWAS) approach Egg production performance is apolygenic trait influenced by the interaction of environmental factors and geneticmechanisms (endocrine mechanisms), of which genetics impacts egg-layingperformance the most (Bello et al., 2022) This is because the genes influence endocrine

mechanisms by regulating hormone levels, influencing follicle growth and development,

and influencing reproductive system development Endocrine research has examined

candidate genes associated with egg-laying performance, which are primarily related toreproductive hormones (Du et al., 2020; Hanafy and Elnesr, 2021)

More than 31 reproductive hormone-based candidate genes have been identified as

significantly associated with egg-laying performance The main endocrine factorsregulating the egg-laying process are Growth Hormone (GH), Follicle-StimulatingHormone (FSH), Luteinizing Hormone (LH), and Prolactin Hormone (PRL) Onespecific candidate gene, PRL, has received significant attention in various studies due toits polymorphic and potential influence on egg-laying traits (Wilkanowska et al., 2014).2.2.1 The structure of the PRL gene

Kansaku et al (2005) have identified sequence information for the PRL gene in

ducks The duck PRL gene consists of five exon regions that code for 229 amino acids,separated by four introns The sequence of the PRL gene in ducks (Anas platyrhynchos)

has been registered in The National Center for Biotechnology Information (NCBI)(Accession Code: AB158611.1) Duck PRZ cDNA is 92.0%, 91.7%, and 91.4%

homologous to chicken, turkey, and quail PRL, respectively Overall PRL in adult ducks

is similar to that in chicken (95.5%), turkey (92.5%), and quail (95.5%)

_ transcription factor j Coding Sequences (CDS) site|

(TATA Boreal io | Intron! Intron2 Intron3 Intron4 |

: H E F ‡ Poly A Tail

SESsl6468825/ %s«sssssIExonÏ ˆ Exon2 Exon3 + Exon4 + Exon5 S34969959656

5'UTR ; 3UTR

ATG/Metionin TAA

(start codon) (stop codon)

Figure 2.2 Illustrations of portions of the prolactin gene in ducks

The information about the positions of the regions on the PRL gene in Figure 2.2:The 5'-flanking region: 1-242 = 242 bp

The 3-flanking region: 6036—6332 = 297 bp

The exon and intron sequences of the PRL gene in poultry species typically extend

over a length of 6 kb, starting from the initial ATG codon and ending at the stop codon

in the translated sequence The 5' flanking sequences of the PRL gene in chicken, turkey,and duck have lengths of 2.6 kb, 2.0 kb, and 0.2 kb, respectively

The gene encoding PRL hormone is located on chromosome No.6 in the human(Owerbach et al., 1981); chromosome No.13 in the mouse genome (Wiemers et al.,

2003); chromosome No.7 in pigs (Vincent et al., 1989); and chromosome No.2 in thebird, chicken, and duck genomes (Alipanah et al., 2011)

2.2.2 The PRL hormone

The PRL hormone is encoded by the PRL gene PRL is a polypeptide hormone as

well as a cytokine that is synthesized in and secreted from specialized cells of the anterior

pituitary gland, where its expression level is highest Moreover, it is also synthesizedwithin the central nervous system, the immunological system, the uterus, and its

associated tissues of conception, and even the milk-producing gland PRL has been

recognized as a multifunctional hormone with more than 300 functions in vertebrates

Therefore, its biological activities are not limited to reproduction but also include manyother biological processes (Freeman et al., 2000).

2.2.2.1 The PRL hormone in mammals

In humans, PRL plays a significant role in both the psychological and physiologicalaspects of reproductive function It has a wide range of physiological functions, but its

two primary responsibilities are milk production and the development of mammary

glands within breast tissues In addition, this hormone has also been reported to have agreat influence on the immune system It has a bioactive function by acting as a cytokinethat can regulate the immune response and has been shown to regulate the activity andnumber of immune cells (Nevalainen and Rui, 2003)

In the mouse, PRL is a key hormone regulating the formation and functioning ofthe corpus luteum and stimulating other necessary processes for a successful pregnancy

and lactation (Bole-Feysot et al., 1998) In rabbits, PRL has responsibility for the

beginning and maintenance of lactation, as well as the synthesis of the principalcomponents of milk (Benedek et al., 2023)

In contrast, in ruminants (bovine and caprine), PRL is not the quintessential lactational hormone as in other mammals In mammals, during lactation, a decrease in

pro-PRL can inhibit or reduce lactation, but in ruminants, despite being mammals, this doesnot occur or happens insignificantly However, PRL is still considered to have a role in

lactation and mammary gland development in dairy ruminants Although it may not be

the primary hormone involved in those processes, its role is not trivial (Ismoyowati and

Sumarmono, 2019)

2.2.2.2 The PRL hormone in avian species

In general, PRL participates in the process of lactation, stimulating milk production

in the mammary glands to nourish the offspring in mammals Conversely, in birds and

poultry that lack mammary glands, PRL plays another important role Some reproductivedifferences between poultry and mammals lead to differences in the role of released PRL

An outstanding behavior observed in poultry is egg incubation This behavior ischaracterized by the release of the PRL hormone The released PRL is associated with

physiological and behavioral changes that facilitate egg incubation and maternal care by

stimulating brood patch formation The brood patch, a featherless patch of skin on the

abdomen of the parent bird, has increased blood flow and specialized glands toeffectively transfer heat toward the eggs and PRL may also contribute to the display of

aggressive and defensive behaviors in brooding poultry (Dobolyi et al., 2020)

It is a well-established fact that elevated levels of PRL in the blood are related tothe complete cessation of egg production (Kulibaba and Podstreshnyi, 2012) Themechanism that controls broodiness has attracted great interest from avian biologists

because broodiness causes the ovary and oviduct to regress, which results in thecessation of egg laying and consequent financial losses for the poultry industry (Ohkubo,

2017)

2.2.3 The PRL hormone receptor

The PRL hormone interacts with multiple receptors located on the cell membrane

of various organs via an endocrine pathway By binding with these receptors, the PRL

hormone exerts its effects, mediated by the prolactin receptor (PRLR), and plays a

crucial role in the signal transduction cascade of PRL The encoded product of the PRLRgene is a receptor protein, specifically a single transmembrane protein that belongs toclass I of the cytokine receptor superfamily The PRLR gene is expressed in various

tissues, including the ovaries, oviduct, testes, deferent ducts, kidneys, and small and large

intestines (Xing et al., 2011)

It is widely recognized that the PRL gene is closely linked to the initiation and

maintenance of broodiness in poultry and could be a potential genetic marker for

breeding programs aimed at increasing performance eggs Meanwhile, the PRLR gene isconsidered a promising candidate genetic marker for reproductive traits due to its

significant role as an important regulator gene in the processes of cell growth anddifferentiation (Wilkanowska et al., 2014)

2.3 Polymerase Chain Reaction

In the 1980s, Kary Mullis invented the molecular biology method known as thePolymerase Chain Reaction (PCR) Since its development, PCR has revolutionized

molecular biology research and evolved into an assemblage of varied methodologies Itbecomes an indispensable methodology in numerous scientific fields, including genetics,

genomics, forensics, diagnostics, and others

PCR, according to the original description, is an in vitro method that essentiallyextends natural DNA replication It allows amplifying a specific DNA segment using a

limited amount of the initial DNA sample in a rapid and very sensitive manner, therebygenerating abundant identical copies sufficient for research PCR also can modify,

remove, and alter DNA sequences The PCR technique involves a series of dependent enzymatic reactions that result in the exponential replication of a targeted

temperature-DNA region (Clark et al., 2019)

A typical PCR assay requires a DNA polymerase enzyme, a DNA template, DNA

primers, nucleotides, a PCR buffer, and a thermal cycler instrument The DNApolymerase synthesizes nucleotides to generate the PCR products The primers have a

specific sequence complementary to the target DNA, determining the size and specificity

of the amplified products The PCR buffer provides optimal conditions for enzyme

activity and DNA amplification The thermal cycler enables precise temperature changesfor repeated cycles of DNA amplification

2.3.1 PCR process

A cycle in the PCR process typically consists of three main steps: denaturation,annealing, and extension In the denaturation step, the reaction solution is heated (94—95°C) for a certain period (20-30 seconds) to separate the double-stranded structure of

the DNA sample into two individual strands In the annealing step, the temperature istypically lowered to a specific temperature (50-65°C) for a certain period (20-30

seconds), allowing the specific primers to bind specifically to their complementarysequences on each DNA strand In the extension step, the temperature is raised again(72°C) for a certain period (1-2 minutes) to enable DNA polymerase to synthesize newDNA strands starting from the primers This three-step cycle is repeated 25—40 times,resulting in a doubling of the number of copied DNA molecules with each repetition

PCR products or amplicons typically range in length from 100 to 3000 base pairs,

although longer amplifications of up to 50,000 base pairs can be achieved under certain

conditions There are two main methods of visualizing the PCR products: Gelelectrophoresis and hybridization with labeled oligonucleotide probes

2.3.2 Advantages and limitations of PCR

PCR has multiple advantages, including high sensitivity, which permits the

detection of samples with low concentrations and the amplification of the desired DNAsequence accurately It is a rapid method for generating many copies of a specific product

for sequencing, cloning, and analysis in a short period PCR is cost-effective andperformed in vitro, eliminating the need for living cells

However, PCR does have some limitations Contamination is a potential issue thatcan lead to misleading results PCR has length limitations, making the amplification of

longer DNA fragments challenging Designing specific and efficient primers for DNAsequences is challenging, and PCR requires prior knowledge of the target DNA

sequence, limiting its applicability to unknown or poorly characterized sequences.Additionally, there is a low rate of error in PCR due to the possibility of base substitution

errors being synthesized by the DNA polymerase (for Taq polymerase, an error rate of

1.8 x 10°), leading to sequence variations in the final products (Potapov and Ong, 2017).

2.4 Molecular markers in animal science

Genetic markers are stable and heritable characteristics that can be quantified or

detected using appropriate methods It serves as a recognizable feature that can be used

to identify specific genetic characteristics or traits in organisms Genetic markers aredivided into two main categories: Conventional markers and molecular markers

2.4.1 Conventional markers

Conventional markers, also known as classical markers, refer to the markers that

were commonly used before the advent of molecular markers Morphological,Cytological, and Biochemical markers are the three primary classifications of

conventional markers

Morphological markers are physical characteristics of an organism used to identify

and distinguish desired traits (such as size, shape, color, or other visible traits) Livestockphenotypes are influenced by both genetics and the environment Evaluating farm animalgenetic resources with morphological markers relies on subjective judgments anddescriptions, which can lead to conclusions that are often not completely accurate A

second type of marker, Cytological markers are related to variations in the numbers,

karyotypes, banding patterns, position, meiotic behavior, and _ constitutive

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heterochromatin of chromosomes They are used to assess the genetic diversity of animalgenetic resources and determine the specific location of a gene on the chromosome, as

well as its position relative to other genes The third type of marker is Biochemical.Biochemical markers focus on variations in protein, blood type, allozymes, and other

biochemical substances, usually detected through electrophoresis or immunoassays.Nevertheless, neither proteins nor allozymes are genetic material and are susceptible to

environmental influences, which restricts their wider application (Samarai and Kazaz, 2015)

Al-Conventional markers still hold value in certain contexts and continue to be utilized

in genetic studies and breeding programs However, they have limitations in terms of

resolution, ability to provide genetic information and may not be directly associated with

specific genes or desired traits In numerous investigations, they have been supplemented

and supported with more advanced molecular markers

2.4.2 Molecular markers

Conventional markers have been criticized for being an indirect and insensitivetechnique for detecting variation in DNA Researchers are interested in markers thatallow direct identification of the genes of interest rather than the products of the genes(Schlötterer, 2004) Advancements in molecular genetics have introduced a wide range

of molecular markers that contribute to better understanding and assist in the selection

allele variations that can be classified into three major groups: differences in the number

of tandem repeats at specific loci (known as simple sequence repeats), segmental

insertions/deletions (InDels), and single nucleotide polymorphisms (SNPs) Due to their

abundance and stability, markers utilizing SNPs have emerged as a prominent focal point

in the field of molecular genetics (Mammadov et al., 2012) However, molecular markers

may not always influence the phenotypic expression or activity of genes These markersmay or may not correlate with how a trait is expressed phenotypically

II

2.4.3 Classification of molecular markers

Molecular markers can be classified into two categories based on genomic

segments: type I and type II Type I markers are associated with genes of known functionand are conserved loci across species Type II markers are characterized by their high

polymorphism and are genomic segments with anonymous or non-coding sequences.Furthermore, molecular markers can also be classified based on the mode of gene action(co-dominant or dominant markers) or detection technique (hybridization-based markers

or PCR-based markers) (Raza et al., 2015)

An ideal molecular marker should possess the following characteristics: Firstly, itmust be co-dominant in expression and have adequate resolution Secondly, it should bepolymorphic and evenly distributed throughout the genome Thirdly, it should generatemultiple independent and reliable markers Fourthly, it should be simple, rapid, and cost-

effective Fifthly, it should require small amounts of tissue and DNA samples Sixth, it

should be associated with distinct phenotypes Lastly, it should not require priorknowledge or information about the genome of the organism It is important to note that

while there is no molecular marker that possesses all these characteristics, ongoingresearch aims to develop markers that come closer to these ideal characteristics (Mondini

2.5 Restriction Fragment Length Polymorphism

A Restriction Fragment Length Polymorphism (RFLP) is a DNA sequence thatcontains specific recognition sites for restriction enzymes at each of its ends, with a

"target" sequence located between them The majority of RFLP markers are type I and

co-dominant, allowing for the distinction between homozygous and heterozygoussamples by detecting both alleles at a particular genetic locus They are based on theidentification of differences in the lengths of DNA fragments generated by the digestion

12

of DNA samples with specific restriction enzymes The formation of RELP as a result ofpoint mutations impacts the presence or distribution of restriction enzyme recognition

sites

2.5.1 Restriction enzymes

Restriction enzymes (REases), also referred to as restriction endonucleases, areenzymes that can recognize particular DNA sequences and cleave the DNA at restrictionsites When a restriction enzyme cleaves the DNA at its specific recognition site, itproduces DNA fragments of defined sizes and ends These DNA fragments generated bythe cleavage of DNA with restriction enzymes could be used as genetic markers

REases are classified into four main types: Type I, Type II, Type II, and Type IV.These classifications are based on their enzymatic properties and mechanisms of action.Type I REases cleave DNA at locations that are relatively random and often far awayfrom the recognition site Type II REases cleave DNA at specific sites within therecognition site Type III REases recognize short nonpalindromic sequences and cleavethe DNA at fixed positions outside of the recognition site Type IV REases cleavemethylated DNA and exhibit weak sequence specificity

Type II restriction endonucleases (REases) represent the largest group of

characterized enzymes and have been extensively studied (Loenen et al., 2014) Theyoffer unparalleled opportunities for advanced genetic research and have revolutionized

fields such as genomics, genetic engineering, and molecular diagnostics

2.5.2 RFLP process

(1) Total DNA Extraction: Isolation of DNA from the sample of interest

(2) Restriction Digestion: Enzymatic cleavage of DNA using restriction enzymes

to generate DNA fragments

(3) Gel Electrophoresis: Separation of DNA fragments based on their size using

agarose gel electrophoresis

(4) Denaturation: Treatment of the gel to denature the double-stranded DNA into

single-stranded DNA

(5) Blotting: Transfer of the single-stranded DNA from the gel to a solid support

membrane, such as nitrocellulose or nylon

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(6) Cross-Linking and Blocking: Blocking the membrane to prevent non-specific

binding and cross-linking the DNA to the membrane

(7) Hybridization and Visualization: Hybridization of the membrane-binding DNA

with a radiolabeled probe specific to the target DNA sequence, followed by detection

and visualization of the hybridized bands using techniques such as autoradiography

laid on the top of it,

transferring the DNA

solution

Q A photographic film laid on top

The paper is exposed toa of the paper is exposed by the

solution containing radioactivity in the bond probe

radioactively-labeled probe to form an image corresponding

to the DNA bands

Figure 2.3 Illustration of RFLP analysis

RFLP has diverse applications in genome mapping, genetic variation analysis,disease association studies, DNA fingerprinting, evolutionary studies, and plant andanimal breeding Although newer techniques have emerged, RFLP has played a

significant role in advancing our knowledge of genetics and laying the foundation formore advanced genetic analysis methods

2.6 Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

In reality, the DNA in a genome is very large, so digesting it with enzymes and

performing Southern blot analysis is costly and complicated and requires large amounts

of high-quality DNA The use of radioactive probes in RFLP poses safety hazards and

increases complexity and cost Access to RFLP analysis may be restricted due to

regulations on radioactive materials Therefore, when the differences in DNA sequenceoccur only in a specific region of the genome, the PCR-based RFLP technique can be

used to differentiate the PCR products

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2.6.1 PCR-RFLP process

The PCR-RFLP method for genotyping is based on SNP markers in candidate

genes and involves the following steps:

Step 1: Selection of SNP markers for each candidate gene by considering theirfunctional relevance, previous literature, genomic databases, and allele frequency

Step 2: Extraction and purification of DNA from the tissue sample

Step 3: Experimental design and performance of the PCR-RFLP reaction Thisinvolves designing primers, optimizing PCR reaction conditions, choosing appropriate

restriction enzymes for digestion, and preparing the necessary reaction buffers

Step 4: Determination of the genotypes based on SNP markers using

electrophoresis A successful PCR-RFLP reaction results in DNA fragments of different

lengths, depending on the genotypic differences caused by the SNP These DNAfragments can then be analyzed using electrophoresis to determine the genotype based

on the size differences of the cut fragments

Extract DNA ep

—————>

| PCR amplification

Blood sample

mmRestriction Enzyme Digest

AIA AIC cic

Figure 2.4 Illustration of PCR-RFLP analysis

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2.6.2 Advantages and limitations of PCR-RFLP and RFLP techniques

PCR-RFLP offers several advantages over traditional RFLP It is a more efficient

method as it amplifies a specific DNA region before restriction enzyme digestion,increasing sensitivity, specificity, and precision while significantly reducing the time

required compared to the traditional RFLP method It also requires smaller amounts ofDNA, making it suitable for samples with limited DNA availability Since no radioactivematerials are required, it is safer

However, there are significant drawbacks to PCR-RFLP Prior knowledge is needed

to plan the experiment, including primer design, optimization of PCR conditions, andselection of appropriate restriction enzymes, which can be complex and time-consuming.PCR-RFLP has limited resolution for detecting genetic variations within specific DNA

segments as well as genetic variations outside of the primer binding sites On the otherhand, RFLP is a well-established technique for genetic analysis It allows for genome-

wide analysis without prior sequence information and is effective at detecting largergenetic variations RFLP is not dependent on primer binding sites, making it suitable for

regions with unknown sequences

2.7 PRL gene research

2.7.1 The research status of Vietnam

Recent years have witnessed a tremendous increase in interest in research on the

PRL gene in poultry Studies have shed light on the role of the PRL gene as a candidategene for supporting genetic selection in various aspects of poultry production andperformance

One notable study, Tran Thi Binh et al (2018) conducted a study to investigate the

polymorphisms in the PRL gene relating to reproductive traits in the indigenous breed

of Lien Minh chickens The researchers found two genotypes for each of the twopolymorphic sites at PRL24 and PRLS The identified genotypes at PRL24 were ID and

DD, while the genotypes at PRLS were CT and TT The results showed that individualswith the ID genotype at PRL24 and the CT genotype at PRLS exhibited significantly

higher average egg weights of 47.57 + 3.11 (g) and 46.91 + 4.29 (g), respectively, with

a significance level of P<0.05 Furthermore, those with the genotypes ID and CT also

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displayed better reproductive characteristics, including the number of eggs and theweight of the first egg.

In 2019, a study by Nguyen Hoang and Nguyen Thi Chau, the polymorphisms of

the PRL gene (including the 24-bp Insertion-Deletion and C2402T) were investigated intwo Vietnamese native chicken breeds, namely Ri and Mia Chicken The study identifiedthree genotypes for the 24-bp Insertion-Deletion polymorphism (DD, ID, and II) and

three genotypes for the C2402T polymorphism (CC, CT, and TT) Notably, genotypes

DD and TT exhibited dominance at their respective polymorphic positions in both the Ri

and Mia chicken populations Research has suggested that polymorphisms of the PRL

gene (24-bp Insertion-Deletion and C2402T polymorphisms) are considered candidategenes for improving reproductive traits in poultry

Pham Thi Nhu et al (2022) evaluated the polymorphism of the PRZ gene incrossbred ducks (Star53xBien) The study found that the PR//Hphl locus was

monomorphic, showing no variation in genotypes At the PRL/Xbal locus, two alleles, Tand G, were identified, with the frequencies of the TT and TG genotypes being 0.78 and

0.22, respectively The T allele and the TT genotype were found to be dominant at thislocus However, the impact of these genotypes on the growth and reproductive traits of

the crossbred duck population has not been investigated yet

Le Tan Loi et al (2022) conducted a study on the PRL gene polymorphism inStar53 crossbred ducks, specifically investigating the exon 5 region at the PRL/Dral,

PRL/Pvull, and PRL/Psfl loci using the PCR-RFLP technique The results of the studyshowed that the PRL/Dral and PRL/Pvull sites were found to be monomorphic.However, the PRL/PstI site exhibited polymorphism, with allele frequencies of 0.85 for

C and 0.15 for T Two genotypes were observed, with genotype frequencies of 0.70 for

CC and 0.30 for CT, respectively

Bui Pham My et al (2022) explored the polymorphisms of the PRL gene on exon

5 and the correlation of the polymorphisms with egg production traits in the firstgeneration of crossbred BT ducks (Bien x TC) The results of the study showed that three

genotypes, CC, CT, and TT, were observed at the PRL/Pstl locus The associationbetween genotypes and phenotypes revealed that the group of ducks carrying the CC

genotype tended to lay their first egg earlier Moreover, they exhibited significantly

17

higher egg production until 38 weeks of age compared to the group of ducks with the CTgenotype (corresponding to 102.8 eggs versus 98.7 eggs, respectively; P<0.05) These

findings suggest a potential relationship between the PRL/Pstl genotypes and eggproduction traits in crossbred BT ducks

In the study conducted by Le Ba et al (2022) on the TB crossbred layer duckpopulation The polymorphism was discovered at the PRL/PstI locus The results

revealed the presence of two identified alleles, C and T, with frequencies of 0.82 and

0.18, respectively The genotype frequencies were found to be 0.66 for CC, 0.32 for CT,

and 0.02 for TT The group of ducks with the CC genotype exhibited significantly higheregg production up to 38 weeks of age, with 99.27 eggs compared to 92.42 eggs in the

group of ducks with the CT genotype (P<0.05)

2.7.2 The research status of worldwide

Since the structure and sequence of the PRL gene in ducks were first studied andpublished by (Kansaku et al., 2005) There has been a continuous increase in the number

of research studies focusing on PRL gene polymorphisms in various duck breeds withthe aim of improving economic traits, especially egg production (Li et al., 2009; Wang

et al., 2011; Chuekwon and Boonlum, 2017;Yurnalis et al., 2019; Bai et al., 2019;Purwantini et al., 2020)

Li et al (2009) conducted a study on the polymorphisms of the PRL gene inGaoyou ducks, a Chinese indigenous breed The study focused on the intron | region ofthe PRL gene (PRL/Dral locus) and identified three observed genotypes: AA, AB, and

BB, resulting from a T/C mutation at the position of 1326 bp The frequencies ofgenotype BB and allele B were the highest The study also examined the associationbetween polymorphisms in PRL/Dral locus and some reproductive traits The results

showed that ducks carrying the BB genotype had significantly higher egg weights at 30weeks of age compared to those with the AB genotype (P<0.01) However, there were

no significant differences in egg number, longest clutch days, and body weight at the

first egg among the three genotypes Therefore, the findings suggest a potentialassociation between the intron | region of the PRL gene and egg weight in ducks

Wang et al (2011) found that a polymorphism resulting from a C5961T mutation

in exon 5 of the PRL gene (PRL/PstI locus) in local Chinese ducks was associated with

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annual egg production and egg weight Based on the association analyses conducted on

the F2 resource population of White Liancheng X White Kaiya breeds, it was concludedthat the C-5961T polymorphism in the PRL/PstI locus is associated with egg productionand egg weight Particularly, ducks with the CC genotype exhibited a greater number of

eggs produced and a higher egg weight compared to ducks with the CT genotype

In Khaki Campbell ducks, a polymorphism was identified at position 359 bp within

intron 1 (the PRL/Xbal locus) due to the C/A mutation of the PRL gene, and itsassociation with egg production after 300 days was investigated At the PRL/Xbal locus,three genotypes were detected: GG, GT, and TT, with the highest frequency observed forthe GG genotype The analysis of the relationship between the polymorphism at the

PRL/Xbal locus and egg production at 300 days of age revealed that the GT genotype

was associated with higher egg production compared to the GG and TT genotypes

(53.32, 37.50, and 36.67 eggs, respectively; P<0.05) (Chuekwon and Boonlum, 2017)

Ghanem et al (2017) surveyed the polymorphism of exon 5 of the PRL gene(PRL/Pstl locus) in 10,000 Pekin ducks and 500 subsequent F2 generation ducks The

polymorphism at the PRL/Ps#l locus revealed three genotypes: CC, CT, and TT.Statistical analysis showed a significant correlation between the genotypes at the

PRL/Pst\ locus and the studied traits Specifically, the CC genotype exhibited higher

values than both the CT and TT genotypes for the examined characteristics of interest.The findings of this study emphasize the potential of the PRZ/PstI locus for diverse

reproductive traits in various duck breeds

Yurnalis et al (2019) investigated the polymorphisms of the PRL gene at thePRL/Xbal and PRL/Dral loci in Bayang ducks and examined their relationship with thebody weight of ducks from 1 to 10 weeks of age The results revealed the presence ofthree genotypes at each locus investigated However, the analysis indicated noassociation between these two types of polymorphisms and duck body weight

The polymorphisms in the PRL gene were investigated in two egg-laying duckbreeds, namely Jinding and Youxian The focus was on the association between thesepolymorphisms and egg production One specific polymorphism (A-412G) wasidentified in intron 1 of the gene by using PCR-SSCP analyses, leading to the presence

of three genotypes: GG, AG, and AA The results from association analyses revealed that

19

ducks with the GG genotype exhibited higher egg weight and greater egg productioncompared to those with the AA and AG genotypes (Bai et al., 2019).

In a recent study, the polymorphisms in the PRL gene were studied in Indonesianindigenous ducks, including Tegal and Magelang ducks (FO), as well as their reciprocalcrosses known as Gallang (F1) and Maggal (F1) The information obtained revealed theoccurrence of a single nucleotide polymorphism (SNP) at position 164 nt (c.164G > A)

in the PRL gene, resulting in three observed genotypes: GG, GA, and AA The results of

this study demonstrate that the SNP genotypes of the PRL gene exhibit polymorphismand have a positive effect on reproductive traits, particularly egg production (Purwantini

et al., 2020)

The results of the aforementioned studies consistently demonstrate the influence of

PRL gene genotypes on reproductive traits, particularly egg production Among the

various regions of the PRL gene, two areas, intron | and exon 5, have been extensively

studied Intron 1 is believed to be associated with egg weight, while exon 5 is associatedwith egg production (Wang et al., 2011; Ghanem et al., 2017)

Exon 5 is known as the exon region that encodes the highest number of amino acids

in the PRL gene Studies have focused on the polymorphic position C-5961T, where a C

to T mutation occurs in exon 5 of the PRL gene, to examine the correlation between

reproductive traits and genetic diversity Therefore, the PRL/PstI locus has prompted

studies investigating the polymorphism at the C-5961T mutation position in the exon 5region of the PRL gene in various duck breeds

Table 2.1 Summary of Genotypic and Allelic Frequencies at the PRL/PstI Locus