Tom tat luan an tieng anh Ứng dụng chỉ thị phân tử trong cải thiện một số chỉ tiêu về năng suất sinh sản của gà Ác (gallus gallus domesticus brisson)

Associations of polymorphisms in Prolactin and Dopamine receptor D2 genes with reproductive traits on Silkie chicken.. Genetic variants of INHA/PstI and VIPR1/HhaI and their relationship

MINISTRY OF EDUCATION AND TRAINING

CAN THO UNIVERSITY

SUMMARY OF DOCTORAL DISSERTATION

BIOTECHNOLOGY Code: 9420201

THIS STUDY WAS ACCOMPLISHED

AT CAN THO UNIVERSITY

Scientific supervisor: Dr Le Thanh Phuong

Scientific co-supervisor: Prof Dr Nguyen Trong Ngu

The thesis will be defend against the council state thesis meeting at the

Place: ……… , Can Tho University

At …… date …… month …… year 2024

The thesis could be found at the library:

- Learning Resource Center, Can Tho University

- National Library of Vietnam

LIST OF PUBLISHED STUDIES OF THE AUTHOR

RELATING TO THE THESIS

1 Tran Trung Tu, Le Thanh Phuong, and Nguyen Trong

Ngu (2023) Associations of polymorphisms in Prolactin and Dopamine receptor D2 genes with reproductive traits on Silkie chicken Online Journal of Animal and Feed Research, 13(5): 321-327

DOI: https://dx.doi.org/10.51227/ojafr.2023.47

2 Trần Trung Tú, Lê Thanh Phương và Nguyễn Trọng

Ngữ (2023) Năng suất sinh sản của gà Ác (Gallus gallus domesticus Brisson) giai đoạn 16-40 tuần tuổi Tạp chí Khoa

học kỹ thuật Chăn nuôi, 286: 13-17

3 Le Thanh Phuong, Tran Trung Tu, Nguyen Trong Ngu

(2023) Genetic variants of INHA/PstI and VIPR1/HhaI and their relationship with reproductive traits in Silkie chicken

(Gallus gallus domesticus Brisson) Veterinary Integrative

Sciences, 21(3): 831-841 DOI; 10.12982/VIS.2023.059

Chapter 1 INTRODUCTION 1.1 The urgency of study

The indicator types used in breeding are morphological, physiological, biochemical, and molecular In the past, to select breeds, people often used morphological indicators for some desired characteristics by observing and performing crosses This method is simple and easy to implement but has limitations in terms of time and accuracy Molecular biology techniques have shortened time, improved accuracy and allowed breeders to produce varieties with a combination of traits (Collard and Markill, 2008) DNA-related markers are superior because they are easy to detect, abundant in the genome, independent

of environmental conditions and can be detected at any stage of an organism's development DNA markers are divided into 3 groups: (i) groups based on hybridization and using DNA markers including DNA restriction fragment length polymorphism (RFLP) and oligonucleotide fingerprinting; (ii) groups based on in vitro of DNA fragment amplification are AFLP, SSR, RAPD and (iii) DNA sequencing groups are SNP

Ac chickens (Gallus gallus domesticus Brisson) is one of the indigenous

chicken breeds raised for a long time in the Mekong Delta In fact, Ac chickens are raised in 2 directions: raising laying hens for commercial eggs and raising chicks for sale as breeders, in which hens continue to be raised for breeding and

roosters are sold at 3-4 weeks old According to Kojima et al (2014), Ac

chickens were a significantly higher source of Carnosine (798.3 mg/100 g breast meat) than white chickens (417.2 mg/100 g breast meat) Carnosine was

a protein found abundantly in the meat and brain of vertebrates, playing an important role in physiological functions such as: pH buffering, anti-aging, anti-inflammatory, anti-oxidant, anti-fatigue and anti-inflammatory properties

neural transmission (Menon et al., 2018; Caruso et al., 2019) Carnosine was

used in medical applications to treat diseases such as diabetes, Alzheimer's,

aging, cancer, and other chronic diseases (Derave et al., 2019)

Besides, Ac chicken eggs were also favored by consumers because they did not have a fishy smell, are fatty, fragrant, have high white protein, high yolk ratio, and had a very attractive dark color (Tran Thi Mai Phuong, 2004) Ac chickens reach sexual maturity early at 113-125 days old (Tran Thi Mai Phuong and Le Thi Bien, 2007), eggs had a weight of 31.3-36.2 g/egg and a laying rate of 52.3-58.1% in the period 23-37 weeks old (Thuy and Ha, 2022)

Ac chickens were raised industrially to produce eggs in Tien Giang and Long

An provinces on a large scale (Nguyen Van Yen, 2014) Research in this field

was also limited and instead focuses on the field of nutrition (Nguyen Nhut

Xuan Dung et al., 2014; Luu Huu Manh et al., 2014, Truong Van Phuoc, 2021)

Thus, the problem of Ac chickens breed in Vietnam has not yet been genetically researched in depth and there have been no publications on the role

of some candidate genes related to the reproductive performance of this chicken breed The application of biotechnology achievements in improving egg productivity in Ac chickens was completely feasible in terms of laboratory equipment as well as practical requirements in the Mekong Delta The success

of this research will open up a new direction for selecting new breeds more accurately and quickly, contributing to promoting crossbreeding and being proactive in breeding issues and thereby bringing practical results to farmers

1.4 The novel aspects from the thesis

This is the first study in the Mekong Delta and in Vietnam to identify six

gene polymorphisms (DRD2 Indel, PRL Indel, IGF1/PstI, INHA/PstI, NPY/DraI and VIPR1/HhaI) in Ac chickens In addition, SNP T829C in the exon 1 region of the INHA/PstI polymorphism is the first result in chickens in

egg productivity than the G0 generation, 4.4 eggs/hen/24 weeks of laying (increased by 6.2%) Besides, the SG0 generation chicken had an egg yield of 149.1 eggs/hen/52 weeks of laying

Chapter 2 OVERVIEW DOCUMENT 2.1 Some molecular markers in animal breeding selection

* Insertion/Deletion marker

Insertion/Deletion polymorphism, often abbreviated Indel, is a type of genetic variation in which a nucleotide can be inserted or deleted in the nucleotide sequence of a gene Indels are mutations that insert or delete multiple nucleotides, while point mutations are deletions, additions or substitutions involving a pair of nucleotides An Indel in the coding region of a gene that is not a multiple of 3 nucleotides leads to frameshift The frameshift and gene transcription sequence can code for a different amino acid or lead to premature inactivation of the codon, changing the structure and function of the protein An Indel can change the DNA sequence and cause a frame shift thereby changing the sequence of amino acids produced leading to abnormal protein production or even no protein being produced Indels are used as genetic markers in populations, especially in phylogenetic evolution studies (Erixon and Oxelman, 2008)

* RFLP marker

When mutations change the nucleotide sequence at the position of the restriction enzyme, these enzymes cannot recognize those sequences In contrast, mutations in normal sequences other than enzyme cleavage sites can create new restriction sites Such mutations will result in different distances between two consecutive restriction sites of the same enzyme The polymorphism resulting from these mutations is called restriction fragment length polymorphism (RFLP) RFLP markers were used as phenotypes in genetic research and standard markers in genetic mapping (Hayder and Mohammed, 2019)

The RFLP polymorphisms can be detected by Southern blot with radiolabeled probes However, with the development of PCR technique, people combine PCR and RFLP into PCR-RFLP technique A pair of primers can be used to amplify a region of DNA that needs to be investigated, then the amplified DNA fragments were cut with cutting enzymes, electrophoresed and analysed on gels stained with Ethidium bromide, and this technique allowed for analysising polymorphism on target DNA segment (Teneva, 2009)

* SNP marker

The SNP involves the substitution, addition or loss of a nucleotide According to Al-Samarai and Al-Kazaz (2015), the use of SNP was increasingly popular due to:

- SNPs appear at high frequency when screening for SNPs in most genomes, about every 225 bp a SNP is found in the chicken genome and about every 1,250 bp is screened for the human genome

- SNPs can appear in the coding gene region and directly impact the traits of interest, very effective in determining the correlation between SNPs and certain traits

- SNPs are more genetically stable than microsatellites and can be used as long-term selection markers

SNP marker was abundant and exhibit low mutation rates, which facilitates genotyping (Chen and Sullivan, 2003) and this supports in mapping underlying diseases in chicken in the future Thus, SNP has become a marker of interest and popular use thanks to its stability, abundance and co-dominant marker, so it can be used to analyse individual genotypes in the population

* Advantages and limitations of molecular marker-based selection

According to Muhammad et al (2017), selection based on molecular

markers had the advantage of selecting individuals with the genotype that determines the desired trait, select individuals right at the young stage, shorten time However, molecular markers had limitations: high technical requirements; modern and expensive equipment

2.2 Some studies on Ac chickens in the world and in Vietnam

The outstanding characteristic of Ac chickens was the melanin pigment

expressed in the skin Dorshorst et al (2010) mapped the link between skin

pigmentation to two main genes: the Id gene located on the Z sex chromosome that inhibited melanin pigmentation in the skin and the Fm gene (fibromelanosis) located on chromosome 20 It was found that TYRP1 and

TYRP2 genes affect skin color (Zhang et al., 2015), EDN3 gene was related to melanin formation, black bone characteristics of Ac chickens (Li et al (2020)

According to Tran Thi Mai Phuong and Le Thi Bien (2007), Ac chickens had a small appearance and scratchy white feathers, five-toed feet, skin, flesh, bones and beak were black The rooster had a dark red comb, the hen had a light red comb At 1 day old, the rooster weighed 18.8 g/bird and the hen weighed 18.5 g/bird At 4 weeks old, roosters reached 128.6 g/bird and hens reached 114.6 g/bird Similarly, at 9 weeks old, the rooster weighed 467 g/bird

and the hen weighed 379 g/bird At 18 weeks old, the rooster weighed 800 g/bird and the hen weighed 630 g/bird The hen's weight at the time of first egg laying was 709 g/bird (16-17.5 weeks old) Ac chickens reached sexual maturity at 113-125 days old, the laying rate was 40.2% and reached 80-105 eggs/hen/year with egg weight of 30-31 g/egg

On the market, although Ac chicken eggs were small, their prices were higher than industrial chicken eggs The meat and eggs of Ac chickens were very popular thanks to their high melanin content and better taste than industrial chickens (Truong Van Phuoc, 2021) Ac chickens also provided a source of Carnosine, a protein that prevents aging and treats complications of

diabetes such as nerve damage, eye disorders and kidney problems Kojima et

al (2014) determined the Carnosine content in the breast meat of Ac chickens

(798.3 mg/100 g of meat) was significantly higher than that of white chickens (417.2 mg/100 g of meat)

In summary, Ac chicken is one of the indigenous chicken breeds widely raised in the Mekong Delta Ac chicken meat and eggs not only bring nutritional value to users but also economic value to farmers Therefore, improving the reproductive performance of this chicken breed is very necessary However, genetic studies and selecting breeds with high reproductive performance have not been carried out Therefore, this study focuses on the field of molecular genetics to identify genes that are closely linked to egg productivity of Ac chickens, thereby applying it in practice to selectively improve egg productivity of the breed chicken

2.3 The scientific basis of poultry breeding selection

* Principle of selection

The purpose of selection was to improve the productivity and quality of livestock The research results showed that, with successful selection, the average selected generation would be better than the average of their parents Therefore, selection is an indispensable method in animal husbandry, the basis

of genetic modification programs (Robin, 2023)

* Selection response

The selection response (R) was the difference in phenotypic value between

the average of the offspring and the parent generation that produced them

before selection Selection response was used to evaluate the degree of change

in the average value of the population over generations of selection (Bui Huu

Doan et al., 2011)

The selection differential (S) was the difference in phenotypic value between the average of the selected individuals retained as the same breed for that entire generation The role of selection differential depended on the size of the population being selected and the phenotypic standard deviation of the trait

Chapter 3 RESEARCH METHODOLOGY 3.1 Material

3.1.1 Time and place

- Time: from December 2021 to May 2023

- Places:

+ The molecular biology analyses were performed at the Faculty

of Animal sciences, College of Agriculture, Can Tho University

+ The indicators of reproductive traits of Ac chickens were monitored and recorded at the chicken farm in Phong Dien district, Can Tho City

3.1.2 Experimental subjects

- The beginning generation (G0): conducted on 400 hens, 120 roosters selected for appearance characteristics from 800 hens and 200 roosters at 9 weeks old

- The selected beginning generation (SG0): conducted on 130 hens and 26 roosters at 16 weeks old selected from G0 with genotypes for high egg yield

- The generation 1 (G1): conducted on 150 hens at 1 day old that origin from the SG0 generation

3.2 Method

3.2.1 Content 1: Identify gene polymorphisms related to reproductive performance of Ac chickens

- Choose and buy 800 hens and 200 roosters at 9 weeks old

- Raise these individuals until 15 weeks old

- Select 400 hens and 120 roosters that qualify the standards of pullets at 16 weeks old

- Extract DNA, determine gene polymorphisms, SNPs in candidate genes (DRD2, IGF1, NPY, PRL, VIPR1, INHA) at 16 weeks old

3.2.2 Content 2: Evaluate reproductive performance and determine the association between candidate genes and reproductive performance of Ac chickens

- Raise a hen in each cage to record reproductive traits at 16-40 weeks old

* Create a chicken flock with high egg productivity

- Mate the family at the ratio of 1 rooster: 5 hens with the same genotype (the gene with high egg yield was selected in Content 2) at 41 weeks old → let the rooster mate directly with the hen → collect the eggs and bring them incubate to create generation 1

* Monitored: egg weight and shape index, age at first egg laying, body

weight at first egg laying, egg productivity, laying rate, survival rate, FCR, feed intake/10 eggs

3.2.4 Evaluate selection effectiveness

Evaluation through G0, SG0 and G1 generation based on data of egg

yij = µ + Ai + ij

(yij: observed traits, µ: mean, Ai: effect of genotype, ij: random error)

- Analyse correlations between reproductive traits using Minitab 16.0 software

Chapter 4 RESULTS AND DISCUSSION 4.1 Identifiy polymorphisms in some potential genes related to the reproductive performance of Ac chickens

4.1.1 The results of DNA extraction from chicken feather samples

The extracted DNA samples were tested for concentration using a Nanodrop machine at 260 nm wavelength The extracted samples must have DNA concentrations greater than 50 ng/µl and have high purity (OD260nm/OD280nm values from 1.8-2.0) These samples did not qualify the standards would have their DNA extracted again from the feather sample Next, the DNA samples were checked for quality through electrophoresis

on a 1% agarose gel at a voltage of 80 V for 30 minutes The results showed that DNA samples obtained were pure and quality for further experiments

4.1.2 The amplify gene segments using PCR technique

Using PCR technique with specific primer pairs for each gene, the study successfully amplified the research gene fragment including: IGF1, INHA, NPY and VIPR1 For the DRD2 and PRL genes, using the Indel technique with specific primer pairs, the PCR and electrophoresis results determined the genotype of this polymorphism The gene fragment amplification results showed that the sizes were 399 bp (IGF1 gene), 409 bp (INHA gene), 248 bp (NPY gene), 434 bp (VIPR1 gene)

4.1.3 Identify mutations in the investigated genes

DRD2 Indel polymorphism: D allele (165 bp) and I allele (187 bp)

corresponding to 3 genotypes: II (187 bp), ID (187 bp and 165 bp) and DD (165 bp)

PRL Indel polymorphism: D allele (130 bp) and I allele (154 bp),

corresponding to 3 genotypes: II (154 bp), ID (154 bp and 130 bp) and DD (130 bp)

IGF1/PstI polymorphism: A allele (399 bp) and G allele (317 bp and 82

bp), corresponding to 3 genotypes: AA (399 bp), AG (399 bp, 317 bp and 82 bp) and GG (317 bp and 82 bp ) SNP analysis showed that the forward strand was a mutation that replaced nucleotide A (A allele) to nucleotide G (G allele) Similarly, in the reverse strand, a mutation has been identified that replaced nucleotide T (T allele) to nucleotide C (C allele)

INHA/PstI polymorphism: C allele (409 bp) and T allele (297 bp and 112

bp), corresponding to 3 genotypes: CC (409 bp), CT (409 bp, 297 bp and 112 bp) and TT (297 bp and 112 bp) SNP analysis showed that the forward strand was a mutation that replaced nucleotide T (T allele) to nucleotide C (C allele) at