VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------oOo------- GRADUATION THESIS TOPIC: “POLYMORPHISM OF PARTIAL D-LOOP MITOCHONDRIAL DNA REGION AND PHYLOGENETICS IN FIVE DUCK BREEDS IN VIETNAM” HA NOI - 2023 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------oOo------- GRADUATION THESIS TOPIC: “POLYMORPHISM OF PARTIAL D-LOOP MITOCHONDRIAL DNA REGION AND PHYLOGENETICS IN FIVE DUCK BREEDS IN VIETNAM” Student : Bui Binh An Code : 637401 Class : K63CNSHE Instructor : MSc. Nguyen Quoc Trung HA NOI - 2023 COMMITMENT The work contained in this thesis has not been previously submitted for a degree or diploma at any other undergraduate education institution. I hereby declare that all results of the thesis were done by me. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where reference is made.
Hanoi, February 10th, 2023 Bui Binh An i ACKNOWLEDGEMENT First of all, I would like to express my gratitude to the Board of Directors of Vietnam National University of Agriculture, the Dean of the Faculty and all the teachers in the Faculty of Biotechnology for creating a great learning environment and provided me the valuable knowledge throughout four and a half academic years. Additionally, I would like to thank Dr. Nguyen Hoang Thinh, Faculty of Animal Science. Nguyen Thi Thuy Hanh, Dr.
Dinh Truong Son, Faculty of Biotechnology supported my thesis. I also express my heartfelt respect, gratitude and sincere appreciation to Msc. Nguyen Quoc Trung, Department of Molecular Biology and Applied Biotechnology, Vietnam National University of Agriculture for his scholastic guidance, constructive criticisms and constant inspiration during the entire period of the study as well as the research, for your time to help me in writing and for provide me the opportunity to carry out my thesis in the laboratory of Department of Molecular Biology and Applied Biotechnology. I would like to thank all professors from Department of Molecular Biology and Applied Biotechnology, who encouraged me to enhance my knowledge and help me get the orientation of thesis.
Along with my supervisor, I would like to thank Cao Thi Thu Thuy and Nguyen Duc Hoang, two of my lab-mates, for all of their assistance throughout the experiment. I appreciated their enthusiastic guidance as I conducted my research and wrote this thesis. Last but not least, I want to express my thanks to my dear parents, family members, and friends for their support, blessings, inspiration, and unwavering sacrifice during the entirety of my academic career. With warm and regards! Hanoi, February 10th, 2023 Bui Binh An ii TABLE OF CONTENTS COMMITMENT.
iv LIST OF TABLE. v LIST OF FIGURE. vi LIST OF ABBREVIATIONS. Duck status in the world.
The importance of duck in Vietnam. Diversity of Vietnamese duck. Mitochondrial DNA as a tool for assessing the genetic diversity. General information of the five duck breeds in this study.
Co Lung duck. Na Tau duck. MATERIALS AND METHODS. Place and time of study.
Place of study. Time of study. PCR amplification and sequencing. RESULTS AND DISCUSSION.
Results of DNA extraction. Amplification of D-loop region and sequencing. Phylogenetic analysis and distribution of haplotypes of all 5 duck breeds in Vietnam. Polymorphism and genetic diversity.
Distribution of haplotypes of 84 samples belonging to 5 duck breeds. Phylogenetic analysis of 84 samples belonging to 5 duck breeds. Phylogenetic analysis and distribution of haplotypes of each breed. Phylogenetic analysis and distribution of haplotypes of Co breed.
Phylogenetic analysis and distribution of haplotypes of Co Lung breed. Phylogenetic analysis and distribution of haplotypes of Na Tau breed. Phylogenetic analysis and distribution of haplotypes of Troi breed. Phylogenetic analysis and distribution of haplotypes of Dom breed.
44 iv LIST OF TABLE Table 2. Price of duck products in Vietnam on 06/09/2022. Sample condition and total number of samples. Primers used for PCR amplification.
Diversity indices and neutrality test results based on the genetic analysis of mtDNA D-loop sequence fragments for the five duck populations studied*. Haplotype distribution table in all 5 duck breeds in Vietnam. Polymorphic analysis from partial sequencing of the mitochondrial D- loop gene segment of 5 duck breeds in Vietnam. Haplotype distribution table in Co duck.
Polymorphic analysis from partial sequencing of the mitochondrial D- loop gene segment of Co breed. Haplotype distribution table in Co Lung duck. Polymorphic analysis from partial sequencing of the mitochondrial D- loop gene segment of Co Lung breed. Haplotype distribution table in Na Tau duck.
Polymorphic analysis from partial sequencing of the mitochondrial D- loop gene segment of Na Tau breed. Haplotype distribution table in Troi duck. Polymorphic analysis from partial sequencing of the mitochondrial D- loop gene segment of Troi breed. Haplotype distribution table in Dom duck.
Polymorphic analysis from partial sequencing of the mitochondrial D- loop gene segment of Dom breed. 38 v LIST OF FIGURE Figures 2. Duck distribution map (FAO, 2010). Population genetics of the duck populations (Jiang et al.
Spot-billed ducks (Anas zonorhyncha). Domestic Cairina moschata. Distribution of duck in Vietnam. Distribution of ducks by region in Vietnam.
Co duck has heterogeneous feathers. Co Lung duck in Ba Thuoc district, Thanh Hoa province. Na Tau duck, also known as short-necked gourd duck. There are many varieties of Troi duck that have been introduced into Vietnam.
Total DNA of 11 Na Tau samples was separated from blood (left) and 11 male Co Lung samples were separated from wing feathers (right). Selected samples were compared with 100ng/nL Lamda DNA giving expected results from 600 to 700bp. Some partial D-loop region sequences. Unrooted Maximum Likelihood phylogenetic tree constructed using the D-loop sequences obtained for the domestic duck breeds in Vietnam considered in the present study.
A phylogenetic tree showing D-loop mtDNA related evolutionary relationships in Co ducks. A phylogenetic tree showing D-loop mtDNA related evolutionary relationships in Co Lung ducks. A phylogenetic tree showing D-loop mtDNA related evolutionary relationships in Na Tau ducks. A phylogenetic tree showing D-loop mtDNA related evolutionary relationships in Troi ducks.
A phylogenetic tree showing D-loop mtDNA related evolutionary relationships in Dom ducks .38 vii LIST OF ABBREVIATIONS Abbreviation Meaning bp base pair DNA Axit deoxyribonucleic MEGA Molecular Evolutionary Genetics Analysis EDTA Axit ethylen diamine tetra acetic PCR Polymerase Chain Reaction rpm revolutions per minute SDS Sodium dodecyl sulfate SNPs Single-nucleotide polymorphisms TE Tris-EDTA UV Ultraviolet µL microlit viii ABTRACT In this study, partial mitochondrial DNA (mtDNA) D-loop sequences of five duck breeds in Vietnam, including Na Tau, Co, Dom, Troi and Co Lung, were analyzed to determine sequence polymorphism and phylogeny among breeds. The D-loop regions of 84 duck individuals were amplified by PCR using primers L78-H774 and sequenced by Sanger method. Sequence alignments of 658 nucleotides at the 5' end were carried out and compared with other Anas spp. using ClustalW of MEGA11 software.
Phylogenetic investigation shows that the Vietnamese ducks have a relatively close genetic relationship with Anas zonorhyncha (GU246018.1) and Cairina moschata (GQ922096. SNPs detection among 5 breeds resulted in 41 nucleotides within D-loop region. This high diversity could be explained by the tendency of farmers to graze and breed freely for a long time. In the phylogenetic analysis, it suggested that five Vietnamese duck breeds have common maternal lineages with Anas zonorhyncha and Cairina moschata.
Keywords: D-loop sequence, maternal lineages, phylogenetic, sequence polymorphism, Vietnamese duck breeds. Introduction Duck is one of the most common domestic poultry in the world, along with chicken, guinea fowl, geese and turkeys. Compare to chicken, duck are more resistant to diseases, have longer egg-production life, eggs are heavier and fetch a better price. Duck meat is also well known for their delicacy and are consummed worldwide.
For many Asia contries, duck production plays an important part in the agricultural economy. The continent alone accounts for 82.6% of the total duck meat produced worldwide (Adzitey and Adzitey, 2011). In Vietnam, duck rearing and farming has been a consistant way for farmers to increase income and reduce poverty, needless to say, duck is essential to Vietnam agriculture. Come from the realization of the importance of duck, 5 Vietnamese duck breeds were selected: Co duck, Co Lung duck, Na Tau duck, Troi duck, Dom duck, to conduct the study: “ Polymorphism of partial D-Loop Mitochondrial DNA Region and Phylogenetics in five duck breeds in Vietnam” with the aim to find origin of the selected duck breeds, opening up a better understanding in genetic diferrences between breeds for serving breeding selection, genetic selection and conservation of rare duck breeds.
One of the main theories about the origin of domestic ducks is to be from the Mallard (Anas platyrhynchos) and Spot-billed Duck (Anas zonorynchos). They are both migratory birds, but may settle in areas with good food and habitat. They also have characteristics that allow them to be easily domesticated. In addition, Mallards may cross with Spot-billed Duck, and the hybrids appear in natural habitats (Jin et al.
In addition, there are also some studies showing that the Muscovy duck (Cairina moschata) is native to Mexico and South America (HM, 1994). They were domesticated and introduced to Asia by European colonists. And became the domestic duck for meat in many Asian 1 countries (Chen et al. Most domestic ducks are difficult to classify using genetic makers because they share these wild duck ancestors (Seo et al.
Mitochondrial DNA (mtDNA) has been used to gain molecular information to identify the origin of breeds. Proteins are not encoded in the D-loop region and this region evolves much faster than other regions of the mtDNA genome. Mitochondrial DNA and especially D-loop sequences have been used in phylogenetic analysis for the past 20 years. Several studies using mtDNA D- loop control region genetic variation have been conducted to elucidate the gene flow of domestic and wild ducks and their genetic relationships in several geographical regions (Sultana et al.
Objective Evaluation on Polymorphism of D-Loop mitochondrial DNA region and phylogenetics of some selected duck breeds. In order to breeding and conservation of local animals genetic resources. Specific objectives In order to achieve the objective of the study, following requirements need to be completed: Collecting blood and bloodfeathers for and extracting DNA from samples. Amplifying and sequencing the mtDNA D-Loop fragments.
Detecting DNA polymorphism in the sequences and identifying haplotypes in the sequences. Analyzing the phylogenetic and haplotype distribution of the five duck breeds in Vietnam. Duck status in the world Duck is the second largest poultry industry that provides income to farmers through egg and meat production. They are particularly numerous in Bangladesh, China and Southeast Asia but are also popular in Egypt, Nigeria and much of Europe, particularly in western France.
Breeds vary greatly depending on location and whether they are specialized for meat, egg or feather production or whether they are multipurpose (FAO). They are raised in a wide range of production systems, from small flocks kept in mixed farms to enormous flocks hauled in lorries to clear paddy fields after rice harvests. Duck distribution map (FAO, 2010) Wild Mallard (Anas boschas) are generally regarded as the ancestor of all breeds of domestic ducks, but there are also another hypothesis stated that domestic ducks orginated from hybrid between the mallard (Anas platyrhynchos) and the Eastern spot-billed duck (Anas p. The domestication process start about 4000 years ago in China and Southeast Asia, though the exact time is still unknown.
Population genetics of the duck populations (Jiang et al., 2021) Nowadays duck has become a important farmed poultry in many country, especially in developing countries thanks to high diseases resistent, high egg production, tasty and nutritious meat, good feed conversion ratio (FCR) and good finished product pricing. In 2021, total duck meat production is estimated at 5 million tons.