VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY THESIS TITLE: REVEALED THE ORIGIN OF THE MONG CAI PIG POPULATION IN QUANG NINH PROVINCE BY STUDYING THE SPECIFIC GENE SEQUENCES HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY THESIS TITLE: REVEALED THE ORIGIN OF THE MONG CAI PIG POPULATION IN QUANG NINH PROVINCE BY STUDYING THE SPECIFIC GENE SEQUENCES Student : Cao Thi Thu Thuy Course : 63 Department : Biotechnology Supervisor : MSc. Nguyen Quoc Trung HANOI – 2022 COMMITMENTS I hereby declare that all the data and results in this thesis are frank and have never been used in any reports yet. I also assure the information cited in my thesis indicated its origin, and all the help is appreciated. Hanoi, December 2022 Student Cao Thi Thu Thuy i ACKNOWLEDGEMENTS First and foremost, I have to express my appreciation to the directory of the Vietnam National University of Agriculture, the board of deans, and lectures in the Faculty of Biotechnology for creating a meaningful learning environment and providing me the precious knowledge throughout my academic years.
In terms of the thesis conducting process, many people helped me a lot to complete this study. Specifically, this is my dear supervisor, MSc. Nguyen Quoc Trung, who brings the most inspiration and aspiration for me to pursue bioscience. Without his support throughout these two years, I would not have gained as many honors as I have at present.
Besides, I cannot neglect the ardent support from my lab-mates in the Laboratory of Molecular Biology and Applied Biotechnology, there is no word to express my gratitude to the whole of you. In addition, I would like to sincerely thank the lecturers and students from the Faculty of Veterinary medicine, VNUA for their help during the sample-collecting trip. This study also gained financial support from the People’s Committee in Quang Ninh province, Vietnam (Conservation of Mong Cai pig genetic resources in Quang Ninh province, 2021-2023). Last but not least, I would like to thank my nearest and dearest, thank Timmy, who was there for me through all my ups and downs, until this point and my academic career way later.
Hanoi, December 2022 Student Cao Thi Thu Thuy ii TABLE OF CONTENTS COMMITMENTS. ii TABLE OF CONTENTS. iii LIST OF ABBREVIATIONS .v LISTS OF TABLES. vi LIST OF FIGURES.
Evolution and domestication of pigs. Molecular study of evolution and domestication of pigs. Studies based on mitochondrial genome. Studies based on nucleus DNA.
Studies based on MC1R sequence. Study on origin and phylogeny of Mong Cai pig breed based on DNA analysis. Study on nucleus DNA in Mong Cai pig. Study on Mong Cai pig phenotype.
Study on Mong Cai pig mitochondrial genome. MATERIALS AND METHODS. Sampling and DNA extraction method. Study on mtDNA methods.
Study on MC1R sequence. RESULTS AND DISCUSSION. Mitochondrial DNA sequence analysis. Phylogeny study on mtDNA D-loop sequence of Mong Cai pig and other 15 pig breeds.
Developing marker set to determine the maternal origin of Mong Cai pig. MC1R gene sequence analysis results. Sequencing and polymorphism detection. Diversity in the Mong Cai pig population.
The potential association between MC1R gene and coat color of Mong Cai pig. Origin of Mong Cai breed. CONCLUSIONS AND PROPOSALS .55 iv LIST OF ABBREVIATIONS Abbreviation Meaning α-MSH α-melanocyte-stimulating Hormone ASP/ ASIP Agouti-signaling Protein BC Before Christ cAMP Cyclic Adenosine Monophosphate D-loop Displacement loop ESR1 Estrogen Receptor 1 EDNRB Endothelin Receptor Type B GPIP Glucose Phosphate Isomerase-processed MC1R Melanocortin Receptor 1 MITF Melanocyte Inducing Transcription Factor MSA Multiple Sequence Alignment mtDNA Mitochondrial DNA PCR Polymerase Chain Reaction SNP Single Nucleotide Polymorphism TYR Tyrosinase TYRP1 Tyrosinase-Related Protein 1 UPGMA Unweighted Pair Group Method using arithmetic Averages VnP Vietnamese native Pig v LISTS OF TABLES Table 3.1: Genbank accession codes for mtDNA pig breeds and the site of D-Loop region 29 Table 3.2: PCR primers and conditions used for amplification of melanocortin 1 receptor (MC1R) gene 32 Table 4.1: The detected variation in the D-Loop region among Mong Cai pigs and 15 other pig breeds in Asia and Europe 34 Table 4.2: PCR primers and conditions used for amplification of fragment in mtDNA D-loop region.3: Expected bands amplified by two primer pairs designed based on mtDNA sequences 38 Table 4.4: Variations of the melanocortin 1 receptor (MC1R) gene and allelic frequency in the European pigs, Asian pigs, and Mong Cai pigs based on coat color phenotype.5: Haplotypes and polymorphism in the melanocortin 1 receptor (MC1R) gene among Mong Cai pigs, European pigs, and Asian pigs.6: Haplotypes distribution in data set of Mong Cai pigs, European pigs, and Asian pigs. 41 vi LIST OF FIGURES Figure 2.1: The domestication of the Eurasian or “Russian” boar resulted in hundreds of breeds of domestic pigs.2: Schematic overview of the history of the pig (Sus scrofa).3: Mitochondrial genome position (source: https://www.4: Pedigree depicting mitochondrial inheritance that shows inheritance is transmitted through the maternal line.5: Neighbor-joining trees of wild boar and domestic pig mitochondrial DNA haplotypes (Giuffra et al.6: Tong Cheng pig .7: Allele frequency of c.699 in ESR1 in Chinese pigs and European pigs (C.
Wang et al.8: Melanin synthesis-biochemical pathway (Robins, 1991; Searle, 1968) .9: The model of the genetics of coat color determination in mice.10: Phenotype and geographical origin of the 20 European pig breeds:. 11: Distribution of some typical Chinese local pig breeds. Wang et al.12: The unweighted pair-group method using arithmetic averages dendrograms based on Nei’s (1972) model (Nguyen Thi Dieu Thuy et al.13: Geographical localizations of eight pig populations analyzed in this study. (Pham Doan Lan et al.14: Genetic relationship among eight studied pig populations.
(Pham Doan Lan et al.15: Multiple correspondence analysis of the characteristics of Vietnamese native pig populations shows the distributions of the various Vietnamese native pig breeds. (Ishihara et al.16: The UPGMA phylogenetic tree of 162 pig complete mitochondrial DNA sequences. (Thuy Nhien Thi Tran et al.1: ML phylogenic tree based on the mtDNA D-loop in the dataset (2 Mong Cai pigs, 15 other pig breeds).2: PCR product of the partial mtDNA D-loop region amplified by Mtmc3 primer .3: PCR product of the partial mtDNA D-loop region amplified by Mtmc4 primer .4: The phenotypes of knotted black saddle (left) and seamless black saddle (right) Mong Cai pigs.5: ML phylogenic tree based on the coding sequence of MC1R gene in the dataset (26 Mong Cai pigs, 20 Asian and European pigs).43 viii ABSTRACT This study focuses on revealing the origin of Mong Cai pig population at present through research on the mitochondrial genome (mtDNA) D-loop sequence and melanocortin 1 receptor (MC1R). A total of 96 individuals were sampled at four swine farms in Quang Ninh province.
The mtDNA D-loop of the Mong Cai pig was aligned with other breeds to design DNA primers that could identify the maternal origin of this breed. Two primer pairs Mtmc3 and Mtmc4 were developed and amplification results showed a total of four band sizes representing four pig breeds for each primer pair. The coding sequences of MC1R gene of 26 selected Mong Cai individuals were sequenced and aligned with 20 other pig breeds from Europe and Asia. Twelve haplotypes were determined using 18 mutations in 46 individuals.
Mong Cai population was distributed into four haplotypes. The phylogenetic tree revealed that Mong Cai pigs belonged to the Asian clade while six Mong Cai individuals formed a distinct cluster. These six individuals carry the nt729G gene, which is prevalent in European pigs. This study provided strong evidence for the Asian origin of Mong Cai pig and showed the diversity in maternal lineage in Mong Cai population at present.
The crossbreeding trend resulted in variation in the typical black saddle coat color phenotype of Mong Cai pigs, and this is linked to the SNP c. INTRODUCTION The Mong Cai pig (Sus scrofa) is one of Vietnam’s unique indigenous pig breeds. It originated in the Mong Cai district of Quang Ninh province, adjacent to the Chinese border, where it was first documented. This pig breed is an important factor in the domestic economic growth in Northern Vietnam due to its better reproductive ability than international pig breeds while requiring inexpensive plant food sources.
With their high fertility characteristic, Mong Cai sows have been bred with western boars as Yorkshire, Landrace, Pietrain, etc. for F1 crossbred products and meat production since the turn of the 20 th century (Lemke et al. The body shape of the Mong Cai pigs is typical of the local pig breed: short body, short neck, small ears, small and short legs, swayback backline, and a drooping shaped belly (Ishihara et al. Especially, the black saddle coat that extends from the hips to the lower abdomen on Mong Cai pigs is distinctive (Duc Nguyen Van, 2006).
With the state of the introduction of exotic breeds as well as crossbreeding at present, many variations in phenotypes of Mong Cai pigs have occurred, obviously the black saddle color phenotype. Therefore, the origin of the Mong Cai population now is important to investigate. First of all, the study of genetic diversity based on mitochondrial DNA (mtDNA) as a maternal line has been a reportedly useful tool as a molecular marker. MtDNA is maternally inherited and experiences nucleotide changes faster than DNA (Brown et al., 1979), which makes mtDNA an ideal tool for studying population genetics (Bailey et al.
In addition, the diversity in black saddle coat color in Mong Cai pigs at the moment is obviously to be seen, which makes it less typical than that in the past. Besides, as a result of human domestic selection, the color of pigs’ coats varies by region. Several important genes affecting pig coat color phenotypes have been identified to date, including KIT (Fang et al., 2009; Johansson Moller et 1 al., 1996; Kijas et al., 1998; Marklund et al., 1998), MITF (Chen et al., 2016), ASIP (Drögemüller et al., 2006), TYRP1 (Ren et al., 2010), and EDNRB (Ai et al. Melanocortin 1 receptor (MC1R) determines dominant black, black- spotted, and red coat colors in both western and Chinese pigs (Fang et al., 2009; Kijas et al.
This is a G protein-coupled receptor that is predominantly expressed in melanocytes and plays a key role in melanogenesis by regulating the production of red/yellow pheomelanin and dark eumelanin. MC1R variants associated with the domestication of Asian and European pig breeds have been identified through characterization (Andersson, 2003). Most genetic studies on the evolution and domestication of pigs were carried out based on both mtDNA and nuclear genes to provide a comprehensive view. Thus, this study was carried out to examine the origin of the Mong Cai pig population in Quang Ninh province based on two specific nucleotide sequences, providing additional information on the genetic resource strategy of this indigenous pig breed in Vietnam.
Objectives: - Clarification of the origin of the Mong Cai pig population in Quang Ninh province based on the mitochondrial genome and MC1R gene Requirements: Study on the maternal lineage of Mong Cai pigs: • Aligning and comparing the mtDNA D-loop region between Mong Cai pigs and other pig breeds that were raised on the same swine farms in Quang Ninh province; • Designing a marker set based on mtDNA D-loop sequence to discriminate Mong Cai pig from other pig breeds; • Surveying the designed marker set by PCR and gel electrophoresis reactions. Study on MC1R gene and the black saddle coat color of Mong Cai pigs: • Amplifying and sequencing the MC1R gene in 26 Mong Cai pigs; 2 • Investigate the haplotype diversity in MC1R sequence among Mong Cai individuals; • Construct the phylogenetic tree to illustrate the relationship between the Mong Cai pig and other pig breeds in the world. Evolution and domestication of pigs The pig (Sus domesticus), also called swine, hog, or domestic pig when distinguishing from other members of the genus Sus, is an omnivorous, domesticated, even-toed, hoofed mammal.