INTRODUCTION
The Mong Cai pig (Sus scrofa) is a unique indigenous breed from Vietnam, specifically the Mong Cai district in Quang Ninh province near the Chinese border This breed plays a crucial role in the economic development of Northern Vietnam due to its superior reproductive capabilities compared to international breeds and its ability to thrive on low-cost plant-based diets Notably, Mong Cai sows, known for their high fertility, have been crossbred with western boars such as Yorkshire, Landrace, and Pietrain since the early 20th century to enhance meat production Characteristically, Mong Cai pigs exhibit a short body, short neck, small ears, short legs, a swayback backline, and a drooping belly.
The distinctive black saddle coat of Mong Cai pigs, which extends from the hips to the lower abdomen, is a notable feature (Duc Nguyen Van, 2006) Due to the introduction of exotic breeds and crossbreeding, there have been significant variations in the phenotypes of Mong Cai pigs, particularly in the black saddle color Consequently, investigating the origin of the Mong Cai population has become essential.
The analysis of genetic diversity through mitochondrial DNA (mtDNA) serves as an effective molecular marker due to its maternal inheritance and rapid nucleotide changes This characteristic makes mtDNA particularly valuable for investigating population genetics.
The current diversity in black saddle coat color among Mong Cai pigs is noticeably greater than in the past, making it less typical Additionally, human domestic selection has led to regional variations in pig coat colors Key genes influencing pig coat color phenotypes, such as KIT, have been identified in recent studies.
Research has identified several key genes related to coat color in pigs, including MITF, ASIP, TYRP1, and EDNRB The melanocortin 1 receptor (MC1R) is crucial for determining dominant coat colors such as black, black-spotted, and red in both western and Chinese pig breeds This G protein-coupled receptor, primarily found in melanocytes, regulates melanogenesis by influencing the production of pheomelanin and eumelanin Variants of MC1R linked to the domestication of Asian and European pig breeds have been characterized, highlighting its significance in pig coloration genetics.
Genetic research on pig evolution and domestication has primarily utilized both mtDNA and nuclear genes for a thorough understanding This study focuses on the Mong Cai pig population in Quang Ninh province, analyzing two specific nucleotide sequences to enhance knowledge of the genetic resources associated with this indigenous Vietnamese pig breed.
- Clarification of the origin of the Mong Cai pig population in Quang Ninh province based on the mitochondrial genome and MC1R gene
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;
• 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
LITERATURE REVIEW
Evolution and domestication of pigs
The pig (Sus domesticus), commonly known as swine or hog, is an omnivorous, domesticated mammal characterized by its even-toed, hoofed structure It is often regarded as a subspecies of the wild boar (Sus scrofa) or as a separate species altogether.
In 1859, Charles Darwin's groundbreaking work, “On the Origin of Species,” established the foundation of evolutionary biology by introducing the theory of natural selection and species diversity In his subsequent publication, “The Variation of Animals and Plants under Domestication,” Darwin identified two primary forms of domestic pigs: the European (Sus scrofa) and the Asian (Sus indicus), which he classified as distinct species due to significant phenotypic differences While it is believed that Asian pigs played a role in enhancing pig breeds during the 18th and early 19th centuries, the extent of their genetic contribution to various European pig breeds remains unclear.
Over the past 10,000 years, pigs have accompanied humans in their global spread, significantly contributing to societal development Extensive zooarchaeological evidence indicates that domestic pigs originated from the Eurasian wild boar, first identified around 8500 BC in the Near East The domestication process provided a safer and more efficient alternative to hunting wild boar, leading to the transformation of the Eurasian boar into the domestic pig (Sus scrofa domesticus) on farms Recent phylogeographic studies suggest that this domestication occurred multiple times across various regions, including Asia, Southeast Asia, India, Africa, and particularly Europe.
Figure 2.1: The domestication of the Eurasian or “Russian” boar resulted in hundreds of breeds of domestic pigs
(Source: Texas A&M, Natural Resources Institute)
Wild boars today pose significant threats to agriculture, water quality, native species, and habitats Human-driven artificial selection has favored traits like high yield, fertility, and intelligence, leading to the domestication of Eurasian boars into various breeds of pigs This transformation has resulted in a diverse array of domestic pigs, showcasing different sizes and colors The domestication process serves as a valuable model for exploring evolutionary questions, particularly regarding the interplay between molecular and morphological changes, and whether these changes stem from reduced natural selection pressures, genetic variation in wild ancestors, or new mutations arising post-domestication.
Figure 2.2: Schematic overview of the history of the pig (Sus scrofa)
Four main events are indicated: 1) Speciation of Sus in ISEA 2) Divergence between
European and Asian S scrofa.3) Independent domestication leading to separate domesticated clades in Europe and Asia.4) Hybridization between domesticated pigs from Asia and Europe (Bosse, 2020)
Molecular study of evolution and domestication of pigs
Recent studies have revealed a discrepancy between archaeological findings and molecular research regarding the domestication timeline of pigs DNA analysis indicates that modern European pigs are descended from European wild boars rather than Near Eastern species, raising questions about the possibility of independent domestication in Europe Additionally, classifying archaeological pig remains as wild, hybrid, or domestic has proven challenging, as skeletal shape alone is insufficient for accurate identification.
2.2.1 Studies based on mitochondrial genome
2.2.1.1 Overview of the function of mitochondrial genome and D-loop region in inheritance
Figure 2.3: Mitochondrial genome position (source: https://www.genome.gov/)
Mitochondrial DNA, a circular chromosome located within the mitochondria in the cytoplasm, plays a crucial role in cellular energy production Mitochondria are the organelles responsible for generating ATP, the primary energy source for cells and various metabolic processes Comprising only 0.1% of the total human genome, the mitochondrial genome is uniquely inherited from mother to offspring.
Genetic changes in mitochondrial DNA (mtDNA) can manifest as alterations in single genes or larger DNA rearrangements These changes can occur at any stage of life, leading to a condition known as heteroplasmy, where cells contain a mix of normal and altered mtDNA While some mtDNA alterations can be inherited through the egg, they are not passed on through sperm, resulting in maternal inheritance This pattern affects both males and females, but the transmission always occurs via the female line, as numerous mitochondria are transferred from ovarian cells to the oocyte, impacting all offspring.
Women affected by the disease have a risk of passing it on to their offspring, which is determined by the proportion of mutant mitochondria they inherit In contrast, affected males do not transmit their mitochondria to their children, ensuring that their offspring remain unaffected.
Figure 2.4: Pedigree depicting mitochondrial inheritance that shows inheritance is transmitted through the maternal line
(Source: NHS National Genetics and Genomics Education Centre)
Furthermore, mtDNA is known for having high acquired mutation rates which are 10 times higher than that of nuclear genomic DNA (Brown et al.,
Mitochondrial DNA (mtDNA) evolves rapidly, making it an effective tool for investigating population genetics and evolutionary processes It helps uncover genetic relationships within closely related populations and identifies instances of population bottlenecks.
The mitochondrial DNA (mtDNA) in pigs features a closed circular structure that includes a complete nucleotide sequence and a non-coding region known as the displacement loop (D-loop), which spans over 1000 base pairs This D-loop region serves as a promoter for both the heavy and light strands of mtDNA, housing crucial transcription and replication factors Recent research has demonstrated the potential of the mtDNA D-loop region in studies focused on maternal lineage and genetic linkage (Purwantini et al., 2013; Tsai et al., 2009).
2.2.1.2 Study of evolution and domestication of pigs based on mitochondrial genome
Research indicates that domesticated pigs with Middle Eastern haplotypes emerged in Northern Europe around 4500 BC (Krause-Kyora et al., 2013) DNA analysis reveals that the Near Eastern ancestors of European domestic pigs largely vanished approximately 3000 years ago due to interbreeding with European wild boar (Frantz et al., 2019) Farmers reported that wild boars frequently infiltrated domestic herds, mating with females and introducing their genes Notably, studies suggest that pigs were not independently domesticated in Europe; instead, herds that roamed freely interbred with wild boar, potentially as an accidental occurrence or a deliberate choice by farmers seeking to enhance their livestock Numerous mtDNA studies have explored the domestication and genetic divergence between European and Asian pigs, with early research by Okumura et al (1996) and Watanabe et al (1986) highlighting genetic differences but lacking a clear timeline for divergence.
In 1999, Giuffa et al made significant advancements in pig genetics, highlighting the importance of genetic diversity conservation in this livestock species Their research provided a comprehensive molecular analysis of domestic pig ancestry by examining wild and domestic pigs from Asia and Europe This analysis included the complete coding sequence of the mtDNA cytochrome B gene and a portion of the mtDNA hypervariable control region The study revealed that domestication of pigs occurred in both Europe and Asia, independent of wild boar subspecies Phylogenetic analysis of the hypervariable D-loop region identified three distinct mtDNA clades: one from Asia and two from Europe.
Recent studies have identified 10 suspected European-origin pigs belonging to the European clade 1, which includes most European wild boar, Israeli wild boar, and many domestic pigs Phylogenetic analysis of cytB sequences revealed a statistically supported Asian clade, while the presence of two distinct European clades lacked statistical significance The divergence of ancestral forms is estimated to have occurred around 500,000 years ago, predating domestication by 9,000 years Historical records suggest that Asian pigs were introduced to Europe in the 18th and early 19th centuries, and molecular evidence supports the hybrid origin of several major "European" pig breeds.
A 2013 study by Krause-Kyora et al revealed that Mesolithic Ertebứlle hunter-gatherers in Northern Germany acquired domestic pigs with diverse sizes and coat colors, possessing both Middle Eastern and European mtDNA ancestry This research demonstrated that these hunter-gatherers not only owned domestic pigs similar to their agricultural neighbors but also that these animals existed in the region approximately 500 years earlier than previously documented.
2.2.2 Studies based on nucleus DNA
In 1999, a study by Giuffra et al analyzed three nuclear genes—melanocortin receptor 1 (MC1R), tyrosinase (TYR), and a glucose phosphate isomerase pseudogene (GPIP)—to explore the genetic divergence between European and Asian pigs The research revealed significant differences in allele frequencies between the two regions, with a notable trend showing greater variation between continents than between wild and domestic pigs within the same continent This evidence supported the theory of independent domestication of pigs in Europe and Asia.
Figure 2.5: Neighbor-joining trees of wild boar and domestic pig mitochondrial DNA haplotypes (Giuffra et al., 2000)
The study analyzed 440 bp of the control region and 1140 bp of the cytB gene from various wild boar samples, including European wild boar (EWB) from Poland and Italy, Israeli wild boar (IWB), and Asian wild boar (AWB) from Ryukyu Island and Japan Additionally, domestic pig breeds were represented by Swedish Landrace (L), Hampshire (H), and Mangalica (Ma).
LW, Large White; Me, Meishan; D, Duroc; Cook, Cook Island
In 2015, C Wang et al studied the genetic basis underlying the morphological and behavioral adaptations of Chinese indigenous pigs The authors did a genome-wide evaluation to screen 196 regions with selective
The study of 12 sweep signals in Tong Cheng pigs, a native Chinese breed, reveals genes linked to lipid metabolism, melanocyte differentiation, and neural development, reflecting the breed's evolutionary changes A significant synonymous SNP, c.669T>C, in the Oestrogen Receptor 1 (ESR1) gene, associated with a key quantitative trait locus for litter size, shows marked differences in allele frequency between Tong Cheng pigs and wild boars The C allele at this locus is prevalent in most Chinese pig populations, indicating strong selection pressure Interestingly, this allele is found in the Large White breed but absent in other European pigs, likely due to historical introgression of Asian pigs into Europe during the 18th and 19th centuries.
The Microphthalmia-associated Transcription Factor (MITF) and Endothelin B receptor (EDNRB) genes are linked to the two-end black coat color phenotype in Tong Cheng pigs Research using SNP microarrays across five Chinese white-spotted pig breeds revealed a consistent genetic signature at these loci, indicating their role in coat color variation among Chinese pigs Through extensive parallel sequencing, candidate positions were identified that respond to both artificial and environmental selections during the domestication of Chinese pigs This study by C Wang et al (2015) provides essential insights for future research on the evolutionary adaptation of Chinese pigs.
Figure 2.7: Allele frequency of c.699 in ESR1 in Chinese pigs and European pigs (C
2.2.3 Studies based on MC1R sequence
2.2.3.1 Overview of melanocortin receptor 1 (MC1R) gene
The domestication of pigs and the artificial selective pressures they have faced have led to gradual changes in coat color across different regions and breeds Additionally, the MC1R gene is crucial in regulating mammalian coat color, prompting extensive research on this gene in pigs to better understand the domestication process.
Study on origin and phylogeny of Mong Cai pig breed based on DNA
2.3.1 Study on nucleus DNA in Mong Cai pig
Nguyen Thi Dieu Thuy et al (2006) conducted a study on the autochthonous pig breeds of Vietnam, analyzing a total of 343 individuals from five indigenous breeds (Muong Khuong, Co, Meo, Tap Na, and Mong Cai) alongside two exotic breeds (Landrace and Yorkshire) and three European commercial breeds (German Landrace, Piétrain, and Large White), as well as the Chinese Meishan and European Wild Boar The research involved genotyping each individual to compare the genetic characteristics of these breeds.
The dendrogram illustrated in Figure 2.12 reveals a clear division of the 12 pig breeds into two distinct branches: one comprising all European breeds along with the European Wild Boar, and the other consisting of Asian breeds This finding aligns with earlier research conducted by Kim et al., which utilized mtDNA polymorphisms from various Asian and European breeds, including the Wild Boar.
In 2002, it was found that Mong Cai pigs represent a unique branch within the Asian pig cluster, establishing a clear genetic topology among both European and Vietnamese indigenous breeds The genetic distances observed align with their geographical distribution, highlighting that Vietnamese indigenous pig breeds, including the Mong Cai breed, possess significant genetic diversity compared to European breeds This finding underscores the importance of bio-conservation efforts for these pig breeds.
The unweighted pair-group method using arithmetic averages dendrograms, based on Nei’s model, illustrates the relationships among various pig breeds This analysis includes individual breeds such as Co, Landrace Germany, Landrace Vietnam, Large White, Mong Cai, and Meo, as well as grouped breeds.
MK = Muong Khuong; MS = Meishan; PI = Pie´train; TN = Tap Na; WB = European Wild Boar;
YV refers to Yorkshire Vietnam, while VB encompasses the Vietnamese breeds Muong Khuong, Co, Meo, and Tap Na XB denotes the exotic breeds Landrace and Yorkshire found in Vietnam, and EB represents the European breeds Landrace, Piétrain, and Large White in Germany.
Since 2013, Pham Doan Lan and colleagues have studied the genetic diversity and structure of five native pig populations (Ha Lang, Muong Te, Mong Cai, Lung, and Lung Pu), two wild boar populations (Vietnamese and Thai), and the Yorkshire breed Utilizing 16 FAO/ISAG-recommended microsatellite markers, they analyzed a total of 236 samples The resulting neighboring-joining dendrogram, based on Nei’s standard genetic distance, categorized the eight populations into four groups: Yorkshire, the two wild boar populations, the Mong Cai population, and a group comprising four other indigenous breeds Their phylogenetic tree indicated that the Mong Cai, the two wild populations, and the Yorkshire breed are closely related.
The Mong Cai pigs exhibited the greatest genetic distance from four other native pig populations, indicating that these indigenous groups likely have at least two distinct founders This separation aligns with the independent domestication of Asian and European pigs from local wild boars Consequently, the Yorkshire breed's genetic divergence from the five indigenous populations, particularly its higher distance from Mong Cai pigs, is evident Additionally, the study highlighted a low allelic diversity in the Mong Cai pig population at that time.
Figure 2.13: Geographical localizations of eight pig populations analyzed in this study (Pham Doan Lan et al., 2014)
Cao Bang province is known for its Ha Lang pigs (HL), while Lai Chau province is home to Muong Te pigs (MT) Phu Tho province features Lung pigs (LU), and Ha Giang Province is recognized for its Lung Pu pigs (LP) Quang Ninh province boasts Mong Cai pigs (MC), and Yen Bai province is famous for Vietnamese wild pigs (VW) Additionally, Ha Tay province is associated with Thai wild pigs (TW), and Hanoi city is known for Yorkshire pigs (YS).
The genetic relationships among eight pig populations were analyzed, revealing distinct connections among Ha Lang (HL), Muong Te (MT), Lung (LU), Lung Pu (LP), Mong Cai (MC), Vietnamese wild pigs (VW), Thai wild pigs (TW), and Yorkshire pigs (YS) (Pham Doan Lan et al., 2014).
2.3.2 Study on Mong Cai pig phenotype
Ishihara et al., 2020 conducted a field survey of Vietnamese native pig
The study analyzed the interrelationships among 15 phenotypic markers across 32 VnP populations from 22 provinces in Vietnam, utilizing data from 1,918 individuals A relational database schema was developed for effective conservation and management Multiple correspondence analyses revealed that most populations were closely related, with the Mong Cai, O Lam, and Chu Prong pigs distinctly separated Notably, the Mong Cai pig was classified as “other” due to its unique characteristics, including a thicker chin and broader forehead, leading to its classification as a separate group from the other VnP populations.
Figure 2.15: Multiple correspondence analysis of the characteristics of Vietnamese native pig populations shows the distributions of the various Vietnamese native pig breeds (Ishihara et al., 2020)
O Lam (AG), Chu Prong (CP), Mong Cai (MC), Hung (HU), Huong (HUO), Ha Lang (HL)
2.3.3 Study on Mong Cai pig mitochondrial genome
In 2016, Thuy Nhien Thi Tran et al successfully sequenced the complete mitochondrial genome of the Mong Cai pig, providing valuable insights into the genetic resources of this breed Their analysis revealed a total genome length of 16,632 base pairs, which includes a non-coding control region (D-loop region), two ribosomal RNA genes, 13 protein-coding genes, and 22 transfer RNA genes.
The authors developed a phylogenetic tree for 162 individuals utilizing the unweighted pairwise group method with arithmetic means (UPGMA) and a Bayesian approach This analysis revealed two primary groups: one for wild boars and another for local and commercial pig breeds from Europe and Asia Indigenous pigs were categorized based on their geographic locations, while several commercial breeds, including Yorkshire, Berkshire, and Large White, were found intermixed within the Asian group, indicating a connection to Asian genetics.
The Mong Cai pig, part of the Asian group, is closely related to the miniature Bama pig from Guangxi province in southern China This close phylogenetic relationship is attributed to their geographical proximity and significant human interaction between North Vietnam and Guangxi province.
Figure 2.16: The UPGMA phylogenetic tree of 162 pig complete mitochondrial DNA sequences (Thuy Nhien Thi Tran et al., 2016)
The two clades represent European and Asian wild boars, local and commercial pig breeds, respectively The Mong Cai pig is indicated with a triangle
A study by Anh Tuan Bui et al (2018) investigated the genetic diversity of mtDNA and the evolutionary origins of six indigenous Vietnamese pig breeds: I, Mong Cai, Muong Khuong, Muong Lay, Huong, and Ha Lang The researchers sequenced the complete 16,556 bp Mong Cai genome using 30 pairs of markers, which was subsequently submitted to Genbank (accession code KU556691) They reconstructed two phylogenetic trees of 33 domestic pig and wild boar breeds from Asian and European clades, revealing that all six Vietnamese breeds are part of the Asian group and closely related to southern Chinese pig breeds and the Chinese Yellow River Valley Notably, Mong Cai, Huong, and Ha Lang pigs share a subbranch with close genetic distances, reflecting similarities in geographic distribution and morphological traits This study's publication of mitochondrial genome sequences significantly contributes to understanding the relationships among native Vietnamese pig breeds and aids in selecting suitable breeds for local pig farming.
MATERIALS AND METHODS
Materials
This study involved Mong Cai pigs sourced from four farms in Quang Ninh province: Thien Thuan Tuong Mining JSC, Quang Ninh Nong Lam Ngu Development MTV Co Ltd, An Loc Organic Agriculture Cooperative, and Van Thanh Phat Cooperative The sampled pigs varied in age from under 6 months to 36 months and included sows, boars, gilts, and piglets.
A total of 96 individuals were selected for the study, comprising 91 Mong Cai pigs, one Hampshire pig, one Meishan pig, and three Huong pigs The phenotypes of these individuals were documented based on the farmers' knowledge of their pedigree information.
Specifically, 26 individuals in this population who had the typical Mong Cai black saddle coat color were selected for study in MC1R gene
The needed chemicals, equipment, and machines were provided by the Laboratory of Molecular Biology & Applied Biotechnology
Figure 3.1 Pig individuals were collected from swine farms in Quang Ninh province
(a) Meishan pig; (b) Hampshire pig; (c) Huong pig
Methods
3.2.1 Sampling and DNA extraction method
A total of 96 ear tissue samples were collected and preserved in 75% ethanol at 4°C Each sample was tagged with a unique four-digit code, indicating the swine farm order followed by a classification.
29 pigs: a male was numbered 1, a female was numbered 2, a sow was numbered 3, and a boar was numbered 4 The last three digits were the sampling order in its farm
Genomic DNA was successfully extracted from ear tissues using the TopPURE ® GENOMIC DNA EXTRACTION KIT (ABT Co Ltd) following the provided protocol The quality of the extracted DNA was confirmed through gel electrophoresis, which was performed at 250V for 20 minutes on a 1% agarose gel.
3.2.2.1 Polymorphism screening and phylogeny analysis based on mtDNA D-loop region
Table 3.1: Genbank accession codes for mtDNA pig breeds and the site of D-Loop region
1 Mong Cai1 KU556691 1 1194 Tran et al., 2016
2 Mong Cai2 KX147100 1 1275 Vo, T.T.B et al., 2017
3 Ha Lang KY800118 1 1285 Vo, T.T.B et al., 2017
4 Huong KY964306 1 1315 Vo, T.T.B et al., 2017
6 Muong Khuong KY432578 1 1244 Vo, T.T.B et al., 2017
7 Muong Lay KX147101 1 1304 Vo, T.T.B et al., 2017
8 Meishan KM998967 1 1274 Ram, M and Chen, B., 2015
9 Chinese wild boar KP681245 1 1254 Yu, P., 2015
11 Rong Chang KM044239 1 1274 Wang, L Y et al., 2014
12 Pietrain KC469587 1 1274 Yu, G et al., 2013
13 Berkshire AY574045 1 1122 Cho, I C et al., 2016
14 Hampshire AY574046 1 1105 Cho, I C et al., 2016
15 Duroc AY337045 1 1145 Cho, I C et al., 2016
16 Yorkshire KF752550 1 1274 Xu, D et al., 2014
17 Landrace NC000845 1 1175 Lin, C S et al., 2021
A comprehensive dataset was created, encompassing the mtDNA D-loop region of Mong Cai pigs alongside 15 other pig breeds, with all sequences sourced from GenBank as detailed in Table 3.1 The selection of these 15 breeds was based on their close genetic relationship to Mong Cai pigs, their shared farming environment in Quang Ninh province, and the clear documentation of their D-loop sequences in GenBank Multiple Sequences Alignment (MSA) was performed using MEGAv11 software, while the analysis of polymorphic sites was conducted with DnaSP version 6.12.03 Additionally, phylogenetic analysis was carried out using MEGAv11, resulting in the construction of a Maximum Likelihood tree based on the Hasegawa-Kishino-Yano model with 300 bootstrap replicates.
A study conducted on swine farms in Quang Ninh province involved the analysis of a mtDNA D-loop dataset for marker design, which included 5 mtDNA D-loop sequences from 4 pig breeds: Mong Cai1 (KU556691.1), Mong Cai2 (KX147100), Hampshire (AY574046), Huong (HY964306), and Meishan (KM998967) Polymorphisms and Indel regions among these sequences were identified using Bioedit software version 7.2.5 The molecular markers were designed based on the variations in D-loop sequences, with the primary goal of amplifying different band sizes across the pig breeds.
The D-loop sequences of Mong Cai pigs were analyzed using the Primer-BLAST tool from NCBI to generate a set of primers These primers were screened for their ability to amplify indel regions, resulting in distinct amplicons for each pig breed Subsequently, the candidate markers underwent quality assessment using the Oligo analyzer (IDT).
31 checked the parameters as: The difference between melting temperatures (Tm) of the Forward and Reverse primers was less than 5°C (as less as possible); the
The GC content ranged from 40% to 60% For self-dimers, hairpins, and heterodimers, the Delta G value must be greater than -9.0 kcal/mole, as positive values suggest that the secondary structure will not form Additionally, there should be minimal 3' complementarity between the two primers to avoid the formation of primer dimers.
Four potential markers were selected and produced by Phusa Biochem Co., Ltd (Table 4.2)
The mtDNA D-loop region was amplified using four specific primer pairs: Mtmc1, Mtmc2, Mtmc3, and Mtmc4, as detailed in Table 4.2, through the Polymerase Chain Reaction (PCR) method Each PCR reaction was performed in a total volume of 40 µL, consisting of 4 µL of template DNA (approximately 25-50 ng/µL), 20 µL of 2X Mytaq Mix PCR Mastermix from Meridian Bioscience, 4 µL of each primer at a concentration of 10 pmol, and nuclease-free water.
PCR amplifications were conducted using a protocol that included an initial denaturation step at 95°C for 3 minutes, followed by 35 amplification cycles consisting of 15 seconds at 95°C, annealing at the melting temperature (Tm) for 15 seconds, and extension at 72°C for 20 seconds The final extension was performed at 72°C for 2 minutes The resulting products were visualized through electrophoresis on a 2% agarose gel stained with GelRed.
The entire coding sequence of the MC1R gene, including introns and both the 5'- and 3'-untranslated regions (UTRs), was amplified using two primer pairs as described by Wu et al (2017) Each PCR reaction was performed in a total volume of 40 µL, incorporating 4 µL of template DNA, which is approximately 25–.
The PCR amplification was performed using a mixture consisting of 50 ng of DNA, 20 µL of 2X Mytaq Mix PCR Mastermix (Meridian Bioscience), 4 µL of primers (10 pmol each), and nuclease-free water The amplification process involved an initial denaturation at 95°C for 3 minutes, followed by 35 cycles of 15 seconds at 95°C, annealing at the melting temperature (Tm) for 15 seconds, and extension at 72°C for 20 seconds, concluding with a final extension at 72°C for 2 minutes The resulting products were analyzed through electrophoresis on a 2% agarose gel stained with GelRed and subsequently purified using the TopPURE® PCR/GEL DNA PURIFICATION KIT (ABT Co Ltd) according to the manufacturer's protocol The purified PCR products were then sent for sequencing via 1st BASE service.
Figure 3.2: Model of melanocortin 1 receptor (MC1R) gene amplificated by the 2 markers MC1R1 and MC1R2
Table 3.2: PCR primers and conditions used for amplification of melanocortin 1 receptor (MC1R) gene
Primer sequence Primer binding region
MC1R full-length sequences were assembledfrom sequences amplified by the two markers MC1R1 and MC1R2 using the CAP3 sequence assembly program (Pôle Rhône-Alpes de Bioinformatique) Subsequently, these
A total of 33 sequences were aligned with the published Sus scrofa MC1R sequence (AF326520) in GenBank to pinpoint the coding region This analysis resulted in an MC1R coding sequence dataset, which includes sequences from 26 Mong Cai pigs and 20 other widely recognized pig breeds from Europe and Asia The MC1R sequences for these additional breeds were sourced from GenBank.
The position and number of polymorphic sites, as well as their corresponding haplotypes, were analyzed with DnaSP version 6.12.03
The MEGAv11 software facilitated the construction of a phylogenetic tree for 46 sequences A Maximum Likelihood tree was generated using the Hasegawa–Kishino–Yano model, with 1000 bootstrap replications performed through MEGA software.
RESULTS AND DISCUSSION
Mitochondrial DNA sequence analysis
4.1.1 Phylogeny study on mtDNA D-loop sequence of Mong Cai pig and other 15 pig breeds
The study identified 46 polymorphisms in the D-loop region sequence of Mong Cai pigs and 15 other European and Asian pig breeds, highlighting distinct mtDNA D-loop genotypes across breeds Notably, two Mong Cai sequences exhibited differences in polymorphism and sequence length Previous research by Thuy Nhien Thi Tran et al (2016) collected samples from Son La province, which is not the native region of the Mong Cai pig, while Anh Tuan Bui et al followed guidelines from the Vietnam National Institute of Animal Science for random sampling It is important to note that boars inherit both male and female mitochondrial genomes, whereas sows possess only a female genome Due to the lack of clarity regarding the accuracy of the results, both reference sequences were utilized for designing molecular markers.
Table 4.1: The detected variation in the D-Loop region among Mong Cai pigs and 15 other pig breeds in Asia and Europe
The article discusses various pig breeds and locations, including the D-loop region, Mong Cai, Ha Long, and Muong Lay It highlights the Chinese wild boar, Meishan, Rongchang, and Qingyu breeds, as well as the Yorkshire, Berkshire, Duroc, and Hampshire breeds These breeds are significant in the context of pig farming and breeding practices.
4.1.1.2 Phylogenetic tree constructed based on D-loop region
A phylogenetic tree was constructed based on the D-loop region of two Mong Cai pigs and other 15 pig breeds in Europe and Asia (Figure 4.1) The
The evolutionary history of pigs was analyzed using the Maximum Likelihood method and the Hasegawa-Kishino-Yano model, revealing that Mong Cai pigs are genetically close to Huong and Ha Lang pigs, which share similar morphological traits and geographical origins in Cao Bang province, near Quang Ninh and the China border These native pigs form a distinct cluster separate from other breeds, while four European breeds (Landrace, Pietrain, Duroc, Hampshire) are grouped together The remaining breeds create separate clusters of Asian, European, and Vietnamese native pigs, showing a closer genetic relationship with the Mong Cai group than with European breeds, supporting previous findings regarding the Asian origin and genetic ties of Mong Cai pigs to Chinese breeds.
Figure 4.1: ML phylogenic tree based on the mtDNA D-loop in the dataset (2 Mong Cai pigs, 15 other pig breeds)
Only values higher than 40% are shown
4.1.2 Developing marker set to determine the maternal origin of Mong Cai pig
Four molecular markers were developed from the indel region of the Mong Cai pig, alongside samples from Huong, Hampshire, and Meishan pigs raised on the same farms, as well as two previous mtDNA sequences Specifically, markers Mtmc1 and Mtmc2 were created based on the aligned results of Mong Cai 2 (KX147100), while Mtmc3 and Mtmc4 were derived from the aligned results of Mong Cai 1 (KU556691).
Table 4.2: PCR primers and conditions used for amplification of fragment in mtDNA D- loop region
Primer binding region (In MC pig)
After conducting a survey, only the marker pairs Mtmc3 and Mtmc4 showed potential for further analysis Markers Mtmc1 and Mtmc2 failed to amplify the target region in the D-loop for some Mong Cai individuals, although DNA bands were still visible in several others This discrepancy may be attributed to crossbreeding among these individuals, which could have introduced mutations at the reverse and/or forward points.
Further research is needed to validate the hypothesis regarding the binding of 38 primers In the study, markers Mtmc3 and Mtmc4 were consistently utilized in PCR reactions across all 96 collected samples, with the target band sizes for four pig breeds detailed in Table 4.3.
Table 4.3: Expected bands amplified by two primer pairs designed based on mtDNA sequences
Pig breed Mtmc3 Mtmc4 Mong Cai 875 bp 885 bp
The Mong Cai population exhibits a diverse range of fragment sizes, varying from approximately 700bp to over 1000bp Analysis of the amplified results from each primer revealed four distinct band sizes, corresponding to the pig breeds Mong Cai, Huong, Hampshire, and Meishan Notably, several Mong Cai pigs share homologous alleles with these three breeds.
The Mong Cai pig population exhibits genetic diversity in the mtDNA D-loop region, demonstrating polymorphism through maternal inheritance Each primer pair identified a group with the same band size, classifying the Mong Cai breed alongside three other breeds.
The observed group sizes varied from the anticipated sizes derived from the D-loop nucleotide sequence, indicating that crossbreeding trends have influenced the mitochondrial genome of Mong Cai progeny.
MC1R gene sequence analysis results
The sequences of each with approximately 1600 bp comprising the coding region and both 5’ and 3’UTR of the MC1R gene were amplified successfully in
26 Mong Cai individuals The complete coding sequence of 26 Mong Cai pigs had a length of 963 bp and encoded 321 amino acids
The sequence alignment revealed 18 polymorphic sites, including two insertion-deletion sites (indel), four synonymous single nucleotide polymorphisms (SNPs), and 12 non-synonymous SNPs (Table 4.4) In addition,
Recent studies have identified 11 genetic variants (c.6T>C, c.51A>G, c.61A>G, c.68_69insCC, c.283A>G, c.305C>T, c.363C>T, c.364G>A, c.370G>A, c.491C>T, and c.729A>G) associated with coat color in pigs (Li et al., 2010; Liu et al., 2016; Lu et al., 2017; Wu et al., 2017) In the Mong Cai population, five specific nucleotide substitutions (c.28C>G, c.152T>G, c.674C>A, c.712T>A, and c.729A>G) were discovered Notably, the 2-bp indel at codon 23 (c.68_69insCC) is a frameshift mutation linked to black and white pig coat color, as highlighted in recent research (Kijas et al., 1998; Klungland et al., 1995; Robbins et al., 1993).
The Mong Cai population analyzed in this study does not display the expected trait Additionally, the chi-square test results (df = 3, P-value < 0.05) indicate a significant association between all polymorphic sites and the coat color trait.
Figure 4.2: PCR product of the partial mtDNA D-loop region amplified by Mtmc3 primer
Figure 4.3: PCR product of the partial mtDNA D-loop region amplified by Mtmc4 primer
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
Table 4.5: Haplotypes and polymorphism in the melanocortin 1 receptor (MC1R) gene among Mong Cai pigs, European pigs, and Asian pigs
Table 4.6: Haplotypes distribution in data set of Mong Cai pigs, European pigs, and Asian pigs
Bamei Xiang Jinhua Tong-Cheng Shengxian-spotted Rong-Chang Qingyu 010 080 12006 12007 12008 13014 13015 32016 32029 41021
Hap2 4 Pietrain Berkshire Large-White Portugal-wild-boar
A total of twelve haplotypes have been identified from 18 mutations across 46 pigs, as detailed in Tables 4.5 and 4.6 Notably, Hap2 to Hap7 represent European original pig breeds characterized by dominant white and two-end black coat colors In contrast, Hap1 includes seven Chinese pig breeds exhibiting diverse coat color phenotypes, along with 18 Mong Cai individuals, while the remaining six Mong Cai members are classified under the Hap12 group.
Figure 4.4: The phenotypes of knotted black saddle (left) and seamless black saddle
The white arrow highlights the knotted position found exclusively in the Mong Cai knotted black saddle coat group, while the red curve indicates the seamless line characteristic of the Mong Cai seamless black saddle coat group.
The nt729 (c.729A>G) mutation, a synonymous variant, distinguishes Hap1 from Hap12 Remarkably, all European pig breeds and six Mong Cai pigs are homozygous for the 729GG allele, while the A allele is present at site 729 in Hap1 and from Hap8 to Hap11.
The constructed phylogenetic tree revealed that European and Asian pig breeds were separated into two distinct clusters, with the Mong Cai population
43 belonging to the Asian clade (Figure 4.5) Interestingly, the six Mong Cai individuals with the Hap12 genotype formed a distinct cluster within the Asian population
The findings reinforce the conclusion that Mong Cai pigs originate from Asia, as supported by previous studies (Anh Tuan Bui et al., 2018; Ishihara et al., 2020) Additionally, the variation in the MC1R gene serves to differentiate between European and Asian pig breeds (Giuffra et al., 2000) However, it is important to note that the other six members of Hap12 may possess a distinct ancestry compared to the overall population.
Figure 4.5: ML phylogenic tree based on the coding sequence of MC1R gene in the dataset (26 Mong Cai pigs, 20 Asian and European pigs)
Only values higher than 50% are shown
All novel sequences were submitted to GenBank with references numbered OP142697 – OP142700.
Discussion
4.3.1 Diversity in the Mong Cai pig population
The mtDNA study of the Mong Cai population in Quang Ninh province reveals that maternal inheritance is significantly influenced by crossbreeding trends The primary maternal origins identified include Huong, Meishan, and Hampshire pigs, which are commonly used as sows and boars in local swine farms This influence is also evident in the phenotypes, particularly the coat color of the piglets Additionally, there is a possibility of other ancestral origins, such as Pietrain and Landrace, which have been prevalent among Vietnamese farmers since the 20th century.
This research identified five substitutions in the MC1R gene of Mong Cai pigs, which is fewer than the nine, seven, and six substitutions found in Min, Qingyu, and Tibetan pigs, respectively (Lu et al., 2017; Mao et al., 2010; Wu et al., 2017) Notably, the substitution c.729A>G was frequently observed in both this study and previous research Additionally, this study revealed five novel substitutions in Mong Cai pigs, comprising four non-synonymous and one synonymous SNP: c.28C>G, c.152T>G, c.674C>A, c.712T>A, and c.729A>G.
Research on the MC1R gene has identified the c.152T>G mutation, an SNP responsible for the p.Leu50Arg substitution, exclusively in the Hap10 population This population includes individual code 12009, a pig characterized by a knotted black saddle that is uneven on both sides, which has been crossed with imported male breeds.
45 has resulted in numerous variations in the saddle-shaped coat areas of Mong Cai pigs, as illustrated by the coat phenotype of Mong Cai pig progeny
The remaining three SNPs identified in the Mong Cai population belong to Hap11, which consists of a single individual with the code 13013 In the
MC1R gene of pigs, these SNPs represent novel substitutions Individual 13013 was a sow with a curved, unbroken head and an extremely short black saddle compared with other Mong Cai individuals
Further research is needed to determine if the novel mutations lead to functional or phenotypic changes Notably, Hap1 and Hap12 are identified as the primary haplotypes in Mong Cai pigs, indicating that this breed may exhibit a slight increase in haplotypic diversity.
4.3.2 The potential association between MC1R gene and coat color of Mong Cai pig
The Extension/MC1R locus is crucial for determining coat color in pigs, as highlighted by Lin and Fisher (2007) Research has shown a significant link between genetic variations in the coding sequences of the MC1R gene and porcine coat color Notably, the c.68_69insCC mutation at codon 23 of the MC1R sequence is associated with a white-and-black-spotted coat color, according to Kijas et al (2001) However, this mutation has not been found in Mong Cai pigs.
The distribution and expression of melanin, which contributes to the black saddle characteristics of the Mong Cai breed, are influenced by various factors The mechanism of melanogenesis begins with the formation of melanosomes, alongside genes related to coat color (Moreiras et al., 2021) Additionally, the biogenesis of melanosomes involves multiple molecules that play a role in intracellular trafficking (Du et al., 2022).
A significant phenotype segregation was observed between haplotypes 1 and 12 concerning saddle coat color, specifically between knotted and seamless types In haplotype 1, 14 out of 18 Mong Cai individuals displayed the knotted black saddle coat phenotype.
The study identified a significant association between haplotypes and coat phenotypes in Mong Cai pigs, with individuals exhibiting either a seamless black saddle coat or a knotted inheritance mode Specifically, five out of six Hap12 individuals displayed a seamless black saddle-shaped coat, while the remaining individuals showed a black saddle coat phenotype The chi-square test results indicate a strong correlation, suggesting that the black saddle coat phenotype may be linked to the MC1R gene.
Table 4.7: The distribution of MC1R haplotype Hap1 and Hap12 with phenotypes in Mong Cai pigs
Hap12 6 16.67 83.33 Χ 2 : Chi-square, df = 1, X 2 = 5.86, P-value = 0.01, ** means the highly significant level
Hap1 owned A at nt729 but it is G in Hap12
The SNP c.729A>G may be linked to variations in coat color among Mong Cai pigs, distinguishing between the knotted black saddle and seamless black types Despite being a synonymous SNP, research suggests that such variations can disrupt cellular functions and lead to different clinical phenotypes (Hunt et al., 2009).
Synonymous SNPs can influence messenger RNA splicing, stability, and structure, as well as protein folding, by modifying the recognition of transcripts by RNA-binding proteins (Hunt et al., 2009) Additionally, mutations in the MC1R gene serve as potential candidate markers for Mong Cai breeding, enabling the differentiation between knotted and seamless black saddles.
4.3.3 Origin of Mong Cai breed
Mong Cai pigs exhibit a close genetic relationship with Bama miniature pigs from Guangxi province, China Additionally, they share characteristics with Luchuan pigs, also native to Guangxi Studies of mtDNA and microsatellites indicate that Mong Cai pigs and other Vietnamese indigenous breeds are closely related to Asian pig breeds in maternal lineage, forming a distinct branch separate from European breeds.
My research confirms that Mong Cai pigs originated in Asia, revealing two distinct coat color variations: a knotted black saddle and a seamless black saddle Notably, the nt729G gene, exclusive to European pigs, is present in the seamless black saddle variant Currently, the majority of Mong Cai pigs exhibit the knotted black saddle, while those with the seamless black saddle phenotype represent a small fraction of the population.
This study demonstrated that the MC1R gene is responsible for the variable coat color phenotype in the Mong Cai pig population, as well as the diversity
48 between indigenous Asian and European pigs In addition, the SNP c.729A>G and the knotted black saddle phenotype identified in my study constitute a significant marker for selecting Mong Cai pig phenotypes
CONCLUSIONS AND PROPOSALS
Conclusions
- Designing a marker set based on mtDNA D-loop sequence to discriminate Mong Cai pig from other pig breeds:
Two marker pairs, Mtmc3 and Mtmc4, were developed from the mtDNA D-loop region of Mong Cai pigs, demonstrating the maternal lineage diversity within the Mong Cai population in Quang Ninh province This finding highlights the ongoing trend of crossbreeding in Vietnamese swine farms While crossbreeding local pigs with exotic breeds can significantly boost meat production and minimize undesirable traits, it poses a significant threat to the preservation of indigenous pig genetic resources.
- Sequencing the MC1R gene in Mong Cai pigs:
For the first time, the MC1R gene in the Mong Cai pig breed was sequenced and analyzed in terms of diversity and haplotype MC1R gene in 26
Mong Cai pigs possess a 963bp exon region that encodes 321 amino acids, providing valuable insights for future research on the unique black coat color of Mong Cai pigs and the domestication of Vietnamese native pig breeds.
- Investigate the haplotype diversity in MC1R sequence among Mong Cai individuals:
The Mong Cai population exhibited five substitutions and four haplotypes, with most individuals sharing a haplotype similar to that of several Chinese pigs The observed divergence in polymorphism and phenotype among Mong Cai pigs may be linked to the SNP c.729A>G This polymorphism serves as a potential marker for selecting specific Mong Cai pig phenotypes.
- Revealing the origin of the Mong Cai pig population in Quang Ninh province:
This research strongly supports the Asian origin of Mong Cai pigs and highlights their close genetic ties to Chinese pig breeds through molecular analysis The MC1R gene, which influences coat color, exhibits significant diversification between Asian and European domestic pigs.
Proposals
This study should be combined with research on microsatellites to provide more evidence and determine the purebred Mong Cai pig
To strengthen the findings on the association between the MC1R gene polymorphism and coat color in Mong Cai pigs, it is essential to conduct a study with a larger sample size.
The journey from gene to protein and ultimately to phenotype is a fascinating process that captivates my interest Although it may take decades to uncover the solutions to these complex questions, my background and passion for this subject drive me to explore it further.
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Appendix 1: The alignment result of mtDNA D-loop region of 4 pig breeds: Mong Cai (KU556691; KX147100); Huong (KY964306); Meishan (KM998967); Hampshire (AY574046)
Appendix 2: Mong Cai pig’s MC1R coding sequences with GenBank association codes OP142697 – OP142700
ATGCCTGTGCTTGGCCCGGAGAGGAGGCTGCTGGCTTCCCTCAGCTCCGCACCCC CAGCCGCCCCCCGCCTCGGGCTGGCCGCCAACCAGACCAACCAGACGGGCCCCC AGTGCCTGGAGGTGTCCATTCCCGACGGGCTCTTCCTCAGCCTGGGGCTGGTGAG CCTCGTGGAGAACGTGCTGGTGGTGGCCGCCATCGCCAAGAACCGCAACCTGCA CTCGCCCATGTACTACTTCGTCTGCTGCCTGGCCGTGTCGGACCTGCTGGTGAGC GTGAGCAACATGCTGGAGACGGCCGTGCTGCCGCTGCTGGAGGCGGGCGCCCTG GCCGCCCAGGCCGCCGTGGTGCAGCAGCTGGACAACGTCATGGACGTGCTCATC TGCGGCTCCATGGTGTCCAGCCTCTGCTTCCTGGGCGCCATCGCCGTGGACCGCT ACGTGTCCATCTTCTACGCGCTGCGCTACCACAGCATCGTGACGCTGCCCCGCGC GGGGCGGGCCATCGCGGCCATCTGGGCGGGCAGCGTGCTCTCCAGCACCCTCTTC ATCGCCTACTACCACCACACGGCCGTCCTGCTGGGCCTCGTCAGCTTCTTCGTGG CCATGCTGGCGCTCATGGCGGTACTGTACGTCCACATGCTGGCCCGGGCCTGCCA GCACGGCCGGCACATCGCCCGGCTCCACAAGACGCAGCACCCCACCCGCCAGGG CTGCGGCCTCAAGGGCGCAGCCACCCTCACCATCCTGCTGGGCGTCTTCCTCCTC TGCTGGGCACCCTTCTTCCTGCACCTCTCCCTCGTCGTCCTCTGCCCCCAGCACCC CACCTGCGGCTGCGTCTTCAAGAACGTCAACCTCTTTCTGGCCCTCGTCATCTGC AACTCCATCGTGGACCCCCTCATCTACGCCTTCCGCAGCCAGGAGCTCCGCAAGA CCCTCCAGGAGGTGCTGCAGTGCTCCTGGTGA
ATGCCTGTGCTTGGCCCGGAGAGGAGGCTGCTGGCTTCCCTCAGCTCCGCACCCCCAGCCGCCCCCCGCCTCGGGCTGGCCGCCAACCAGACCAACCAGACGGGCCCCCAGTGCCTGGAGGTGTCCATTCCCGACGGGCTCTTCCTCAGCCTGGGGCTGGTGAGCCTCGTGGAGAACGTGCTGGTGGTGGCCGCCATCGCCAAGAACCGCAACCTGCACTCGCCCATGTACTACTTCGTCTGCTGCCTGGCCGTGTCGGACCTGCTGGTGAGCGTGAGCAACATGCTGGAGACGGCCGTGCTGCCGCTGCTGGAGGCGGGCGCCCTGGCCGCCCAGGCCGCCGTGGTGCAGCAGCTGGACAACGTCATGGACGTGCTCATC
TGCGGCTCCATGGTGTCCAGCCTCTGCTTCCTGGGCGCCATCGCCGTGGACCGCT ACGTGTCCATCTTCTACGCGCTGCGCTACCACAGCATCGTGACGCTGCCCCGCGC GGGGCGGGCCATCGCGGCCATCTGGGCGGGCAGCGTGCTCTCCAGCACCCTCTTC ATCGCCTACTACCACCACACGGCCGTCCTGCTGGGCCTCGTCAGCTTCTTCGTGG CCATGCTGGCGCTCATGGCGGTACTGTACGTCCACATGCTGGCCCGGGCCTGCCA GCACGGCCGGCACATCGCCCGGCTCCACAAGACGCAGCACCCCACCCGCCAGGG CTGCGGCCTCAAGGGCGCGGCCACCCTCACCATCCTGCTGGGCGTCTTCCTCCTC TGCTGGGCACCCTTCTTCCTGCACCTCTCCCTCGTCGTCCTCTGCCCCCAGCACCC CACCTGCGGCTGCGTCTTCAAGAACGTCAACCTCTTTCTGGCCCTCGTCATCTGC AACTCCATCGTGGACCCCCTCATCTACGCCTTCCGCAGCCAGGAGCTCCGCAAGA CCCTCCAGGAGGTGCTGCAGTGCTCCTGGTGA
ATGCCTGTGCTTGGCCCGGAGAGGAGGCTGCTGGCTTCCCTCAGCTCCGCACCCCCAGCCGCCCCCCGCCTCGGGCTGGCCGCCAACCAGACCAACCAGACGGGCCCCCAGTGCCTGGAGGTGTCCATTCCCGACGGGCTCTTCCTCAGCCGGGGGCTGGTGAGCCTCGTGGAGAACGTGCTGGTGGTGGCCGCCATCGCCAAGAACCGCAACCTGCACTCGCCCATGTACTACTTCGTCTGCTGCCTGGCCGTGTCGGACCTGCTGGTGAGCGTGAGCAACATGCTGGAGACGGCCGTGCTGCCGCTGCTGGAGGCGGGCGCCCTGGCCGCCCAGGCCGCCGTGGTGCAGCAGCTGGACAACGTCATGGACGTGCTCATCTGCGGCTCCATGGTGTCCAGCCTCTGCTTCCTGGGCGCCATCGCCGTGGACCGCTACGTGTCCATCTTCTACGCGCTGCGCTACCACAGCATCGTGACGCTGCCCCGCGCGGGGCGGGCCATCGCGGCCATCTGGGCGGGCAGCGTGCTCTCCAGCACCCTCTTCATCGCCTACTACCACCACACGGCCGTCCTGCTGGGCCTCGTCAGCTTCTTCGTGGCCATGCTGGCGCTCATGGCGGTACTGTACGTCCACATGCTGGCCCGGGCCTGCCAGCACGGCCGGCACATCGCCCGGCTCCACAAGACGCAGCACCCCACCCGCCAGGGCTGCGGCCTCAAGGGCGCAGCCACCCTCACCATCCTGCTGGGCGTCTTCCTCCTCTGCTGGGCACCCTTCTTCCTGCACCTCTCCCTCGTCGTCCTCTGCCCCCAGCACCCCACCTGCGGCTGCGTCTTCAAGAACGTCAACCTCTTTCTGGCCCTCGTCATCTGCAACTCCATCGTGGACCCCCTCATCTACGCCTTCCGCAGCCAGGAGCTCCGCAAGACCCTCCAGGAGGTGCTGCAGTGCTCCTGGTGA
ATGCCTGTGCTTGGCCCGGAGAGGAGGGTGCTGGCTTCCCTCAGCTCCGCACCCCCAGCCGCCCCCCGCCTCGGGCTGGCCGCCAACCAGACCAACCAGACGGGCCCCCAGTGCCTGGAGGTGTCCATTCCCGACGGGCTCTTCCTCAGCCTGGGGCTGGTGAGCCTCGTGGAGAACGTGCTGGTGGTGGCCGCCATCGCCAAGAACCGCAACCTGCACTCGCCCATGTACTACTTCGTCTGCTGCCTGGCCGTGTCGGACCTGCTGGTGAGCGTGAGCAACATGCTGGAGACGGCCGTGCTGCCGCTGCTGGAGGCGGGCGCCCTGGCCGCCCAGGCCGCCGTGGTGCAGCAGCTGGACAACGTCATGGACGTGCTCATCTGCGGCTCCATGGTGTCCAGCCTCTGCTTCCTGGGCGCCATCGCCGTGGACCGCTACGTGTCCATCTTCTACGCGCTGCGCTACCACAGCATCGTGACGCTGCCCCGCGCGGGGCGGGCCATCGCGGCCATCTGGGCGGGCAGCGTGCTCTCCAGCACCCTCTTCATCGCCTACTACCACCACACGGCCGTCCTGCTGGGCCTCGTCAGCTTCTTCGTGGCCATGCTGGCGCTCATGGCGGTACTGTACGTCCACATGCTGGCCCGGGCCTGCCAGCACGGCCGGCACATCTCCCGGCTCCACAAGACGCCGCACCCCACCCGCCAGGGGTGCGGCGTCAAGGGGGCAGCCACCCTCACCATCCTGCTGGGCGTCTTCCTCCTCTGCTGGGCACCCTTCTTCCTGCACCTCTCCCTCGTCGTCCTCTGCCCCCAGCACCCCACCTGCGGCTGCGTCTTCAAGAACGTCAACCTCTTTCTGGCCCTCGTCATCTGCAACTCCATCGTGGACCCCCTCATCTACGCCTTCCGCAGCCAGGAGCTCCGCAAGACCCTCCAGGAGGTGCTGCAGTGCTCCTGGTGA
Appendix 3: Figures of 26 Mong Cai pigs which were studied in analysing about MC1R gene
Result of part 4.1.2 has been accepted for poster presentation and published in proceeding of the Vietnam National Conference on Biotechnology 2022
Time: November 04 th , 2022 Place: Tay Nguyen University
DESIGNING THE DNA MARKERS FOR DETERMINING THE MATERNAL LINEAGE ORIGIN OF THE MONG CAI PIG BREED
Thuy Thi Thu Cao 1 , Giang Huong Nguyen 1 , Khoa Van Nguyen 1 , Mai Thi Thanh Nguyen 1 , Trung Thanh Ngo 2 , Trung Quoc Nguyen 1 *
1 Faculty of Biotechnology, Vietnam National University of Agriculture
2 Faculty of Veterinary Medicine, Vietnam National University of Agriculture
The Mong Cai pig (Sus scrofa) is Vietnam's most popular indigenous breed, known for its high fertility and distinctive black saddle coat However, the introduction of exotic breeds has altered its phenotypic traits This study analyzed the mitochondrial genome of the Mong Cai pig to create DNA primers for identifying its maternal lineage Samples included 91 Mong Cai pigs and individuals from Hampshire, Meishan, and Huong breeds Two primer pairs, Mtmc3 and Mtmc4, were developed to amplify DNA segments between 700bp and 1000bp, revealing four alleles corresponding to the different breeds Results indicated that some Mong Cai pigs have maternal ancestry from Hampshire, Meishan, and Huong breeds, reflecting farmers' efforts to enhance production traits The markers demonstrated significant polymorphism and are valuable for identifying the maternal lineage of the Mong Cai breed, warranting further research to preserve its pure breeding.
Keywords: marker, D-loop, mtDNA, Mong Cai pig, maternal origin
*Author for corresponding: Tel +84 97 6588239; Email: nqtrung@vnua.edu.vn