Sequencing and analysis of complete mitochondrial genome of 6 indigenous pigs in some northern provinces of Vietnam

oint or by the out group. Chapter 2. SUBJECTS AND RESEARCH METHODS 2.1. Subjects and research locations Pigs of 6 indigenous pig breeds in Vietnam (eg, Mong Cai, Mong Khuong, Muong Lay, Huong and Ha Lang) were randomly selected among pig herds in some localities: Dien Bien, Lao Cai, Cao Bang, Hai Phong and Thanh Hoa. 2.2. Research methods 2.2.1. Survey method based on pig appearance Observe the outside of the animal by groups of fur, skin color and evaluation criteria for the shape, characteristics of the pig's body parts, weight measurement, indicators: body length, round chest and high shoulders. Evaluation criteria against slices of livestock breeds in Vietnam and Monograph on conservation and exploitation of Vietnamese domestic animal genetic resources. 2.2.2. Sequencing mitochondrial DNA genome DNA extraction, mtDNAfragment amplification using PCR, Sequencing mitochondrial genome by shotgun sequencing follow by Sanger's mothod. 2.2.3. Methods for assembling, predicting and annotating genomes 2.2.3.1. Genome assembly Sequence data was assessed and edited on BioEdit v7.2.5 software. Contig segments obtained from the cutting process will be assembled based on the overlapping sequences at the ends of each segment using DNA Dragon v1.6.0 (SequentiX) and EditSeq (DNASTAR) software. 5 2.2.3.2. Multiple alignment The D-loop region sequences and the coding sequences of the assembled mitochondrial genome will be separated. The sequence of short iterations before and after 5 '-CGTGCGTACA-3' is determined by the number of repetition units and discarded. The mitochondrial genome sequence of pig breeds in the world is arranged in a multi- sequence, using the MUSCLE algorithm. Identify the most appropriate evolutionary model using MEGA7 software. 2.2.3.3. Genome analysis and annotation Analysis and annotation of genome and tRNA genes of indigenous pigs in Vietnam using Dogma and Mitos Web Server online. All annotations were checked by BLAST tool on GenBank. 2.2.4. Sequence analysis and method of determining the level of sequence similarity In order to obtain the sequence we analyzed the basic indices for asymmetric ratio of nucleotide types such as percentage of nucleotides using DAMBE v6.3.17 software ( .uottawa.ca /), along with two deviations: GC skew and AT skew are calculated using the following formula: AT skew = (A-T)/(A+T) GC skew = (G-C)/(G+C) 2.2.5. Phelogenetic reconstructiong and analysis method 2.2.5.1. Evolutional distance The evolutional distance (p-distance) between sequence pairs was calculated using the two-parameter algorithm of Kimura in MEGA software 2.2.5.2. Phylogenetic analysis The sequence of the Dloop region and the entire coding region of the mitochondrial genome are used separately as input data to 6 reconstruct the corresponding phylogenetic trees. Bayes method is used in BEAST v1.8.3 software, set up Yule process and MCMC 10000000 to calculate prior probability. The optimal tree was found using Tree Annotater v.1.8.4. The root of the tree was determined by a method of using the out-group. Finally, Figure Tree v1.4.2 software is used to read the output file and reconstruct the phylogenetic tree. Chapter 3. RESULTS AND DISCUSSION 3.1. Sample selection and collection Up to now, in Vietnam, to identify indigenous pig breeds, breeders have not had any molecular database but mainly bases and set of morphological indicators have been published and approved. To ensure reliability in sampling for research, we surveyed appearance characteristics based on 3 groups of criteria: (1) Characteristics of fur and skin; (2) stature, weight and (3) body shape, number of nipples. a. Group of characteristics characteristics of fur and skin In Muong Lay breed, it is observed that the skin color distribution is often accompanied by some appearance indicators to form a smaller subgroup (name: group A +). The number of pig individuals observed in skin color was classified into A +, A and B groups in descending order of breed characteristics. Only individuals with full breed criteria (group A or A +) can be selected for screening in the next target group. Table 3.1. Survey results on fur and skin characteristics of 6 indigenous pig breeds Characteristics on the colour of fur and skin Total N Group % I pigs Fur is thin and coarse. Skin is black but not glossy 9 9 A 100 7 Mong Cai pigs - The body is black and white, black head, adjacent to the black and white hairs have a blur 100 82 B 82,0 - The body is black and white, the black head in the middle of the forehead has white spots, adjacent to the black and white hairs have blurred space 18 A 18,0 Muong Khuong pigs - Fur is pure black. The fur is thin and soft. 100 77 B 77,0 - Black fur has white spots on the tail and legs, the fur is soft and thin. 23 A 23,0 Huong pigs - Body and 4 legs are white with black skin on the buttocks and scalp. The juxtaposition between black and white is about 2-3 cm wide, on which the skin is black, the white fur 38 22 B 57,8 - There are additional characteristics: in the middle of the forehead is a white point, the four legs are white. 16 A 42,1 Ha Lang pigs - The belly is white and has a white shoulder strap, with a black strip similar to a saddle. 41 13 B 31,7 - There are additional characteristics: The forehead has a white point almost like a wedge 28 A 68,3 Muong Lay pigs - Black, straight back, nipple head 10-15cm above the ground 70 35 B 50,0 - There are more white spots on the legs, forehead, tail, back, straight nipples 10-15 cm from the face 8 A 11,4 2 - There is more back slightly hammock, slightly saggy belly, nipple head are not sag. 27 A+ 38,5 (N: Number of selected individuals with skin color characteristics suitable for Slices; %: percentage of individuals with suitable characteristics on the total number of observed individuals) Only individuals in groups A and A + will be selected to conduct surveys in the criteria group of stature and weight. b. Characteristics of dimensions of dimensions and mass 8 Table 3.2. Survey results of weight and size of 6 indigenous pigs Pig breeds Mass Long body Chest High shoulders ± SD ± SD ± SD ± SD I 40,34 ± 0,92 85,76 ± 1,32 83,93 ± 1,44 39,58 ± 0,91 Mong Cai 51,47 ± 0,32 92,41 ± 1,35 90,77 ± 1,46 45,25 ± 0,93 Huong 40,55 ± 0,22 86,73 ± 1,33 81,31 ± 1,42 42,56 ±0,36 Ha Lang 42,68 ± 042 87,12 ± 1,33 84,34 ± 1,43 43,68 ±0,98 Muong Khuong 52,64 ± 0,92 93,94 ± 1,32 90,52 ± 1,46 46,14 ±0,96 Muong Lay 40,43 ± 0,95 85,71 ± 1,38 83,97 ± 1,46 43,25±0,94 (Unit: Mass: Kg; Dimension: cm) : Average value ± SD: standard deviation The results showed that 100% of the individuals surveyed on the group of criteria of stature and body size have the characteristics of each breed. c. Survey on body shape criteria group Table 3.3. Survey on body shape characteristics of 6 indigenous pigs Body shape characteristics (number of nipples) Total N % I pigs - Moderately large head, near-flat forehead, wrinkled face, sagging neck and saggy cheeks when fat, short muzzle - The belly is less saggy, the body, legs are long and taller than the I Mo pigs 9 9 100 Mong Cai pigs - Large head, small muzzle and long, small and pointed ears, big and short wrinkles in the mouth. - The neck is large and short, the chest is broad and deep, the back is long, slightly sagging, the belly is slightly saggy, the buttocks broad and down. 18 15 83,3 Muong Khuong pigs - Muzzle long straight or slightly arched. The forehead is smooth, the ear cup is hanging forward. 23 20 86,9 9 - Four big strong legs. The back is slightly curved, the belly is big but not saggy to the ground, butt is slightly sloping. Huong pigs - The head is black and rough, there is a white spot between the forehead. - - Big feet, moderately large and not touching the ground, back hammock but not broken, four legs are white, sloping butt, shoulders broad, chest deep. 16 10 62,5 Ha Lang pigs - Muzzle short, wrinkly face. - Short legs. Back hammock, abdomen does not touch the ground 28 25 89,2 Muong Lay pigs - Muzzle straight, moderately long, wrinkled forehead, large and thick ears - Body is elongated, slightly back hammock, large and moderately high legs - The nipple is even, when pregnant and nursing, the nipple does not sag, do not touch the ground. 27 13 48,1 (N: Number of individuals selected to have a body shape that is consistent with Slic;%: percentage of individuals with an appropriate feature out of the total observed.) The number of individuals belonging to 6 pig breeds after being selected through 3 groups of appearance indicators has reached all the typical criteria of each breed. Blood samples from these individuals were randomly selected for each of the 6 samples for total DNA extraction for research purposes. 3.2. Mitochondrial genome sequence of 6 indigenous pigs Complete sequences of mitochondrial genomes of 6 indigenous pigs, including I, Mong Cai, Muong Khuong, Muong Lay, Huong and Ha Lang, have been identified and registered on GenBank with access numbers: KX094894, KU556691, KY432578 , KX147101, KY964306 and KY800118. The work of the author Tran Thi Thuy Nhien et al (2016) conducted an independent study also announced the complete sequence of the mitochondrial genome of Mong Cai pigs (access code GenBank: KU556691). The genome size of Mong 10 Cai pigs' genome published by this group is 16,632 bp, shorter than the sequence in our study results of 79 bp, the difference is mainly in the D-loop region, which has the number of repetitive motifs 'CGTGCGTACA'. 3.3. Mitochondrial genome analysis 3.3.1. Analysis of mitochondrial genome composition The composition ratios of nucleotide types are listed in Table 3.5. Table 3.5. Base component ratio in mitochondrial genome of 6 indigenous pigs in Vietnam Pigs breed A(%) C(%) G(%) T(%) G+C(%) I 34,64 26,24 13,33 25,75 39,57 Mong Cai 34,70 26,20 13,30 25,79 39,50 Muong Khuong 34,68 26,19 13,31 25,81 39,50 Ha Lang 34,67 26,20 13,32 25,78 39,55 Huong 34,65 26,22 13,352 25,78 39,57 Muong Lay 34,70 26,19 13,32 25,79 39,51 The results show that the deviation of the mitochondrial nucleotide genome composition of the studied pig breeds is in the direction of A + T rich (60.43 - 60.50). Some indicators specific to the mitochondrial genome sequence such as the percentage of base type G and type C (% GC), GC and AT deviations (GC skew and AT skew) are listed for analysis and comparison. between indigenous pigs in Vietnam and other pigs in the world in Table 3.6. Table 3.6. The sequence composition of groups of pigs in the world Region Pig breeds Complete sequence D-loop sequence %GC GC skew AT skew %GC GC skew AT skew Vietname se indigeno us Pigs I 39,35 -0,33 0,15 39,91 -0,32 0,16 Mong Cai 39,23 -0,33 0,15 38,77 -0,33 0,16 Ha Lang 39,24 -0,33 0,15 38,47 -0,34 0,16 Huong 39,25 -0,33 0,15 39,25 -0,30 0,14 Muong Khuong 39,25 -0,33 0,15 38,79 -0,33 0,16 Muong Lay 39,29 -0,33 0,15 39,59 -0,31 0,16 North Jeju - - - 38,55 -0,35 0,17 11 East Asia Korean native pig - - - 38,76 -0,35 0,15 WB-China northeast 39,25 -0,33 0,15 38,2 -0,34 0,15 WB-Japan - - - 39,09 -0,33 0,16 WB-Korea 39,19 -0,33 0,15 38,59 -0,33 0,16 Yellow River Valley Bamei 39,21 -0,33 0,15 38,69 -0,34 0,16 Huzu 39,2 -0,33 0,15 38,69 -0,34 0,16 European Country Berkshire 39,28 -0,33 0,15 38,47 -0,34 0,16 Duroc 39,32 -0,33 0,15 38,66 -0,35 0,16 Hampshire 39,33 -0,33 0,15 38,27 -0,36 0,16 Iberian 39,29 -0,34 0,15 38,37 -0,35 0,16 Landrace 39,28 -0,33 0,15 38,47 -0,35 0,15 Large White 39,27 -0,33 0,15 38,47 -0,35 0,16 Pietrain 39,28 -0,34 0,15 38,52 -0,35 0,16 WB-European 39,28 -0,34 0,15 38,56 -0,35 0,17 Mekong Region Banna mini 39,28 -0,33 0,15 39,16 -0,34 0,16 Dahe 39,24 -0,33 0,15 38,92 -0,33 0,16 Thailand indigenous pig - - - 38,7 -0,33 0,16 WB-Malaysia 39,18 -0,33 0,15 38,59 -0,38 0,19 WB-Vietnam 39,26 -0,33 0,15 38,69 -0,34 0,16 WB-Yunnan 39,28 -0,33 0,15 39,27 -0,31 0,15 South China Lantang 39,22 -0,33 0,15 38,57 -0,34 0,16 Lanyu 39,25 -0,30 0,12 38,55 -0,34 0,16 WB-Fujian 39,19 -0,33 0,15 38,71 -0,33 0,16 WB-Hainan 39,19 -0,33 0,15 38,68 -0,33 0,15 Yangtze River Region Aba 39,21 -0,33 0,15 38,69 -0,34 0,16 Bihu 39,81 -0,31 0,14 38,69 -0,34 0,16 Jinhua 39,22 -0,33 0,15 38,46 -0,34 0,16 Kele - - - 38,68 -0,34 0,16 Taoyuan - - - 38,66 -0,34 0,16 WB-Jiangxi 39,22 -0,33 0,15 38,59 -0,34 0,16 Wei 39,2 -0,33 0,15 38,69 -0,34 0,16 Xiang pig 39,18 -0,33 0,15 38,59 -0,33 0,16 "-" : The complete sequence has not been announced. GC skew indices are all negative for pigs, AT skew is positive. Thus, in terms of evolution, the trend of changing the nucleotide composition among pigs is no big difference. For the complete sequence, all six studied pigs were similar in GC skew and AT skew values with values of -0.33 and 0.15 respectively. 12 In Huong and Muong Lay breeds with Chinese WB-Yunnan, the complete sequence has the least difference between C and G respectively 0.30 and 0.31. The pigs Huong breed has the lowest deviation between the two types of nucleotides A and T (0.14). Comparison between the D-loop sequence and the complete sequence, it can be seen that the D-loop region in most pigs has higher AT skew, that is, the level of nucleotide conversion of type A and T higher. The average value of the GC skew value for Vietnamese native pigs (-0.32) is higher than the average for other pigs in the world (-0.34). Theoretically, these deviation indicators also allow a partial assessment of genetic diversity and indirectly identify the relationships of phylogenetic. However, the difference is not large enough between the research subjects, so it is not possible to draw any specific scientific reasoning in the two directions from the GC skew and AT skew indicators. 3.3.2. Annotation of mitochondrial genome structure Results of the interpretation of the mitochondrial genome structure of the studied pigs consisted of 37 genes including 2 genes encoding RNA ribosome, 13 genes encoding protein, 22 genes encoding RNA transport and a control area D -loop. There are also twelve small non-coding regions scattered throughout the mitochondrial genome. Results of the genome annotation using the online tool GenomeVx showed that the native pigs of Vietnam have the double-chain, closed-loop mitochondrial genome, with the sizes of the following: Mong Cai pigs: 16,711 base pairs (bp) , Muong Lay pigs : 16,740 bp, Muong Khuong pigs : 16,679 bp, Ha Lang pigs : 16,722 bp, Huong pigs : 16,753 bp and I pigs : 16,731 bp. Comparison of sequences of previous and next sequence 13 according to motif 5'-tacacgtgcg-3 'in the D-loop showed that there were big differences between the groups of Vietnamese pigs and those of European and Asian pigs. Results of structural analysis, organization of the mitochondrial genome of the 6 indigenous pigs in Vietnam showed similarities with the structure of the mitochondrial genome of other mammals. The base types in the mitochondrial genome of all 6 pigs are towards A + T richness (over 60%), similar to other Asian pigs. 3.3.3. Structure and composition of transport RNA genes Information about the structural composition of tRNA genes is listed in Table 3.13. Table 3.13. Nucleotide composition of 22 tRNA genes STT Genes Strand (+/-) Length (bp) Nucleotides composition GC skew AT skew A G C T 1. tRNA Phe + 70 26 13 16 15 -0,10 0,27 2. tRNA Val + 68 26 10 14 18 -0,17 0,18 3. tRNA Leu2 + 75 23 14 17 21 -0,10 0,05 4. tRNA Ile + 69 28 11 8 22 0,16 0,12 5. tRNA Gln - 73 16 20 8 29 0,43 -0,29 6. tRNA Met + 70 20 13 20 17 -0,21 0,08 7. tRNA Trp + 68 26 12 14 16 -0,08 0,24 8. tRNA Ala - 68 19 15 8 26 0,30 -0,16 9. tRNA Asn - 75 21 18 11 25 0,24 -0,09 10. tRNA Cys - 66 18 16 12 20 0,14 -0,05 11. tRNA Tyr - 65 21 15 11 18 0,15 0,08 12. tRNA Ser2 - 69 17 20 11 21 0,29 -0,11 13. tRNA Asp + 68 23 12 9 24 0,14 -0,02 14. tRNA Lys + 67 20 14 16 17 -0,07 0,08 15. tRNA Gly + 69 25 10 13 21 -0,13 0,09 16. tRNA Arg + 69 29 6 9 25 -0,20 0,07 17. tRNA His + 69 29 8 8 24 0,00 0,09 18. tRNA Ser1 + 59 20 9 12 18 -0,14 0,05 19. tRNA Leu1 + 70 27 13 11 19 0,08 0,17 20. tRNA Glu - 69 17 17 8 27 0,36 -0,23 21. tRNA Thr + 68 22 12 18 16 -0,20 0,16 14 22. tRNA Pro - 65 13 19 10 23 0,31 -0,28 Total in length 1509 486 297 264 462 Average 68,59 22,09 13,50 12,00 21,00 0,06 0,02 22 tRNA genes had an overall size of 1509 bp and an average length of 68.6 bp, ranging from 59 bp (tRNAPro) to 75 bp tRNALeu (CTA). The nucleotide composition of all 22 tRNA genes was in the direction of deviation towards AT content with the ratio of nucleotide types: 32.2% A, 30.6% T, 19.6% G and 17.5% C , in order A> T> G> C. The value of GC skew index among 22 tRNA genes had 10 negative values (0.45%), 8 tRNA genes had a negative AT skew index (36%) and the average value of AT skew, GC values skew are both positive values (0.06 and 0.02%). This proves that the presence of type A nucleotides on DNA strands is greater than that of T in most tRNA genes and that the G and C difference ratio is almost the same among the 22 tRNA genes of the mitochondrial genomes of varieties. Indigenous pigs. Only the tRNA His gene has a frequency G equal to C with a GS skew index of 0. 3.3.4. Phân tích cấu trúc bậc hai của các tRNA The results of predicting the secondary structure of 22 tRNA show that the structure of 21 tRNAs except tRNASer-1 has typical clover forms made up of three lobes dihydrouridine DHU, pseudouridin (TψC) and anti-codon. All 21 tRNAs had amino acid acceptor and aniticodon triad (7 bp and 5 bp), with the exception of tRNA Ser-1 with branch length of triplets is 6 bp. Particularly for tRNA Ser-1 due to its short genetic size, the dihydrouridine lobes (DHU) do not form a sustainable structure. 3.4. Compare polymorphic sequences Because of differences in structural characteristics and the role of evolution between two sequence regions, the results of sequence 15 polymorphic analysis are shown in two result groups: D-loop region polymorphism and complete coding region polymorphism. 3.4.1. D-loop region 3.4.1.1. Similarity The result of comparing the similarity coefficient in the D-loop region was the lowest in pigs (<0.951), followed by Muong Lay pigs (<0.971), thereby reflecting the characteristics of genetic of these two native pigs. Besides, it can be seen that there is a high degree of similarity in the sequence between Huong and Ha Lang (the highest similarity index reached: 0.999). Comparison of similarities in the Vietnamese Lang pig group (Huong, Ha Lang and Mong Cai) shows that there is a higher level of similarity within the group than other native Vietnamese pigs and other world's pigs breeds. 3.4.1.2. Difference There are 25 polymorphic positions out of a total of 1184 positions (contig length), of which 5 locations have at least 2 variants. The results of comparison of D-loop differences are presented in Table 3.15. Table 3.15. SNP sites in the D-loop region of 6 studied pigs Ord Breed Location I Muong Khuong Muong Lay Mong Cai Ha Lang Huong 1 24 G/A - - - - - 2 183 T/C - - - - - 3 215 T/C - - - - - 4 242 T/C T/C T/C T/C - - 5 280 - C/T C/T - - - 6 407 - - - T/C T/C T/C 7 454 - C/T C/T C/T C/T C/T 8 503 A/G - - - - - 9 562 T/C - - - - - 10 706 G G - G - - 11 714 A - A - - - 12 736 - - A - - - 16 13 734 G/G/A - - - - - 14 744 A - - - - - 15 746 - - A - - - 16 754 G/G/A - - - - - 17 756 - - A - - - 18 764 G/G/A - - - - - 19 766 - - A - - - 20 774 A - - - - - 21 776 - - A - - - 22 791 C - - - - - 23 813 - T/T/C - - - - 24 1032 A/G - - - - - 25 1165 - - - - A/C/A A/C/A When comparing D-loop sequence differences, we can see some positions, for example at the 454 nucleotide position (Figure 3.6.), All groups of Vietnamese pigs except I pigs are different from most remaining pigs in this variant (C / T). This sequence of I pigs has a lot of differences with the other 5 indigenous pigs in Vietnam and with other pigs in the world. Figure 3.6. The position is different in the D-loop sequence of indigenous pigs in Vietnam and pigs in the world 3.4.2. Complete coding sequence of mtDNA genome 3.4.2.1. Similarity 17 The Vietnamese pig breeds in the Lang pigs group (Ha Lang, Huong and Mong Cai) have quite similar levels. The two indigenous breeds in Cao Bang province (Huong and Ha Lang) show a high level of genetic intimacy with a high similarity index (0.999). Compared to the D-loop region, the coding region sequence of the I breed does not represent a high level of specificity. 3.4.2.2. Difference The difference usually falls into the regions of the genes encoding proteins and tRNAs, while the rest are quite conservative. In addition, among the notable SNP variants is the 2250 variation located on the gene encoding 12S rRNA. All indigenous Vietnamese breeds except I breed with the present of (C/T) variant in this region, while most other pig breeds are present, the result is illustrated in Figure 3.8. Figure 3.8. The sequence variation at the nucleotide position 2250 encodes the mitochondrial genome of pigs The results of comparing this sequence variation show the similarity between the I pigs and Banamini (in the Mekong region) 18 and between the five groups of Vietnamese pigs (Huong, Mong Cai, Muong Lay, Muong Khuong and Ha. Lang) with the Lantang pigs (in Southern China) allowed us to make predictions about the close genetic relationship between these pigs. 3.5. Phylogenetic relationship analysis In order to enhance the reliability of the origin and the phylogenetic relation, the D-loop region data and the complete sequence data are independently analyzed to reconstruct the different phylogenetic trees. For Mong Cai pigs, in addition to the results of the thesis, we also use the order of the author Tran Thi Thuy Nhien (2016) (denoted on the phylogenetic tree type MongCai_TN) to reconstruct the phylogenetic tree as well as analysis of evolutionary relationships. 3.5.1. Phylogenetic tree based on D-loop sequence data The three pigs in the Northeastern region (Mong Cai, Ha Lang, and Huong pigs) share the sister taxa pair with the Lantang pigs (in southern China, the p distance from 0.0006 - 0.0016). Two pigs of the Northwest region (Muong Khuong pigs and Muong Lay pigs) have close genetic relationship with Bamei pigs (Yellow river region with p distance of 0.0012). The results of phylogenetic relationship presented on the tree are consistent with the prediction about the closeness between Huong and Ha Lang varieties when they have similar morphological and geographical distribution. A relatively close p distance exists among the group of Vietnamese pigs is 0.0039 ± 0.00112. The Vietnamese I pigs breed is in the same branch as the Banna Mini pigs (in the Mekong region, with a p distance of 0.0006, the bootstrap value is 99%). The p distance of the I breed 19 is the farthest from the remaining 5 indigenous pigs in Vietnam, specifically the I pig breed has the p distance from Huong and Ha Lang is 0.0081; the distance with 3 breeds Mong Cai, Muong Khuong and Muong Lay is 0.00782. The relationship among these pigs is clearly shown in the phylogenetic tree in Figure 3.11. For Mong Cai pigs, the taxon MongCai_TN has the mtDNA sequence published by Tran Thi Thuy Nhien, et al. Other Asian pigs are located very far from the taxon from our findings. 2.2.6. Phylogenetic tree based on complete coding sequence data The close relationship of Vietnamese pigs (Huong, Ha Lang and Mong Cai) was once again confirmed in trees based on the complete sequence (Figure 3.12). Along with the similarities in morphological characteristics between Huong and Lantang pigs in Guangdong province, southern China. From this phylogenetic relation, it is possible to make an assumption about the common origin of the Vietnamese native pigs group with Lantang pigs stemming from past traffic activities between Guangdong Province of China and the provinces of the mountainous border areas, North Vietnam. The average p distance determined is relativel