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MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY - Bui Anh Tuan SEQUENCING AND ANALYSIS OF COMPLETE MITOCHONDRIAL GENOME OF INDIGENOUS PIGS IN SOME NORTHERN PROVINCES OF VIETNAM Major : Biotechnology Code : 94 20 20 SUMMARY OF BIOLOGY DOCTORAL THESIS Ha Noi - 2020 The research was completed in Graduate University of Science and Technology - Vietnam Academy of Science and Technology Scientific supervisor 1: Prof., Dr Nghiem Ngoc Minh Scientific supervisor 2: Assoc Prof., Dr Vo Thi Bich Thuy Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be defended in front of the graduate university's doctoral thesis evaluation council, meeting at Graduate University of Science and Technology - Vietnam Academy of Science and Technology At … ’, date of … 2020 The thesis can be found at: - Library of Graduate University of Science and Technology - Vietnam National Library INTRODUCTION The necessity of the thesis Under current breeding conditions, native pig breeds are declining in number, losing a local and national precious gene source, some breeds have become extinct or on the brink of extinction To date, there has not been a complete scientific research on the genome of indigenous pigs in Vietnam, thereby clarifying the origin and relationships arising from the species, in order to serve the conservation The establishment of molecular database on genetic resources of these pig breeds has not yet been fully implemented and exploited From the above urgent issues, we conduct research on the subject: “Sequencing and analysis of mitochondrial genome of indigenous pig breeds in some Northern provinces of Vietnam" Research purpose - Collecting complete data of mitochondrial genome of indigenous pig breeds in Vietnam (I, Mong Cai, Muong Lay, Huong, Muong Khuong and Ha Lang pigs) and deposited on the Genebank - Determine the composition, structure of mitochondrial genome, compare the difference in sequence, identify specific genetic characteristics of the six indigenous pig breeds, thereby contributing to the database to identification and conservation - Analysis genetic relationships, determine the origin and type of indigenous pig breeds in Vietnam The main research content - Investigating and surveying breeds, places of residence, collecting blood samples of the six indigenous pig breeds studied - Complete sequencing of mitochondrial genome of six indigenous pig breeds Assemble, determine the complete sequence of the mitochondrial genome, and annotate - Analysis of the composition and structure of the genome - Studying sequence polymorphism, comparing mitochondrial genome sequences of these pig breeds with some pig breeds in Asia and Europe - Reconstructing a phylogenetic tree based on the D-loop region sequence and the complete sequence of the mitochondrial genome, analyzing the phylogenetic relationship between Vietnamese indigenous pig breeds and some other pig breeds in the world Chapter LITERATURE REVIEW 1.1 Origin, classification and domestication of domestic pigs The ancient ancestors of pigs, wild boars, were hunted to provide food for the lives of primitive people All current pig breeds are considered to be the forms of Sus scrofa domestica Evidence of phylo-geographic showing that the domestication of pigs occurs many times in many parts of the world There is a view that today the ancestors of pigs are identified as primitive wild boars and their native land is Southeast Asia 1.2 Application of mitochondrial genome in the study of phylogenetic and traceability Determining the relationship of phylogenetic using mtDNA sequences based on the principle: information about evolution can be obtained through analysis of sequence data mtDNA has been widely used for phylogenetic research for the following reasons: First, the evolution of mtDNA in mammals takes place primarily from the replacement of individual nucleotide pairs, which is very rare when rearranging the major parts of the genome Second, the evolution rate of mtDNA is thought to be 10 times faster than that of nuclear DNA Third, mtDNA is inherited by the maternal line, is haploid and does not occur recombinant 1.3 Phylogenetic tree reconstruction and analysis 1.3.1 Phylogenetic tree A phylogenetic tree is a branched graph that represents the evolutionary relationship between living organisms, giving a hypothesis about how tree organisms are related The topological geometry of a tree determines the relationship of the entities represented on the derived tree Only one branch is connected between the two nodes Nodes represent taxonomies (taxons that are specific DNA or protein sequences), the node is the intersection or end point of two or more branches An operative taxon unit (OTU) is an existing taxon present at an outermost node or leaf 1.3.2 Phylogenetic analysis Analysis of molecular phylogenetic is divided into five steps: (1) Collecting and selecting sequences: collecting sequences from a database containing thousands of families of eukaryotes, or results from the BLAST tool (2) Aligning sequences: Use the sequence alignment of a group of homologous sequences.(3) Selecting substitution models in DNA and amino acid sequence (4) Tree reconstruction: There are four main methods for building trees: based on distance, maximum parsimony, maximum likelihood, and Bayes inference MrBayes evaluates an a priori probability distribution, which is the ability of a tree to satisfy observed data (5) Phylogenetic tree analysis: The accuracy of the tree was assessed by bootstrap analysis.Bootstrap describes the power of topology of a tree Determining the root of the tree helps to assess the overall direction of change The root can be determined based on the midpoint or by the out group Chapter SUBJECTS AND RESEARCH METHODS 2.1 Subjects and research locations Pigs of 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 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 '-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 multisequence, 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 (http://dambe.bio) 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 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 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 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 indigenous pig breeds Characteristics on the colour of fur and skin I pigs Fur is thin and coarse Skin is black but not glossy Total N Group A % 100 Mong Cai pigs - The body is black and white, black head, adjacent to the black and white hairs have a blur - 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 100 82 B 82,0 18 A 18,0 77 B 77,0 23 A 23,0 22 B 57,8 16 A 42,1 13 B 31,7 28 A 68,3 Muong Khuong pigs - Fur is pure black The fur is thin and soft 100 - Black fur has white spots on the tail and legs, the fur is soft and thin Huong pigs - Body and 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 - There are additional characteristics: in the middle of the forehead is a white point, the four legs are white 38 Ha Lang pigs - The belly is white and has a white shoulder strap, with a black strip similar to a saddle - There are additional characteristics: The forehead has a white point almost like a wedge 41 Muong Lay pigs - Black, straight back, nipple head 10-15cm 70 35 B 50,0 above the ground - There are more white spots on the legs, 11,4 A forehead, tail, back, straight nipples 10-15 cm from the face - There is more back slightly hammock, 27 A+ 38,5 slightly saggy belly, nipple head are not sag (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 Table 3.2 Survey results of weight and size of indigenous pigs Pig breeds Mass Long body ± SD I Mong Cai Huong Ha Lang Muong Khuong Muong Lay Chest ± SD High shoulders ± SD ± SD 40,34 ± 0,92 51,47 ± 0,32 40,55 ± 0,22 42,68 ± 042 85,76 ± 1,32 92,41 ± 1,35 86,73 ± 1,33 87,12 ± 1,33 83,93 ± 1,44 90,77 ± 1,46 81,31 ± 1,42 84,34 ± 1,43 39,58 ± 0,91 45,25 ± 0,93 42,56 ±0,36 43,68 ±0,98 52,64 ± 0,92 93,94 ± 1,32 90,52 ± 1,46 46,14 ±0,96 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 indigenous pigs Body shape characteristics (number of nipples) Total N % 9 100 18 15 83,3 23 20 86,9 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 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 Muong Khuong pigs - Muzzle long straight or slightly arched The forehead is smooth, the ear cup is hanging forward 11 East Asia Korean native pig WB-China 39,25 -0,33 0,15 northeast WB-Japan WB-Korea 39,19 -0,33 0,15 Yellow Bamei 39,21 -0,33 0,15 River Huzu 39,2 -0,33 0,15 Valley Berkshire 39,28 -0,33 0,15 Duroc 39,32 -0,33 0,15 Hampshire 39,33 -0,33 0,15 European Iberian 39,29 -0,34 0,15 Country Landrace 39,28 -0,33 0,15 Large White 39,27 -0,33 0,15 Pietrain 39,28 -0,34 0,15 WB-European 39,28 -0,34 0,15 Banna mini 39,28 -0,33 0,15 Dahe 39,24 -0,33 0,15 Mekong Thailand indigenous pig Region WB-Malaysia 39,18 -0,33 0,15 WB-Vietnam 39,26 -0,33 0,15 WB-Yunnan 39,28 -0,33 0,15 Lantang 39,22 -0,33 0,15 South Lanyu 39,25 -0,30 0,12 China WB-Fujian 39,19 -0,33 0,15 WB-Hainan 39,19 -0,33 0,15 Aba 39,21 -0,33 0,15 Bihu 39,81 -0,31 0,14 Yangtze Jinhua 39,22 -0,33 0,15 River Kele Region Taoyuan WB-Jiangxi 39,22 -0,33 0,15 Wei 39,2 -0,33 0,15 Xiang pig 39,18 -0,33 0,15 "-" : The complete sequence has not been announced 38,76 -0,35 0,15 38,2 -0,34 0,15 39,09 -0,33 38,59 -0,33 38,69 -0,34 0,16 0,16 0,16 38,69 -0,34 0,16 38,47 38,66 38,27 38,37 38,47 38,47 38,52 38,56 39,16 38,92 38,7 38,59 38,69 39,27 38,57 38,55 38,71 38,68 38,69 38,69 38,46 38,68 38,66 38,59 38,69 38,59 0,16 0,16 0,16 0,16 0,15 0,16 0,16 0,17 0,16 0,16 0,16 0,19 0,16 0,15 0,16 0,16 0,16 0,15 0,16 0,16 0,16 0,16 0,16 0,16 0,16 0,16 -0,34 -0,35 -0,36 -0,35 -0,35 -0,35 -0,35 -0,35 -0,34 -0,33 -0,33 -0,38 -0,34 -0,31 -0,34 -0,34 -0,33 -0,33 -0,34 -0,34 -0,34 -0,34 -0,34 -0,34 -0,34 -0,33 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 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 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 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 10 11 12 13 14 15 16 17 18 19 20 21 Genes tRNA Phe tRNA Val tRNA Leu2 tRNA Ile tRNA Gln tRNA Met tRNA Trp tRNA Ala tRNA Asn tRNA Cys tRNA Tyr tRNA Ser2 tRNA Asp tRNA Lys tRNA Gly tRNA Arg tRNA His tRNA Ser1 tRNA Leu1 tRNA Glu tRNA Thr Strand (+/-) + + + + + + + + + + + + + + Length (bp) 70 68 75 69 73 70 68 68 75 66 65 69 68 67 69 69 69 59 70 69 68 Nucleotides composition A 26 26 23 28 16 20 26 19 21 18 21 17 23 20 25 29 29 20 27 17 22 G 13 10 14 11 20 13 12 15 18 16 15 20 12 14 10 13 17 12 C 16 14 17 8 20 14 11 12 11 11 16 13 12 11 18 T 15 18 21 22 29 17 16 26 25 20 18 21 24 17 21 25 24 18 19 27 16 GC skew AT skew -0,10 -0,17 -0,10 0,16 0,43 -0,21 -0,08 0,30 0,24 0,14 0,15 0,29 0,14 -0,07 -0,13 -0,20 0,00 -0,14 0,08 0,36 -0,20 0,27 0,18 0,05 0,12 -0,29 0,08 0,24 -0,16 -0,09 -0,05 0,08 -0,11 -0,02 0,08 0,09 0,07 0,09 0,05 0,17 -0,23 0,16 14 22 tRNA Pro Total in length Average - 65 1509 68,59 13 486 22,09 19 297 13,50 10 264 12,00 23 462 21,00 0,31 -0,28 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%), 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 3.3.4 Phân tích cấu trúc bậc hai 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 bp), with the exception of tRNA Ser-1 with branch length of triplets is bp Particularly for tRNA Ser-1 due to its short genetic size, the dihydrouridine lobes (DHU) 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 (