Xác định và phân tích hoàn chỉnh trình tự hệ gen ty thể của 6 giống lợn bản địa tại một số tỉnh miền bắc việt nam tt tiếng anh

From the above urgent issues, we conduct research on the subject: “Sequencing and analysis of mitochondrial genome of 6 indigenous pig breeds in some Northern provinces of Vietnam".. Res

SUMMARY OF BIOLOGY DOCTORAL THESIS

Ha Noi - 2020

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

Technology

At … ’, date of … 2020

T he thesis can be found at:

- Library of Graduate University of Science and Technology

- Vietnam National Library

INTRODUCTION

1 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 6 indigenous pig breeds in some Northern provinces of Vietnam"

2 Research purpose

- Collecting complete data of mitochondrial genome of 6 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 6 indigenous pig breeds in Vietnam

3 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 6 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 6 Vietnamese indigenous pig breeds and some other pig breeds in the world

Chapter 1 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 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

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 (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 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

I pigs

Fur is thin and coarse Skin is black but not

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,

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

- 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

- There are more white spots on the legs,

forehead, tail, back, straight nipples 10-15 cm

from the face

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

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

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

- 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

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(%)

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

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

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-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

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