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Morphological and mitochondrial genome characterization of indigenous Dong Khe pig in rural areas of Northeast Vietnam

Bui Thi Thom, Tran Van Phung, Cu Thi Thuy Nga, Duong Thi Khuyen1, Dao Thi Hong Chiem1 and Nguyen Minh Duc2

Thai Nguyen University of Agriculture and Forestry, Tan Thinh Ward, Thai Nguyen City, Vietnam
nmduc@igr.ac.vn
1 Institute of Life Sciences - Thai Nguyen University, Tan Thinh Ward, Thai Nguyen City, Vietnam
2 Institute of Genome Research - Vietnam Academy of Science and Technology, No. 18, Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam

Abstract

Dong Khe pig is an indigenous pig breed mainly distributed in Vietnam, has delicious quality meat and the ability to adapt to hard natural conditions. However, at present, this pig is decreasing in number and only a few small population numbers. Therefore, our study focuses on the phenotypic evaluation, production attributes, and identification of the overall mitochondrial genome sequencing of these pigs. Over 60 randomly selected swine farms rearing village pigs in Thach An district, Cao Bang province were used for the study. Of which, two villages of Trong Con and Duc Thong accounted for the highest proportion, 28.27%, and 28.06%, respectively. Morphological traits and reproductive performance were measured. With 481 Dong Khe pigs of 1,332 pigs (36.11%) raised in Thach An district. Among 120 livestock households surveyed, the ratio of hygiene and periodic vaccination households accounts for 74.17%. The coats of Dong Khe pig are relatively thick and coarse, with a nonspecific black-and-white cavity. The hair and skin on the head, ears are black, with white streaks shaped from the forehead to snout. With the tenderloin color of Dong Khe pig together with the average value of the Minolta L* index (luminosity) from 48.02 to 48.95 and bright red, Dong Khe pork is considered to meet the export requirements into the most demanding markets such as Japan and Europe. In addition, the results also showed that the genome length of Dong Khe pigs was 16,755 base pairs, with 2 rRNA genes (12S and 16S), 22 tRNA and 13 mRNA genes, and %AT, GC skew, and AT skew were 60.39, -0.33 and 0.15, respectively. Phylogenetic trees were established based on the complete mitochondrial genome sequences of the Dong Khe breeds and some indigenous pig breeds in Vietnam and the world. The Dong Khe breed is closely related to the Lantang pig in the South of China and the Mong Cai breed in Vietnam. Our results are completely consistent with previous studies and contribute to providing complete information about the Dong Khe pig breed in Vietnam.

Keywords: complete mitochondrial genome, Dong Khe pig, morphological descriptors, phylogenetic trees


Introduction

In Vietnam, Thach An is a district located in the southeast of Cao Bang province, with a natural area of about 690.79 km2. The population of the district is about 30,850 people with about 6 main ethnic groups living together, of which about 90% of the population lives in rural mountainous areas and mainly works in agriculture. In Thach An, pig farming plays an important role in the life, economy, and society of ethnic minorities. The local pig breed in Thach An district (Dong Khe pig, or Lai Pheng pig) has a long-standing attachment to the lives of ethnic minorities. Currently, this breed of pig has disappeared a lot, only a few are distributed in communes such as Trong Con, Quang Trong, and Duc Thong, where geographical conditions are remote, mountains are dangerous and special. The transportation system is not convenient, so mixing with other imported breeds and domestic pigs from other areas in the region is almost impossible. Pigs have a compact body with coarse color having black and white streaks, thick coarse long hair, and a short tail. These pigs have many similarities with the Mong Cai pig, the relatively straight back (not saggy back like Mong Cai pig), the small legs, the small ears pointing up, small head, the straight face, straight nose bridge with white hairstreak nasal bridge. Mature pigs have a weight of about 70 - 80 kg. Another special advantage is that the quality of fatty meat is very delicious and popular with consumers. However, due to the low productivity and efficiency of breeding, while the selling price is not different from the hybrid pigs, many people do not want to raise this breed. Moreover, due to the social differences and low educational attainment of the people in remote areas, the Dong Khe pig quality for breeding has not been improved. During the development process, along with the breed advances introduced into production practices, the number of Dong Khe pigs is decreasing day by day. From that point of view, the risk of extinction of the local pig, which has long been associated with the life of the ethnic minorities is increasing.

Additionally, The mammalian mitochondrial genome is a closed-circular, double-stranded DNA molecule of about 16.6 kb. The two strands on the double strand of mitochondrial DNA are distinguished into heavy (H) and light (L) strands based on their proportions of nucleotides (Kasamatsu and Vinograd 1974). Most of the information is encoded on the heavy strand, with genes coding for 2 rRNAs, 14 tRNAs, and 12 proteins. The light strand encodes 8 tRNAs and a large protein. All protein products are components of the enzyme complexes involved in oxidative phosphorylation (Chomyn et al 1986). Several protein-coding genes overlap in many cases, and a part of the ending trilogy that is unencrypted is generated after transcription by a post-transcription polyA-tag (Ojala et al 1981). Although very small in the overall size of an organism's whole genome, the mitochondrial genome still being considered one of the most common molecular diversity markers in animals for decades (Avise and Arnold 1987; Moritz et al 1987).

However, up to now, there is no literature on the physical characterization and mitochondrial genome of Dong Khe pig. Therefore, the present study was carried out to study the phenotypic traits, production performance, and mitochondrial genome sequence of this pig. The complete mitochondrial genome sequences obtained were used for analysis to find the genetic relationship of Dong Khe pig with some indigenous pig breeds in Vietnam and the world.


Material and method

Morphological characteristics, growth performance and reproductive of the Dong Khe pig
Investigation and survey

Based on the methods of Henk Vander Akker (1998), including (1) Secondary data and documents of the authorities: Collected from economic reports, agricultural development policy documents of Department of Agriculture and Rural Development of Cao Bang province; Thach An District People's Committee; Department of Agriculture and Rural Development of Thach An district; People's Committees of communes in Thach An district. (2) Primary data: Through questioner on the basis of breed survey and participatory rural rapid assessment (PRA): Village survey: in 8 pig-raising communes of Thach An district; Household survey: In some communes with the high distribution density of Dong Khe pigs, the total number of questionnaires was 120.

Using the questionnaires prepared in advance according to each survey content and open questionnaire for people to fill in. Participants in the interview to collect data included leaders of localities (sectors), all levels, farming households, and households trading veterinary services. The main investigation indicators: distribution of Dong Khe pigs, number and structure, the density of the herd compared to other domestic animals in the region; breeding scale; breeding methods of the people; results of animal husbandry in the population, veterinary situation, and veterinary work in the locality.

Morphological characteristics and growth performance

Conducting research directly on locally raised Dong Khe pigs (100 pigs), description of hair color, texture, and appearance characteristics. All data collected from the questionnaire survey were coded and entered in Excel spreadsheets (MS EXCEL-2007) and analyzed using the Statistical Analysis Software (SAS 9.1). The body weights in kilogram were recorded with the aid of a weighing scale while the linear body measurements in centimeters were recorded using a tape. Mean values and standard error for body weight and linear body measurements were calculated using the General Linear Model Procedure of SAS software. The simple Linear Correlation procedure of SAS was used to establish the strength of linear relationships and the association between the different linear body measurements together with the body weight. Linear and multiple regression analyses were performed using the PROC REG procedures, and the stepwise model selection option. Prediction equations that related to body weight and linear body measurements were chosen based on values of the adjusted R2 (R-squared) and RSD (Residual Standard Deviations).

Mitochondrial genome analysis
Sample collection and DNA extraction

Five ml blood sample from the jugular vein of Dong Khe pigs was collected and transferred into blood tubes supplemented with EDTA anticoagulants. The samples were stored at 4C by ice during transfer to the laboratory. According to the standard phenol-chloroform method of Sambrook and Rusell, 2011, total DNA from blood samples was extracted.

Primer design and amplification of mtDNA by the polymerase chain reaction (PCR)

The list of 30 primers was designed based on the genomic sequence in GenBank and references (Hieu et al 2017; Thuy et al 2018; Wu et al 2007). PCR process for the whole mitochondrial genomic sequencing was performed and PCR product was examined by electrophoresis on 1% agarose. Purification of PCR product was performed using QIAquick PCR Purification Kit (Qiagen).

Sequencing, Structure and Phylogenetic analysis

The ABI3500 DNA sequencer system was used for sequence analysis. Reading and editing were done on the software BioEdit v7.2.5. DNA Dragon v1.6.0 software ( http://www.dna-dragon.com/) was used to identify and join the overlapping parts. The final sequence obtained was the complete mitochondrial sequence and was saved as a FASTA file. The GC skew and AT skew were calculated by the following formula: GC skew = (G–C)/(G+C) and AT skew = (A–T)/(A+T) using DAMBE v6.3.17 software, MITOS, and DOGMA. The D-loop sequence and the whole mitochondrial genome sequence along with the published pig breeds sequences were coupled through the MUSCLE algorithm of MEGA6 software to identify similar locations. Bayesian analysis algorithm on BEAST v1.8.3 software with initial setup Yule process was used for calculation, and Figure Tree v1 software 4.2 was then used to draw phylogenetic trees.


Results and discussion

In terms of distribution, Thach An district (Cao Bang province) has 16 villages and towns, however, Dong Khe pigs can only be raised in 8 villages and Dong Khe town (Trong Con: 28.27%; Duc Thong: 28.06%; Quang Trong: 12.26%; Others: 6.23 – 11.01%) (Table 1). The results show that Dong Khe pigs are distributed mainly in upland communes, the living area of ​​ethnic minorities, where the infrastructure and transportation are difficult, income is mainly from agriculture (Hao and Thanh 2010).

In addition, the rate of breeding in the wild form (free-range, 100% forage by themselves) is low (5.83%). 23/120 households supplemented refined food to pigs in the wild breeding form (19.17%). The number of households raising semi-wild, grazing during the day and locked up at night, is 30.83%. Dong Khe pigs are completely kept in cages (44.17%) mainly in households in Dong Khe, Trong Con and Duc Thong (Table 2). Free-range farming practices in forests and villages are also adopted by ethnic minorities in Son La province in pig breeding (Thuy and Hung 2008).

Table 1. Numbers and distribution of Dong Khe pig

Item

Numbers

Ratio (%)

Total investigated individuals

1,332

100

Dong Khe breed

481

36.11

Other breed

851

63.89

Distribution in Villages

Trong Con

136

28.27

Duc Thong

135

28.06

Quang Trong

59

12.26

Dong Khe

53

11.01

Duc Xuan

36

7.48

Minh Khai

30

6.23

Thuy Hung

24

4.99

Duc Long

8

1.66



Table 2. Husbandry practices for Dong Khe pig

Item

Number

Ratio
(%)

Total households surveyed

120

100

Wild farming

7

5.83

Wild farming with supplementary food

23

19.17

Semi-wild farming

37

30.83

Captive condition

53

44.17

Households have veterinary hygiene and periodic vaccination

89

74.17

Households have veterinary hygiene but not using vaccine

31

25.83

Dong Khe pigs have the ability to use a variety of raw and locally available feed such as sweet vegetables, wild vegetables, banana stem (green food) or refined feed such as rice bran, flour. cassava, maize. In which, green food is used the most. As a result, the source of Dong Khe pig feed is plentiful, diversified, inexpensive and easy to find. The initiative of local food availability is very significant in the event of an escalation in animal feed prices. However, in order to promote the economic value and adaptability of indigenous pigs, further investment in knowledge of husbandry as well as management skills for the people is essential.

In terms of morphological

The coat of Dong Khe pig is relatively thick and coarse, with a nonspecific black-and-white cavity, divided into three groups. The first group has white streaks covering the entire abdomen and 4 legs, with black spots on the back (Figure 1). This group has 214 pigs out of a total of 481 Dong Khe pigs (44.49%). The second group consists of 185 pigs (38.46%), with white extending all the way through the abdomen, most of the back on the black, white streaks extending from the forehead to the nose (Figure 2). The final group accounted for 17.05% (82/481 pigs), with white extending from the back to the abdomen and 4 legs, black spots on the buttocks and back, white streaks extending from the forehead (Figure 3). Comparison of coat color of Dong Khe pig with some other indigenous pig breeds shows a clear difference. For example, the Mong Cai pig has a black head, back and rump; a wedge-shaped white spot in the middle of the forehead; the shoulders with a white leather band extending down the entire abdomen and 4 legs, giving the black back and hips a saddle shape; a translucent outline between the black and white part, in which having white skin and black coat; sparse and coarse coat (Duc et al 2005). However, there is a clear difference in Dong Khe pig that there is no boundary between the black and white part, the white skin, the black coat like Mong Cai pig. In addition, the white streak on the forehead is quite long. In general, the coat color of Dong Khe pigs is very diverse.

Figure 1. The morphological of Dong Khe pigs


Table 3. The coat color of body parts (n= 100)

Body
part

Character

Number

Ratio
(%)

Head

The hair and skin of the head, ears are black, with white streaks shaped from forehead to snout

100

100

The hair and skin of the head and ears are different

0

0

Snout

The hair and skin are white, pointed and long

100

100

Back

White fur and skin with black spots

48

48

Coat and skin of back has black color

32

32

The fur and skin on the back are white.

20

20

Abdomen

White hair and skin on the abdomen

100

100

Leg

White fur, skin on legs, nails; small legs

96

96

Black fur and skin on legs

4

4

Tail

Black color

100

100

n- No. of animals

Reproductive traits

Of Dong Khe sows, compared with some other breeds of pigs, Dong Khe sows when in heat often manifest clearly and quietly, such as: fewer screams, less red and swollen, wet water, no oily water vagina (Van and Ha 2005).

Table 4. Mean performances for reproductive traits of Dong Khe pigs

Parameters

Unit

n

Average SE

Age at first mating

day

20

148.55 2.35

Body weight at first mating

kg

20

19.57 0.45

Time mating

day

20

4.96 0.13

Estrous cycle

day

20

21.07 0.41

Gestation period

day

20

113.94 0.29

Time for mating again after weaning

day

20

18.73 0.39

Age of boars at first mating

day

5

225 0.65

Weight of boars at first mating

kg

5

43.50 1.25



Table 5. Reproductive productivity of Dong Khe sows

Index surveyed

Unit

First Parity
(n=6)

Second Parity
(n=6)

Three Parity
(n=6)

Average

Piglets /litter

Pig

6.98a0.07

7.79b0.37

8.55c 0.13

7.77 0.59

Survival piglets after 24 hours / litter

Pig

6.54a0.07

7.63b 0.47

8.32c 0.12

7.50 0.55

Weaner /litter

Pig

6.54a0.07

7.63b 0.47

8.32c 0.12

7.49 0.53

Weight at birth/pig

g

504.43a12.65

514.28a9.90

505.56a9.54

508.099.16

Weight at birth/litter

kg

3.53a0.18

4.00b 0.15

4.36c 0.32

3.96 0.22

Weaning time

Day

60.13a0.22

60.00ab0.25

59.07b0.56

59.73 0.32

Weaning weight/pig

kg

3.76a0.11

3.94a 0.09

3.98a 0.17

3.89 0.44

Weaning weight /litter

kg

24.59a1.40

30.06b1.62

33.11c1.23

29.25 2.12

Parity /year

litter/year

1.82 0.04

ab : Means in the same rows with varying superscript differ significantly (p< 0.05)

The number of weaned pigs/litter is one of the criteria to evaluate the reproductive performance and efficiency of sows. This value depends on the vitality of the piglets during the nursing period of mother pigs, the nurturing properties, and the care and nurturing conditions of the breeding facilities for the mother and piglets. In addition, these values also affect piglet weight at weaning. The follow-up results showed that in 3 litters, the average rate of piglets surviving 24 hours maintained until weaning was 6.56 piglets/litter. In contrast, the study of (Ton et al 2007) on F1 hybrid sows (Yorkshire x Mong Cai) showed that the number of newborn piglets alive after 24 hours was higher than the number of piglets weaned. After 24 hours, the average number of surviving piglets of the first 3 litters in F1 hybrid sows was 11.32 piglets/litter, while the number of weaned piglets was only 10.37 piglets/litter. With a weaning time of 59.73 days, Dong Khe piglets had an average weaning weight over the first 3 litters of 3.89 kg/piglet.

Meat quality of Dong Khe pigs

After investigation, we conducted the quality assessment and analyzed some chemical components of the finished meat from Dong Khe pigs. The tenderloin color of Dong Khe pig with the average value of the Minolta L* index (luminosity) ranged from 48.02; 48.95; 48.85 and bright red. The toughness of pig tenderloin between groups was equivalent 4.55 - 4.66 - 4.75 kg/cm2. The results of chemical composition analysis of the experimental pork showed no difference in the ratio of meat chemical components, especially the protein ratio of pork. Lean rump dry matter was higher for lean shoulder meat and lean rump meat in Lot 2 (24.84%) and lower than in first-parity (26.69%) and third-parity (26.55%). The protein ratio in the buttock was higher than that of the shoulder meat in the difference litters. The difference between the shoulder meat and the buttocks was quite clear (P <0.05). The percentage of fat in shoulder meat was higher than butt meat. Total lipids in the shoulder and buttocks in second-parity (8.13%; 2.65%) were both low compared to first-parity (10.14% - 4.08%) and third-parity (8.59% - 4.15%).

Table 6. Results of evaluation of experimental pork quality

Parameters

First-Parity
(n=3)

Second-Parity
(n=3)

Third-Parity
(n=3)

Color parameters value and Carcass traits

Color of meat - Minolta lightness (L*)

48.02 1.16

48.95 1.44

48.85 1.32

Meat toughness (kg/ cm2)

4.55 1.25

4.66 1.53

4.75 1.24

pH of the slaughtered carcass

6.8 0.31

7.1 0.14

7.2 0.14

pH of carcass at 45 minutes after slaughter

5.8 0.21

5.6 0.24

5.5 0.26

Cholesterol (mmol/L)

2.62 1.21

2.75 0.95

2.86 1.82

Triglyceride (mmol/L)

2.35 1.39

2.51 0.92

2.63 1.15

Chemical composition of Dong Khe pork (% in fresh meat)

Dry matter

Ham

26.69 0.03

24.84 0.04

26.55 0.21

Shoulder

29.69 0.12

28.34 0.08

28.65 0.16

Total Protein

Ham

20.88 0.17

20.74 0.11

20.77 0.17

Shoulder

18.06 0.16

18.64 0.13

18.59 0.11

Total Lipit

Ham

4.08 0.28

2.65 0.09

4.15 0.32

Shoulder

10.14 0.36

8.13 0.08

8.59 0.41

Total mineral

Ham

1.37 0.09

1.34 0.02

1.35 0.42

Shoulder

1.25 0.02

1.31 0.04

1.24 0.61

Gene annotation

The mitochondrial genome sequence of Dong Khe pig was 16,775 base pairs (bp) in length (Figure 2). The base compositions were 34.63%, 26.24%, 13.36% and 25.76% for A, C, G and T, respectively, and the AT content accounted for 60.39%. Based on structural analysis data, Dong Khe pig mitochondrial genome included 2 genes encoding ribosome RNA, 13 genes encoding proteins, 22 genes coding for transport RNA, and a control area of D-loop, and distributed in both two heavy and light mitochondrial fibers. The D-loop region had a length of 1,336 bp and was located between two genes encoding tRNA Phe and tRNA Pro.

Figure 2. The structure of the Dong Khe pig mitochondrial genome built by the GenomeVX

There were also nineteen small noncoding regions scattered across the mitochondrial genome ranging in length from 1 to 32 bp and four regions where the gene sequences overlapped from 1 to 40 bp. The lengths of the rRNA 12S and 16S genes were 960 and 1,570 bp, respectively. Two genes coding for transport RNAs ranged in size from 59 to 113 bp, including two Ser tRNA (AGC and TCA anti-codons) and two Leu tRNAs (anti-codon CTA and TAA). The 14 genes were on the heavy strand, and eight were on the light strand of the mitochondrial genome. The total length of the thirteen genes coding for the proteins was 11,382 bp with genes ranging in length from 201 to 1,818 bp, of which the longest gene was ND5, the gene with the shortest size was ATPase8. Of the thirteen genes coding for proteins, except for the ND4l and ND6 genes, which had the initial trilogy of GTG and ATT, the remaining genes had a starting code of ATG (COX1, COX2, COX3,ATPase6, ATPase8, ND1, ND4, and Cytb) or ATA (ND2, ND3, and ND5). The termination codes for these genes are either TAG or TAA, in addition, there were five genes (COX2, COX3, ND1, ND4, and ND4l) that carried the incomplete T-- terminating trilogy, which was assumed to form the trio terminates TAA after undergoing poly A-tagging during post-transcription (Table 7).

Table 7. The organization of the whole mitochondrial genome

Gene

Codon

Strand

GC
skew

AT
skew

Position

Size
(bp)

Space
(bp)

Start

End

Anti-codon

Start

End

D-loop

-

-

-

H

-

-

1

1336

1336

0

tRNA Phe

-

-

TTC

H

-

-

1337

1406

70

0

12S rRNA

-

-

-

H

-0.15

0.25

1407

2366

960

0

tRNA Val

-

-

GTA

H

-

-

2372

2439

68

5

16S rRNA

-

-

-

H

-0.11

0.24

2440

4009

1570

0

tRNA Leu2

-

-

TAA

H

-

-

4010

4084

75

0

ND1

ATG

T--

-

H

-0.4

0.13

4087

5040

954

2

tRNA Ile

-

-

ATC

H

-

-

5042

5110

69

1

tRNA Gln

-

-

CAA

L

-

-

5108

5180

73

-3

tRNA Met

-

-

ATG

H

-

-

5182

5251

70

1

ND2

ATA

TAG

-

H

-0.49

0.24

5252

6292

1041

0

tRNA Trp

-

-

TGA

H

-

-

6294

6361

68

1

tRNA Ala

-

-

GCA

L

-

-

6368

6435

113

6

tRNA Asn

-

-

AAC

L

-

-

6437

6511

75

1

tRNA Cys

-

-

TGC

L

-

-

6544

6609

66

32

tRNA Tyr

-

-

TAC

L

-

-

6610

6674

65

0

COX1

ATG

TAA

-

H

-0.2

0

6676

8217

1542

1

tRNA Ser2

-

-

TCA

L

-

-

8224

8292

69

6

tRNA Asp

-

-

GAC

H

-

-

8300

8367

68

7

COX2

ATG

T--

-

H

-0.31

0.12

8368

9054

687

0

tRNA Lys

-

-

AAA

H

-

-

9056

9122

67

1

ATPase8

ATG

TAA

-

H

-0.54

0.2

9124

9324

201

1

ATPase6

ATG

TAA

-

H

-0.46

0.11

9285

9962

678

-40

COX3

ATG

T--

-

H

-0.32

0.05

9965

10747

783

2

tRNA Gly

-

-

GGA

H

-

-

10749

10817

69

1

ND3

ATA

TAA

-

H

-0.48

0.16

10818

11162

345

0

tRNA Arg

-

-

CGA

H

-

-

11165

11233

69

2

ND4l

GTG

T--

-

H

-0.33

0

11234

11527

294

0

ND4

ATG

T--

-

H

-0.5

0.14

11524

12900

1377

-4

tRNA His

-

-

CAC

H

-

-

12902

12970

69

1

tRNA Ser1

-

-

AGC

H

-

-

12971

13029

59

0

tRNA Leu1

-

-

CTA

H

-

-

13030

13099

70

0

ND5

ATA

TAA

-

H

-0.46

0.15

13100

14917

1818

0

ND6

ATT

TAA

-

L

-0.56

0.35

14907

15431

525

-11

tRNA Glu

-

-

GAA

L

-

-

15432

15500

69

0

Cytb

ATG

TGA

-

H

-0.38

0.09

15505

16641

1137

4

tRNA Thr

-

-

ACA

H

-

-

16645

16712

68

3

tRNA Pro

-

-

CCA

L

-

-

16712

16775

64

0

Notes: bp: base pairs; rRNA: ribosomal RNA; 16S rRNA & 12S rRNA: large & small rRNA subunit; tRNA: transfer RNA and italic words are replaced by one amino acid code; ND1-6 and ND4l: genes encoding nicotinamide dinucleotide dehydrogenase subunits 1 to 6 and 4l; ATPase6 and 8: genes encoding adenosine triphosphatase subunits 6 and 8; COX1 to 3: genes encoding cytochrome c oxidase subunits 1 to 3; Cytb: gene encoding cytochrome b. T-- indicates the incomplete termination codon

Analytical data on mitochondrial genome length, the nucleotide composition of the sequence, position and length of genes, starting and ending triplet codes for proteins coding for genes, and triadic coding genes encode for the transport RNAs, showed that our results were similar to in other pig breeds (Shen et al 2016; Zhang and Xie 2015). In addition, the GC skew and AT skew index showed the degree of deviation in the nucleotide composition of the mitochondrial genome sequence. These indexes were derived from the hypothesis of an unbalanced substitution between two DNA strands (Sueoka 1995). Most of the genes coding for proteins and rRNA were on a heavy strand, these genes had negative GC skew and positive AT skew, which indicated that the cytosine and adenine content on the same strand were higher than that of Guanine and Thymine. The gene with the lowest GC skew value was ND6 (-0.56) and the gene with the highest GC skew was rRNA 16S (-0.11), ND6 had an AT skew of 0.35, which was the gene having the highest difference between A and T. Our results of calculating GC skew and AT skew of the genes showed that all genes, except COX1 and ND41 (AT skew = 0), had a certain bias in nucleotide composition. This might be due to the accumulation of mutants and selection during the resulting strain. In order to find out the derivative type relationship, we compared these parameters between Dong Khe pig with international and Vietnamese indigenous breeds. Because of the impossible to compare individual genes of pig breeds to each other, we decided to select the entire mitochondrial sequence including both extensive and antagonistic to construct the phylogenetic tree.

To find the differences in sequence composition for Dong Khe pigs, sequence-specific indices of the percentage AT, GC skew and AT skew were compared between the sequence of Dong Khe pigs and other pigs. The values of the ratios of percentage AT, GC skew, and AT skew of the whole mitochondrial gene of Dong Khe pigs were 60.39; -0.33 and 0.15, respectively, and the D loop sequences were 60.75; -0.33 and 0.16, respectively. The values ​​of the percentage AT, GC skew and AT skew indices of the published sequences were (60.19; 60.89), (-0.34; -0.30) and (0.12; 0.15), respectively. The percentage AT, GC skew and AT skew values ​​of the published D loop sequences were (60.73; 61.84), (-0.38; -0.31) and (0.15; 0.19), respectively. Generally, it can be seen that GC skew was negative and AT skew was positive in both sequence regions. Thus, the tendency to change nucleotide composition in the sequence regions and between pig breeds is not different. At the same time, if comparing the indices between the two D-loop sequence regions and the complete mtDNA sequence, it could be seen that the D loop in most pig breeds was more AT and had a higher degree of variation in nucleotide composition than the whole mtDNA genome sequence. However, the comparison showed that the D-loop sequence region of Dong Khe pigs had higher AT content than the AT percentage of the whole sequence, the difference between the Cytosine and Guanine ratio of D-loop was also different when compared with the complete sequence. Comparing Dong Khe pigs with other breeds, the percentage AT of the entire mitochondrial genome and the D-loop region of this breed was determined to be the lowest (60.39% and 60.75%, respectively). The D-loop of Dong Khe pigs also showed less GC variation than most other pigs (Table 8).

Table 8. Geographical area and sequencing parameters of pig breeds in Vietnam

Place

Species

GenBank
ID

Complete sequence

D-loop sequence

Ref.

%AT

GC skew

AT skew

%AT

GC skew

AT skew

Vietnam

Dong Khe

-

60.39

-0.33

0.15

60.75

-0.33

0.16

I

KX094894

60.65

-0.33

0.15

60.09

-0.32

0.16

Hieu et al 2017

Muong Khuong

KY432578

60.76

-0.33

0.15

61.34

-0.34

0.15

Ha lang

-

60.46

-0.32

0.15

60.84

-0.34

0.15

Tuan et al 2018

Huong

KY964306

60.77

-0.33

0.15

61.44

-0.35

0.16

Thuy et al 2017

Muong Lay

KX147101

60.89

-0.33

0.15

61.23

-0.34

0.15

Thuy et al 2018

Mong Cai

-

60.49

0.33

0.15

60.87

-0.33

0.16

Thuy et al 2019

- : The complete sequence has not been announced

Phylogenetic analysis

The phylogenetic tree was built based on 14 published sequences of different pig breeds, including 7 native Vietnamese pig breeds and several breeds of pigs in the world: Duroc and Berkshire (Europe); Banna mini (Mekong river area); Lantang (South China); Aba, Bihu and Bamei (Changjiang River area) which was got from GenBank with code (Figure 3).

Figure 3. The phylogenetic tree from maximum likelihood analysis of mitochondrial sequences

Based on the complete mitochondrial genome sequence, our results showed a clear ramification between Asian and European pig breeds. The Asian swine group had cleavage to form three main separate clades. In which, Dong Khe and Lantang pigs formed a group, the Mong Cai breed was said to have a close relationship with the two above breeds.


Conclusions


Conflict of interest

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.


Acknowledgement

The authors gratefully acknowledge funding of the Ministry of Science and Technology, Vietnam. We also would like to thanks to Institute of Life Science, the Thai Nguyen University to facilitate the experiment.


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