| Livestock Research for Rural Development 33 (6) 2021 | LRRD Search | LRRD Misssion | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
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
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.
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.
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).
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 4°C 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.
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).
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.
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.
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 |
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 |
|||
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 |
Second Parity |
Three Parity |
Average |
|
|
Piglets /litter |
Pig |
6.98a±0.07 |
7.79b±0.37 |
8.55c ± 0.13 |
7.77 ± 0.59 |
|
|
Survival piglets after 24 hours / litter |
Pig |
6.54a±0.07 |
7.63b± 0.47 |
8.32c ± 0.12 |
7.50 ± 0.55 |
|
|
Weaner /litter |
Pig |
6.54a±0.07 |
7.63b± 0.47 |
8.32c ± 0.12 |
7.49 ± 0.53 |
|
|
Weight at birth/pig |
g |
504.43a±12.65 |
514.28a±9.90 |
505.56a±9.54 |
508.09±9.16 |
|
|
Weight at birth/litter |
kg |
3.53a±0.18 |
4.00b ± 0.15 |
4.36c ± 0.32 |
3.96 ± 0.22 |
|
|
Weaning time |
Day |
60.13a±0.22 |
60.00ab±0.25 |
59.07b±0.56 |
59.73± 0.32 |
|
|
Weaning weight/pig |
kg |
3.76a±0.11 |
3.94a ± 0.09 |
3.98a ± 0.17 |
3.89 ± 0.44 |
|
|
Weaning weight /litter |
kg |
24.59a±1.40 |
30.06b±1.62 |
33.11c±1.23 |
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.
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 |
Second-Parity |
Third-Parity |
|||
|
|
|
|
||||
|
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 |
|||
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 |
AT |
Position |
Size |
Space |
||||
|
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 |
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 |
|||||||||
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.
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
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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