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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 4  |  Page : 412-419

Evaluation of genetic diversity and origin of song ma village dogs in Vietnam


1 Institute of Tropical Ecology, Vietnam - Russia Tropical Centre, Nguyen Van Huyen, Cau Giay, Hanoi, Vietnam
2 Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam

Date of Submission26-Aug-2021
Date of Acceptance14-Oct-2021
Date of Web Publication14-Dec-2021

Correspondence Address:
Dinh Duy Vu
Institute of Tropical Ecology, Vietnam-Russia Tropical Centre, 63 Nguyen Van Huyen, Cau Giay, Hanoi
Vietnam
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_202_21

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  Abstract 


Background: Vietnam's Song Ma village dog is a breed of indigenous dog found along the Ma River in Vietnam. They have many excellent traits, like their intelligence, agility, friendliness toward humans, and more importantly, they are very easy to train, and have the instinctive hunting capabilities of their wild canine ancestors. However, the exploration of genetic diversity and the origins of Song Ma village dogs in Vietnam have a serious lack of information. It is very difficult to promote the conservation of these dogs. Therefore, urgently needed in order to uncover and better understand the genetic architecture of Song Ma village dogs. Methods: We used 100 blood samples collected in Vietnam to estimate genetic diversity by sequencing the hypervariable-1 region. Results: We reported high levels of genetic diversity in the Song Ma village dog (Pi = 0.00912, Hd = 0.969, and Kt = 5.456). A total of 51 different haplotypes were identified in four haplogroups (A, B, C, and E). Furthermore, Song Ma village dogs were discovered in rare groups such as B1, B5, B6, B10, C2, and E1. Notably, no one in the haplogroup has the haplotypes (D and F). There were 49 single nucleotide polymorphisms, including 48 nucleotide base substitution or insertion changes and six nucleotide indel mutations found in the Song Ma village dog. A phylogenetic tree showed that Song Ma village dogs have a close relationship with dogs that originated from East Asia. Conclusions: This study has provided a valuable platform for breeding and conservation and management of the species in Vietnam.

Keywords: Genetic diversity, haplogroup, haplotype, hypervariable-1 (D-loop region), phylogenetic tree, Song Ma village dog


How to cite this article:
Bui XP, Pham TH, Tran HC, Phung TT, Ngo QD, Dinh T, Dam QT, Vu DD. Evaluation of genetic diversity and origin of song ma village dogs in Vietnam. Biomed Biotechnol Res J 2021;5:412-9

How to cite this URL:
Bui XP, Pham TH, Tran HC, Phung TT, Ngo QD, Dinh T, Dam QT, Vu DD. Evaluation of genetic diversity and origin of song ma village dogs in Vietnam. Biomed Biotechnol Res J [serial online] 2021 [cited 2023 Jun 10];5:412-9. Available from: https://www.bmbtrj.org/text.asp?2021/5/4/412/332456




  Introduction Top


The dogs are derived from the gray wolf (Canis lupus) in Eurasia at least 15,000–40,000 years ago.[1] They were bred in many locations in Asia, and then spread throughout Europe and to Africa, Oceania, and the Americas.[2],[3] These dogs live as human commensals. In the domestication process over centuries, specific traits such as size, shape, physiology, and behavior were developed. Therefore, they developed geographical characteristics of genetic differentiation.[4] The mitochondrial DNA (mtDNA) was investigated in 654 dogs in the world and showed previous mtDNA of domestic dogs from Eurasian wolves and the highest mtDNA diversity in East Asian dogs.[2] The Bali dogs' high genetic diversity was observed using microsatellites and single nucleotide polymorphism (SNP) markers.[5],[6] Boyko et al. have used the mitochondrial D-loop, 300 SNPs, and 89 microsatellite markers to analyze complex population structure in 318 African dogs. The results showed that African dogs are a mosaic of indigenous dogs descended from early migrants to Africa, and non-native, breed-admixed dogs and similar mtDNA haplotype diversity in African and East Asian village dogs.[7] Shannon et al. confirmed a large-scale survey of autosomal, mitochondrial, and Y chromosome diversity in 4,676 purebred dogs from 161 breeds and 549 village dogs from 38 countries using a semicustom 185,805-marker genotyping array. They demonstrated both isolation and gene flow had shaped genetic diversity in village dogs in relation to geographic structure. Many dog populations in Vietnam, India, and Egypt had minimal evidence of European admixture.[8]

In Vietnam, dogs are commonly bred around human settlements. However, many dogs can be complex mixtures of both indigenous dogs and nonnative breeds. Indigenous dogs contain genetic variants that are not found in nonnative dogs. A study on the genetic variation of indigenous dogs in Vietnam has not been reported to date. However, a study on the genetic frequency of ridgeback mutations in Phu Quoc ridgeback dogs was determined using real-time polymerase chain reaction (PCR) with four primer pairs.[9] Tran et al. showed the high frequency of rare halotypes E4 and E1 in Phu Quoc ridgeback dogs based on mtDNA analysis. The difference in length between E4 and E1 haplotypes is mainly due to the difference in motif repeat frequency in the control region. In order to preserve indigenous dogs, at present, we have a serious lack of information on genetic variants. It is very difficult to promote the conservation of these dogs.[10] Mitochondrial DNA is strongly conserved, has few duplications, evolves and has very short intergenic regions. It is widely used in studies of genetic diversity and systematics.[11],[12] Mutations often accumulate in the D-loop gene. Thus, this gene was chosen to study genetic structure and phylogenetics in animals.[13],[14]

This study investigated the genetic diversity of 100 individuals of Song Ma village dogs from four populations in two provinces (Thanh Hoa and Hanoi) in Vietnam, using nucleotide sequencing of the hypervariable-1 (HV1) region in the D-loop gene. The results provided a scientific basis for proposing solutions for breeding, breeding, and sustainable development of this species in Vietnam.


  Methods Top


Collection of samples

The blood samples of Song Ma village dogs were collected from Thanh Hoa province and Hanoi city in Vietnam. A total of 100 dogs were sampled; among them, 32 samples were collected from Muong Lat district, 32 samples were collected from Quan Hoa district; 32 samples were collected from Ba Thuoc district of Thanh Hoa province, and four were collected from Ha Noi City [Figure 1] and [Table 1]. Ethical statesmen to collect these samples, we had the permission of the Ministry of Agriculture and rural development-Vietnam and followed the regulations of administration of Muong Lat, Quan Hoa, and Ba Thuoc districts, Thanh Hoa province. We only collect blood samples of Song Ma village dogs for research purposes, not a commercial business. The study was approved by the Ethics and Research Committee of the Vietnam - Russia Tropical Centre.
Table 1: Sampling location Song Ma village dog in Vietnam

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Figure 1: Song Ma village dog in Thanh Hoa province, Vietnam (Photo: Dr. Huu Coi Tran)

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DNA extraction

Total genomic DNA was extracted using the QIAamp DNA Blood mini Kit (Qiagen-Germany) from blood samples obtained from scientific collections [Table 1] and [Figure 1]. The total DNA purity and integrity were tested by the Nanodrop ND-2000 spectrophotometer (NanoDrop Technologies, USA) and then diluted to a concentration of 20 ng/μl.

Polymerase chain reaction amplification

PCR reaction was performed in a 25 μl volume: 2 μl of genomic DNA (total 40 ng), 12.5 μl master mix 2X (Thermo Scientific) and 9.5 μl H2O deionized. The PCR amplifications were carried out on a Mastercycler X50s Eppendorf, under the following thermal cycler: Initial denaturation at 94°C for 2 min, followed by 40 cycles consisting of 1 min at 94°C for denaturation, 30 s alignment at the annealing temperature for each primer pair at 56°C and 1 min at 72°C for extension and 10 min at 72°C for the final cycle to complete the extension of any remaining products before holding the samples at 4°C until they were analyzed. The mitochondrial HV1 region was PCR amplified using the primer pairs DF: 5'-GCACCCAAAGCTGAGATTC-3'; DR: 5'-ACCTTGATTTTATGCGTGAGTT-3'.[15]

Sequencing of the HV1 region

The PCR products were purified by QIAquick PCR Purification Kit and directly sequenced using BigDye Terminator kits (Applied Biosystems, Foster City, CA, USA) on an automated sequencer. Sequencing of the 100 studies used the primers DF/DR. The DNA sequences were edited and concatenated using Chromas Pro (Technelysium Pty Ltd, Helensvale, Queensland, Australia) and aligned.[16]

Data analysis

Identification of haplotypes

The haplotypes types of H'mong dogs were identified, based on characters of sequences HV1 (D-loop) using an identification tool.[17]

Genetic diversity

Genetic parameters were analyzed with a proportion nucleotide diversity (Pi), haplotypes diversity (Hd), and a mean number of nucleotide differences (Kt) on the DnaSP.[18]

Phylogenetic tree

Chromas Pro 2.1.6 software was used to edit the sequences. Sequence alignments were made with Bioedit.[16] We used maximum likelihood (ML) and Bayesian inference (BI) to build phylogenetic trees.[19],[20] In ML and BI analyses, the best model for each partition of HV1 region was based on a general time-reversible model with a gamma shape parameter (G: 0.1002 in ML and 0.0332 in BI). The strength of nodal support in the ML tree was determined using 1000 replicates of the bootstrap (MLBS). We used Mega to analyze the p-distance between Song Ma village dogs.[21]


  Results Top


Characteristics of a dataset

The mitochondrial HV1 region was sequenced for 100 Song Ma village dogs. The total length of mitochondrial fragments was 670 bp after removing primer sequences. The sequences of mitochondrial HV1 of Song Ma dogs were deposited in GenBank (MZ505107-MZ505206). The obtained dataset contains 41 variable positions, including 38 sites carrying parsimony evolutionary information. The average nucleotide composition of analyzed the HV1 region with nucleotide T/U predominates at 30.8%, followed by type A with 26.8% and C accounting for 26.5%, and the lowest was G with 16%. The average substitution rate for Song Ma village was 1.2%. Compute MCL transitions/transversion with the Tamura-Nei model using a statistical method. The overall transition/transversion bias was R = 469.118.

Identification of haplotypes

Song Ma village dogs were grouped into four (A, B, C, and E) of six haplogroups (from A to F) based on the HV1 sequences. Among 100 Song Ma village dogs, 90 individuals (90%) revealed 20 haplotypes belong to haplogroup A, including A8, A9, A17, A29, A38, A44, A73, A84, A85, A105, A116, A121, A130, A132, A137, A138, A140, A143, A200, A223 and 24 new haplotypes (An1 to An24). Six individuals carried the haplotype B1, B5, B6, B10 of haplogroup B. Three individuals belong to haplogroup C with haplotype C1 and one new haplotype (Cn1) and one individual belongs to haplogroup E with haplotype E1 [Table 2] and [Table 3]. Haplotypes belong to common haplogroups (A, B, and C) (90/100; 99%). Furthermore, archaic haplotypes such as B1, B5, B6, and B10 (6/100; 6%) and C2, Cn01 were found in Song Ma village dogs (3/100; 3%). The most common haplotype type was A29, A73, which shared 17% (17/100) of the dogs. We identified 24 new haplotypes in haplogroup A and one new haplotype (Haplogroup C), while no new haplotypes were found in haplogroup D and E.
Table 2: Identification of different haplotypes of 100 sampled Song Ma village dog

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Table 3: Forty-nine polymorphic haplotypes in HV1 region of Song Ma village dog

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Genetic diversity of Song Ma village dog

There were 39 single nucleotide polymorphisms detected in the Song Ma village dog, comprising 49 nucleotide base substitutions or base insertions and six nucleotide indel mutations. Almost all mutations (48/49) were transversions, with only one nucleotide transition change (T15639A) [Table 3]. The genetic diversity of Song Ma village dogs was recorded with Pi = 0.00912, Hd = 0.969 and Kt = 5.456.

Genetic relationship of Song Ma village dog

We created a genetic relationship diagram of dog breeds using ML and BI methods to overview the genetic relationships between 100 individuals from Song Ma village dog in this study. The genetic distance between 100 individuals of Song Ma village dog ranged from 0 to 2.4%. The topologies with (-lnL) =1504.591 and 1956,637 were generated by the ML and Bayesian analyses, respectively. Phylogenetic analyses employing ML and BI methods yielded slightly different topologies only among referenced species, and only the ML tree is presented in [Figure 2]. Phylogenetic analysis grouped all dog sequences into previously described six distinct haplogroups (A–F). Song Ma village dogs were nested into four haplogroups (A, B, C, and E). Individuals have close relationships belonging to the same haplogroup and fall into distinct groupings on the tree diagram. In haplogroup B, the samples ML4, BT14, ML30, ML28, and BT10 were closely related to the breed dogs Saint Bernard (AY656743), Malamute Husky HM561540, Jack Russell (HM561530) with 98% MLBS and 100% Bayesian posterior probabilities (BPP). While, individual QH14 (Haplotype E1) formed a separate branch in haplogroup E and has a close relationship with the breed dogs Phu Quoc Ridgeback (KF757290, KF757297), Chinese Shar-Pei (GU079513), Chinese Gray Wolf (AY916815), Japanese Shiba (D83632), Japanese Jindo (AB622528), Korean Pungsan (EU789662), Korean Jindo (AF531741) with (MLBS = 65% and BPP = 68%). In haplogroup C, the individuals ML2, ML1, and ML32 nested in the same branch contained Dalmatian dogs (AY240092) from Croatia, Anatolian Shepherd Dogs (AY240068) from Turkey, and Cocker Spaniel (AY240078) from the United States with high supported values (MLBS = 100% and BPP = 91%). There were 90/100 individuals nesting in group haplotype A. There are no Song Ma village dogs carrying haplogroup D and F. The phylogenetic tree analysis showed that the Song Ma village dogs were closely related to dogs from East Asia (China, Japan, and Korea).
Figure 2: Phylogenetic tree of Song Ma village dog on mitochondrial HV1 region. The numbers above and below the branches represent the bootstrap 1000

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  Discussion Top


The researchers identified six haplogroups (A, B, C, D, E, and F) of dog breeds from around the world based on the SNP sequence of the D-loop region while 71.3% of dogs were haplogroup A and 95.9% were haplogroup A, B, or C. All three haplogroup (A, B, and C) were distributed equally throughout the world (97.4%) (except haplogroup C, which is not found in the Americas).[1],[22],[23] In contrast, three haplogroups (D, E, and F) were rare (less than 3%) found in Turkey and Spain (E and F), Scandinavia (group D); Japan, Korea, and China (E), Siberia (F).[22],[24],[25],[26] There are numerous distinct haplotypes within each haplogroup due to the variation of a single nucleotide. It demonstrates that the phenotypic distinctions between dog breeds worldwide are the consequence of migration and breeding between breeds. This explains why various breeds can have many different haplotypes and why one haplotype can be found in many different dog breeds.[10] Our research also demonstrates that out of 100 individuals of the Song Ma village dog, 51 haplotypes were discovered in four groups: A, B, C, and E, consistent with previous studies, and this information will help clarify the origins of Vietnam's Song Ma village dog.

This study revealed 51 distinct polymorphic haplotypes in Song Ma village dogs, including 44 haplotypes in haplogroup A (86.3%), four haplotypes in haplogroup B (7.8%), two haplotypes in haplogroup C (3.9%), and one haplotype in haplogroup E (1.9%). The number of different polymorphic haplotypes was found in 100 individuals of Song Ma village dogs (51 haplotypes) in the HV1 region, higher than Phu Quoc ridgeback dogs (16 haplotypes) and H'Mong Bobtail (25 haplotypes). Inbreeding was probably frequent in Phu Quoc ridgeback dogs on the isolated Phu Quoc island in Vietnam. The haplotype diversity of Phu Quoc ridgeback dogs was limited, whereas the normal dogs living on the mainland had more chances to mate with individuals harboring different haplotypes.[15],[27]

The primary criteria utilized in the exploration of genetic diversity such as Pi, Hd, and Kt. Hd measures the uniqueness of a particular haplotype in the population, which reflects an abundance of haplotype Pi (Fu, 1997) and Kt are haplotype mutation.[28],[29] The Song Ma village dogs had relatively high genetic diversity in species level (Hd = 0.9569 and Kt = 5.456), which was higher compared to Tibetan mastiffs (Hd = 0.808; Kt = 3.917), African dogs (Hd = 0.59 ± 0.11), Iberian breed dogs (Hd = 0.52 ± 0.11), Iberian village dogs (Hd = 0.46 ± 0.08), Phu Quoc ridgeback dogs (Hd = 0.9 ± 0.04).[26],[30],[31]

There are different theories about the domestication of dogs. According to Dayan et al.(1994), the Geometric Kebaran wolves were domesticated in Israel.[32] Pennisi et al. reported Italy as the origin of dogs.[33] Pang et al. stated that dogs might originate in the Yangtze River area.[21] Tanabe suggested East Asia as the origin of domestic dogs.[34] Serpell et al. claimed eastern Europe or western Asia as the origin of dog domestication because of archeological evidence.[35] The Wayne and Holdt study revealed the nuclear genome of dogs provided essentially by Middle Eastern or European wolves, which was supported by more consistent with the archeological records. Moreover, IGF1 gene sequencing analysis, which had been targeted at a small population size, further supported the above hypothesis.[36] Recently, researchers have developed a consensus on the domestication of dogs from wolves, which is parallel to the current study's findings. Our data revealed that the sequences of gray wolves were not formed in a monophyletic group but distributed among domestic dogs. In particular, each gray wolf constituted a sister group with Groups A, B, C, E, and F, respectively. This suggested that A, B, C, E, and F groups might have originated from different gray wolf groups. Vila et al. reported that the first DNA analysis results showed that the wolf was the ancestor of the dogs. None of the dog sequences differed from any wolf sequences by more than 12 substitutions, whereas dogs differed from coyotes and jackals by at least 20 substitutions and two insertions which clearly showed the wolf ancestry for dogs.[37] Our results suggest that domestic dogs originated from different groups of wolves. This is further supported by the fact that 86,3% haplotypes of Song Ma village dogs belong to haplogroup A, and a small percentage of haplotypes from other haplogroups (B, C, and E) appearing due to crossbreeding. Many studies have proven that it is possible to establish the origin of dog breeds based on rare haplotypes.[10],[22],[26] Analysis of haplotypes of dogs from East Asia showed that few haplotypes of haplogroup E could be found in dog breeds such as Shiba (Japan), SharPei (China), and Jindo (South Korea).[1],[23],[38],[39],[40] Our study indicated that Song Ma village dogs originated in East Asia (Vietnam, Thailand, China, Japan, and Korea), such as Phu Quoc Ridgeback, Korean Pungsan, Korean Jindo, Japanese Shiba, and Chinese Shar-Pei.


  Conclusions Top


Four haplogroups (A, B, C, and E) were identified in 100 individuals of Song Ma village dogs in Vietnam. None of the individuals belonged to two haplogroups (D and F). Among 51 recorded haplotypes, 25 new haplotypes were found in the current study. Furthermore, Song Ma village dogs were found in rare groups, such as B1 (ML4), B5 (BT14, ML28, ML30), B6 (BT10), B10 (ML11), C2 (ML2, ML15), and E1 (QH4). The current study indicated that Song Ma village dogs maintained high levels of genetic diversity. Our findings will provide a platform for breeding and conservation of Song Ma village dogs.

Acknowledgments

This research is funded by the Vietnam-Russia Tropical Center basis project: Select and improve the quality of native wolf dogs (Song Ma village dogs), which are recognized as national breed standards, serving security and defense (2020-2022). We are grateful to the administration of Muong Lat, Quan Hoa, and Ba Thuoc districts, Thanh Hoa province. Special thanks to local people who supported our survey.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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