MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND TRAINING AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES  THAI KE QUAN EVALUATION OF GENETIC DIVERSITY OF PHU QUOC RIDGEBACK DOGS BASED ON MITOCHONDRIAL DNA HYPERVARIABLE-1 REGION Speciality: Biotechnology Code: 9420201 SUMMARY OF DOCTORAL DISSERTATION HO CHI MINH CITY - 2019 This dissertation has been completed in Ho Chi Minh city Supervisors: Assoc Prof Dr Tran Hoang Dung Dr Chung Anh Dung Reviewer 1: Reviewer 2: Reviewer 3: The dissertation will be presented to the Institute-level Dissertation Defense Committee at the Institute of Agricultural Science for Southern Vietnam on , 2019 This dissertation can be found at: National library of Vietnam Library of Vietnam academy of agricultural sciences Library of Institute of Agricultural sciences for Southern Vietnam PREAMBLE Introduction Phu Quoc ridgeback dog, a valuable dog breed originating in Phu Quoc Island (Kien Giang province, Vietnam), is one of the three dog breeds in the world harboring a ridge of hair running along the back in the opposite direction from the rest of the coat Currently, Phu Quoc ridgeback dog has not yet been officially identified as a dog breed It is said that the Phu Quoc ridgeback dog originated from the Thai ridgeback dog because of the same characteristic of the ridge However, no scientific evidence has been provided to prove this hypothesis Mitochondrial DNA (mtDNA), especially the control region (CR), has a high mutation rate, leading to genetic differences between individuals The project "Evaluation of genetic diversity of Phu Quoc ridgeback dogs based on mitochondrial DNA hypervariable-1 region" assesses the genetic diversity in the CR of the Phu Quoc ridgeback dog population to understand the genetic relationships between Phu Quoc ridgeback dog with other dog breeds in the world, thereby serving as a basis for tracking the origin of this special dog breed Aim of the study Tracking the origin of Phu Quoc ridgeback dog based on information about the mitochondrial DNA hypervariable-1 region variation The new contributions and implications of the study - For the first time in the world, a web-based tool has been developed for haplotyping the mtDNA HV1 of dog - The genetic diversity of mtDNA HV1 of Phu Quoc ridgeback dog was evaluated successfully - A hypothesis about the origin of Phu Quoc ridgeback dog was given based on information collected from this study The meaning of the study 4.1 Scientific meaning Examined and identified the genetic diversity of mtDNA HV1 of Phu Quoc ridgeback dog, this study has provided scientific information supporting the tracking of the origin of Phu Quoc ridgeback dog A simple protocol for DNA extraction from dog hairs was also established, which therefore can be applied to other studies on genetic of dog The web-based tool for quick haplotyping of mtDNA HV1 has been available for public using via world wide web 4.2 Practical meaning This study has provided initial information and scientific data for tracking the origin of Phu Quoc ridgeback dog The study has also provided the information on genetic diversity of Phu Quoc ridgeback dog, which can be a premise for the genetic resources conservation of this valuable Vietnamese dog breed Subject and scope of the study 5.1 Subject This dissertation has carried out the study on 582 bp fragment in the mtDNA HV1 of dogs Totally, 200 individuals (100 Vietnamese village dogs and 100 Phu Quoc ridgeback dogs) have been examined in this study 5.2 Scope This study focuses on the 582 bp fragments in the mtDNA control region of 100 Vietnamese village dogs and 100 Phu Quoc ridgeback dogs Other dog breeds, which is used in determining the genetic relationships with Phu Quoc ridgeback dog, are used based on the availability of data and the information about the migration route of dog Not all dog breeds on the world are included in this study CHAPTER LITERATURE REVIEW 1.1 Phu Quoc ridgeback dog 1.1.1 Introduction to Phu Quoc ridgeback dog Ridgeback dog living on Phu Quoc island in Kien Giang province is a dog breed characterized by many valuable characteristics There are currently three dog breeds on the world harboring the ridgeback trait Of those, the Rhodesian ridgeback dog in South Africa and the Thai ridgeback dog are recognized by the Federation Cynologique Internationale (FCI), while Phu Quoc ridgeback dog of Vietnam has not been approved Many historical evidences have been reported to prove the special origin of Phu Quoc ridgeback dog, but the persuasion of these data is not high So far, the origin and the genetic relationship of this dog breed with others has not been thoroughly studied 1.1.2 Taxonomy of the Phu Quoc ridgeback dog 1.1.3 Morphological and behavioral characteristics of the Phu Quoc ridgeback dog 1.1.4 Studies on the origin and genetics of Phu Quoc ridgeback dog So far, there has been no study on the origin and genetics of Phu Quoc ridgeback dog outside of Vietnam Tran Hoang Dung et al (2017) initially analysed DNA and proved the genetic relationship between the Phu Quoc ridgeback dog harbouring the E4 haplotype with the Pungsang dog in North Korea 1.2 Inheritance of the ridgeback trait The ridge trait in ridgeback dog is caused by the autosomal dominant allele (R) on chromosome 18 Besides, dogs harbouring the homozygous dominant alleles (RR) have a higher risk of getting Dermoid sinus (DS), which negatively affect the dog’s health (Table 1.1) Table 1.1: Correlation between genotype and ridge, DS Genotype Ridge DS RR yes high risk Rr yes low risk rr none none 1.3 Mitochondrial DNA of dog 1.3.1 Structure of mitochondrial DNA of dog 1.3.2 Structure and inheritance of mtDNA control region The mtDNA control region of dog consists of three small regions, in which, the hypervariable region (HV1) and hypervariable region (HV2) are highly polymorphic The genetic diversity of HV1 is usually evaluated to study the origin of dog breeds Figure 1.4: Position and structure of mtDNA control region 1.4 Studies on the origin, phylogeny and genetic diversity of dogs 1.4.1 Studies using archaeological remains 1.4.2 Studies using modern genetic methods 1.4.2.1 Molecular markers Many different target DNAs have been used in genetic research in dogs such as microsatellite, Y chromosomes, COI, CytB Especially, many studies in the world have focused on the polymorphism of 582 bp fragment on mtDNA to learn about the origin and history of many different dog breeds 1.4.2.2 Studies on the origin of domestic dogs On the phylogenetic tree based on the 582 bp fragments of HV1, dogs’ and wolves’ haplotypes are grouped into six distinct haplogroups A, B, C, D, E, F There are totally 72.34% of dogs worldwide belonging to haplogroup A; 97.40% of dogs belongs to haplogroup A, B, C The distribution of haplogroups D, E, F are more geographic specificity which can be found only in particular regions These haplogroups are rare and believed to be the result of postdomestication wolf-dog bybridization Based on the distribution of haplotypes, Wang et al (2016) unraveled the place of origin and the migration route of dog on earth Figure 1.8: The place of origin and the migration route of domestic dog 1.4.2.3 Studies on the genetic diversity of dog breeds The genetic diversity of different dog breeds are varied, implicating the place and date of origin of dog breeds The haplotype diversitys of dog breeds living in isolated regions are low, such as Portuguese Castro Laboreiro dog (0.1) while the haplotype diversity of Shiba dog in Japan, which was bred from three bloodlines from three different regions in Japan, is quite high (0.8161) 1.5 DNA databank GenBank and the mtDNA HV1 data 1.5.1 GenBank 1.5.2 mtDNA HV1 data on GenBank mtDNA HV1 data is stored vastly on GenBank As in many previous publications, there is a lot of inaccurate or inconsistent information that needs to be corrected and reconciled 1.6 Perception on the study on the genetic diversity of the Phu Quoc ridgeback dog - The 582 bp fragments of mtDNA HV1 will show the genetic diversity of the Phu Quoc ridgeback dog population - A reliable and accurate dataset on worldwide dog haplotypes should be developed for the comparison and studying of the genetic diversity of the Phu Quoc ridgeback dog - The genetic diversity of the Phu Quoc ridgeback dog, along with the genetic relationship with other dog breeds, will be the basis for tracking the origin of this dog breed CHAPTER CONTENT AND RESEARCH METHOD 2.1 Research contents - Content 1: Building the database and the web-based tool for haplotyping the 582 bp fragment of mtDNA HV1 of dog - Content 2: Identifying the nucleotide sequence of 582 bp fragment of mtDNA HV1 of 100 Phu Quoc ridgeback dogs and 100 Vietnamese village dogs - Content 3: Evaluating the genetic diversity of Phu Quoc ridgeback dog based on the 582 bp fragments of mtDNA HV1 - Content 4: Assuming the origin of Phu Quoc ridgeback dog 2.2 Research methods 2.2.1 Building the database and the web-based tool for haplotyping the 582 bp fragment of mtDNA HV1 of dog 2.2.1.1 Building the database of 582 bp fragment of mtDNA HV1 Database of 582 bp fragments of mtDNA HV1 is designed using the relational model of data, consisting of tables with logical connections Each table in the database contains distinct data about the haplotype, nucleotide sequence, taxonomy data… Nucleotide sequences collected from Genbank using an in-house developed computer program Information of sequences are extracted from GenBank records and stored in the database 2.2.1.2 Nucleotide numbering and mutation illustration 2.2.1.3 Haplotyping and data reconcilation Assigned haplotypes and the haplotype of a sequence would be identified by following rules: 12 CHAPTER RESULT AND DISCUSSION 3.1 Building the database of 582 bp fragments of mtDNA HV1 Using BLAST against the GenBank nucleotide database, 5567 nucleotide sequences originating from individuals of Canis lupus species with high similarity to the reference mtDNA HV1 sequence (GenBank accession: U96639.2) were collected and stored in the in-house developed database Of these, 1921 entries are the 582 bp partial sequences which are not sufficient for haplotyping; 414 entries were correctly haplotyped and annotated with haplotype information using A-F system, 3232 entries were annotated with wrong haplotype identification or with the haplotyping information using other systems or did not contain any information about haplotype The lack of haplotype information and inconsistencies derived from the GenBank were modified or corrected in our database Web-based tool for rapid haplotyping of the 582 bp fragment of mtDNA HV1 (namely Haplotype identifier) was developed in Perl programming language, and freely available via the Internet at http://chd.vnbiology.com Testing on 50 random sequences in the database showed that Haplotype identifier was working correctly, resulting in a more appropriate and faster haplotyping than the same work done with phylogenetic trees The advantages of haplotypings with the Haplotype identifier compared to those with phylogenetic tree can be summarized in the following table (Table 3.5) 13 Table 3.5: Comparison between haplotyping with Haplotype identifer and with Phylogenetic tree Haplotype identifier Sequence called identical with corresponding reported exactly Phylogenetic tree the could not haplotype haplotype Sequence identical not called exactly with haplogroup the called exactly the haplogroup reported haplotype Running time fast (about second/sequence) long, depend on the method used (ranging from 10 minutes to hours) Specialized no yes software Experience The experience on User must be familiar haplotyping is required not with methods of inferring Phylogenetic tree 14 3.2 Sequencing the mtDNA HV1 of Phu Quoc ridgeback dogs and Vietnamese village dogs 3.2.1 Establishing the protocol for DNA extraction from dog hairs After examining the conditions of sample incubation (temperature, time), using common chemicals in laboratory of molecular biology, a protocol for DNA extractrion from dog hairs was established It can be summarized as follows (Figure 3.5) Figure 3.5: Protocol for DNA extraction from dog hairs Using this extraction process, total DNA (including nuclear DNA and mtDNA) from a source of about 40 dog hairs can be obtained The extracted DNA may be used in further studies 3.2.2 DNA extraction from dog hairs Two hundred samples of dog hairs (100 Phu Quoc ridgeback dogs and 100 Vietnamese village dogs) were used to extract DNA using the established protocol The quality of obtained DNAs were good, which can be used in subsequent PCR 15 3.2.3 Amplification of mtDNA HV1 The extracted DNA was used as materials for the amplification of mtDNA HV1, and the PCR products in turn were used in nucleotide sequencing The purity of PCR products was checked through electrophoresis using 1% agarose gel The signals of the DNA bands are bright, clear, located between the 1000 bp band and 1500 bp band of 1Kb DNA ladder, matching the expected size of DNA products (about 1200 bp) 3.2.4 Sequence editing PCR products were sequenced using 15412F and 16114R primers The chromatogram files representing DNA sequencing results were read by FinchTV software For each sample, two nucleotide sequences primed by 15412F and 16114R primers were compared to verify and result in a final sequence Sequencing result of 200 DNA sequences in this study are clear, especially in the region of 582 bp 3.2.5 Haplotyping of Phu Quoc ridgeback dogs’ and Vietnamese village dogs’ sequences All 200 examined sequences were submitted into the Haplotype identifier for quick haplotyping Most of sequences belong to common haplogroups (A, B, and C) 197 out of 200 sequences are identified harbouring haplotypes which are previously reported Besides, three new haplotypes - An1 (haplogroup A), Cn1, Cn2 (haplogroup C) - have never been reported so far In Phu Quoc ridgeback dogs, the proportion of haplogroup E’s haplotypes ís 16 notably high while these haplotypes are rare in the world’s dog population 3.3 Genetic diversity of mtDNA HV1 in Phu Quoc ridgeback dogs 3.3.1 Nucleotide diversity In the Phu Quoc ridgeback dogs, 100 examined sequences were classified into 19 groups based on the identity of nucleotide sequences, corresponding to 19 reported haplotypes Through the length of 582 bp fragment of Phu Quoc ridgeback dog, there are totally 33 polymorphic sites Of these, 31 sites are nucleotide substitutions and sites are observed with insertion or deletion (indel) Most of polymorphic sites are with transition (30/31 sites), only sites (the nucleotide 15479 and 15639) are with transversion (G/C substitution; T/A,G substitution) Nucleotide at position 15639 can be observed with both transversion and transition In Vietnamese village dogs, a total of 34 polymorphic sites were recorded Of these, 32 sites are nucleotide substitutions and sites are with indels All nucleotide substitutions are transitions, except for the site 15639, as seen in the Phu Quoc ridgeback dogs, that can be observed with both transversion and transition Compared to other dog breeds in the world, the nucleotide diversity of Phu Quoc ridgeback dogs and Vietnamese village dogs are quite high (0.014588 ± 0.007534 and 0.014035 ± 0.007270; respectively) 3.3.2 Haplotype diversity One hundred 582 bp sequences of Phu Quoc ridgeback dogs were sequenced and identified as 19 different haplotypes, including a new unpublished haplotype All 18 remaining haplotypes were 17 previously seen in the world dog population 77% of sequences harbour haplotypes in common haplogroups A, B, C, none of sequences harbours haplotype in rare haplogroup D and F Notably, 23% of sequences harbour haplotypes in the rare haplogroup E which only accounts for a very low proportion in the world (along with haplogroup D and F accounts for less than 3%) With 19 haplotypes detected among 100 examined samples, the haplotype diversity of Phu Quoc ridgeback dog is up to 0.9042 ± 0.0127, meaning that the probability to take two different haplotype in the samples is about 90% Compared with some other dog breeds in the world, the haplotype diversity of Phu Quoc ridgeback dog is lower than that of Thai dogs However, the haplotype diversity is still high, similar to those of Pungsang dogs (Korea), Vietnamese village dogs and much higher than the Portuguese sheepdogs, German shepherd dogs, Tibetan Mastiff dogs, Kangal dogs (Turkey), or Shiba dogs (Japan) and Jindo dogs (Korea) 3.3.3 Genetic relationship between Phu Quoc ridgeback dogs and other dog breeds 3.3.3.1 Genetic relationship between Phu Quoc ridgeback dogs and other dog breeds AMOVA analysis of mtDNA HV1 data shows that although the differences among examined dog breeds are small, they are statistically significant (Table 3.11) 18 Table 3.11: AMOVA analysis of some dog breeds in the world Source of variation Among Sum of squares Variation Percentage of variation F value P value groups of dog 512.086 0.55663 13 0.13001 0.01369 111.69 0.348993 8.15 0.09369 2538.619 3.37582 78.85 0.21152 3162.395 4.28145 100 population Among dog populations Within populations Total It is notable that Thai dog is not too genetically close to the Vietnamese village dog (Figure 3.13) The genetic distance between the Phu Quoc ridgeback dog and Thai dog is 2.27668, nearly times higher than that between the Phu Quoc ridgeback dog and the Vietnamese village dog (0.33899) This is the basis for asserting Phu Quoc ridgeback dog originating from Vietnamese village dogs, not from Thai dogs as previously believed 19 Figure 3.13: Genetic distance among some world dog breeds 3.3.3.2 Genetic relationship between Phu Quoc ridgeback dogs and Vietnamese village dogs AMOVA analysis showed a small and unsignificant difference between Phu Quoc ridgeback dog and Vietnamese village dog (Table 3.12) Table 3.12: AMOVA analysis of dog populations in Vietnam Source of variation Between two breeds Among populations Within population Total Sum of squares Variation Percentage of variation F value P value 21.120 0.19002 4.38 0.04378 0.10655 6.923 -0.04919 -1.13 -0.01185 0.84360 818.857 4.19927 96.76 0.03245 0.06647 451.278 4.31726 100% According to the calculation of Savolainen et al (2009), unit of genetic distance between two populations (or mutation rate) in the mtDNA HV1 of dogs is substitution per about 40000 years 20 Thus, considering the entire examined dog populations in this study, with the genetic distance of 0.33899, Phu Quoc ridgeback dog and Vietnamese village dog differentiated from a common ancestor about 6780 years ago 3.3.3.3 Genetic relationship between Phu Quoc ridgeback dogs and Vietnamese village dogs living in Kien Giang province The similarity of the haplotype compositions of Phu Quoc ridgeback dogs and Vietnamese village dogs living in Kien Giang, especially the common haplotypes with high ratio in the population, indicates the phenomenon of gene flow from Vietnamese mainland to Phu Quoc island (Figure 3.14) Hình 3.14: The similarity of haplotypes between dogs living on the mainland and on the Phu Quoc island AMOVA analysis of mtDNA HV1 data also showed no significant difference between two groups of dogs living on the mainland and on the island in Kien Giang province (Table 3.13) 21 Table 3.13: AMOVA analysis of dog populations living in Kien Giang province Source of variation Between two groups Among populations Within population Total Sum of squares Variation Percentage of variation F value P value 10.204 0.14484 3.38 0.03383 0.34018 2.970 -0.03645 -0.85 -0.00881 0.52590 438.104 4.17242 97.47 0.02532 0.13392 451.278 4.31726 100% Notably, the genetic distance between Vietnamese village dogs living on the mainland of Kien Giang and Phu Quoc ridgeback dogs living on the Phu Quoc island (0.36176) is similar to that between Vietnamese village dogs and Phu Quoc ridgeback dog in general (0.33899) In a meanwhile, the genetic distance between Vietnamese village dogs and Phu Quoc ridgeback dogs living on the Phu Quoc island is much smaller (0.07154) Based on the mutation rate in this mtDNA HV1 region, the time point of differentiation of these two populations is about 1430 year before (0.07154 x 40000/2) 3.4 Assuming the origin of Phu Quoc ridgeback dog Based on the analyses of this study, along with previous studies on the migration route of dogs on earth, and the origin of the Australian dingo, as well as the genetic characteristics of the ridge trait, the origin of Phu Quoc ridgeback dog can be hypothesized as follows: The domestic dog originated in the Vietnam - China border and migrated to different lands, resulting in the diversity and wide distribution of current dogs Beside a group of dogs moving west to 22 Europe, and a group of dogs moved north, at this place of domestication, a group of dogs moved south, which are the ancestors of current Vietnamese dogs During the migration to the south, the mating between the male dog and female wolf harbouring haplotype E has added this gene into the dog's gene pool About 6780 years before, a group of Vietnamese village dogs separated from populations, forming a new deme and migrating towards the land currently known as Kien Giang today Until about 1430 years before, the human migration process from the mainland (Kien Giang province) brought a group of Vietnamese village dogs carrying haplotypes A, B, C and E to Phu Quoc island These dogs did not have dorsal hair ridge, they carried two alleles r/r on chromosome 18 Under the condition of geological isolation and the limitation of nutrient sources, allele r was mutated into allele R in dogs harbouring different haplotypes The advantage of haplotype E harbouring individuals in the habitat, together with inbreeding due to geographical isolation, has contributed to raising the frequency of haplotype E and allele R in the population of Phu Quoc ridgeback dogs 23 CONCLUSION – RECOMMENDATION I Conclusion From 5567 sequences stored in GenBank, a database and a tool for mtDNA HV1 haplotyping were developed A protocol for DNA extraction from dog hairs was established 582 bp fragments of 100 Phu Quoc ridgeback dogs and 100 Vietnamese village dogs were sequenced, analysed and edited In the population of Phu Quoc ridgeback dogs, 19 haplotypes belonged to different haplogroups were detected Of these, 23 dogs harbours rare haplotype E In the population of Vietnamese village dogs, 24 haplotypes belonged to different haplogroups were detected Of these, dogs harbours rare haplotype E The genetic diversity of Phu Quoc ridgeback dogs is high, with the haplotype diversity of 0.9042 ± 0.0127 and the nucleotide diversity of 0.014588 ± 0.007534 The genetic diversity of Phu Quoc ridgeback dogs is higher than that of many other dog breeds on the world Phu Quoc ridgeback dog is genetically close to the Vietnamese village dog, with the genetic distance of 0.33899, while the genetic distance between Phu Quoc ridgeback dog and Thai dog is quite high (2.27668) The above scientific data initially asserted that Phu Quoc ridgeback dog originated from Vietnamese village dog and did not originate from Thai dogs as previously stated The natural conditions 24 on Phu Quoc island and the inbreeding in the closed population have caused the appearance and improved the frequency of R allele as well as haplotype E in the population, which gradually developed the population of Phu Quoc ridgeback dogs, a dog breed which have the ability to hunt and survive well in unfavorable conditions II Recommendation For the conservation of genetic resource of Phu Quoc ridgeback dog, studies on the correlation among haplotype E, nuclear DNA and morphological characteristics should be implemented, so that the characterized phenotype, genotype of Phu Quoc ridgeback dog can be defined They will be criteria for selective breedings of this valuable dog breed LIST OF PUBLICATIONS Thái Kế Quân, Nguyễn Văn Tú, Huỳnh Văn Hiếu, Nguyễn Thành Cơng, Trần Hồng Dũng (2016), “Quy trình tách chiết DNA đơn giản hiệu từ lơng chó” Tạp chí Sinh học, 38(1): 124-132 Thai Ke Quan, Nguyen Van Tu, Tran Ngoc Trinh, Huynh Van Hieu, Chung Anh Dung, Tran Hoang Dung (2016), “Evaluation of genetic diversity of Phu Quoc ridgeback dogs based on mitochondrial DNA Hypervariable-1 region” Vietnam Journal of Biotechnology, 14(1A): 245-253 (Số đặc biệt Tạp chí Cơng nghệ Sinh học, Việt Nam) Trần Hồng Dũng, Thái Kế Qn, Nguyễn Thành Cơng, Huỳnh Văn Hiếu, Chung Anh Dũng (2016), “Phân tích nguồn gốc chó lưng xốy Phú Quốc dựa trình tự vùng D-loop ty thể” Tạp chí Sinh học, 38(2):269-278 Quan Thai Ke, Van-Hieu Huynh, Anh-Dung Chung, Hoang- Dung Tran (2016), “Evaluation of genetic diversity of Vietnamese dogs based on mitochondrial DNA hypervariable-1 region”, Research result, Belgorod State National Research University, Russia 3(9), 45-49 Quan Ke Thai, Dung Anh Chung, Hoang-Dung Tran (2017), “Canis mtDNA HV1 database: a web-based tool for collecting and surveying Canis mtDNA HV1 haplotype in public database”, BMC Genetics 18(1), 60 ... 1.4.2.3 Studies on the genetic diversity of dog breeds The genetic diversity of different dog breeds are varied, implicating the place and date of origin of dog breeds The haplotype diversitys of... Huỳnh Văn Hiếu, Chung Anh Dũng (2016), “Phân tích nguồn gốc chó lưng xốy Phú Quốc dựa trình tự vùng D-loop ty thể Tạp chí Sinh học, 38(2):269-278 Quan Thai Ke, Van-Hieu Huynh, Anh- Dung Chung, Hoang-... as average nucleotide differences using Arlequin 3.5 2.2.3.1 Haplotype diversity Haplotype diversity is defined as the probability that two randomly chosen haplotypes are different in population