Origin, Phylogenetic Analysis And Genetic Diversity Of Tibetan Mastiff

Domestic dog is the closest friend of mankind and was probably the first animal to be domesticated. As the intimate relationship with people grew, the origin, domestication and breed formation of the dog had become a major topic of studies among the scholars in the world. With regard to the origin, there has been a confirmed conclusion that all the domestic dogs originated from the grey wolf. However, about the region and time of origin, there were greatly differing views. So the analysis of the most archaic extant domestic dog breeds in the world at present will be an effective method to reveal the region and time of domestic dog origin. Tibetan Mastiff is one of the most archaic and fierce large breed of domestic dogs, so the research about the origin of Tibetan Mastiff and its relationship between other large breed dogs will contribute to reveal the origin, origin time and origin regions. At present the study about Tibetan Mastiff is lagged, there have been few reports on the origin of the Tibetan Mastiff and its relationship with other domestic dogs. Therefore, we sequenced the complete mitochondrial genome of Tibetan Mastiff and analyzed its characteristics in comparison to those of other Canidae(domestic dogs, grey wolves and coyotes) and some mammals. Based on the mitochondrial genome (mtDNA) of 12 protein-coding genes, COI gene, DNA barcoding, D-loop and its hypervariable region I sequence, we analyzed the origin, taxonomic status of Tibetan Mastiff and its relationship between other domestic dogs, speculated the origin time of Tibetan Mastiff and domestic dog; we identified the structure characters of control region in Tibetan Mastiff, analyzed the genetic diversity based on the control region and hypervariable region 538bp sequence. The results followed as below:1. Sequencing and analyzing the complete sequence of the Tibetan Mastiff mitochondrial genomeThe complete sequence of the Tibetan Mastiff mitochondrial genome (mtDNA) was determined (GenBank No. HM048871) for the first time. The complete nucleotide sequence of the Tibetan Mastiff mtDNA was 16710 bp, and included 22 tRNA genes, 2S rRNA gene,13 protein-coding genes, and one non-coding region (D-loop region), the constitution, arrangements and location of genes is consistent with other mammalian mitochondrial genomes. The base composition of the Tibetan Mastiff mtDNA is 28.7%T,25.6%C,31.6% A, and 14.1% G. The AT content (60.3%) is significantly higher than the GC content (39.7%). The total length of the Tibetan Mastiff mtDNA that encodes the 13 protein-coding genes is 11410bp, in which the longest gene is ND5 (1821bp), the shortest gene is ATPase8 (204bp), The genome uses four initiation codons:eight genes use ATG; NADH2, NADH3, and NADH5 use ATA; NADH6 uses AAT; and NADH4 uses GTG. The stop codons of 11 genes are complete termination codons (TAA, TAG, or AGA) and four genes (COⅢ, NADH3, NADH4, and NADH6) use incomplete termination codons; The complete length of the D-loop region in the Tibetan Mastiff is 1250bp, and is located between the tRNA-Pro and tRNA-Phe genes; there were two conserved sequenced blocks (CSB), that is, CSB I and CSB II in the mitochondrial control region, and a short tandem repeat region between the two CSBs, which was composed of 30 repeated units (5’-GTACACGT(G/A)C-3’).22 tRNA lengths range from 66 to 75bp, the total length of the Tibetan Mastiff mtDNA that encodes the tRNA genes is 1521bp, showing 99.08% homology with domestic dog (GenBank No. U96639) and 4 variable sites. The lengths of the 12s rRNA and 16s rRNA genes are 954bp and 1580bp respectively, showing 99.96% homology with domestic dog (GenBank No. U96639) and one variable site.2. Origin of Tibetan Mastiff and its phylogenetic analysis with other domestic dogsAccording to the methods of NJ, MP and network, based on the 5 sequences of 12 protein-coding genes in H chain, COI gene, DNA barcoding (648bp), control region and hypervariable region I (582bp) respectively, combining the sequences sequenced by us with the corresponding sequences of domestic dogs, grey wolves and coyotes gathered by GenBank, using coyotes as the outgroup, we constructed the phylogenetic trees and haplotypes network among Tibetan Mastiff, domestic dogs and grey wolves. Phylogenetic trees and network also showed that Tibetan Mastiff, domestic dogs and grey wolves clustered together, while the coyotes clustered alone, which suggesting that Tibetan Mastiff, as other domestic dogs, originated from grey wolves;the sequence divergence, based on the 5 types of sequences, between Tibetan Mastiff and domestic dogs were smaller than that of between Tibetan Mastiff and grey wolves but bigger than that of interspecies, so the Tibetan Mastiff could be considered as one species of domestic dogs, which belonged to the Carnivora, Canidae, Canis, Canis lupus, Canis lupus familiaris in the animal taxonomic status. Tibetan Mastiff and domestic dogs clustered into four clades, implying at least four maternal origins, which consistent with the before reports. Tibetan Mastiff and some large breed dogs such as Saint Bernard and the Old English Sheepdog belonged to clade A, the sequence divergence and genetic distance between the Tibetan Mastiff and these domestic dogs was smallest, indicating that Tibetan Mastiff appears to be closely related to large breed dogs such as the Saint Bernard and the Old English Sheepdog and confirming the point many of famous domestic dogs in the world such as Saint Bernard were possible of having blood lineage of Tibetan Mastiff at molecular level. According to the method of Savolainen et al (2002) based on the sequences of mitochondrial 12 protein-coding genes, COI gene, DNA barcoding, control region and hypervariable region I, we calculated the rate of base transition, speculated the divergence time between Tibetan Mastiff and grey wolves and between domestic dogs and grey wolves. The results showed that:considering the four clades synythetically, the divergence time between domestic dogs and grey wolves were 16000 YBP,32000 YBP,65000 YBP and 50000 YBP respectively, which was in the middle of the earliest originated time (100000 YBP) and the latest originated time (15000 YBP) of domestic dogs; we considered, synthetizing various factors,the originated time of domestic dogs was about at least 16000 YBP, which consistent with the results speculated by Savolainen et al (2002) and Pang et al (2009) (15000 YBP and 16300 YBP respectively) and fossil record (26000 YBP); the origin time of the Tibetan Mastiff was about 18000 YBP,33000 YBP,68000 YBP and 56000 YBP, which were earlier than the originated time of domestic dogs speculated by corresponding sequences, phylogenetic analysis showed that Tibetan Mastiff clustered with some archaic domestic dog (such as Old English Sheepdog), there was small sequence divergence and genetic distance between Tibetan Mastiff and these archaic domestic dogs, Tibetan Mastiff shared the haplotype of DNA barcoding with Old English Sheepdog, which suggesting that Tibetan Mastiff was one of the most archaic breed of domestic dog.3. Genetic diversity of Tibetan Mastiff populationWe sequenced the complete sequences of mitochondrial D-loop of 40 Tibetan Mastiff, analyzed the structure of control region and genetic diversity of control region and its 538bp hypervariable region I sequences and compared the genetic diversity with other 7 population of Canids (Golden Retriever, Labrador Retriever, Illyrian sheepdog, Kangal, Chinese wolf, grey wolf and coyote). The results showed that:the length of D-loop in Tibetan Mastiff populations ranges from 1240bp to 1320bp, of which 1270bp was the primary length types, there were 13 (32.5%) individuals owning 1270-bp length. Further analysis found that the length diversity mainly due to the different number of repeated units (5’-GTACACGT(G/A)C-3’). In 40 Tibetan Mastiff individuals,75 sites were polymorphic, taking up 5.68% of total sits, of which 14 (18.67% variable sites) was singleton polymorphic sites and 61 (81.33% variable sites) was parsimony informative polymorphic sites. There were two types of mutation of nucleotide such as transition, insertion/deletion, there was no transversion, showing strong transition bias. The haplotype diversity, nucleotide diversity and average number of nucleotide differences of Tibetan Mastiff popultion was 0.990,1.201 and 13.082 respectively, which were higher than that of Golden Retriever population and Labrador Retriever population, but compared with other mammals nucleotide diversity was lower. In 8 populations of Canids, the haplotype diversity, nucleotide diversity and average number of nucleotide differences of 538bp control region I sequence in wild populations (Chinese wolf, European wolf and coyote) were obviously higher than that of domestic dog breeds; in 5 breeds of domestic dog, the most variable sites (21 variable sites) happened in kangal (3.90%), following the Tiebtan Mastiff and Illyrian sheepdog with also 19 variable sites (3.53%); 289 individuals of 5 domestic dogs breeds were defined 36 haplotypes in which 29 haplotypes were the characteristic of their corresponding breeds and 7 haplotypes were the sharing haplotypes among breeds; haplotype diversity of Golden Retriever was smallest (0.471) and haplotype diversity of Tibetan Mastiff was highest (0.854), but the nucleotide diversity and average number of nucleotide differences in Tibetan Mastiff was smallest (0.845 and 4.519 respectively), the nucleotide diversity and average number of nucleotide differences in Illyrian sheepdog was highest (1.427 and 7.661 respectively). In Tibetan Mastiff population, the high haplotype diversity and low nucleotide diversity suggested the extant Tibetan Mastiff population probably formed and rapidly increased from a small and effective population. From these, we concluded that the genetic diversity of Tibetan Mastiff was relative spareness.