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Animal (2011), 5:12, pp 1868–1873 & The Animal Consortium 2011 animal doi:10.1017/S1751731111001042

The origin of the Tibetan and species identification of Canis based on mitochondrial cytochrome c oxidase subunit I (COI) gene and COI barcoding

- - Y. Li 1, X. Zhao2,Z.Pan1, Z. Xie1, H. Liu1,Y.Xu1 and Q. Li1

1College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, ; 2College of Animal Science and Technology, State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China

(Received 28 December 2010; Accepted 24 April 2011; First published online 11 July 2011)

DNA barcoding is an effective technique to identify species and analyze phylogenesis and evolution. However, research on and application of DNA barcoding in Canis have not been carried out. In this study, we analyzed two species of Canis, Canis lupus (n 5 115) and Canis latrans (n 5 4), using the cytochrome c oxidase subunit I (COI) gene (1545 bp) and COI barcoding (648 bp DNA sequence of the COI gene). The results showed that the COI gene, as the moderate variant sequence, applied to the analysis of the phylogenesis of Canis members, and COI barcoding applied to species identification of Canis members. Phylogenetic trees and networks showed that domestic had four maternal origins (A to D) and that the originated from Clade A; this result supports the theory of an East Asian origin of domestic dogs. Clustering analysis and networking revealed the presence of a closer relative between the Tibetan Mastiff and the , Newfoundland, and Saint Bernard, which confirms that many well-known large breed dogs in the world, such as the Old English sheepdog, may have the same blood lineage as that of the Tibetan Mastiff.

Keywords: Tibetan Mastiff, cytochrome c oxidase subunit I, COI barcoding, origin, species identification

Implications subunit I (COI) gene could distinguish different species at a molecular level, and considered that the COI gene was the In this paper, the cytochrome c oxidase subunit I (COI) gene suitable DNA barcode for Animalia, being the first to apply this sequences of 119 individuals of Canis, including the Tibetan DNA barcoding to research. Subsequent to this, scientists Mastiff, were determined. The intra- and interspecies phy- widely used the COI gene and found that COI barcoding can logenetic relationships and sequence differences between identify species exactly and effectively (Hebert et al., 2004), Canis members were analyzed using COI and COI barcoding. distinguish amphibolous species in traditional taxonomy The results will play an important role in the protection of the (Janzen et al., 2005) and recognize species variation of diff- Tibetan Mastiff, will be useful for studying the worldwide erent geographies (Yassin et al., 2010). speciation of large dogs and the origin of the domestic , The Tibetan Mastiff is a special domestic that and will help to identify Canis species. mainly inhabits high and cold zones with 3000 to 5000-m altitudes, belongs to the second-protection group of animals in China and is the only canine ‘living fossil’ of the snowfield, Introduction unchanged by time and environment (Ren et al., 2009; Li The use of short DNA sequences for the standardized identi- et al., 2011). However, large profits can be made from the fication and phylogenetic analysis of organisms has recently sale of Tibetan Mastiffs, and the outflow of Tibetan Mastiffs gained attention under the term DNA barcoding. Owing to its has become a serious problem since the 1980s. Therefore, quick identification and handy operation, DNA barcoding has the purebred Tibetan Mastiff is at the edge of extinction (Li been widely used in the fields of ecology, systems biology and et al., 2008). Being the only large breed dog in China and conservation biology (Kress and Erickson, 2008; Derycke et al., one of the most idioplasmatic resources worldwide, the 2010). In 2003, Hebert et al. (2003) found that the 50-end Tibetan Mastiff must be protected and rapidly researched. of the 648 bp DNA sequence of the cytochrome c oxidase No studies of the origin of domestic dogs and species identification of Canis according to the COI gene and COI - E-mail: [email protected] barcoding have been found. In this study, we analyzed the

1868 COI, COI coding in Tibetan Mastiff and Canis phylogenetic relationships among Canis members, including and sequencing, and the sequence length was 1545 bp. the Tibetan Mastiff, using COI sequences and COI barcoding The length, base composition and codon distribution of the (nt 58 to 705 in COI, 648 bp) in order to offer a reference for COI gene in the Tibetan Mastiff, and protecting the idioplasmatic resource of the Tibetan Mastiff and Borzoi were consistent with those in other species further studying the origin and domestication of domestic dogs. (Bjo¨ rnerfeldt et al., 2006). For research animals, we chose Canis lupus as the domestic dog There were 119 variable sites in the Tibetan Mastiff and most closely related to human beings, and Canis latrans as the 76 domestic dogs, and the percentage of variable sites was closest genetic relative to C. lupus. The grey and sheepdog 7.70%. A total of 23 haplotypes were defined in the COI of C. lupus are closer to C. latrans in appearance and body form. gene sequence of the Tibetan Mastiff and 76 domestic dog We analyzed the intra- and interspecies sequence differences breeds (Table 1), and the haplotype percentage was 21.50%. between members of Canis and discussed the feasibility of The polymorphism was lower than that based on the mito- using DNA barcoding to identify Canis species. chondrial displacement loop (D-loop; especially hypervari- able region 1; Vila` et al., 1997; Savolainen et al., 2002; Luo, 2003), but higher than that based on the mitochondrial Material and methods Cytb gene (Li et al., 2008). Vila` et al. (1997) found 27 vari- able sites and identified 26 haplotypes in the 261-bp D-loop Samples and DNA extraction region with a 10.34% polymorphism loci percentage and an The samples included 119 individuals of Canis,including 18.57% haplotype percentage. Savolainen et al. (2002) 102 domestic dogs (77 breeds), 13 grey (C. l. lupus) found 128 variable loci and identified 109 haplotypes in the and four coyotes (C. l. latrans; Table 1). Blood samples (5 mL) 582-bp D-loop region with a 21.84% polymorphism loci were obtained from the ear arteries of four Tibetan Mastiffs percentage and a 16.67% haplotype percentage. Luo (2003) (two females and two males), one German shepherd (male) found 113 variable loci and identified 184 haplotypes in the and one Borzoi (male) from the Base of Xigang, 597-bp D-loop with an 18.93% polymorphism loci percen- Nanjing. Genomic DNA was extracted by the standard phenol– tage and a 15.11% haplotype percentage. Li et al. (2008) chloroform method. The rest of the individual resources were found 88 variable loci in a 2525 bp mitochondrial sequence, searched for in the database and tracked literature. with a 3.39% polymorphism loci percentage. Therefore, the COI gene, as the moderate variant mitochondrial DNA PCR amplification and sequencing sequence, applied to the phylogenetic analysis among Primers were designed by Primer Premier 5.0 software (Premier domestic dogs. There exist 636 conserved sites and 12 vari- Biosoft International, Palo Alto, CA, USA) using the mitochon- able sites in the COI barcoding (nt 58 to 705, 648 bp) of the drial genome sequence of the domestic dog (Accession no. Tibetan Mastiff and 76 domestic dogs, and the percentage of 0 U96639). Primer sequences were UCOI:5-CAC TGC CTT GAG variable sites was 1.85%. On the basis of the 12 variable sites, 0 0 CCT CCT CAT C-3 and DCOI:5-GGG GAG GTT GCG TCC TGT 10 haplotypes were defined in the COI barcoding sequence of 0 AAT C-3 . Amplification conditions consisted of initial denatur- the Tibetan Mastiff and 76 domestic dog breeds, with a 9.85% alization at 948C for 5 min, 35 cycles of denaturalization at 948C haplotype percentage. Thus, compared with the COI gene, COI for 30 s, annealing at 608C for 45 s, extension at 728Cfor1min barcoding of domestic dogs, which offers limited polymorph- and a final extension at 728C for 10 min. The purified PCR pro- ism, could not be applied to the analysis of the phylogenesis ducts were obtained using a V-gene Kit (Tiangen, Beijing, China) among domestic dog breeds. and sequenced by Shanghai Invitrogen Co. (Shanghai, China). Two criteria are available to test the validity of DNA bar- coding. One is whether two species possess the same Data analysis sequences, and the other is whether the interspecific differ- The analyses of the sequence alignment and sequence diver- ence is markedly larger than the intraspecific difference gence were carried out by DNAStar 5.02 software (DNAStar Inc., (Hebert et al., 2004; Yoo et al., 2006). On the basis on the Madison, WI, USA). The base transition, transversion, proportion complete sequence of the COI gene, we found a shared of distance (p-distance) and genetic distance among the differ- sequence in domestic dogs and grey wolves, but this ent sequences were calculated by MEGA4.1 software (Kumar instance did not exist between domestic dogs/grey wolves et al., 2008). The haplotype was determined by DNASP 5.0 and coyotes. The mean genetic distance was 0.58% in software (Librado and Rozas, 2009). Phylogenetic trees were domestic dogs/grey wolves, 0.26% in coyotes and 4.45% constructed by Neighbor-Joining (NJ) methods of the MEGA4.1 between domestic dogs and grey wolves. The interspecific software. The minimum-spanning network was received by difference was 8 to 17 times the intraspecific difference. In Network4.5 software (Polzin and Daneschmand, 2003). the 648 bp COI barcoding, we found that shared sequences existed in domestic dogs/grey wolves and coyotes, but not between domestic dogs/grey wolves and coyotes. The mean Results and discussion genetic distance was 0.40% in domestic dogs/grey wolves, Mitochondrial COI gene and COI barcoding in Canis but was 0.31% in coyotes; the mean genetic distance was We obtained the complete COI gene sequence of the Tibetan 4.73% between domestic dogs/grey wolves and coyotes. Mastiff, German Shepherd and Borzoi by PCR amplification The interspecific difference was larger than the intraspecific

1869 Li, Zhao, Pan, Xie, Liu, Xu and Li

Table 1 Experimental samples used in this study COI DNA barcoding Sample Species haplotype haplotype size Individuals and GenBank no.

Domestic dog D01 DD01 14 Akita (EU408245), Airedale (AY656748), Frise (EU408250), (EU408253), Cavalier King Charles (EU408263), Corgi (EU408265), (EU408271), (EU408274), (FJ817362), (AY656751), Italian (EU408280), (AY656738), Miniature (DQ480498), (EU408294) D02 DD02 1 Brittany Spaniel (EU408257) D03 DD03 1 (EU408262) D04 DD01 1 Basenji (AY656737) D05 DD01 23 (EU408246), Cocker Spaniel (DQ480495, EU408268), (EU408284), Toy (EU408302), Old English Sheepdog (AY656742), Chihuahua (EU408262), Cockapoo (EU408259), English Shepherd (EU408273), Keeshond (EU408284), (AY656747), (EU408249), (EU408264), (AY656744), Jack Russell (EU408281), Miniature (EU408286), Newfoundland (EU408287), Vizsla (EU408305), Chinese Crested (AY656754), Irish Soft-Coated Wheaten Terrier (DQ480496), Keeshond (EU408282), (EU408297), Irish Setter (AY656753) D06 DD04 5 (AY656750), French (EU408275), (EU408276), Schnauzer (EU408299), (AY656752) D07 DD05 1 West Highland White Terrier (DQ480497) D08 DD01 2 Rottweiler (EU408295, EU408296) D09 DD01 5 Irish Setter (DQ480491, AY656741), (EU408289), Neapolitan Mastiff (EU408290), Saint Bernard (AY656749) D10 DD01 2 Tibetan Mastiff (*), Tibetan Mastiff (EU408300) D11 DD06 3 Tibetan Mastiff (*), Tibetan Mastiff (*), Tibetan Mastiff (*) D12 DD01 1 Siberian Husky (DQ480499) D13 DD01 1 Dachshund (EU408272) D14 DD01 1 German Shepherd (*) D15 DD07 1 (AY729880) D16 DD08 1 Australian Shepherd (EU408248) D17 DD01 3 Dachshund (EU408270), Great Pyrenees (EU408277), Sapsaree (AY656755) D18 DD01 1 Borzoi (*) D19 DD01 25 Australian Terrier (EU408247), Basset (EU408254, EU408255, EU408256), (EU408252), Cockapoo (EU408258), Cardigan Corgi (EU408260), Cocker Spaniel (EU408268), Doberman Pinscher (EU408269), English Springer Spaniel (AY656745), Flat-Coated Retriever (DQ480490), Golden Retriever (FJ817363, FJ817364), Great Pyrenees (EU408278), Kerry Blue Terrier (AY656740), Labradoodle (EU408285), Poodle (EU408292, AY656739, DQ480494), Saint Bernard (AY656743), (DQ480500), Standard Schnauzer (AY656752), (EU408301), Walker Hound (EU408307), Yorkie Chihuahua (EU408308) D20 DD09 2 German Shepherd (DQ480489), Terrier (EU408293) D21 DD09 4 (DQ480493), Blue Heeler (EU408251), Cocker Spaniel (EU408267), Havanese (EU408279) D22 DD09 1 Pomeranian (EU408291) D23 DD10 3 Jamthund (DQ480492, DQ480502), Norwegian Elkhound (EU408288) Grey wolf W01 WD01 2 Grey wolf (AM711902, NC_009686) W02 WD02 1 Grey wolf (DQ480508) W03 WD02 3 Grey wolf (NC_008092, DQ480505, DQ480503) W04 WD02 1 Grey wolf (DQ480507) Coyote W05 WD03 1 Grey wolf (DQ480506) W06 WD04 1 Grey wolf (DQ480504) W07 WD02 2 Grey wolf (NC_011218, FJ032363) W08 WD05 2 Grey wolf (NC_010340, EU442884) C01 CD01 3 Coyote (DQ480509, DQ480511, NC_008093) C02 CD02 1 Coyote (DQ480510)

COI 5 cytochrome c oxidase subunit I. *Sequences produced during this work. There is GenBank no. in the parenthesis.

1870 COI, COI coding in Tibetan Mastiff and Canis

Figure 1 Saturation analysis of nucleotide substitution based on cytochrome c oxidase subunit I gene (a) and DNA barcoding (b) among 77 domestic dog breeds. The abscissa is the proportion of distance and the vertical coordinate is the base transition and transversion. difference by 12 to 15 times. Thus, COI barcodes, compared group (BP 5 57%; Clade B); D20 to D22 were clustered with the complete sequence of the COI gene, are easier to into one group (BP 5 71%; Clade C); and D23 was alone amplify and more suitable for Canis species identification. (Clade D). The two haplotypes of the Tibetan Mastiff (D10 and D11) belonged to Clade A. The network technique is the The origin of domestic dogs and the Tibetan Mastiff most effective method to study the phylogenetic relation- Domestic dogs originate from the grey wolf, a finding that is ships among domestic animals. In this study, the genetic well established and recognized by both biologists and relationships among domestic dogs were analyzed using the sociologists. However, disputes remain regarding the exact network technique (Figure 3a). From Figure 3a, we found domestication and origination processes. According to the that the network relationship among domestic dogs was complete mitochondrial sequence and part control region consistent with that of the phylogenetic tree (Figure 2a); that (hypervariable region), domestication of domestic breed 23 haplotypes were divided into four clades; and that D05 dogs was not an isolated event. All sequences of domestic was the most important and primordial haplotype in domestic dogs are primarily clustered in four clades, considering that dogs. The phylogenetic trees (Figure 2b) and haplotype net- the domestic breed dogs had four maternal origins. Vila` et al. work (Figure 3b) of the Tibetan Mastiff and domestic dogs (1997) found that Clades A, B, C and D contained 19, 1, 3 constructed on the basis of COI barcoding were consistent and 3 haplotypes, respectively, among 26 haplotypes. The with the COI gene. According to the NJ tree and network earliest domestication is estimated at about 100 000 years constructed by the COI gene sequence, we found 23 haplo- before the present (YBP) and originated in Europe, South- types clustered into four clades; the numbers of haplotypes west Asia and East Asia, considering its multiregion and (17, 73.91%) and individuals (66, 64.71%) of Clade A were asynchronous domestic events. Savolainen et al. (2002) more than those of Clades B and C, which is consistent with found that Clades A, B and C included 71.3%, 13.7% and previous reports (Vila` et al., 1997; Savolainen et al., 2002). 7.2% domestic dogs, respectively. The haplotype distribu- These results support the view of the East Asian origin of tions of various geographic areas showed that Clade A ori- domestic dogs and the Clade A origin of the Tibetan Mastiff, ginated from East Asia and that haplotypes of Europe and which are consistent with the results according to the complete Southwest Asia were formed by a subset of East Asian types. sequence of the mitochondrial genome (Li et al., 2011). In the same way, Clade B originated from East Asia. For Clade C, the pattern is less clear, but an East Asian origin for Phylogenetic relationship between the Tibetan Mastiff and this clade is also possible. The estimated origins of Clades A, other large breed dogs B and C were 40 000, 13 000 and 17 000 YBP, respectively. If On the basis of the historical data, the closest ancestor of Clades A, B and C are simultaneously considered, the origin the Tibetan Mastiff may be the Qiang dog. Following the of domestic breed dogs was approximately 15 000 YBP. Mongolian army (Tibetan Mastiff corps; it is said that this Saturation analysis of base substitutions (made with the corps owned 30 000 Tibetan Mastiffs) of Genghis Khan, the scattergram p-distance as the abscissa and the base transi- Tibetan Mastiff came into Europe and played an important tion and transversion as the vertical coordinate; Figure 1) role in the breed formation of large breed dogs, such as revealed that the COI gene and COI barcoding of domestic the Saint Bernard and English Shepherd, which are close to the dogs shows a strong transition preference. The phylogenetic Tibetan Mastiff in blood lineage. In this study, the NJ tree and trees of the Tibetan Mastiff and domestic dogs were con- minimum-spanning network constructed by the COI gene and structed by the NJ method based on the COI sequence COI barcoding showed that, in large breed dogs, the Tibetan (Figure 2a). The Tibetan Mastiff and domestic dogs were Mastiff clustered with the Old English Shepherd, Rottweiler clustered with the grey wolf (bootstrap percentages (BP) 5 and Saint Bernard, and possessed the least sequence diver- 99%), and coyotes were clustered alone (BP 5 99%). The gence. Therefore, we considered that there was one closest Tibetan Mastiff and domestic dogs were obviously divided relative between the Tibetan Mastiff and Old English Shep- into four groups: D01 to D017 were clustered initially herd, Rottweiler and Saint Bernard, and that these large breed (BP 5 74%; Clade A); D18 to D19 were clustered into one dogs probably had the blood lineage of the Tibetan Mastiff in

1871 Li, Zhao, Pan, Xie, Liu, Xu and Li

Figure 2 Phylogenetic tree of domestic dogs and grey wolves was constructed based on cytochrome c oxidase subunit I gene (a) and DNA barcoding (b) by Neighbor-Joining methods. D and DD represent domestic dogs, W and WD represent grey wolf, C and CD represent coyote. The nodal parameters support the 1000 times repetition analysis on sampling examination.

Figure 3 Minimum-spanning Networks based on cytochrome c oxidase subunit I gene (a) and DNA barcoding (b) among domestic dogs. Grey circles represent haplotypes; the digitals on the line between yellow circles represent mutation times.

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