SUPPLEMENTARY MATERIAL FOR:
MHC variability supports dog domestication from a large number of wolves
- high diversity in Asia.
Summary
This supplementary material includes additional laboratory methods, one supplementary figure and three supplementary tables. Supplementary Figure S1 is a map of sampling regions and Supplementary Table S1 a list of Asian dog samples used in this study. Supplementary table S2 is a list of the dog samples from Kennedy et al. (2007b) by breed and region of origin. Supplementary table S3 includes all dog and wolf DRB1-alleles obtained from GenBank database or reported in this study.
Methods
DNA techniques
DNA was extracted from blood, hair or buccal cell swabs preserved on FTA-cards (Whatman International, UK). DNA extraction from hair was performed according to Hopgood et al. (1992) using two to five dog hairs for each individual extraction, from blood using NucleoSpin Blood Mini Kit (Clontech, California, USA) and from FTA-cards according to the manual provided with the kit.
Amplification of the second exon of the DLA-DRB1 gene was carried out with a semi-nested PCR (Mullis and Faloona, 1987) for increased target specificity. An initial reaction with forward primer 1 (5’- CGA ACC GTG GGT GCT GGT -3’) and reverse primer 1 (5’- TGT GTC ACA CAC CTC AGC ACC A -3’ (Wagner, et al., 1996), was followed by a second reaction with an inner forward primer 2 (5’- GGG TGC TGG TGG TTG GG 3’) and the same reverse primer as in the initial reaction with a T7-tail (underlined) attached (5’- TAA TAC GAC TCA CTA TAG GG TGT GTC ACA CAC CTC AGC ACCA -3’). The forward primers' design was based on the dog sequence on Vega dog website (http://vega.sanger.ac.uk/Canis_familiaris). PCR amplification was carried out using a ThermoHybaid MBS 02S thermal cycler (Thermo Electron Corporation, Massachusetts, USA). The initial PCR mixture contained DNA template (either 5 ml of hair DNA extract, 1 ml of blood DNA extract or 1 punch from the FTA-card), 0.2 mM of each primer, 2 mM MgCl2, 20 mM Tris-HCl pH 8.4, 200 mM of each dNTP and 1 U of Platinum Taq DNA Polymerase (Invitrogen) in a total volume of 50 ml. The initial PCR program consisted of a predenaturation step of 2 min at 94°C, 20 cycles of denaturation (94°C, 30 s), primer annealing (60°C, 30 s) and extension (72°C, 1 min), followed by a final extension at 72°C for 10 minutes. For the secondary PCR, the mixture was identical to the initial PCR mixture, except that 1 ml of the initial reaction was used as a template and 0.1 mM of both inner primers were added. The secondary PCR program was identical to the initial PCR program, with the exception that 35 cycles were performed.
Both strands were sequenced using the inner forward PCR primer on the leading strand and a T7 universal primer on the lagging strand as sequencing primers giving full reading of the sequence in both directions. For the cycle sequencing reaction, the final amplification product was diluted with MilliQ-water: 1:3 for the forward primer reaction and 1:1 for the reverse primer reaction. One ml of diluted PCR product was mixed with 17,5 ml 1x Cycle Sequencing buffer (26 mM Tris pH 9.0, 6.5 mM MgCl2), 0,5 ml Big Dye Terminator (ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit v2.1, Applied Biosystems, California, USA) and 0.25 mM primer, for a total reaction volume of 20 ml. Cycle sequencing reaction was run using a PTC thermal cycler (MJ Research Inc., Massachusetts, USA). The program consisted of 35 cycles of denaturation (96° C, 10 s), primer annealing (55° C, 15 s) and extension (60° C, 4 min). Cycle sequencing products were analysed with ABI 3700 instrument (Applied Biosystems) according to the manufacturer's instructions.
Cloning and allele verification
DNA sequences were edited using Sequencing Analysis (Applied Biosystems), and assembled into contigs and further edited in Sequencher 4.1 (Gene Codes, Michigan, USA). Individual dogs were identified as either homozygous or heterozygous from the presence of double peaks in the contigs. To identify the alleles from heterozygous individuals, all heterozygotes were cloned using the GenetJETTM PCR Cloning Kit (Fermentas, Burlington, Canada). The cloning was done according to the manual, except that the reaction volume was reduced by 75%. Individual clones were picked and used as PCR templates first using the primer provided with the cloning kit and then using the primers of the secondary PCR described above. Due to inherent problems in cloning multi-template PCR products (Keohavong and Thilly, 1989, Thompson, et al., 2002) we followed the standard rules for cloned PCR products to identify MHC alleles. All alleles identified by cloning were verified by at least two separate clones, which were sequenced in both directions. Furthermore, all novel alleles identified in this study were verified by a second independent analysis (PCR, cloning and DNA sequencing) in which the DNA sample was amplified in a new PCR reaction, using another set of PCR primers (forward 5’- GATCCCCCCGTCCCCACAG -3’, reverse 5’- CGCCCGCTGCGCTCA -3’ (2007a)) normally used in previous studies. This was done to verify (i) that the novel alleles were not created by PCR error, and (ii) that the novel alleles had not been missed in previous studies (due to primer mismatch) because of the choice of PCR primer.
Title and legend to supplementary figure
Supplementary Figure S1. Sampling regions and the number of samples from each region. For more detailed information about geographical origin, see Supplementary Table S1. A: Siberia (n = 16 samples), B: China (n = 18), C: Japan (n = 17), D: Southeast Asia (n = 18), E: Western Southwest Asia (n = 10), F: Persian plateau (n = 18), G: India (n = 12), H: General Southwest Asia (n = 19). The green line indicates the division of Asia into "North or East Asia" (N/E Asia) and "South or West Asia" (S/W Asia).
Supplementary Table S1. The Asian dog samples used in this study by breed and region of origin. For non-breed dogs geographical origin refers to the location of the population, for
breed dogs it refers to the historical origin of the breed, not the location of sampling. Note, the region Siberia includes the adjacent regions NE Russia and Tajikistan, and China includes Nepal.
Breed/Type / Geographical region (#) / Country, Region/Province / n
Chinese crested / China (B) / China / 1
Chow-chow / China (B) / China / 6
Lhasa Apso / China (B) / China / 1
Pug / China (B) / China / 1
Shar-pei / China (B) / China / 1
Shih tzu / China (B) / China / 1
Tibetan Mastiff / China (B) / China, Tibet / 3
Tibetan Terrier / China (B) / China, Tibet / 3
Village dog / China (B) / Nepal / 1
Akita / Japan (C) / Japan / 1
Hokkaido / Japan (C) / Japan / 5
Iwate Matag / Japan (C) / Japan / 1
Japanese spitz / Japan (C) / Japan / 1
Kai / Japan (C) / Japan / 1
Kawakami / Japan (C) / Japan / 1
Kisyu / Japan (C) / Japan / 1
Minamidaitojima / Japan (C) / Japan / 1
Satsuma / Japan (C) / Japan / 1
Shiba / Japan (C) / Japan / 2
Shikoku / Japan (C) / Japan / 1
Tsushima dog / Japan (C) / Japan / 1
Village dog / SoutheastE Asia (D) / Cambodia / 2
Village dog / Southeast Asia (D)SE Asia / Thailand / 2
Phuquoc / Southeast Asia (D)SE Asia / Vietnam, Phuquoc Island / 12
Village dog / Southeast Asia (D)SE Asia / Vietnam / 2
Aboriginal sledge dog / Siberia (A) / Russia, Siberia / 4
Chukotka sledge dog / Siberia (A) / Russia, NE Siberia, Chukotka / 1
East Siberian Laika / Siberia (A) / Russia, East Siberia / 2
Samoyed / Siberia (A) / Russia, NE Russia/NW Siberia / 2
Siberian Husky / Siberia (A) / Russia, NE Siberia, Chukotka / 1
West Siberian Laika / Siberia (A) / Russia, West Siberia / 2
Village dog / Siberia (A) / Russia, Siberia / 3
Village dog / Siberia (A) / Tajikistan / 1
total / 69
South and West Asia
Breed/Type / Geographical region / Country, Region/Province / n
Saluki/Tazi / General Southwest Asia (H) / 19
Village dog / India (G) / India, Tamil Nadu / 12
Afghan / Persian plateau (F) / Afghanistan / 4
Village dog / Persian plateau (F) / Iran, across western part / 14
Canaan dog / Western Southwest Asia (E) / Israel / 6
Kangal / Western Southwest Asia (E) / Turkey / 4
total / 59
# Index of the region from Supplementary Figure S1.
Supplementary Table S2. The dog samples from Kennedy et al. (2007b) by breed and region of origin.
breed / Country, region / n
Airedale terrier / United Kingdom / 2
Basset hound / United Kingdom / 5
Beagle / United Kingdom / 67
Bearded collie / United Kingdom / 4
Bernese mountain dog / Switzerland / 3
Bichon frise / France/Belgium / 22
Border collie / United Kingdom / 22
Border terrier / United Kingdom / 10
Bouvier / Belgium/France / 1
Boxer / Germany / 41
Bulldog / United Kingdom / 5
Bullterrier / United Kingdom / 1
Cairn terrier / United Kingdom / 12
Cavalier king charles spaniel / United Kingdom / 14
Clumber spaniel / United Kingdom / 1
Cocker spaniel / United Kingdom / 30
Collie / United Kingdom / 15
Corgi / United Kingdom / 4
Croatian Pudl (medium) / Croatia / 3
Croatian Pudl (mini) / Croatia / 2
Dachshund / Germany / 4
Dachshund (long haired) / Germany / 3
Dachshund (miniature) / Germany / 1
Dachshund (miniature short haired) / Germany / 11
Dachshund (short haired) / Germany / 5
Dalmatian / Croatia / 4
Dandie dinmont terrier / United Kingdom / 1
Deerhound / United Kingdom / 1
Doberman / Germany / 183
English setter / United Kingdom / 41
Flat-coated retriever / United Kingdom / 4
Fox terrier / United Kingdom / 1
German langhaar / Germany / 1
German shepherd / Germany / 27
German spitz / Germany / 1
Golden retriever / United Kingdom / 31
Gordon setter / United Kingdom / 5
Great dane / Germany / 5
Hungarian vizsla / Hungary / 2
Hovawart / Germany / 4
Ibizan hound / Spain / 51
Supplementary Table S2. Continued.
Irish setter / Ireland / 3Irish wolfhound / Ireland / 1
Jack russell terrier / United Kingdom / 39
Labrador / United Kingdom / 88
Lagotto romagnolo / Italy / 1
Lakeland terrier / United Kingdom / 1
Leonberger / Germany / 1
Lurcher / Ireland/United Kingdom / 4
Maltese terrier / Mediterranean region / 1
Mongrel / Europe / 87
Norwegian elkhound / Norway / 1
Norwich terrier / United Kingdom / 1
Old English sheepdog / United Kingdom / 6
Papillon / France/Belgium / 3
Petit basset griffon vendeen / France / 1
Pointer / United Kingdom / 2
Polish lowland sheepdog / Poland / 1
Pomeranian / Germany / 2
Poodle / France / 4
Poodle (Miniature) / France / 17
Poodle (Toy) / France / 5
Pyrenean mountain dog / France / 1
Rottweiler / Germany / 8
Schnauzer / Germany / 1
Schnauzer (miniature) / Germany / 13
Scottish terrier / United Kingdom / 2
Shetland sheepdog / United Kingdom / 4
Soft coated wheaten terrier / Ireland / 1
Springer spaniel / United Kingdom / 12
Staffordshire bull terrier / United Kingdom / 19
St Bernard / Switzerland / 1
Weimaraner / Germany / 3
Welsh springer spaniel / United Kingdom / 1
West highland white terrier / United Kingdom / 22
Yorkshire terrier / United Kingdom / 44
total / 1051
Supplementary Table S2. Continued.
breed / Country, region / n
chow chow / China / 6
Siberian husky / Russia, Siberia / 1
Japanese akita / Japan / 1
West siberian laika / Russia, Siberia / 13
Lhasa apso / China / 5
Samoyed / Russia, Siberia / 15
Shih tzu / China / 13
Tibetan terrier / China, Tibet / 6
total / 60
South and West Asia
breed / Country, region / n
Afghan hound / Afghanistan / 1
Caucasian shepherd dog / Caucasus / 6
Caucasian wolfdog / Caucasus / 12
Middle Asian shepherd dog / Turkmenistan/Central Asia / 42
total / 61
Africa
breed / Country / n
Pharaoh hound / Egypt/Malta / 1
Rhodesian ridgeback / Southern Africa / 82
Mongrel Tansania / Tanzania / 27
total / 110
North America
breed / Country / n
Alaskan malamute / USA / 12
Newfoundland / Canada / 75
Chesapeake bay retriever / USA / 1
American cocker spaniel / USA / 2
total / 90
South America
breed / Country / n
Mongrel Brasilia / Brazil / 112
total / 112
Supplementary table S3. All full length (270 bp) dog and wolf DLA-DRB1 alleles obtained from GenBank database or reported in this study. Geographical distribution is shown, if the allele has been found in this study or in the study by Kennedy et al. (2007b). The canine species in which the allele was first identified: C.f. = Canis familiaris and C.l. = Canis lupus.
Asia / NE
Asia / Africa / North
America / South
America / Australia
DLA-DRB1*03101 / AF336108 / C.lC.l.
DLA-DRB1*03501 / AF336109 / C.lC.l.
DLA-DRB1*03601 / AF336110 / C.lC.l.
DLA-DRB1*03701 / AF343738 / C.lC.l.
DLA-DRB1*03801 / AF343739 / C.lC.l.
DLA-DRB1*03901 / AF343740 / C.lC.l.
DLA-DRB1*03301 / AF343737 / C.fC.f. / X
DLA-DRB1*04001 / AF343741 / C.fC.f. / X / X / X
DLA-DRB1*04601 / AF343747 / C.fC.f. / X / X
DLA-DRB1*04701 / AF343748 / C.fC.f.
DLA-DRB1*00301 / AJ003012 / C.fC.f.
DLA-DRB1*01502 / AJ003013 / C.fC.f. / X / X / X
DLA-DRB1*01503 / AJ003014 / C.fC.f. / X / X / X
DLA-DRB1*01201 / AJ003015 / C.fC.f.
DLA-DRB1*02301 / AJ003016 / C.fC.f. / X / X
DLA-DRB1*00501 / AJ003017 / C.fC.f.
DLA-DRB1*02401 / AJ003018 / C.fC.f.
DLA-DRB1*02501 / AJ003019 / C.fC.f. / X / X
DLA-DRB1*02601 / AJ003020 / C.fC.f.
DLA-DRB1*01601 / AJ012454 / C.fC.f. / X