1471-2156-6-6 1471-2156 Research article <p>Genetic variation analysis of the Bali street dog using microsatellites</p> Irion N Dawn dnirion@ucdavis.edu Schaffer L Alison alison@ucdavis.edu Grant Sherry sherrygrant@grantland.com Wilton N Alan a.wilton@unsw.EDU.AU Pedersen C Niels ncpedersen@ucdavis.edu

Veterinary Genetics Laboratory, Center for Veterinary Genetics, School of Veterinary Medicine, University of California, Davis, California 95616, USA

Yayasan Yudisthira, Sanur, Bali 80228, Indonesia

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia

 BMC Genetics 1471-2156 2005 6 1 6 http://www.biomedcentral.com/1471-2156/6/6 15701179 10.1186/1471-2156-6-6 10 8 2004 08 2 2005 08 2 2005 2005 Irion et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Approximately 800,000 primarily feral dogs live on the small island of Bali. To analyze the genetic diversity in this population, forty samples were collected at random from dogs in the Denpasar, Bali region and tested using 31 polymorphic microsatellites. Australian dingoes and 28 American Kennel Club breeds were compared to the Bali Street Dog (BSD) for allelic diversity, heterozygosities, F-statistics, GST estimates, Nei's DA distance and phylogenetic relationships.

Results

The BSD proved to be the most heterogeneous, exhibiting 239 of the 366 total alleles observed across all groups and breeds and had an observed heterozygosity of 0.692. Thirteen private alleles were observed in the BSD with an additional three alleles observed only in the BSD and the Australian dingo. The BSD was related most closely to the Chow Chow with a FST of 0.088 and also with high bootstrap support to the Australian dingo and Akita in the phylogenetic analysis.

Conclusions

This preliminary study into the diversity and relationship of the BSD to other domestic and feral dog populations shows the BSD to be highly heterogeneous and related to populations of East Asian origin. These results indicate that a viable and diverse population of dogs existed on the island of Bali prior to its geographic isolation approximately 12,000 years ago and has been little influenced by domesticated European dogs since that time.

Background

Bali, a province of the Republic of Indonesia, is an island just 87 km from north to south and 142 km from east to west and home to more than 2.9 million people 1. Approximately 800,000 stray dogs (Fig. 1) also live on the island based on a survey conducted by the Bali Street Dog Foundation (personal communication). Only a small percentage of these dogs live in homes or are provided routine veterinary care 2.

<p>Figure 1</p>

Typical Balinese street dogs

Typical Balinese street dogs. Their phenotypic appearance is similar to that described for randomly breeding feral dog subpopulations in other parts of the world.

More than 90% of the residents of Bali are Hindu 3 with myth and ritual playing a vital part of daily life 1. The dog is also an important part of Balinese life and mythology. A popular tale from the Mahabharata 4 describes King Yudisthira's journey to Heaven's Gate, and his love for a dog that befriended him on his arduous and tragic journey (Fig. 2). As a direct result of such mythology, BSDs are treated with a degree of reverence and are often provided ceremonial food offerings 2. The deliberate killing of street dogs is not typically practiced, because Balinese people believe that all things should be allowed to die naturally 2. These cultural mores have contributed to the current overpopulation of dogs on the island.

<p>Figure 2</p>

The Story of Yudisthira [4]

The Story of Yudisthira [4].

As a result of overpopulation, many BSDs suffer from chronic skin diseases, internal parasites, parvo- and distemper-virus infections, and malnutrition. In an effort to reduce the dog population and to care for their medical needs, the Bali Street Dog Foundation (Yayasan Yudisthira Swarga) was founded in 1998 2. They provide emergency care, treatment for skin disease and parasites, sterilization, public education on the plight of feral dogs, and improved veterinarian training. Twenty to 30 dogs are sterilized each day, with more than 9,000 dogs sterilized to date.

The BSD population is of interest for both its genetic diversity and historical relationships. It is also a population that has bred more or less randomly for thousands of years with limited genetic influx, due mainly to geographic barriers and a strict rabies control program in effect since 1926. The present study is concerned with the genetic diversity of this unique canine population and its relationship to other canine subpopulations in Asia and throughout the world. Data presented herein was derived from the DNA testing of 40 BSD samples from the Denpasar city region of Bali with 31 polymorphic microsatellite loci. The genetic diversity of the BSD was compared to that of the Australian dingo and 28 American Kennel Club (AKC) breeds.

Results

Locus diversity

Analysis of locus diversity across all 30 subpopulations revealed that the number of observed alleles ranged from six to 20 with a total of 366 for all loci (Table 1). Overall heterozygosity of the loci was high, with an average of 0.779, and all but four loci having HT values greater than 0.700. Average HS was 0.577 for the 30 subpopulations, with all but three loci having HS values greater than 0.500. The HS and HT values were closest for C23.123 and farthest for C22.279 and C10.404. HWE analysis revealed that all but one locus had at least one population out of equilibrium for the 30 populations sampled. C01.424, C31.646 and CPH16 had 7 populations out of HWE and AHT130 did not have any populations with p values below 0.05. The level of locus diversity attributable to subpopulation structure was evaluated with two statistics – RST and FST. Both statistics gave similar average values at 0.230 and 0.236 respectively. However, RST ranged from 0.098 to 0.486 while FST ranged from 0.179 to 0.328.

<p>Table 1</p>

Observed number of alleles, average total heterozygosity (HT), average subpopulation heterozygosity (HS), number of populations out of HWE, average p values, RST, FST, RST/FSTratio, GSTand pairwise FSTvalues for 31 loci.

BSD Pairwise FST by Locus

Chr.

Num. Observed Alleles

HT

HS

Num. Loci with p value <0.05

Average p value

RST

FST

RST/FST

× Dingo

× Chow

CPH16

CFA20

11

0.829

0.610

7

0.407

0.098

0.233

0.420

0.005

0.132

C08.618

CFA08

9

0.744

0.553

4

0.481

0.148

0.229

0.646

0.071

0.185

FH2001

CFA23

13

0.791

0.593

4

0.433

0.167

0.225

0.741

0.083

0.100

C20.446

CFA20

10

0.729

0.553

3

0.541

0.173

0.215

0.804

0.123

0.105

C01.424

CFA01

9

0.716

0.476

7

0.475

0.258

0.320

0.807

0.125

0.110

CPH02

CFA32

9

0.693

0.520

4

0.499

0.194

0.223

0.871

0.148

0.066

FH2004

CFA11

18

0.809

0.611

3

0.522

0.187

0.214

0.873

0.057

0.060

AHT137

CFA11

14

0.861

0.672

2

0.427

0.176

0.197

0.893

0.064

0.188

C03.877

CFA03

12

0.730

0.509

1

0.524

0.268

0.275

0.977

0.244

0.114

C06.636

CFA06

12

0.652

0.483

6

0.435

0.233

0.237

0.982

0.069

0.024

AHT121

CFA13

18

0.865

0.632

1

0.511

0.255

0.251

1.016

0.098

0.063

VIASD10

CFA07

9

0.759

0.555

2

0.525

0.249

0.233

1.066

0.268

0.030

C31.646

CFA31

14

0.814

0.566

7

0.410

0.301

0.281

1.075

0.088

0.036

RVC1

CFA15

9

0.774

0.569

4

0.458

0.270

0.242

1.119

0.355

0.216

LEI002

CFA27

11

0.737

0.551

4

0.534

0.259

0.229

1.127

0.107

0.149

LEI004

CFA37

13

0.667

0.509

4

0.510

0.246

0.219

1.128

0.133

0.051

C28.176

CFA28

10

0.735

0.546

6

0.400

0.270

0.234

1.152

0.249

0.013

C22.279

CFA22

11

0.836

0.529

2

0.441

0.262

0.214

1.226

0.201

0.109

PEZ02

Unlinked

12

0.762

0.600

1

0.567

0.232

0.187

1.242

0.134

0.027

FH2054

CFA12

10

0.848

0.654

4

0.524

0.251

0.199

1.261

0.055

0.069

C23.123

CFA23

8

0.766

0.636

4

0.402

0.351

0.278

1.263

0.110

0.002

CPH08

CFA19

11

0.765

0.582

4

0.465

0.284

0.222

1.276

0.082

0.056

C14.866

CFA14

10

0.840

0.604

3

0.473

0.330

0.255

1.293

0.180

0.112

PEZ08

CFA17

17

0.859

0.684

5

0.465

0.235

0.179

1.312

0.121

0.079

AHT130

CFA18

11

0.829

0.614

0

0.539

0.313

0.235

1.331

0.116

0.105

AHT111

CFA02

11

0.785

0.582

4

0.371

0.348

0.246

1.414

0.214

0.006

C10.404

CFA10

13

0.865

0.558

3

0.526

0.486

0.328

1.479

0.177

0.160

C09.250

CFA09

10

0.830

0.582

1

0.491

0.412

0.272

1.513

0.016

0.042

FH2140

CFA05

20

0.795

0.621

2

0.560

0.297

0.192

1.546

0.119

0.072

AHT139

CFA15

6

0.664

0.508

4

0.452

0.330

0.206

1.604

0.085

0.078

CPH03

CFA06

15

0.815

0.612

2

0.526

0.394

0.229

1.724

0.017

0.180

All

366

0.779

0.577

108

0.481

0.230

0.236

1.135

0.126

0.088

Bali street dog diversity

Overall, the BSD was the most genetically diverse population surveyed here, displaying 239 total alleles out of the 366 seen in all 30 subpopulations or 65.3% of the total observed alleles (Table 2). The Australian dingo displayed 144 alleles and the AKC breeds displayed 138.8 on average. Analysis of expected (HE) and observed (HO) heterozygosities (Table 2) revealed that the BSD had a 44.0% higher HE than the Australian dingo (0.736 vs. 0.511) and a 28.4% higher HE than the average AKC breed (0.573). HO was also highest in the BSD at 0.692, versus 0.426 in the Australian dingo and 0.563 in the average AKC breed.

<p>Table 2</p>

Total number of alleles (NA) observed, range of the lowest and highest number of observed alleles per locus, expected heterozygosity (HE), observed heterozygosity (HO), FIS, number of loci out of HWE and average p values for all 31 loci for the BSD, a bootstrapped sampling of the BSD, the Australian dingo, the American Kennel Club breeds and for all subpopulations.

NA Observed

NA Range

HE

HO

FIS

Num. Loci with p value <0.05

Average p value

Bali Street Dog

239

3 – 14

0.736

0.692

0.097

4

0.357

Bali Street Dog20

214.6

---

0.727

0.692

---

---

---

Australian dingo

144

2 – 9

0.511

0.426

0.194

12

0.284

AKC Breeds

138.8

2.2 – 7.8

0.573

0.563

0.137

3.29

0.492

All Populations

366

6 – 20

0.577

0.562

0.136

3.60

0.492

In order to evaluate the bias of sampling twice the number of BSDs, twenty samples were taken at random from the total pool of 40 for bootstrap determinations, and the number of observed alleles, HE, and HO were calculated. This process was repeated for 10,000 iterations and the average value for each measurement was determined. The average bootstrap value for the number of observed alleles for 20 BSDs was 214.6. The average bootstrapped HE and HO values for the BSD were 0.727 and 0.692 respectively.

To understand the loss of approximately 24 observed alleles after bootstrapping, the allele frequencies for the BSD at each locus was examined. While the BSD had the highest number of alleles, they also had the highest number of alleles with a frequency below 5% (67 out of 239, data not shown).

FIS estimates were calculated to assess the level of inbreeding for each subpopulation (Table 2). The BSD had the lowest value at 0.097 and the Australian dingo had the highest at 0.194 with the average of AKC breeds at 0.137.

Private alleles

Allele frequency analysis also revealed that 10 private alleles were observed in the BSD as well as three alleles shared only with the Australian dingo (Table 3). The majority of private alleles in the BSD were below 5% in frequency with the exception of AHT121 where the private alleles had a combined frequency of 18.75%. The BSD and the Australian dingo also shared three alleles not seen in any AKC breed at locus C10.404 with a combined frequency of 16.25% in the BSD and 75% in the Australian dingo.

<p>Table 3</p>

Private alleles for the BSD and Australian dingo subpopulations relative to the 28 comparison AKC breeds.

Locus

Allele

Pop

Freq.

Pop

Freq.

AHT111

92

BSD

0.013

AHT121

82

BSD

0.075

AHT121

90

BSD

0.113

C06.636

158

BSD

0.013

C10.404

168

BSD

0.038

Dingo

0.075

C10.404

170

BSD

0.063

Dingo

0.450

C10.404

172

BSD

0.063

Dingo

0.225

C10.404

174

BSD

0.038

C23.123

154

BSD

0.013

FH2140

160

BSD

0.013

FH2140

171

BSD

0.025

PEZ02

144

BSD

0.013

VIASD10

94

BSD

0.013

Asian alleles

Several additional unique alleles were found only in the BSD, Australian dingo, Chow Chow and Akita; demonstrating a closer relationship of the BSD to Asian versus non-Asian dogs (data not shown). Appearing at the highest frequency was allele 201 of locus CPH08 in the BSD, Australian dingo and Chow Chow at frequencies of 21.3%, 10% and 20%, respectively. Further, the BSD, Australian dingo, Chow Chow and Akita share allele 113 of locus C22.279 at frequencies of 23.8%, 67.5%, 15% and 5%, respectively. Results for locus PEZ08 demonstrate a lack of influence of European alleles where a high frequency of deviations from n+4 alleles were observed in the AKC breeds sampled yet no deviation from n+4 alleles was observed in the BSD or the Australian dingo.

A pairwise FST analysis was performed for each locus between the BSD and the two closest subpopulations: the Australian dingo and Chow Chow (Table 1). The BSD was most similar to the Australian dingo at locus CPH16 with a FST of 0.005 and to the Chow Chow at locus C23.123 with a FST of 0.002. The BSD was most dissimilar to the Australian dingo and Chow Chow at locus RVC1 with a FST of 0.355 and 0.216 respectively. Several loci had similar distances for both population pairs, such as locus C01.424 or locus C20.446 and may indicate areas of the genome that are neutral to either environmental or human selection.

Genetic distance relationships

Further distance analysis was performed for all 31 loci between all 30 subpopulations using both Nei's DA distance and pairwise FST estimates (Table 4). Across all loci, the BSD shared allele frequencies most closely with the Chow Chow (DA = 0.242, FST = 0.088) and the Australian dingo (DA = 0.242, FST = 0.126), and least closely with the Airedale Terrier (DA = 0.454, FST = 0.258).

<p>Table 4</p>

Nei's DA distance (lower triangle) and mean FSTestimates (upper triangle) between each pair of 9 dog subpopulations represented graphically in Figure 3.

BSD

Dingo

Chow

Akita

AES

AS

AT

BCO

BLT

MBT

BMD

BS

BT

BU

BX

BZ

DP

GH

GR

JRT

KE

LR

NE

PG

PM

PPN

PWC

RR

WM

YT

BSD

0.13

0.09

0.13

0.14

0.14

0.26

0.19

0.26

0.25

0.21

0.15

0.17

0.18

0.33

0.18

0.26

0.17

0.14

0.11

0.17

0.17

0.18

0.23

0.13

0.11

0.15

0.15

0.16

0.13

Dingo

0.24

0.23

0.26

0.27

0.27

0.41

0.32

0.42

0.40

0.37

0.30

0.29

0.29

0.50

0.31

0.41

0.30

0.26

0.25

0.31

0.30

0.31

0.39

0.27

0.25

0.29

0.29

0.29

0.25

Chow

0.24

0.40

0.19

0.22

0.21

0.34

0.26

0.33

0.32

0.29

0.24

0.23

0.24

0.41

0.26

0.34

0.26

0.21

0.17

0.23

0.25

0.25

0.31

0.20

0.17

0.22

0.22

0.23

0.17

Akita

0.29

0.41

0.36

0.19

0.19

0.33

0.26

0.35

0.32

0.31

0.20

0.22

0.25

0.40

0.23

0.30

0.24

0.18

0.17

0.23

0.22

0.26

0.30

0.20

0.17

0.20

0.20

0.22

0.19

AES

0.28

0.43

0.43

0.37

0.10

0.29

0.16

0.33

0.30

0.23

0.14

0.16

0.19

0.32

0.24

0.24

0.13

0.12

0.10

0.14

0.16

0.18

0.25

0.12

0.10

0.17

0.13

0.16

0.13

AS

0.27

0.42

0.38

0.36

0.20

0.27

0.15

0.32

0.29

0.24

0.16

0.17

0.18

0.35

0.21

0.23

0.16

0.13

0.10

0.17

0.16

0.19

0.23

0.13

0.10

0.16

0.13

0.17

0.12

AT

0.45

0.55

0.50

0.45

0.40

0.36

0.35

0.46

0.44

0.37

0.34

0.32

0.34

0.50

0.31

0.46

0.32

0.29

0.26

0.34

0.32

0.32

0.42

0.27

0.28

0.34

0.32

0.31

0.28

BCO

0.35

0.49

0.48

0.44

0.26

0.25

0.45

0.39

0.37

0.24

0.21

0.21

0.25

0.41

0.29

0.30

0.22

0.21

0.15

0.24

0.23

0.25

0.30

0.17

0.16

0.20

0.22

0.21

0.16

BLT

0.43

0.55

0.51

0.52

0.46

0.42

0.51

0.54

0.10

0.43

0.35

0.40

0.35

0.49

0.40

0.44

0.36

0.30

0.25

0.34

0.32

0.35

0.46

0.33

0.27

0.36

0.36

0.36

0.30

MBT

0.41

0.52

0.48

0.48

0.41

0.38

0.48

0.50

0.09

0.54

0.52

0.54

0.38

0.46

0.50

0.52

0.48

0.43

0.36

0.42

0.41

0.34

0.44

0.30

0.26

0.35

0.34

0.33

0.29

BMD

0.39

0.51

0.47

0.49

0.36

0.35

0.45

0.36

0.54

0.42

0.23

0.28

0.30

0.42

0.35

0.36

0.28

0.23