The facts about dingo DNA testing – reliability and accuracy.

There are several options to carry out ancestry testing on dingoes in Australia, however, not all tests are accurate and there are several limitations that users should be aware of when considering DNA test results – as pet owners, captive breeding sanctuaries or wildlife managers. Here I answer some common questions about DNA testing technology and the limitations of these methods.

What's the problem and why do people do DNA testing on dingoes?

There has been concern about the introgression of domestic dog genes into wild dingo populations through dingo x dog hybridisation for decades. In the 1970s-1980s, scientists developed a method of skull morphology assessment that could be used to estimate the likelihood that an animal was a dingo, dingo x dog hybrid or domestic dog. In the late 1990s Dr Alan Wilton developed a method of genetic ancestry estimation that could be used to DNA test dingoes. Since then improvements have been made to the ‘dingo DNA test’, including by Dr Danielle Stephens (Zoolgenetics) and it is widely used by dingo owners, conservation groups and wildlife managers. Over the last 20 years more than 5000 DNA tests have been conducted on wild and captive dingoes.

Some commercial companies also offer DNA testing, focused on estimating the breed make up of domestic dogs. It is based on a similar technology, but these options may not compare directly to known dingoes. Skull morphology testing is now regarded as having limited value because A) dingoes with dog ancestry maintain a dingo-like skull morphology and B) estimates were based on the assumption that dingoes form a single population. We now know that there are multiple geographically subdivided and evolutionarily distinct lineages of dingo across Australia (see Cairns et al 2018 and Koungoulos 2020).

What type of genetic technology is used in most dingo DNA tests?

A majority of DNA tests either offered specifically for dingoes or for domestic dogs rely upon microsatellite markers, these are short repeating regions in the genome. Microsatellites are useful for DNA testing because the size of the repeat region is highly variable. A DNA test uses the length of these repeat regions to distinguish between species or breeds or individuals. Some newer testing methods use genome-wide SNPs or single nucleotide polymorphisms, these tend to be more informative about evolutionary history, but more markers are generally needed. SNP based technology can be more expensive because it is based on newer technology, but this can depend on the number of markers used and laboratory methods.

A microsatellite allele from the Wilton dingo DNA test

The DNA test developed by Dr Alan Wilton, currently offered by UNSW and Zoolgenetics, relies predominately on a set of 21 microsatellite and 2 SNP markers. Dr Kylie Cairns is leading a research project exploring the utility of genome-wide SNP using 1000s of markers to assess ancestry in dingoes.

Microsatellite markers differentiate between dingoes and dogs by comparing the frequency of different alleles (markers) between dingoes and dogs. For example, allele 2, 3 and 5 are found more commonly in dingoes rather than dogs, whilst alleles 9, 10 and 13 are more common in dogs than dingoes. Some alleles are observed in both dogs and dingoes, so more data is needed to estimate the ancestry reliably. SNP technology that looks at 1000s of markers may provide a more reliable and informative assessment of ancestry.

Allele frequencies in dogs versus dingoes at CXX2175 from Cairns et al (2019).

An example of an informative SNP marker

How do DNA tests distinguish between dingoes, dingo hybrids and domestic dogs?

The microsatellite DNA testing method relies upon comparing an unknown sample (ie your dingo’s DNA) to a set of known pure dingoes and domestic dogs, called reference populations. Most commercial tests rely on reference populations made up of domestic dog breeds. A sample is identified as having ancestry from a particular breed, dogs or dingoes if they share markers common to a particular reference population. Reliance on reference populations can be an issue for genetic testing methods. When doing dingo DNA testing it is important that the dingo reference population contains examples of dingoes from all regions of Australia, particularly now that we know there are multiple evolutionary lineages of dingo in Australia. If a reference population does not have an even representation then this may bias the result, particularly if comparing a sample from a region not included in the reference population. At the moment, we don't know what impact the regional variation in dingoes might be having. It may be sensible to compare dingoes to reference populations from the same region and be mindful of this limitation when considering the results of DNA testing.

With commercial dog breed testing providers, it is important to check that they have a dingo reference population. Zoolgenetics has both dingo and dog reference populations, however their dingo reference population is predominately from WA, NT, SA and QLD. The Alan Wilton (UNSW) dingo test method has a dingo reference population from predominately NSW, VIC and captive dingoes.

Some more modern SNP-based DNA tests do not rely as heavily on strict reference populations, instead using larger numbers of markers and complex computer modelling programs to estimate ancestry. If you want to know more about my ongoing research project (or contribute samples) then please get in touch!

A PCA comparison of dingoes, dogs and wolves from genome-wide SNP data (Smith et al. 2019)

Can you tell if a dingo is pure based on physical appearance ie photographs?

No, it is difficult to detect domestic dog ancestry in dingoes based on photographs or physical appearance because dingoes, including those with dog ancestry, have a strongly dingo-like appearance. Dingoes may have coat colours such as ginger, black & tan, white, black (with white tips) and sable. The colours brindle and patchy may be an indication of historical domestic dog ancestry but not necessarily recent hybridisation. It is also possible that these colours are ancestral.

If a dingo has recent domestic dog ancestry it may be more likely to have physical features that are dog-like, for example: floppy ears, unusual coat colours, different tail set, short muzzle, broad chest, shortened legs etc. Unusual coat colours may be mottled (like an Australian Cattle Dog), merle, brown colour or solid black (with no markings).

Whilst it may be difficult to detect dog ancestry in dingoes, it is reasonably easy to distinguish between domestic dogs and dingoes.

Photos from Michelle J Photography, The Australian Dingo Foundation and ABC News

Can ‘dingo DNA testing’ tell me if my dingo is 99% or 97% dingo?

The currently available methods are not sensitive enough to detect if an animal is 99% or 97% dingo, particularly if based on 23 (or fewer) markers.

Microsatellite based testing provides a broad estimate that an animal is:

1. a dingo with no dog ancestry

2. a dingo with likely no dog ancestry

3. a dingo with dog ancestry (>75% dingo)

4. a dingo with dog ancestry (65-75% dingo)

5. a dingo with dog ancestry (50-64% dingo)

6. a feral dog with dingo ancestry (<50% dingo)

7. a feral dog with no dingo ancestry

Newer SNP based methods may be able to estimate a specific percent of dog ancestry based on larger numbers of genetic markers, computer modelling and comparison to a wider set of domestic dogs.

How accurate and reliable is my DNA test result?

The accuracy and reliability of DNA testing depends upon the number or type of genetic markers, as well as the quality of the comparison (or reference population) data.

The type of marker and number of markers is very important in assessing the reliability of a DNA test result. The detection limit of the ‘dingo DNA’ microsatellite test is generally considered to be 4 generations ie it may only be capable of detecting dog ancestry if it occurred within 4 generations (Cairns et al 2011). SNP genotyping is likely to provide more reliable and accurate estimates of dingo ancestry because it uses many more markers across the whole genome (1000s compared to 23), there is also a larger capacity to compare to large collections of domestic dog data to increase the accuracy of ancestry estimates.

If less than 23 markers are used, this limits the reliability and accuracy of a microsatellite DNA test result. Both Stephens et al 2015 and Cairns et al 2019 only consider a result to be reliable if more than 14 markers have been successfully tested, out of the 23 markers available. In short, more markers mean a more reliable result.

The next consideration is what animals are used in the reference populations. Now that it is known that dingoes form several distinct lineages, it is important to consider what impact this may have on ancestry DNA testing. It may become best practice to compare a dingo from a specific region to other dingoes from that region, or to ensure the reference population includes dingoes from as many regions as possible. This might improve the reliability of a DNA test result. Another consideration might be the number of dingoes within the reference population, a larger and more diverse sample will provide better reliability and accuracy in modelling.

Dingo reference population (Stephens et al 2015)

Take home message

Dingo DNA test users should carefully weigh the reliability of a result. A DNA result is only as reliable as the data it is based upon and a test result is an estimate not a definitive answer.

Questions that you may wish to ask the DNA testing provider

1. How many markers do you use in your DNA testing method and what type of markers are they?

2. What reference populations do you use in your analysis and what implications does this have on the reliability of a result? (It is important to check that they do have a dingo reference population, some providers do not).

3. Do you have any scientific publications on dingoes?

I am always happy to provide dingo owners and conservation organisations with advice about DNA testing and interpretation of results - please contact me at kylie@kyliecairns.com or k.cairns@unsw.edu.au

Further reading

1. Cairns, K.M., Wilton, A.N., and Ballard, J.W.O. (2011) The identification of dingoes in a background of hybrids. In 'Advances in genetics research.' (Ed. KV Urbano) pp. 309–327. (Nova Science Publishers: New York)

2. Cairns, K.M., Shannon, L.M., Koler-Matznick, J., Ballard, J.W.O., and Boyko, A.R. (2018) Elucidating biogeographical patterns in Australian native canids using genome wide SNPs. PLoS ONE 13(6), e0198754.

3. Cairns, K.M., Nesbitt, B.J., Laffan, S.W., Letnic, M., and Crowther, M.S. (2019) Geographic hot spots of dingo genetic ancestry in southeastern Australia despite hybridisation with domestic dogs. Conservation Genetics 21, 77-90.

4. Koungoulos, L. (2020) Old dogs, new tricks: 3D geometric analysis of cranial morphology supports ancient population substructure in the Australian dingo. Zoomorphology 139(2), 263-275.

5. Parr, W.C.H., Wilson, L.A.B., Wroe, S., Colman, N.J., Crowther, M.S., and Letnic, M. (2016) Cranial shape and the modularity of hybridization in dingoes and dogs; hybridization does not spell the end for native morphology. Evolutionary Biology 43(2), 171-187.

6. Stephens, D., Wilton, A.N., Fleming, P.J.S., and Berry, O. (2015) Death by sex in an Australian icon: a continent-wide survey reveals extensive hybridization between dingoes and domestic dogs. Molecular Ecology 24(22), 5643-5656.

7. Wilton, A. (2001) DNA methods of assessing Australian dingo purity. In 'A Symposium on the dingo.' (Eds. CR Dickman and D Lunney) pp. 49 - 55. (Royal Zoological Society of New South Wales: Sydney)

8. Wilton, A.N., Steward, D.J., and Zafiris, K. (1999) Microsatellite variation in the Australian dingo. Journal of Heredity 90, 108-111.