DNA testing and profiling came of age less than 50 years ago – providing for a myriad of opportunities, including forensics, screening for predisposition to disease, and paternity testing.
Paternity testing, by common definition: Typically, a DNA-based test to determine whether a given male could be the biological parent of a given offspring.
Parentage, of course, includes the putative mother of the offspring. Most questions of parentage revolve around the determination of the offspring’s father as typically births are witnessed, and the mother is known. However, there are many documented cases of animal offspring being adopted by surrogate mothers and mistakenly attributed to the incorrect, non-biological mother.
Important Fact: Parentage testing is a test of exclusion, not inclusion. Putative parents are excluded, and each genetic marker used is assessed for its probability of making an exclusion. (For our math enthusiasts, read more here.
When parentage testing was first adopted within the canine community, DNA testing was new enough that limited information about the process was available, resulting in some concern and suspicion that the DNA collected may be used for purposes beyond parentage testing. Education campaigns and disclaimers followed, and slowly dog breeders accepted the process and welcomed the power provided to verify pedigrees.
However, there is still not widespread use of parentage verification within the canine community, and most applications are limited to resolving dual sired litters, frozen/fresh semen breedings, and the dreaded accidental breeding. Some national registries, such as the American Kennel Club (AKC), have also implemented compliance programs that literally ‘spot check’ random breeding programs or follow up on suspect errors in record keeping.
Interestingly, at this time, most domestic livestock registries (e.g., horses, cattle, goats, etc.) require parentage testing of all offspring as a condition of registration. These registries have implemented sophisticated software that not only allows for individual tracking but will flag matings for which offspring exhibit traits, such as inconsistent coat color, that is not genetically compatible with the parents-of-record traits, based on known modes of inheritance. These practices ensure pedigree integrity.
The question often arises as to why dog registries, with some exceptions, have not chosen to require parentage testing with all registrations? Most likely, this is due to simple economics – horses and cows typically have 1-2 offspring, whereas dog matings customarily result in multiple offspring, adding a significant cost to the breeder. Further, should a racehorse or a top producing bull not actually have the correct identity/pedigree, the financial losses have been historically significant. However, the cost of producing and raising puppies continues to rise. Breeding management is becoming more sophisticated as technology intersects art, underscoring that pedigree integrity should be just as essential for dog breeders as for other species.
DNA Parentage testing can be achieved through a variety of methods. Early techniques included RFLP (Restriction Fragment Length Polymorphisms), a reliable method that required a considerable DNA sample and could be slow to process. RFLP was a precursor to STR (Short Tandem Repeat) analysis. An STR is a microsatellite genetic marker with repeat units that are two to seven base pairs in length. The number of these repeats varies among individuals; thus, they are ideally suited for individual identification and relatedness assays.
Microsatellite application proliferated across all species, offering a rapid, economical, and mostly reliable source, for parentage testing and individual identification (DNA fingerprinting). Microsatellite/STR methodology is still being used by some commercial laboratories. The International Society for Animal Genetics (ISAG) offers an every-two-year forum for laboratories worldwide to compare and standardize testing so that individual results can be easily shared between laboratories and parentage assessed with no retesting required. Currently, most canine testing facilities still use microsatellites. However, informative (polymorphic) microsatellite marker availability can be limited, especially across closed, inbred populations typical of purebred dog breeds, which may lead to inconclusive results. When a parentage question cannot be resolved via the standard ‘panel’ of select microsatellites, typically, a back-up panel is employed. But there are a fair number of cases that still cannot be resolved even with unusually large numbers of microsatellites. Common scenarios involve closely related individuals, for example, putative sires who are siblings or father/son situations where both may have covered the same female.
More recently, advanced technology has provided for rapid, higher resolution parentage determination. A different type of genetic marker, known as single nucleotide polymorphisms (SNPs), provides another measurement of genetic variation. Hundreds to thousands of SNPs can be tested in one assay. This allows for dense and comprehensive panels with a broad selection of genetic markers to effectively inform across hundreds of breeds. Two other advantages are that SNPs have lower mutation rates than STRs and also perform well on degraded or lower quality DNA samples.
Most livestock parentage testing has long since embraced SNP testing; cattle testing employs hundreds of SNPs and has led the way in refining testing and reporting. As mentioned, ISAG oversees international comparison tests, and this coming year will be the second time ISAG will include an SNP comparison test alongside the STR comparison test. Roughly 200 SNPs are being evaluated for an internationally accepted primary panel. Should testing go well, an internationally accepted canine SNP panel may become the standard allowing for SNP testing results to be shared among laboratories.
There remain some exceptions, however, as some registration bodies have elected not to participate in result sharing. Dog breeders and owners should always check directly with the registry of interest for specific regulations and requirements of parentage testing.
Embark is participating in the international consortium conducting the laboratory comparison tests. Embark, of course, has included the ~200 SNPs being considered by the ISAG committee in our routine test. Notably, every dog tested with an Embark DNA test is screened by over 230,000 SNPs – easily providing for tens of thousands of select genetic markers to be applied to parentage testing for an even higher resolution of parentage exclusion.