Ann T. Bowling
Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis, CA

The genetic heritage of a registered horse is described by its pedigree. Owners highly value the ancestry records to provide information about their horses’ expected special potential as performance animals and as breeding stock. Recognizing that assurance of pedigree accuracy is central to their mission, all the major horse breed registries have adopted parentage testing programs.

The first generation of parentage testing was based on blood typing (blood groups and protein polymorphisms), using a series of reliable, low-cost and technologically simple testing routines, now provided by laboratories worldwide. While these tests have been highly effective in providing solutions to paternity questions and sorting out switched foal scenarios, three limitations have occasionally, but repeatedly, provided frustrations for owners and registries. First, the tests can only be performed with fresh blood, thus excluding the possibility to use other kinds of samples, especially those that might be available from dead horses. Second, the requirement of a blood sample may necessitate the assistance (and expense) of a veterinarian, special shipping containers, handling conditions and difficulties in the international transport of samples. Third, if a solution could not be obtained from the available blood typing tests, for example to exclude one of two stallions known to have covered a mare in a single breeding season, no other effective tests were available to extend the test battery.

Now, breed registries and owners can take advantage of horse research that has developed assays of genetic information that are not restricted to blood samples. Parentage verification is accomplished through analysis of inherited variants of selected DNA regions (microsatellites), not only from blood samples of living horses but also from hair roots and from teeth or other materials that might be available from dead horses. The sample of choice for routine testing is hair roots, which an owner can easily obtain by pulling mane hair and sending it to the laboratory with no special shipping or handling requirements. While the DNA-based genetic marker testing is more effective for parentage verification than blood typing, the cost is the same as that for blood typing. In the highly unlikely event that a unique solution is not found using the routine DNA test for a parentage analysis case (with multiple likely possibilities), additional tests using the same methods are available that can extend the microsatellite panel until a single set of qualifying parents is identified.

Looking at DNA

Genetic information is encoded in DNA by means of a linear string of four chemicals symbolized A, C, G and T. Each gene is encoded by thousands of these letters comprising a unique sequence that allows the precise production of proteins controlling inherited traits. While the blood typing tests identify genetic characteristics of selected proteins, the DNA tests identify inherited differences within a class of DNA sequences known as simple tandem repeats (STRs) or microsatellites.

A microsatellite is a DNA segment characterized by simple repeat motifs. Thousands of microsatellites are predicted to be present in the genetic material of the horse. While research during the last several decades has defined many of the functional motifs within DNA, we do not know the function of microsatellites (in fact, they have been labeled as "junk" DNA). In any case, we have no evidence of association for the microsatellites we use with traits that may be of interest to horse breeders, but they form the basis of a very effective parentage test.

The horse microsatellites in our test can be represented as simple two-letter repeats. These repeats are embedded in the more typical mixed-letter sequence, in a pattern such as:
 
 

In the example here, the microsatellite pattern is 11 consecutive "CA" motifs flanked by unique letter sequences. The length of the repeat is a reliably inherited trait but can be highly variable within a breed. For example, the systems selected for the horse parentage test each have from 8 to 16 length variants, depending on breed. The unique sequence containing a microsatellite can be rapidly amplified into millions of copies from a very small initial quantity of DNA using polymerase chain reaction (PCR) technology. During the amplification process, fluorescent dye labels are incorporated into the products so that length variation can be assayed with laser detection methods. For simplicity of record keeping and exchange of data between laboratories, each length variant is assigned a letter code.
 
 

DNA parentage test for horses

The horse DNA parentage test developed and used by the Veterinary Genetics Laboratory consists of 15 microsatellites discovered by research programs in laboratories worldwide. Considering the number of systems tested and the number of variants seen for each system, it is highly unlikely that any two horses except identical twins will have the same type. The efficacy of this test to detect incorrect parentage is very high—calculated for a diverse group of breeds to be 99.99% or greater. The test can be performed using a variety of biological samples, but the routine test is set up for hair (root) samples. For efficiency of processing, three different dye labels are used, allowing for each sample the simultaneous detection of the length variants for all the microsatellites tested. Computer programs analyze the laser-detected fluorescent marker data and perform the parentage analysis.

The following example with fictitious data illustrates the parentage analysis process using microsatellites. The top line provides the name of the microsatellite (only six are shown in this example). The letters on the left are D for dam, O for offspring and S for sire. For each microsatellite and each horse, the results of the length variation are provided in alphabetic code.