Validation of microsatellite markers for routine horse parentage testing.

A parallel testing of 4803 routine Quarter Horse parentage cases, using 15 loci of blood group and protein polymorphisms (blood typing) and 11 loci of dinucleotide repeat microsatellites (DNA typing), validated DNA markers for horse pedigree verification. For the 26 loci, taken together, the theoretical effectiveness of detecting incorrect parentage was 99.999%, making it extremely unlikely that false parentage would fail to be recognized. The tests identified incorrect parentage assignment for 95 offspring (2% of cases). Despite fewer loci, DNA typing was as effective as blood typing and, in parentage exclusion cases, provided more systems to substantiate the genetic incompatibility. Five offspring presented potential genetic incompatibilities with their parents in only a single microsatellite system, but the parentage exclusions could not be confirmed with discordant results at additional loci. Two of these five incompatibilities could be explained as consequences of a null allele and three as fragment size increases or decreases (putative mutations). Provided that an exclusion assignment was based on at least two systems of genetic incompatibility, such rare genetic events did not lead to false exclusions. Notwithstanding the near 100% effectiveness estimations for either typing panel alone to identify incorrect parentage, this validation test showed an actual effectiveness of 97.3% for blood typing and 98.2% for DNA typing. The DNA-based test, however, may feasibly achieve higher efficacy than reported here by adding selected systems to the parentage test panel.

A single base transversion in the flanking region of an equine microsatellite locus affects amplification of one allele.

The equine dinucleotide microsatellite HMS7 is part of a microsatellite panel utilized in a parentage verification programme at the Veterinary Genetics Laboratory (Davis, California, USA). Apparent non-Mendelian inheritance was noted when a Quarter Horse mare was excluded as the parent of two offspring based on analysis of the HMS7 locus. The mare's DNA type qualified her as a parent of the offspring at an additional 20 microsatellite loci. The three animals appeared homozygous for HMS7 with each possessing an allele different from that of the other two animals. Polymerase chain reaction primers designed to bind outside the published primer-binding sites amplified an additional shared allele in all three horses, which qualified the mare as the dam of the two offspring. Sequencing of this newly detected allele revealed a C to A transversion in one of the published primer-binding regions. Apparent non-Mendelian inheritance at the HMS7 locus has been encountered in an additional 26 Quarter Horse parentage cases. In all instances, the lack of amplification and resultant 'null' allele was shown to be caused by the same transversion.