INTRODUCTION: The male specific region of the Y-chromosome is only paternally
inherited, therefore Y-chromosomal polymorphisms are a useful tool to investigate
paternal lineages. For domestic horses, in contrast to other domestic animals, only
one Y-chromosomal polymorphism has been described so far.
MATERIAL AND METHODS: By sequencing four equine Y-chromosomal BAC
clones, eleven new microsatellites (Eca.YE2, Eca.YE3, Eca.YNO1, Eca.YNO2,
Eca.YNO3, Eca.YNO4, Eca.YP10, Eca.YP11, Eca.YP12, Eca.YP13) were detected.
The newly isolated microsatellites were tested to confirm their Y-chromosomal origin
and their variability. Eighty-one male domestic horses from thirty different breeds and
three male Przewalskis horses were typed, three female domestic horses were used
as controls. Eight microsatellites were confirmed to be Y-chromosomal. Variation was
examined using fluorescence-labeled primers and capillary electrophoresis,
amplifying the microsatellites in two 4-Plex PCRs. Artifacts occurred for Eca.YNO4,
so a separate PCR to amplify this single marker was performed. To detect eventually
existing sequence variations, High Resolution Melting (HRM) analysis of the eight
microsatellites was performed in a smaller number of horses.
RESULTS: Seven microsatellites could be amplified in all horse breeds, but the locus
Eca.YNO4 did not produce legible genotypes in ten horses. No variation was
detected between or within the horse breeds using DNA fragment size analysis. The
haplotype of the Przewalskis horse differed from the domestic horse at one locus
(Eca.YP13). The allele in all investigated domestic horses was 118 bp long, whereas
the Przewalskis horses showed a 114 bp allele. At the locus Eca.YP12 two alleles
(104 bp, 100 bp) within the Przewalskis horse were detected.
Using HRM analysis no variations were detected between the breeds as well. The
length polymorphism between the Przewalskis horse and the domestic horses,
shown in the DNA fragment size analysis, could not be detected by HRM analysis.
DISCUSSION: Fourteen equine Y-chromosomal microsatellites are described so far,
but only one Y-chromosomal polymorphism in the domestic horse was found. A
possible cause for the monomorphy of the equine Y-chromsome is a strong sex bias
in breeding. The polymorphism at the locus Eca.YP12 in the Przewalskis horse shows, that at least two paternal lineages have survived until today, despite the
genetic bottleneck the population have had undergone.
Overall, only one Y-chromosomal haplotype in the European horse breeds was
identified after the investigation of eight new microsatellites.