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Simple Summary  Once present in the entirety of Europe, mouflon (wild sheep) became extinct due to intense hunting, but remnant populations survived and became feral on the Mediterranean islands of Corsica and Sardinia. Although now protected by regional laws, Sardinian mouflon is threatened by crossbreeding with domestic sheep causing genetic hybridisation. The spread of domestic genes can be detrimental for wild populations as it dilutes the genetic features that characterise them. This work aimed to identify diagnostic tools that could be applied to monitor the level of hybridisation between mouflon and domestic sheep. Tens of thousands of genetic markers known as single nucleotide polymorphisms (SNPs) were screened and we identified the smallest number of SNPs necessary to discriminate between pure mouflon and sheep. We produced four SNP panels of different sizes which were able to assess the hybridisation level of a mouflon and we verified that the SNP panels efficacy is independent of the domestic sheep breed involved in the hybrid. The implementation of these results into actual diagnostic tools will help the conservation of this unique and irreplaceable mouflon population, and the methodology applied can easily be transferred to other case studies of interest.Abstract  Hybridisation of wild populations with their domestic counterparts can lead to the loss of wildtype genetic integrity, outbreeding depression, and loss of adaptive features. The Mediterranean island of Sardinia hosts one of the last extant autochthonous European mouflon ( Ovis aries musimon ) populations. Although conservation policies, including reintroduction plans, have been enforced to preserve Sardinian mouflon, crossbreeding with domestic sheep has been documented. We identified panels of single nucleotide polymorphisms (SNPs) that could act as ancestry informative markers able to assess admixture in feral  x  domestic sheep hybrids. The medium-density SNP array genotyping data of Sardinian mouflon and domestic sheep ( O. aries aries ) showing pure ancestry were used as references. We applied a two-step selection algorithm to this data consisting of preselection via Principal Component Analysis followed by a supervised machine learning classification method based on random forest to develop SNP panels of various sizes. We generated ancestry informative marker (AIM) panels and tested their ability to assess admixture in mouflon  x  domestic sheep hybrids both in simulated and real populations of known ancestry proportions. All the AIM panels recorded high correlations with the ancestry proportion computed using the full medium-density SNP array. The AIM panels proposed here may be used by conservation practitioners as diagnostic tools to exclude hybrids from reintroduction plans and improve conservation strategies for mouflon populations.

Identification of Ancestry Informative Marker (AIM) Panels to Assess Hybridisation between Feral and Domestic Sheep