LAUSR

Gene editing and subsequent cloning techniques offer great potential not only in genetic disease correction in domestic animals, but also in livestock production by enhancement of desirable traits. The existence of the technology, however, leaves it open to potential misuse in performance-led sports such as horseracing and other equestrian events. Recent advances in equine gene editing, regarding the generation of gene-edited embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer, has highlighted the need to develop tools to detect potential prohibited use of the technology. One possible method involves the characterisation of the mitochondrial genome (which is not routinely preserved during cloning) and comparing it to the sequence of the registered dam. We present here our approach to whole-mitochondrial sequencing using tiled long-range PCR and next-generation sequencing. To determine whether the background mutation rate in the mitochondrial genome could potentially confound results, we sequenced ten sets of dam and foal duos. We found variation between duos but none within duos, indicating that this method is feasible for future screening systems. Analysis of WGS data from over one hundred Thoroughbred horses revealed wide variation in the mitochondria sequence within the breed, further displaying the utility of this approach.