Final Report Summary - NGP (Deciphering the domestication and speciation of horses through next-generation paleogenomics using Pleistocene specimens, modern breeds and the domesticated donkey) The retrieval of DNA templates, aDNA, from dead specimens offers a unique perspective on evolutionary biology by providing information from extinct organisms. Such ancient data give access to frozen-state along history with major application fields, such as paleovirology, paleoecology, conservation biology, domestication and human evolution. Although first limited to the sequencing of reasonable amount of short DNA fragments, paleogenetics has now entered into the genomic era by delivering complete genomes of extinct species, such as woolly mammoths, ancient humans and archaic hominid.The IEF project "Deciphering the domestication and speciation of horses through next-generation paleogenomics using Pleistocene specimens, modern breeds and the domesticated donkey" (acronym NGP) had several ambitious goals. The main aim was to obtain the oldest genome from a mammal species using the latest massively parallel sequencing technologies available. It was achieved on a middle pleistocene horse sample (700 KY-BP old) using breakthrough new techniques such as 3rd generation sequencing providing a genome at 1.12X coverage on the horse reference. The corresponding paper "Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse" was published the 4th July 2013 in Nature, (Orlando et al., 2013) where both the scientific coordinator, Ludovic Orlando and the fellow are the two first authors. In this study, we analyzed not only the 700 KY-BP horse sample, but also 6 modern genomes as it was planned initially in the project. Four individuals from the following domestic breeds: Arabian, Icelandic, Norwegian Fjord and Standardbred. We added from the initial plan the Thoroughbred by resequencing at high coverage the individual Twilight that was used to generate the horse reference (EquCab2, Wade et al. 2009). The Przewalski's horse was sequenced to determined its relationship with domestic breeds and potential admixture. This information is of main relevance for conservation purposes together with the estimation of the genetic diversity of the critically endangered Przewalski's horses population.A modern donkey that was also sequenced and de novo assembled for comparison as out-group. The main results were:the paleogenome allowed us to recalibrate the time of the most recent common ancestor of horses and donkey was between 4 to 4.5 Myr, twice the time that was commonly used.the Przewalski's horse diverged from domestic horses 38-72 KY BP and did not show recent sign of admixture with the 5 horse breeds sequenced. Moreover, this individual shown a genetic diversity comparable to the domestic breeds, justifying the conservation efforts and reintroduction into the wild of Przewalski's horses.The authenticity of the genomes was confirmed by the basal phylogenetic position of ancient specimens based on autosomal, Y and mitochondrial data, amino acid bone signatures and both amino acids and nucleic acids degradation. The latter was performed using the tool published by the fellow and colleagues, mapDamage (Ginolhac et al. 2011, Jónsson et al. 2013).We demonstrated that accessing paleogenomes from the middle pleistocene is feasible. Recently, two studies confirmed that such time range was accessible. The mitogenomes of both a cave bear (Dabney et al. 2013, PNAS) and the iconic Homo heildelbergensis (Meyer et al. 2013, Nature) from northern Spain shown that early middle pleistocene specimens are not out of reach even from warmer regions.