Highlight: A Massively Parallel Sequencing Approach Uncovers Ancient Origins and High Genetic Variability of Endangered Przewalski’s Horses

For millions of years, the stout, muscular Przewalski’s horse freely roamed the high grasslands of Central Asia. By the mid-1960s, these, the last of the wild horses, were virtually extinct: a result of hunting, habitat loss, and cross breeding with domestic horses. Recovering fromatinypopulationof12 individualsand only fourpurebredfemales, therearenownearly2,000Przewalski’s horsesaround the world. Once again, the light-coloredhorses, standing about 13 hands, or 1.3 meters, tall, are beginning to graze on the Asian steppe, thanks to captive breeding and reintroduction programs. ProtectingPrzewalski’shorses, listedascriticallyendangered by the International Union for Conservation of Nature, will require far more than protecting their habitat. Understanding and safeguarding their genetic diversity is key, said Kateryna Makova,anevolutionarygenomicistatPennsylvaniaStateUniversity. In a new study (Goto et al. 2011), Makova and her colleagues Hiroki Goto, Oliver Ryder, and others report on the most complete genetic analysis of Przewalski’s horses to date, clarifying previous genetic analyses that were inconclusive. Because Przewalksi’s horses are the only remaining wild horses, many people have hypothesized that they gave rise to modern domestic horses. The Australian Brumbies or the American Mustangs, sometimes referred to as wild horses, are actually feral domestic horses, adapted to life in the wild. Przewalski’s horses are not the direct progenitors of modern domestic horses, Makova and her colleagues conclude, but split approximately 0.12 Ma. Horses were likely domesticated several times on the Eurasian steppes. It is not known where and when the first event took place. Recent excavations in Kazakhstan indicate humans were using domestic horses as early as 5,500 years ago. The team base their findings on a complete sequencing of the mitochondrial genome and a partial sequencing, between 1% and 2%, of the nuclear genome. They used one horse from each of the historical matrilineal lines. After processing the DNA samples with massively parallel sequencing technology, they compared the Przewalski’s horses to each other, to domestic Thoroughbred horses, and to an outgroup, the Somali wild ass. Their results carry several implications for breeding strategies. Przewalski’s horses and domestic horses come from different evolutionary gene pools, so breeders should avoid crosses with domestic horses, they advise. Przewalski’s horses and domestic horses have a different number of chromosomes (66 for the former, compared with 64); yet their offspring are fertile (with 65 chromosomes). The hybrids are viable because they differ only by a centric fusion translocation, also called a Robertsonian translocation. The process of pairing chromosomes during meiosis is not disrupted. Cross breeding should be a last resort, if too few Przewalski’s horses are available. Their analysis also suggests that, since diverging, Przewalski’s and domestic horses have both retained joint ancestral genes and swapped genes between populations. One of the two current major blood lines, the ‘‘Prague’’ line, is known to have a Mongol pony as one of its ancestors. The other primary line, the ‘‘Munich’’ line, is believed to be pure. However, because the two groups have historically mixed, keeping ‘‘pure’’ Przewalski’s horses from Przewalski’s horses with known domestic horse contributions might not be necessary, the authors write. Additionally, a full sequencing of the mitochondrial genomes from the four surviving maternal lineages revealed surprising diversity: three mitochondrial haplotypes, a set of genes generally inherited as a single unit. Haplotypes I and II were very similar, whereas haplotype III stemmed from a division that is between 0.12 and 0.19 Myr old; this division is at least a hundred thousand years before horses were first domesticated. ‘‘Przewalski’s horses have a lot more diversity than anyone was expecting,’’ said Makova. The Przewalski horses used in