Scientists in Denmark have done the seemingly unthinkable, mapping the genetic makeup of a horse that lived 700,000 years ago.
The scientists broke the record for mapping the genome of a prehistoric creature by more than 600,000 years. A genome is the entire hereditary information of an organism.
Researchers at the Centre for GeoGenetics at the Natural History Museum of Denmark, part of the University of Copenhagen, sequenced and analyzed short pieces of DNA molecules preserved in bone remnants from a horse that had been kept frozen for the last 700,000 years in the permafrost of Yukon, Canada.
By tracking the genomic changes that transformed prehistoric wild horses into domestic breeds, the researchers have revealed the genetic make-up of modern horses with unprecedented detail. The spectacular results are now published in the international scientific journal, Nature.
DNA molecules can survive in fossils well after an organism dies – not as whole chromosomes, but as short pieces that could be assembled back together, like a puzzle.
Sometimes, enough molecules survive so that the full genome sequence of extinct species ccan be resurrected and, over the last years, the full genome sequence of a few ancient humans and archaic hominins has been characterized. But so far, none dated back to before 70,000 years.
Now, Dr Ludovic Orlando and Professor Eske Willersle, from the Centre for GeoGenetics, have beaten this DNA-record nearly ten-fold. The two researchers – in collaboration with Danish and international colleagues – have been able to track major genomic changes over the last 700,000 years of evolution of the horse lineage.
First, by comparing the genome in the 700,000-year-old horse with the genome of a 43,000-year-old horse, six present-day horses and a donkey, the researchers could estimate how fast mutations accumulated through time and calibrate a genome-wide mutation rate.
This revealed that the last common ancestor of all modern equids was living about 4 million to 4.5 million years ago.
Therefore, the evolutionary radiation underlying the origin of horses, donkeys and zebras reaches back in time twice as long as previously thought.
Additionally, this new clock revealed multiple episodes of severe demographic fluctuation in horse history, in phase with major climatic changes such as the Last Glacial Maximum, some 20,000 years ago.
The results also puts to an end the long debate about the Przewalski’s Horse from the Mongolian steppes. This horse population was discovered by the Western world in the second half of the 19th century and rapidly became threatened.
It almost became extinct in the wild by the 1970s, but has survived until now following massive conservation efforts. The evolutionary origin of this horse, that shows striking physical differences compared to domesticated horses, as well as an extra pair of chromosomes, remained a mystery.
The researchers reveal now that the Przewalski’s horse population became isolated from the lineage leading to the present day domesticated horses about 50,000 years ago. As the scientists could detect similar levels of genetic diversity within the Przewalski’s Horse genome to genomes of several domestic breeds, this suggests that the Przewalski’s Horses are likely genetically viable and therefore worthy of conservation efforts.
The geological context and dating information available was very strong and was built on about 10 years of field work and research, carried out by Dr Duane Froese, of the University of Alberta.
Additionally, cold conditions, such as those from the Arctic permafrost, are known to be favourable for DNA preservation. But even so, sequencing the first genome from the Middle Pleistocene was by no means straightforward, says Dr Orlando, who, with his team, spent most of the last three years on the project.
The researchers first got excited when they detected the signature of the amino acids most abundant in collagen, as this could indicate that proteins had survived in the fossil. They became more excited when they succeeded in directly sequencing collagen peptides.
When they detected blood proteins, it really started looking promising because those are rarely preserved. At that stage, it seemed possible that the ancient DNA could also be preserved.
Indeed, DNA was present – in tiny amounts, as the vast majority of sequences generated actually originated from environmental micro-organisms living in the bone. But with Helicos true Single DNA Molecule Sequencing, the researchers managed to identify molecular preservation niches in the bone and experimental conditions that enabled finishing the full genome sequence.
This was methodologically challenging, but clearly some parameters worked better than others, Willerslev says.
But sequencing was just half the task.
Willerslev continues: “Because of 700,000 years of evolution and damage, it is not something that comes without any modification to the DNA sequence itself. We had to improve our ability to identify modified and divergent ancient horse sequences by aligning them to the genome of present-day horses.”
It was quite a computational challenge.
Dr. Orlando explains: “Levels of base modifications were extremely high, for some regions even so high that every single cytosine was actually damaged. This, and the phylogenetic position of the ancient horse outside the diversity of any horse ever sequenced, provided clear evidence that the data was real.”
Willerslev adds: “The results of the studies and the applied techniques open up new doors for the exploration of prehistoric living creatures.
“Now, with genomics and proteomics, we can reach 10 times further back in time compared to before. And new knowledge about the horse’s evolutionary history has been added – a history which is considered as a classical example in evolutionary biology and a topic which is taught in high schools and universities.”
The project was made possible through the collaboration with researchers from Denmark, China, Canada, the United States Switzerland, Britain, Norway, France, Sweden and Saudi Arabia, and with financial support from the Danish National Research Foundation.