Pristine DNA recovered from 1,600-year-old sheep mummy

A lone sheep leg, likely discarded by hungry mine workers, lay hidden in a salt mine in Iran for over a thousand years, during which time the salinity of the surrounding environment naturally mummified the limb. Now, scientists have extracted pristine DNA from the mummified leg and dated the sample to the fifth or sixth century.

The DNA molecules were "so well preserved and not fragmented, despite their age," senior study author Kevin Daly, a research fellow at the Smurfit Institute of Genetics at Trinity College Dublin, told Live Science. This immaculate preservation not only allowed the team to examine DNA from the sheep, but also the genetic material of salt-loving microbes that grew on the specimen, the team reported in a new study, published July 13 in the journal Biology Letters.

Archaeologists first recovered the sheep leg from an ancient salt mine located near the village of Chehrabad in northwest Iran; the same mine has also shielded a number of human bodies from decay over the millenia. Since 1993, eight salt-cured human mummies have been recovered from the mine, several with skin and hair intact; these so-called "saltmen," as they're known, range between about 1,300 and 2,500 years old, scientists estimate.

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"This site is something truly remarkable," said first author Conor Rossi, a doctoral student at the Smurfit Institute of Genetics. The high-salt, low-moisture mine not only preserves skin and hair, but also helps protect DNA from destruction by saprophytic microbes — microorganisms that gorge themselves on dead and decaying organic matter, he said.

Enzymes that break down DNA typically rely on water to execute those chemical reactions, and saprophytes use such enzymes to break down flesh, Rossi said. After an organism dies, enzymes within its own cells also begin hacking away at the DNA and altering its chemical structure, according to a 2013 report in the journal Cold Springs Harbor Perspectives in Biology. These enzymes also use water to split chemical bonds within DNA strands.

But in the Chehrabad salt mine, salt molecules bind to water in the environment, making it unavailable for these enzymes. Meanwhile, soft tissues from human and animal carcasses completely dry out in the extreme environment of the mine. "It's a really rare case of what's known as natural mummification," Rossi said. 

The team first got access to the rare, mummified sheep specimen through their collaborator and co-author Marjan Mashkour, an archaeozoologist with the National Museum of Natural History in France and the University of Tehran in Iran. "She had brought in this small, little plastic pouch just a little cutting of the skin of this leg — what we thought was a goat leg, actually," Daly said. 

The skin sample measured roughly 0.6 square inches (4 square centimeters), providing the team with plenty of DNA to work with. Using carbon dating, the group estimated that the leg was about 1,600 years old, give or take 30 years.

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To draw DNA from the desiccated flesh, the team had to first completely dissolve the skin and isolate any genetic material released as a result. The researchers identified about 25% to 30% of the sample as sheep DNA, with the rest being bacterial or archaeal DNA. "The bacteria and archaea that we could characterize were really dominated by these extremophiles, and largely archaea, that were salt-loving," Rossi said. "This was really unusual … it wasn't the kind of typical, garden-variety saprophytic bacteria that you'd find on remains of animals." 

Zooming in on the sheep DNA, the team found that the genetic material showed remarkably low levels of fragmentation compared with Iranian sheep bone samples of a similar age. The sample also showed extremely low levels of deamination, a process by which enzymes remove so-called amino groups from molecules that make up the rungs of the DNA double helix. Ancient DNA samples often come heavily scarred by deamination, but not in this case. 

The researchers then examined genes within the sheep DNA that in modern sheep breeds influence the wooliness of the animals' coats. They found that the sheep carried one copy of the "woolly" gene variant and one copy of an ancestral "hairy" gene variant, which is a dominant genetic trait; this suggests the sheep had a hairy coat, "so it wasn't necessarily specialized, as a kind of a wool sheep, as we think of them today," Daly said. 

Instead, the animal likely came from a herd raised for meat or milk production, which would align with previous findings that portions of the Chehrabad mine served as stables for sheep and goats that the miners consumed, the authors noted in their report.

Beyond this study, Daly, Rossi and their labmates use ancient genomes to investigate the history of how humans domesticated livestock like cattle, sheep and goats. Regarding the new study, "One thing that we were limited by was the fact that this is just a single individual," so the team can't use the DNA to make broad inferences about what was happening with sheep domestication at the time, Rossi said.

However, "This sample, it was something really exceptional, just how well preserved the DNA was," Rossi said. "I think it's fair to say we weren't expecting to see that level of preservation."

Originally published on Live Science.