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Genetic Analysis Confirms Skeleton as Belonging to Richard III

Skull of Richard III

NEW YORK (GenomeWeb) – Putting to rest a more than 500-year-old forensic case, an international team led by researchers from the University of Leicester has used genealogical data and genetic analyses to confirm that a skeleton discovered under a British parking lot belongs to King Richard III.

"It is exciting to have access to genetic data from any known historical individual, let alone a king of England," Simon Chaplin, director of culture and society at the Wellcome Trust, which partially funded the work, said in a statement. "Adding this information to a wealth of existing material about Richard III further highlights the ways in which studying human remains can inform our understanding of the past, and we look forward to learning more about Richard for many years to come."

Richard III assumed the English monarchy in 1483 and died two years later during the Battle of Bosworth, making him the last British king to die in battle. Historical records indicate that his body was brought to Leicester and buried in the medieval church of the Grey Friars, but in 1538 the friary was dissolved and most of its buildings were torn down in the following years.

Despite rumors that Richard III's remains were disinterred and thrown into the river Soar in Leicester, most historians believed that the monarch remained undisturbed where the friary once stood. Yet the exact location of his grave remained unknown.

In 2012, a skeleton was excavated at a site believed to be the site of the Grey Friars friary, with archeological and radiocarbon dating all suggesting that it belonged to Richard. Further, an analysis of the skeleton showed it to have belonged to a male between 30 and 34 years old with severe scoliosis and numerous battle injuries — all consistent with what is known about the king.

To confirm once and for all that the skeleton, known at the time of its discovery as Skeleton 1, did indeed belong to Richard III, the University of Leicester-led investigators conducted DNA analyses of both the remains and living relatives of the former king, focusing on several key genetic markers including complete mitochondrial genomes, which are inherited through the maternal line, and Y-chromosomal markers, which pass through the paternal line.

The scientists sequenced Y-chromosome SNPs on Skeleton 1 using on-array DNA hybridization capture of 24 amplified Illumina sequencing libraries, followed by sequencing on a single 100 SE Illumina Hiseq 2000 sequencing lane, according to their report, which appeared in Nature Communications. The arrays contained probes generated to cover SNPs relevant to the major European Y lineages.

Further typing was performed using targeted PCR with the amplification products sequenced on an Ion Torrent PGM, while an STR haplotype was generated using the Promega PowerPlex Y23 system.

When they compared their findings to those from analyses of five confirmed male-line relatives of Richard III, they found no genetic relation. But in light of the possibility of a false-paternity, which is estimated to be as much as 16 percent over the number of generations between Richard III and his descendants, the researchers did not consider this a highly significant factor.

Noting that, for obvious reasons, the possibility of false maternity is far less likely than false paternity, the research team next examined mitochondrial DNA (mtDNA) from Skeleton 1 and compared it with samples from two individuals 19 and 21 generations removed from Richard III, respectively, on the female line.

They first sequenced both strands of the mtDNA control region in duplicate from both relatives using Sanger sequencing, and found no sequence differences between either duplicated samples from the same individual or individuals.

Three hypervariable sections of the mtDNA control region of Skeleton 1 were sequenced from two independent extractions carried out in two different laboratories specializing in ancient DNA analyses, the investigators wrote in Nature Communications. Sanger sequencing of cloned PCR products was also performed and no sequence differences were observed except for two that can be attributed to DNA damage patterns found in ancient DNA, they added.

A perfect match was discovered between Skeleton 1 and Richard's descendants, which the researchers stated is consistent with these individuals all being matrilinear relatives at the genealogical time depth considered.

To determine the full mtDNA similarity, they next carried out complete mitochondrial genome sequencing on all three samples. For the modern samples, the entire mitochondrial genome was amplified via two long-range PCRs in duplicate, followed by sequencing on an Ion Torrent PGM.

Meanwhile, whole-genome sequencing of the mtDNA sequence of Skeleton 1 was carried out using on-array DNA hybridization capture on 24 sequencing libraries generated from 16 extracts using probes generated from the mtDNA sequence of the two modern relatives, followed by sequencing on a single 100 SE Illumina Hiseq 2000 sequencing lane.

This revealed a perfect whole-genome sequence match between Skeleton 1 and one of Richard's relatives, and a single base difference with the other relative that was deemed consistent with these individuals being matrilinear relatives over the time period considered. A review of a database of 26,127 European complete mtDNA control region sequences and a database of 1,832 British Isles samples confirmed that the perfect mtDNA match was highly unlikely to result from chance.

Finally, eye and hair color DNA typing was carried out on Skeleton 1 and showed it had a 96 percent probability of having blue eyes and a 77 percent probability of having blond hair, which matches one of the earliest of the few surviving portraits of Richard III.

"Our paper covers all the genetic and genealogical analysis involved in the identification of the remains of Skeleton 1 from the Grey Friars site in Leicester and is the first to draw together all the strands of evidence to come to a conclusion about the identity of those remains," Turi King, lead author of the Nature Communications study, said in a statement. "Even with our highly conservative analysis, the evidence is overwhelming that these are indeed the remains of King Richard III, thereby closing an over 500-year-old missing persons case."

"The combination of evidence confirms the remains as those of Richard III," senior author Kevin Schurer added. "Especially important is the triangulation of the maternal line descendants. The break in the Y-chromosome line is not overly surprising given the incidence of non-paternity, but does pose interesting speculative questions over succession as a result."

Indeed, as the paper's authors pointed out, one or more false paternity events occurring in the years before Richard III's ascension to the throne would call into question the legitimacy of a number of claims to the British monarchy following Richard III's death.