Dna Markers Reveal Genetic Associations Among 11,000-Year-Old Scots Pine (Pinus Sylvestris L.) Found In The Baltic Sea With The Present-Day Gene Pools In Lithuania

We aimed to extract DNA and amplify PCR fragments at the mitochondrial DNA Nad7.1 locus and 11 nuclear microsatellite loci in nine circa 11,000-year-old individuals of Scots pine found at the bottom of the Baltic sea and test the genetic associations with the present-day gene pool of Scots pine in Lithuania. We followed a strict anticontamination protocol in the lab and, simultaneously with the aDNA specimens, tested DNA-free controls. The DNA was extracted by an ATMAB protocol from the ancient wood specimens sampled underwater from Scots pine stumps located circa 20-30 m deep and circa 12 km ashore in western Lithuania. As the references, we used 30 present-day Lithuanian populations of Scots pine with 25-50 individuals each. The aDNA yield was 11-41 ng/mu L. The PCR amplification for the mtDNA Nad7.1 locus and the nDNA loci yielded reliable aDNA fragments for three and seven out of nine ancient pines, respectively. The electrophoresis profiles of all the PCR tested DNA-free controls contained the sizing standard only, indicating low likelihood for contamination. At the mtDNA Nad7.1 locus, all three ancient Scots pine individuals had the type A (300 bp) allele, indicating postglacial migration from the refugia in Balkan peninsula. The GENECLASS Bayesian assignment tests revealed relatively stringer and consistent genetic associations between the ancient Scots pine trees and the present-day southern Lithuanian populations (assignment probability 0.37-0.55) and several wetlands in Lithuania. Our study shows that salty sea water efficiently preserves ancient DNA in wood at the quality levels suitable for genetic testing of trees dated back as far as 11,000 years before present.