Seed remains of common millet from the 4th (Mongolia) and 15th (Hungary) centuries: AFLP, SSR, and mtDNA sequence recoveries

Seed remains of common millet (Panicum miliaceum L.) were excavated from sites of AD 4th-century Darhan (Mongolia), and AD 15th-century Budapest (Hungary). Because the 15th-century medieval grains looked so intact, a germination test was carried out under aseptic conditions, which resulted in swelling of the grains but no cell proliferation or germination. Ancient DNA (aDNA) was extracted from the aseptic grains; analysed for amplified fragment length polymorphisms (AFLP), simple sequence repeats (SSR) and mitochondrial DNA (mtDNA); and compared with the modern millet cultivar ‘Topaz’. AFLP analysis revealed that extensive DNA degradation had occurred in the 4th-century ancient millet, resulting in only 2 (1.2%) AFLP fragments (98.8% degradation) amplified by MseCAA–EcoAGT, compared to the 15th-century medieval millet, with 158 (40%) fragments (60% degradation), and modern millet cultivar ‘Topaz’ with 264 fragments (100%). EcoAGT–Mse- CAA was found to be the most effective selectiveprimer combination for the analysis of medieval and modern millet. Eight AFLP fragments were sequenced after re-amplification and cloning. Microsatellite (SSR) analysis at the nuclear gln4, sh1, rps28 and rps15 loci revealed one SNP (single nucleotide polymorphism) at the 29th position (A ! G) of rps28 locus, compared to modern millet. An mtDNA fragment (MboI), amplified at the 18S–5S ribosomal DNA (rDNA) locus in the medieval millet, showed no molecular changes compared to modern millet. The results underline the significance of aDNA extraction and analysis of excavated seeds for comparative analysis and molecular reconstruction of ancient and extinct plant genotypes.