DNA analysis in charred grains of naked wheat from several archaeological sites in Spain

In the present work we attempt to recover endogenous ancient DNA from cereal grains preserved under different conditions: charred, partially charred and waterlogged. A total of 126 grains from naked wheat and 18 from barley from different sites on the Eastern Iberian Peninsula ranging from the beginning of agriculture in the region to the turn of the Common Era, were studied. Two different extraction protocols were used, a standard phenol–chloroform method and a silica-based DNA extraction procedure implemented for artificially charred seeds. Amplifications were directed to three markers: the large subunit of ribulose 1,5 biphosphate carboxylase (rbcL) and the microsatellite WCT12 in the chloroplast genome and the x and y subunits of the high molecular weight glutenin gene (Glu-1) in the nucleus. The first two were used to assess the preservation status of the samples, while with the third we tried to identify the wheat grains at species level. It was possible to obtain eleven positive amplifications in 8 partially charred seeds but only two amplifications of the Glu-1 gene from a single sample of the Early Bronze age were genome-specific. Different contamination sources were identified and reported. Cloning and alignment of sequenced clones showed a correspondence of the amplified fragment to modern wheat D genome haplotypes. This result suggests that the sample corresponds to hexaploid wheat (Triticum aestivum L.), thus being the first ancient DNA evidence to date for the cultivation of hexaploid wheat in the prehistoric agriculture of the Iberian Peninsula. Moreover, obtained results highlight contamination problems associated to the study of ancient archaeobotanical charred seeds suggest that the combination of a silica-based extraction method together with the amplification of specific targets is a good strategy for recovering endogenous ancient DNA from this kind of material.