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.     

Ancient DNA analysis of desiccated wheat grains excavated from a Bronze Age cemetery in Xinjiang

Wheat has been one of the most important crop in Eurasia since the Neolithic period. Understanding the spread of wheat cultivation is crucial to understanding the spread of agriculture as a whole and the interactions between prehistoric populations across the Eurasian continent. However, the routes by which wheat cultivation spread eastwards have been poorly understood to date, due to the scarcity of plant remains recovered from archaeological sites. Desiccated wheat grains excavated from the Xiaohe cemetery in Xinjiang, and dated to the early Bronze Age, show excellent DNA preservation. Here we present an ancient DNA (aDNA) analysis of wheat (Triticum sp.) grains excavated from Xiaohe and provide the first definitive evidence for bread wheat in China during the Bronze Age. The nuclear ribosomal DNA internal transcribed spacer regions (ITS1 and ITS2) and the intergenic spacer region (IGS) were amplified. The IGS region within the D genome of wheat has a 71 bp insertion that is absent from corresponding regions in the A and B genomes. The results showed that the Xiaohe wheat showed most sequence similarity to hexaploid bread wheat (Triticum aestivum), including the characteristic insertion into the D genome. The presence of bread wheat at the Xiaohe cemetery is discussed in relation to it having spread into Xinjiang by the Bronze Age, providing new insight into the origins of bread wheat in East Asia.     

Next generation sequencing of DNA in 3300-year-old charred cereal grains

We investigated whether ‘next generation’ methods can be used to sequence ancient DNA molecules in charred cereal grains. We prepared a DNA extract from a mixed sample of barley, einkorn, emmer and broomcorn millet, taken from a 3300-year-old assemblage of charred cereal grains from Assiros Toumba, Greece. Using the SOLiD 5500 system, we obtained 21,112,844 unique sequence reads. Of these, 178,779 had a 75% or greater nucleotide sequence similarity with one or more entries in the full nucleotide sequence database; 496 of these matches were to previously reported sequences from barley, einkorn, emmer, broomcorn millet or related species such as hexaploid wheat. The unique reads were also compared with a database comprising only wheat sequences. This analysis identified 1658 charred grain sequences that had 90% or greater similarity with segments of the wheat genome. The presence of barley, wheat and millet sequences in the next generation dataset confirms the presence of ancient DNA in this charred grain assemblage. Enrichment of extracts by hybridization capture or equivalent methods is likely to enable sequences to be obtained for entire genes and other genomic regions of interest.     

Archaeogenetic study of prehistoric rice remains from Thailand and India: evidence of early <Emphasis Type="Italic">japonica</Emphasis> in South and Southeast Asia

We report a successful extraction and sequencing of ancient DNA from carbonized rice grains (Oryza sativa) from six archaeological sites, including two from India and four from Thailand, ranging in age from ca. 2500 to 1500 BP. In total, 221 archaeological grains were processed by PCR amplification and primary-targeted fragments were sequenced for comparison with modern sequences generated from 112 modern rice populations, including crop and wild varieties. Our results include the genetic sequences from both the chloroplast and the nuclear genomes, based on four markers from the chloroplast and six from the nuclear genome. These markers allow differentiation of indica rice from japonica rice, the two major subspecies of Asian rice (O. sativa) considered to have separate geographical origins. One nuclear marker differentiates tropical and temperate forms of subspecies japonica. Other markers relate to phenotypic variation selected for under domestication, such as non-shattering, grain stickiness (waxy starch) and pericarp colour. Recovery and identification of sequences from nuclear markers was generally poor, whereas recovery of chloroplast sequences was successful, with at least one of four markers recovered in 61 % of archaeological grains. This allowed for successful differentiation of indica or japonica subspecies variety, with japonica identified in all the Thai material and a mixture of indica and japonica chloroplasts in the two Indian assemblages. Rice subspecies was also assessed through conventional archaeobotanical methods relying on grain metrics, based on measurements from 13 modern populations and 499 archaeological grains. Grain metrics also suggest a predominance of japonica-type grains in the Southeast Asian sites and a mixture of japonica and indica in the Indian sites with indica in the minority. The similar results of grain metrics and ancient DNA (aDNA) affirm that grain measurements have some degree of reliability in rice subspecies identification. The study also highlights the great potential of ancient DNA recovery from archaeological rice. The data generated in the present study adds support to the model of rice evolution that includes hybridization between japonica and proto-indica.     

Remains of the day-preservation of organic micro-residues on stone tools

Here I report on the decay processes of microscopic organic residues left on stone tool surfaces after their use. Residue analysis on ancient stone tools facilitates reconstruction of past activities. This study enables predictions about the circumstances under which ancient residues preserve. Experimental tool sets with modern residues were buried for a year in separate deposits at Sterkfontein, Sibudu (South Africa) and Zelhem (the Netherlands) whose pH and geomorphology varied, they were then analysed using light microscopy. Biological weathering mainly causes residue decay. In unstable environments rich in microbes and micro-organisms, residues decay quickly. From an archaeological perspective this means that sites that are stable, desiccated, waterlogged, extremely acidic or alkaline and extremely cold or hot sites. Different residue types have different preservation optima and this may lead to a preservation and perhaps interpretation bias. The preliminary predictive models presented in this paper could aid in the considered selection of sites and samples.     

Re-evaluating the history of the wheat domestication gene NAM-B1 using historical plant material

The development of agriculture is closely associated with the domestication of wheat, one of the earliest crop species. During domestication key genes underlying traits important to Neolithic agriculture were targeted by selection. One gene believed to be such a domestication gene is NAM-B1, affecting both nutritional quality and yield but with opposite effects. A null mutation, first arisen in emmer wheat, decreases the nutritional quality but delays maturity and increases grain size; previously the ancestral allele was believed lost during the domestication of durum and bread wheat by indirect selection for larger grain. By genotyping 63 historical seed samples originating from the 1862 International Exhibition in London, we found that the ancestral allele was present in two spelt wheat and two bread wheat cultivars widely cultivated at the time. This suggests that fixation of the mutated allele of NAM-B1 in bread wheat, if at all, occurred during modern crop improvement rather than during domestication. We also discuss the value of using archaeological and historical plant material to further the understanding of the development of agriculture.     

Recovery of DNA from archaeological insect remains: first results,problems and potential

We report the recovery of short fragments of PCR amplifiable ancient DNA from exoskeletal fragments of the grain weevil Sitophilus granarius (L.) extracted from Roman and medieval deposits in Northern England. If DNA preservation in archaeological insect remains is widespread then many applications in the spheres of evolutionary studies and archaeology can be conceived, some of which are outlined.     

Bone Preservation and Ancient DNA: The Application of Screening Methods for Predicting DNA Survival

Given the technical difficulties associated with ancient DNA research, any methods that help to identify samples that will yield amplifiable DNA will be of great value. This study examined the relationships between gross preservation, histological preservation, bone size and the ability to amplify short fragments of mitochondrial DNA in 323 goose humeri from the Anglo-Saxon site at Flixborough. Bone size was not a good predictor of the presence of amplifiable DNA, but there was a significant association between both gross and histological preservation and DNA survival. This suggests that it is worthwhile to preferentially select morphologically well-preserved bones for ancient DNA studies. Our results with ancient avian bone mirror those previously obtained with mammalian archaeological bone, although the relationship between DNA survival and histological preservation was stronger for the latter.     

DNA in cremated bones from an early bronze age cemetery cairn

The discovery of polymeric DNA in charred wheat grains and other burnt plant remains prompted us to examine a set of cremated bones for the presence of ancient DNA. Extracts of cremated bones from an early Bronze Age cemetery cairn were analysed by hybridization probing and the polymerase chain reaction. Hybridization with two probes targeting human DNA resulted in strong positive signals. Polymerase chain reactions directed at a multicopy sequence in human nuclear DNA were successful, extracts of five burials giving amplification products of the expected size. Exploitation of ancient DNA in cremated bones could be of great value in archaeology as it may enable information pertaining to sex and kinship to be obtained from fragmented material.     

Variations in the C/C ratios of modern wheat grain, and implications for interpreting data from Bronze Age Assiros Toumba, Greece

Variations in the ¹³C/¹²C ratios of wheat grain at different spatial and temporal scales are examined by analysis of modern samples, including harvests of einkorn and durum wheat from Greece, and serve as a guide to interpreting data for Bronze Age grains from Assiros Toumba. The normal distribution and low variability of δ¹³C values of einkorn from 24 containers in the Assiros storerooms are consistent with pooling of local harvests, but less likely to represent the harvest of several years or include grain imported from further afield. Correlation between emmer and spelt δ¹³C values provides strong support for other evidence that these were grown together as a maslin crop. ¹³C discrimination (Δ) for the Bronze Age samples is estimated to be 2.5‰ larger than at present, and would be consistent with an intensive, horticultural regime of cereal cultivation, possibly involving some watering.     

Molecular markers for the discrimination of Triticum turgidum L. subsp. dicoccum (Schrank ex Schübl.) Thell. and Triticum timopheevii (Zhuk.) Zhuk. subsp. timopheevii

The persistent uncertainty on the classification of the “new” glume wheat found in Neolithic and Bronze Age sites from Greece and other European settlements might be resolved only through analysis of its ancient DNA. Tools able to discriminate among different Triticum species on the basis of scarce, very damaged DNA, are therefore essential. While current attempts concentrate on DNA fragments sequencing and comparison, in some instances PCR-based selective amplification techniques might offer a cheaper and quicker alternative. The purpose of this research was therefore the identification of species-specific primers, able to distinguish caryopses of Triticum timopheevii subsp. timopheevii from those of Triticum turgidum subsp. dicoccum. Primers and their working conditions were defined and optimized using DNA from modern accessions. The ribosomal primers ITS1 tim and ITS2 tim, and the nuclear primer acetyl-coenzyme A tim clearly discriminated the sequences of Triticum timopheevii from other species. Finally, Neolithic charred wheat grains found in the sites of Sammardenchia (Pozzuolo del Friuli, Udine) and La Marmotta (Lago di Bracciano, Roma), belonging to the “new” wheat type or to emmer, were tested with the three selected primers. However, the results were not conclusive, because the samples analysed were apparently too degraded to yield useful DNA.     

COMPLEX RELATIONSHIPS BETWEEN MITOCHONDRIAL AND NUCLEAR DNA PRESERVATION IN HISTORICAL DNA EXTRACTS*

‘Historical’ DNA obtained from specimens preserved in natural history collections has proven useful for addressing a wide variety of questions, such as the spread of domesticated species or changes in genetic diversity. With the development of high‐throughput sequencing techniques, there is an increasing focus on acquiring genetic information encoded by single‐copy nuclear DNA from historical DNA extracts. The development of efficient techniques to determine the level of nuclear DNA preservation in candidate specimens is necessary to maximize the data obtained from these analyses. Although current evidence suggests that a sample's mitochondrial DNA preservation predicts its nuclear DNA preservation, we show that the relationships between mitochondrial and nuclear DNA preservation are complex.     

Evidence for Differential Ancient DNA Survival in Human and Pig Bones from the Norse North Atlantic

Current models of DNA degradation and previous research on Icelandic human skeletons predict ancient DNA preservation in the Norse North Atlantic faunal remains to be excellent. In contrast, we found that DNA preservation in Viking‐Age pig remains was poor. We posit that this discrepancy in DNA survival between human and faunal remains is due to differing taphonomies. Our results highlight that DNA degradation is strongly dictated by micro‐environmental taphonomic processes even in regions where the climate is conducive to DNA survival. Due to these differences, DNA preservation in animal remains may not be suitable proxies for DNA preservation in associated human remains. Copyright © 2012 John Wiley & Sons, Ltd.     

Early Processed Triticeae Food Remains in The Yanghai Tombs,Xinjiang, China

Visible food remains can provide evidence regarding ancient food processing, the spread of cereals and cultural communication. Some desiccated food remains were discovered in the Yanghai Tombs, Turpan district, in Xinjiang, China (2600–2900 bp ). These food remains were analysed by Fourier transform infrared (FT–IR) spectroscopy combined with plant microfossils, including starch grains and cross cells of pericarp from the cereal bran fragments. The results showed that these food remains were cooked dough food made from wheat (Triticum aestivum) and barley (Hordeum spp.). The cross‐sections of these remains look very dense, not porous under a microscope, which suggests that no fermentation had happened, so these foodstuffs may be some kind of flatbread. Although wheat and barley had been introduced into China by at least the third millennium bc , these remains are still the earliest known direct evidence that wheat and barley were ground into flour and then processed as foodstuffs in north‐western China.     

Preservation of DNA in ancient Egyptian mummies

The presence of DNA has been demonstrated in the cell nuclei of an ancient Egyptian mummy fragment. When extracted, this DNA proved to be degraded to a considerable extent and chemically modified. However, the preservation of nucleic acids in this specimen suggests that applying recombinant DNA techniques to the study of ancient mummified tissues might prove to be a fruitful future area of research.     

Ancient olive DNA in pits: preservation,amplification and sequence analysis

The olive tree (Olea europaea) was domesticated by vegetative propagation of selected wild individuals with superior fruit. Later, new cultivars were established repeatedly from feral trees or from crosses between wild, feral, and domesticated trees. Thus the genetic background of many contemporary domesticated lines is a mixture of ancient cultivars and local wild trees. Ancient DNA may illuminate the complicated process of olive domestication because such DNA sequences provide data about ancient genomes that existed closer to the domestication events. Well preserved DNA must be available for such studies, even though in the Mediterranean region, where olive cultivation took place, the climatic conditions are not favorable for DNA preservation. To select for well preserved pits we measured their proportions of lignin by IR spectroscopy, and correlated this with parameters of DNA quality such as template length in an olive-specific repeat array, and template quantity as determined by real-time PCR amplification. Archaeological pits that passed these tests did contain high quality ancient DNA. We present the first ancient olive DNA sequences and compare them to modern wild, feral and domesticated lines.     

Isotopic Determination of Seasonality in Diet and Death from Nubian Mummy Hair

Sections of human hair from naturally desiccated Sudanese Nubian mummies representing X-Group (AD 350-550) and Christian (AD 550-1300) periods in the Wadi Halfa area have been analysed for their isotope ratios. Because the carbon in hair represents food intake over the growth period of this tissue, analysis comparing segments taken next to the scalp with those further along the shaft can indicate diet just prior to death as well as any recent shift in food consumption. Modern populations in this area practise seasonal crop scheduling which involves the cultivation of C₃ plants (wheat, barley and most fruits and vegetables) in the winter and the hardier C₄ plants (sorghum and millet) in the summer. Shifting isotopic values along the length of hair strands of the ancient inhabitants of this area demonstrate that this practice has its roots at least as far back as the Initial Phase of Intensive Agriculture. The most common season of death appears to have been summer, the time of greatest climatic, nutritional and physiological stress for both ancient and modern inhabitants.     

Bone preservation and DNA amplification

The use of ancient DNA has increased during the past two decades in several scientific disciplines. However, the underlying mechanisms of DNA degradation in bone tissue are poorly understood. Here we address the importance of hydroxyapatite and collagen for DNA preservation in bone. We used two series of bones and teeth, one set of modern experimentally degraded bovid bones and one set of ancient horse bones/teeth. From these samples, we measured crystallinity, DNA presence and extracted collagen. The mtDNA fragments, parts of cytochrome b and the D–loop were amplified and sequenced. Our results show that presence of DNA was strongly related to the crystallinity in the hydroxyapatite and to the amount of collagen. This suggests that the hypothesis that hydroxyapatite has a crucial role in DNA preservation in calcified tissue is valid; and hydroxyapatite and collagen can be used to indicate whether DNA is present in the material. This is what would be expected if DNA is adsorbed to and stabilized by hydroxyapatite in calcified tissue, and collagen is part of the complex system that preserves DNA in bone tissue. Further, since collagen is the preferred material for radiocarbon dating, such bones may be a starting–point for a DNA analysis.     

Characterising the potential of sheep wool for ancient DNA analyses

The use of wool derived from sheep (Ovis aries) hair shafts is widespread in ancient and historic textiles. Given that hair can represent a valuable source of ancient DNA, wool may represent a valuable genetic archive for studies on the domestication of the sheep. However, both the quality and content of DNA in hair shafts are known to vary, and it is possible that common treatments of wool such as dyeing may negatively impact the DNA. Using quantitative real-time polymerase chain reaction (PCR), we demonstrate that in general, short fragments of both mitochondrial and single-copy nuclear DNA can be PCR-amplified from wool derived from a variety of breeds, regardless of the body location or natural pigmentation. Furthermore, although DNA can be PCR-amplified from wool dyed with one of four common plant dyes (tansy, woad, madder, weld), the use of mordants such as alum or iron leads to considerable DNA degradation. Lastly, we demonstrate that mtDNA at least can be PCR-amplified, cloned and sequenced from a range of archaeological and historic Danish, Flemmish and Greenlandic wool textile samples. In summary, our data suggest that wool offers a promising source for future ancient mitochondrial DNA studies.     

Preliminary report on the archaeobotanical investigations at Tell Abraq with special attention to chaff impressions in mud brick

Examination of impressions from chaff-tempered mud brick proved rewarding, given the poor preservation of carbonised seed and grain material at Tell Abraq. Individual chaff elements were found to be recognisable from the impressions and it was shown that naked wheat and barley chaff were used as a tempering medium. Charcoal samples indicate that two species of mangrove were present, one of which is no longer found in the area. Tamarisk and Ziziphus were also present. Date charcoal was common. This study remains preliminary, and further sampling and analyses have been planned for the future.