DNA in charred wheat grains from the Iron Age hillfort at Danebury, England.

DNA in charred wheat grains from the Iron Age hillfort at Danebury, England. The genetic history of wheat is the story of the world'stemperate staple food. Archaeologically, charred grains are the commonway wheat is preserved. Study of burnt spelt wheat from the British IronAge In the British Isles, the Iron Age lasted from about the 7th century BC until the Roman conquest and until the 5th century in non-Romanised parts. This period is also called the era of Celtic Britain<ref name=>Celtic Britain (The Iron Age) c. shows DNA DNA:see nucleic acid. DNAor deoxyribonucleic acidOne of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. is present, and begins to shows the wheat'scharacter.DNA and ancient wheatGenetic change, always central to studies of prehistoric agriculture,still operates as the principal criterion for designating certain plantand animal taxa as 'domesticated' (Harris & Hillman 1991)rather than wild. To this end, much bio-archaeological research intoearly agriculture has examined the gross morphology of plant and animalremains for phenotypic features that on the one hand seem to correspondto agricultural selection pressures, and on the other possess evidenceof a direct genetic basis. However, it has frequently proved difficultto consolidate these two requirements. Features relating to larger, moreaccessible fruits and seeds, or to smaller, more docile animals, haveoften been judged as resulting from selection, but in many cases thegenetic basis to the trait is uncertain. In contrast, phenotypes with aclear genetic foundation, such as the ploidy-dependent features of wheatchaff (Kislev 1984; Gordon Hillman pers. comm.), are often difficult toaccount for in terms of agricultural selection pressures. In otherwords Adv. 1. in other words - otherwise stated; "in other words, we are broke"put differently , we have clear evidence of past phenotypic change consistent withwhat we assume to have been agricultural selection pressures, and weknow in broad terms that many of those changes are due, directly orindirectly, to genetic events, but our existing methods constrain usfrom tying these two strands strongly together.The discovery of preserved DNA in a range of archaeological materialscould transform our approach. If the obstacles of survival, degradation,contamination and taphonomy ta��phon��o��my?n.1. The study of the conditions and processes by which organisms become fossilized.2. The conditions and processes of fossilization. can be overcome, then such DNA as survivesin ancient plants and animals has the potential to provide a direct linkbetween genotype, phenotype and the cultural context. Not only wouldthis open the way to precision, it could also liberate our thinking fromthe gross 'genetic events' of 'domestication' to theperspective of a continuous evolutionary dynamic, in which the constantrestructuring in human society through space and time is reflected in anequally continuous process of phylogenetic response, sometimesminuscule, sometimes substantial, in the plants and animals with whichhumans have been most closely associated.We are attempting to start towards this goal with one of the mostabundant archaeological resources of agricultural relevance: preservedwheat seeds. In archaeological contexts, plants are preservedprincipally by one of four mechanisms: the partial or complete reductionto carbon by heat; the exclusion of water in desiccating environments;the exclusion of oxygen in anoxic an��ox��i��a?n.1. Absence of oxygen.2. A pathological deficiency of oxygen, especially hypoxia.[an- + ox(o)- + -ia1. , often waterlogged environments; andpartial mineralization by calcium phosphate, calcium carbonate or, lesscommonly with archaeological remains, iron sulphide (pyrites). We do notknow which types of preservation might be compatible with retention ofDNA, but intuitively one might expect biomolecular decay to be retardedin dry and/or anoxic settings. This conjecture is supported by reportsof ancient DNA in anoxically preserved plant remains from Miocenedeposits at Clarkia, Idaho (Golenberg et al. 1990; Golenberg 1991;Soltis et al. 1992), and in maize cobs preserved through variouscombinations of carbonization car��bon��i��za��tion?n.1. The process of carbonizing.2. The destructive distillation of bituminous coal, done in the absence of air in order to obtain coke and other fractions having a greater percentage of carbon than the and desiccation des��ic��ca��tionn.The process of being desiccated.desic��ca (Rollo et al. 1991;Goloubinoff et al. 1993), the latter papers also demonstrating thephylogenetic inferences that are possible. Wheat and other Old Worldcereals have been encountered in all four preservation states, but thedifferent states vary in their geographical and temporal coverages.Anoxically preserved and dry preserved cereals have sometimes beenencountered in rich and impressive assemblages (e.g. Jacomet &Schlichterle 1984; Rowley-Conwy 1991), but the fullest spatial andtemporal coverage is associated with carbonized remains: carbonizedremains are therefore the principal source material on which an ancientDNA approach to wheat bio-archaeology must be based. We have examinedcharred seeds of spelt wheat from the Iron Age hillfort at Danebury,England (Jones 1984; Nye & Jones 1991), directly dated by theconventional radio-carbon method to the second half of the 1stmillennium BC (Cunliffe 1984), for the presence of ancient DNA.Procedures and resultsAncient DNA studies are almost entirely dependent on the polymerasechain reaction (PCR PCRpolymerase chain reaction. PCRabbr.polymerase chain reactionPolymerase chain reaction (PCR); Saiki et al. 1988), a biochemical technique thatresults in amplification of ||micro~gram~ quantities of DNA from minutetraces of starting material. The relatively large amounts of DNAprovided by PCR are sufficient for genetic analysis by, for example,nucleotide sequencing (for a review of ancient DNA techniques see Brown& Brown 1992). In a PCR experiment the genetic region to beamplified is selected by two short, synthetic DNA molecules that areadded to the reaction mixture. These molecules attach to the substrateDNA at either side of the target region and prime the amplification. Inour experiments we targeted a 246 base pair segment of DNA from apolymorphic region, linked to the genes for the high-molecular-weight(HMW HMW High Molecular WeightHMW health, morale, and welfare (US DoD)HMW Hazardous Material WarningHMW How might weHMW Highly Modular Workplan ) glutenin proteins, chosen partly on technical and partly onscientific grounds. The technical advantage is that there are two pairsof HMW glutenin genes per genome in wheat, so hexaploid spelt wheat(with three genomes) has a total of 12 targets. Multiple targetsincrease the chances of a successful amplification from small amounts ofstarting DNA. In scientific terms the region is attractive because ofits polymorphic nature. Sixteen different allelic forms of the HMWglutenin genes have been identified in modern European cultivars ofwheat, various combinations occurring within a single genotype (Payne1987), and many additional alleles are known in wild populations (Ciaffiet al. 1993). Six alleles have been characterized by nucleotidesequencing, each recognizable by diagnostic sequence features within theregion targeted for amplification. Sequence analysis of the moleculesobtained by PCR of ancient DNA might lead to identification of the HMWglutenin alleles present. Complete allelic typing of a single specimenwould be extremely time-consuming, but even a partial allele set mightallow broad phylogenetic comparisons to be made between differentspecimens. In addition, the role of the HMW glutenins in determining theviscoelastic Adj. 1. viscoelastic - having viscous as well as elastic propertiesnatural philosophy, physics - the science of matter and energy and their interactions; "his favorite subject was physics" properties of dough prepared from the grain (Flavell et al.1989) has led to identification of modern alleles that confer'good' or 'poor' bread-making qualities (Payne1987). There is a certain appeal to the possibility that allelesconferring defined bread-making qualities might be identifiable inarchaeological grain.We subjected the charred grain to a standard nucleic acid extractionprocedure, previously shown to be applicable to preserved material(Rogers & Bendich 1985). Initially the extracts were analysed byhybridization hybridization/hy��brid��iza��tion/ (hi?brid-i-za��shun)1. crossbreeding; the act or process of producing hybrids.2. molecular hybridization3. probing, the results suggesting that nucleic acid might bepresent (results not shown). Subsequently a PCR with the Daneburyextract gave rise to amplification products of the expected size. DNAresulting from the PCR was cloned, and two nucleotide sequencesobtained, both clearly derived from wheat HMW glutenin genes. Neithersequence was an exact match with any of the known alleles, though one(DANEB1) displayed just a single difference to the 5(X) allele.Single-position sequence changes can occur during PCR, this effect beingmore frequent when the substrate DNA is chemically-damaged, which isalmost certainly the case with ancient DNA (Paabo 1989; Lindahl 1993).It is therefore possible that DANEB1 and 5(X) are identical. The secondsequence, DANEB2, displayed eight nucleotide differences to the mostclosely similar of the known sequences. DANEB2 therefore appears torepresent a novel HMW glutenin allele.The major problem with ancient DNA studies lies with the difficultyin establishing that amplification products derive from DNA preserved inthe specimens and not from modern contamination (reviewed by Brown &Brown 1992). PCR is so sensitive that DNA present in the air and workingmaterials can easily give false-positive results. With archaeologicalmaterial the problem is magnified by the possibility of contaminationduring the relatively uncontrolled conditions of the excavation. Thereis no mechanism for proving beyond doubt that an amplification productfrom ancient material is authentic; all that can be done is to takesuitable precautions to minimize the chances of contamination and tocarry out control experiments. Our project included precautions andcontrols equivalent to those adopted by other groups working withancient DNA. We have also taken the added precaution in this pilot studyof working with particularly rich and well-sealed assemblages fromDanebury that were removed en bloc and immediately enclosed withinaluminium foil, rather than being put through sieving or flotation. Theonly potential contamination on site would be from modern wheat pollen,which the collection regime was designed to avoid. In fact, theextraction procedure does not give significant yields of DNA frompollen, probably because of difficulties in disrupting the toughsporopollenin spo��ro��pol��len��in?n.A polymer that constitutes the outer wall of spores and pollen grains.sporopollenin?An organic polymer that is extremely resistant to degradation. coat. Our laboratory technique incorporated standardprecautions for avoiding contamination in PCR experiments, including theuse of specialized equipment, autoclaved solutions, and a geographicalseparation between the rooms used for DNA preparation and PCR analysis.Our basic technique has proved reliable in projects involving PCR ofhuman material, where contamination with personal DNA can be a majorproblem. The results of two control experiments are shown in FIGURE 2.With the 'water blank', a PCR with water rather than DNAextract gave no amplification product, showing that cross-contaminationof samples did not occur during the PCR experiment. In the secondcontrol, no amplification product was seen when an 'extractblank' was used in the PCR. The extract blank was prepared bycarrying out the entire extraction procedure without seeds, providing arigorous test for poor technique and contamination of working solutions.DiscussionGiven these precautions and the results of the control experiments,we believe that an acceptable degree of confidence can be assigned tothe results with the Danebury seeds. Not every extraction has succeeded,suggesting perhaps that not all seeds in the sample contain amplifiableDNA. In contrast, a single extraction with a second sample of charredgrain -- 3300-year-old emmer wheat from Assiros Toumba, Greece (Jones etal. 1986; Jones 1987) -- has provided an amplification product of thecorrect size (results not shown). We are modifying the extraction andPCR techniques in attempts to improve the success rate with the Daneburyseeds, and are extending the work to a brooder range of archaeologicalwheats in order to assess the overall potential of ancient DNA analysis.To obtain information from which phylogenetic inferences can be made, itwill be necessary to study polymorphic sequences that enable differentpopulations of archaeological wheats to be distinguished. The gluteninalleles may be suitable, though complete allelic typing of just onespecimen would require a substantial number of cloning and sequencingexperiments. Our current work will determine whether a phylogeneticanalysis based on the glutenin genes is technically feasible.Despite its archaeological importance, our knowledge of the chemicalcomposition of carbonized plant tissue remains poor, and probablyinferior to that of its geological counterpart, fusain (Scott 1989).While transformation by heat is clearly instrumental in creating amicroenvironment microenvironment/mi��cro��en��vi��ron��ment/ (-en-vi��ron-ment) the environment at the microscopic or cellular level. hostile to soil biota biota/bi��o��ta/ (bi-o��tah) all the living organisms of a particular area; the combined flora and fauna of a region. bi��o��tan.The flora and fauna of a region. and therefore perhaps permittingDNA preservation, visual inspection of carbonized specimens indicates arange of transformation levels, from burst, distorted specimens throughto specimens more or less retaining their original shape and cellularstructure, and with several deviations from the soot-black colour offull carbonization. As a working hypothesis we assume that such DNA aspersists in carbonized specimens is present in uncarbonized domains thatsurvive encased in transformed and thus protective tissue. An importantstep for future work is to corroborate, or otherwise, this hypothesis. Abetter understanding of the material may enable us to use the appearanceof specimens as a means of preselecting individual seeds likely tocontain ancient DNA. Analysis of individual seeds is desirable as mostarchaeological assemblages are probably not genetically homogeneous. ThePCR technique is sensitive enough to amplify ancient DNA from a singleseed, but this approach will be extremely time-consuming if there is noway of preselecting suitable specimens.Acknowledgements. A preliminary report of this work has beenpublished as part of a review article (Brown et al. 1993). We are verygrateful to Glynis Jones, University of Sheffield The University of Sheffield is a research university, located in Sheffield in South Yorkshire, England. ReputationSheffield was the Sunday Times University of the Year in 2001 and has consistently appeared as their top 20 institutions. , for supplying theAssiros Toumba grain. We also thank Gordon Hillman (University ofLondon For most practical purposes, ranging from admission of students to negotiating funding from the government, the 19 constituent colleges are treated as individual universities. Within the university federation they are known as Recognised Bodies ), Alan Clapham (University of Cambridge), Chris Howe (Universityof Cambridge), Paul Sims (UMIST UMIST University of Manchester Institute of Science and Technology (UK)), Robert Sallares (UMIST) and Keri Brown(UMIST) for helpful discussions. This work was begun with fundingprovided by the Biomolecular Palaeontology Special Topic of the NaturalEnvironment Research Council, and subsequently supported by a researchgrant from the Science-Based Archaeology Committee of the Science andEngineering Research Council (body) Science and Engineering Research Council - (SERC) Formerly the largest of the five research councils funded by the British Government through the Office of Science and Technology. .ReferencesANDERSON, O.D., F.C. GREENE, R.E. YIP, N.G. HALFORD, P.R. SHEWRY& J.-M. MALPICA-ROMERO. 1989. Nucleotide sequences of twohigh-molecular-weight glutenin genes from the D-genome of a hexaploidbread wheat, Triticum aestivum L. cv Cheyenne, Nucleic Acids Research Nucleic Acids Research or NAR is a peer reviewed scientific journal published by Oxford University Press. NAR publishes research on Nucleic Acids, such as DNA and RNA, and related work. 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