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Gossip in the Grain: Epigenetic evolution tracked through the barley archaeogenome

Excavations at Qasr Ibrim, a frontier outpost near the Egyptian / Nubian border in southern Egypt, have recovered samples of cultivated crops from multiple periods of occupation. Many of these samples show an unusually high level of preserved genetic material for archaeological specimens, most likely due to the extreme aridity of the depositional environment1. Samples of barley grain (Hordeum vulgare) are of particular interest due to an unusual archaeobotanical dataset that belies their genotype2, 3.

While this discrepancy may be inconsequential due to fruit underdevelopment as opposed to null development, the combination of this and hostile paleoclimate suggests that a stress-induced genetic or epigenetic response may have occurred. If epigenetic in nature, the response would be mediated by the action of microRNAs (miRNA) and short interfering RNAs (siRNA); 19-24nt regulatory RNA molecules which regulate gene expression without altering the underlying genetic code. Epigenetic stress-response in barley is well documented4, although the critical mechanism by which these responses are inherited transgenerationally is not well understood.

High-throughput sequencing of the Qasr Ibrim barley microRNA has revealed a number of potentially revealing epigenetic differences, when compared to a recently-cultivated 6-row barley from the same geographic region. We have identified significant discrepancies in known miRNAs known to play an active role in plant growth and development between the two samples. These miRNAs act along the class II TCP domain-containing transcription factor (miR319), auxin (miR167)5, PHV / PHB (miR166)5 and GAMYB (miR319; miR159)6 pathways, suggesting that the ancient barley epigenome is subject to differential expression of developmental microRNAs.

Qasr Ibrim was occupied continually throughout multiple cultural phases over around 3,000 years (Napatan, Meroitic, Roman, Christian and Islamic)1. Considering the relatively low grain yield from the local variety as opposed to high-yielding varieties, which were in existence in the immediate geographical area throughout this period, it is unclear why successive occupiers of the site continued to grow at a lower yield. The reason may a simple inability to cultivate a sufficient quantity of higher-yielding barley (amongst other cereals) under extreme selection pressure, forcing the inhabitants to re-plant from grain harvested in situ. Considering the unusual purpose and isolated nature of the site, it is intriguing how Qasr Ibrim highlights the combination of paleoclimate, adaptive biology and military politics in shaping the diet of a population.

While so far only samples from the two most recent periods (strata: Islamic, ~600-200 years BP and Late Christian, ~200 - 900 years BP) have been analysed, the similar genetic and archaeobotanical profiles of grain from earlier strata allow us to anticipate that these will exhibit a similar epigenetic profile. Provided the integrity of nucleic acid remains acceptably high between strata, as fluorescence quantitation has shown, successful sequencing will provide evidence of epigenetic transgenerationalism under environmental stresses. We hope to show that viable data from archaeological material will promote study of the archaeoepigenome as a viable area of research.

In addition, we also observed an interesting accessory genome in the archaeological material - barley stripe mosaic virus (BSMV), which can be confirmed as ancient due to genetic distance from the standard reference genomes and evidence of postmortem base modification. Incredibly, we were able to reconstruct the entire genome to around ~97% coverage using a combination of typically-sized small RNA fragments and more obvious, larger degradation products. Even more interestingly, coverage 'spikes' of certain sequence motifs along the genome which fit with standard siRNA-sized molecules are highly indicative of a plant-mediated viral defense mechanism known as RNA interference (RNAi). These ta-siRNA molecules seem to target viral genes involved with propagation (RNA-dependent RNA polymerase) and trans-membrane movement (beta-D protein), suggesting a clear strategy of defense.


1 Copley, M.S., Rose, P.J., Clapham, A., Edwards, D.N., Horton, M.C., Evershed, R.P. (2001) Processing palm fruits in the Nile Valley: biomolecular evidence from Qasr Ibrim. Antiquity 75(289): 538-542.

2 Komatsuda, T., Pourkheirandish, M., He C., Azhaguvel, P., Kanamori, H., Perovic, D., Stein, N., Graner, A., Wicker, T., Tagiri, A., Lundqvist, U., Fujimura, T., Matsuoka, M., Matsumoto, T., Yano, M. (2007) Six-rowed barley originated from a mutation in a homeodomain-leucine zipper 1-class homeobox gene. PNAS 104(4): 1424-1429

3 Palmer, S.A., Moore, J.D., Clapham, A., Rose, P., Allaby, R.G. (2009) Archaeogenetic Evidence of Ancient Nubian Barley Evolution from Six to Two-Row Indicates Local Adaptation. PloS ONE 4(7):e6301. doi:10.1371/journal/pone.0006301

4 Kantar, M., Unver, T., Budak, H. (2010) Regulation of barley miRNAs upon dehydration stress correlated with target gene expression. Funct. Integr. Genomics 10(4): 493-507.

5 Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP. (2002) Prediction of plant microRNA targets. Cell. 110(4):513-20.

6 Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP. (2002) MicroRNAs in Plants. Genes Dev. 2002 Jul 1;16(13):1616-26.


Robin G. Allaby

Contact Details

r dot g dot allaby at warwick dot ac dot uk