Project title: Epigenetic soft-selection as a driver for local adaptation: unravelling ancient methylomes
using ancient DNA
Project code:
Host institution: University of Warwick
Theme: Evolution & Ecosystems
Key words: Ancient DNA, epigenetics, NGS, archaeology
Supervisory team (including institution & email address):
Robin Allaby, University of Warwick (r.g.allaby@warwick.ac.uk)
Logan Kistler, University of Warwick (logankistler@gmail.com)
Currently there are two major projects underway in
our group studying the archaeogenomics of maize and
sorghum, particularly with reference to local
adaptation. This project will focus on epigenetic
aspects of these crops in conjunction with these
projects.
DNA methylation of cytosine residues is an epigenetic
process and is one of the signatures of gene silencing,
often occurring in response to biotic or environmental
stresses. We have previously shown the possibility
that these modifications in archaeological barley
direct local adaptation to such stresses, and
consequently may be a driver for long-term
evolutionary change. In contrast to mammalian
epigenetics, the potential combination of methylation
‘islands’ in plants is significantly higher and requires a
slightly different approach to previous aDNA methods.
The plasticity of archaeobotanical genome
methylation is demonstrated by a link between
genomic hypermethylation and short interfering RNA
(siRNA) activity in archaeological material under biotic
stress, demonstrating in vivo activity in
archaeogenome.
Hydrolytic deamination of cytosine to uracil is a welldocumented process in the breakdown of DNA over
time. Since uracil shows up as thymine using current
sequencing technology, the original state of
deaminated bases must be inferred by molecular or
statistical methods. However there is evidence to
suggest that methylated cytosines are more prone to
deamination than their unmethylated counterparts.
Methods exist however to isolate deaminated
cytosine from deaminated 5-methyl-cytosine in
ancient DNA, allowing reconstruction of functional
methylomes. The purpose of this project is to apply
these methods to pertinent loci of plant
archaeogenomes, such as transposable elements and
functional architecture genes, in order to infer
adaptation to environmental stress.
50
Genomic methylation (%)
Overview: The Allaby lab is currently at the forefront
of archaeogenetic research, having recently shown
the applicability of epigenetic information from
archaeological material to biotic and abiotic stress
responses.
Uninfected methylation
(genomic %)
Late Christian
(actual)
45
Late Christian infected
methylation (genomic %)
40
35
30
Islamic
25
Late Christian
(adjusted)
20
15
10
Meroitic
Napatan
5
0
0
500
1000
1500
2000
2500
3000
Sample Age (yBP)
Figure 1: Diagenetic demethylation of the barley
genome according to time, including evidence of
genomic hypermethylation of barley under biotic
stress (Smith et al., 2014 ; go.nature.com/dCWBHQ)
Methodology:
This project will involve a combination of approaches
including next-generation sequencing, methylationspecific sequencing, bioinformatic methods and
statistical analysis. Briefly, the project aim will be to
first identify functional / stress-related loci within the
archaeogenomes of sorghum and/or maize (e.g.
transposable elements, plant architecture-associated
alleles, transcription factors), and isolate these loci
using a DNA-capture method (e.g. Mycroarray or
Agilent Sureselect) from ancient DNA samples. These
loci will then be sequenced, using the Illumina
platform, before and after bisufite treatment and
further using uracil-DNA-glycosylase treatment to
distinguish C>U from 5mC>T modifications. Loci will
then be reconstructed in silico using bioinformatics
methods. Using a combination of comparative preand post-bisufite treatment reads and coverage depth
analysis, we hope to reconstruct a) the in vivo
epigenetic state of functional alleles, b) diagenetic
demethylation from these alleles, and c) calibration of
5mC>T modifications (as opposed to C>U
modifications) as a secondary measure of hydrolytic
deamination.
Training and skills:
CENTA students will attend 45 days training
throughout their PhD including a 10 day placement. In
the first year, students will be trained as a single
cohort on environmental science, research methods
and core skills. Throughout the PhD, training will
progress from core skills sets to master classes specific
to the student's projects and themes. The candidate
will already possess a background in molecular biology
from his/her prior education. They will receive full
training in molecular methods pertinent to ancient
DNA, the use of Next-Generation Sequencing (NGS)
platforms, and advanced data analysis. To this end,
the candidate will also receive initial and continuing
training in programming, scripting and statistical skills
that are fast becoming vital tools for high-throughput
data analysis. The candidate’s experience may be
further augmented by archaeological fieldwork, to
fully appreciate the complete archaeogenetic process.
Partners and collaboration (including CASE):
There are a range of collaborators involved in the
sorghum and maize projects, including archaeologists
and genome scientists from a range of institutions.
Possible timeline:
Year 1: Sample collection and optimisation of
molecular methods. The nature of ancient DNA will
require optimisation of DNA extraction, DNA capture,
bisulfite treatment methods. Illumina library
construction may require further optimisation due to
the use of several untested methods in this context,
specifically thymine-skipping polymerases (resulting in
increased abasic sites) and bisulfite-treated aDNA.
Year 2: Completion of method optimisation and
straight on to Illumina data generation. Data analysis
to start, initially focusing on allelic reconstruction and
threefold alignments from ‘standard’, bisulfite-treated
and uracil-skipped datasets.
Year 3: Continuation of data analysis to reconstruct
differential genome methylation at loci of interest.
Bioinformatic prediction of gene network interactions
to predict in vivo outcome of allelic / epigenetic
profile in the archaeological sorghum and maize.
Further reading:
Briggs, A. W., U. Stenzel, M. Meyer, J. Krause, M.
Kircher and S. Pääbo (2010). "Removal of deaminated
cytosines and detection of in vivo methylation in
ancient DNA." Nucleic Acids Research 38(6): e87.
Gokhman, D., E. Lavi, K. Prüfer, M. F. Fraga, J. A.
Riancho, J. Kelso, S. Pääbo, E. Meshorer and L. Carmel
(2014). "Reconstructing the DNA Methylation Maps of
the Neandertal and the Denisovan." Science
344(6183): 523-527.
Smith, O., A. Clapham, P. Rose, Y. Liu, J. Wang and R.
G. Allaby (2014). "A complete ancient RNA genome:
identification, reconstruction and evolutionary history
of archaeological Barley Stripe Mosaic Virus." Sci. Rep.
4.
Smith, O., A. J. Clapham, P. Rose, Y. Liu, J. Wang and R.
G. Allaby (2014). "Genomic methylation patterns in
archaeological barley show de-methylation as a timedependent diagenetic process." Sci. Rep. 4.
Further details:
Robin Allaby (r.g.allaby@warwick.ac.uk)