Table 1.
Girth, National Grid Reference and species of the oak samples.
Table 2.
The dendrochronological analysis performed on the oak samples.
Table 3.
DNA extraction buffers.
Fig 1.
PCR-inhibitory effect of heartwood DNA extracts.
A) PCR inhibitory effect of heartwood DNA extracts obtained with STE 2% SDS 6% PVP, STE 0.2% SDS 6% PVP, aDNA buffer with and without DTT, PTB buffer. Amplifiable sapwood DNA extract (s) was titrated with heartwood DNA extract (h) at 1:4 (a), 2:4 (b), 3:4 (c), 3.25:4 (d), 3.5:4 (e). Sapwood-heartwood extract DNA was amplified by PCR for 40 cycles. The two panels of this figure were taken from different gels under the same experimental conditions. B, C) Concentration of phenolic compounds and ellagic acid derivatives in DNA extracts obtained from oak heartwood (B) and from the washing surnatants of oak sapwood and heartwood (C). Error bars represent the standard deviation. D) Concentration of the tested compound: a) 1.0 mM, b) 0.1 mM, c) 0.01 mM, d) 0.001 mM. Sapwood (+), 50% diluted sapwood (w), PCR negative control (-). Hb: hydroxybenzoic acid, dhb: dihydroxybenzoic acid. Ellagic acid is dissolved in methanol which is why controls with the addition of only methanol to the PCR were carried out (99%, 9.9%, 0.9%, 0.09%). The upper, middle, and lower panels of this figure were taken from different gels under the same experimental conditions.
Fig 2.
Interactions between ellagic acid, solvents and DNA.
A) dsDNA model with labels (1 to 40) for the nucleobases. Cytosine is in red, adenine in yellow, thymine in cyan, guanine in green, and phosphorus atoms in orange. An exemplar minor-groove site (viii) is shown in blue and an exemplar major-groove site (IX) is shown in purple, with the relevant distances for the proximity calculation of ellagic acid. B) Density maps of ellagic acid atoms in interaction with dsDNA in water and in methanol. The isosurfaces correspond to a density of 0.5 Å−3. C) Average proximity occurrences in the minor groove sites (top) and in the major groove sites (bottom). D) Ellagic acid with labelled oxygens. E) Average number of hydrogen bonds between ellagic acid oxygens and water (blue) and methanol (red) oxygens, averaged for each pair of equivalent ellagic acid oxygens. F) Pair correlation function (g(r)) of ellagic acid oxygens and water (blue) and methanol (red) oxygens, averaged for each pair of equivalent ellagic acid oxygens as labelled in the ellagic acid model. G) A representative molecular dynamics snapshot of the intercalated ellagic acid within DNA bases. H) An example of ellagic acid unbinding trajectory obtained with metadynamics. This image was made with VMD and is owned by the Theoretical and Computational Biophysics Group, NIH Center for Macromolecular Modeling and Bioinformatics, at the Beckman Institute, the University of Illinois at Urbana-Champaign (www.ks.uiuc.edu).
Fig 3.
Analysis of the factors affecting the success of DNA library preparation.
A) Success of WGS library preparation performed from Dulwich Woods and GLOR01–07 trees (Table 2). Two distinct samples were collected from GLOR07, one was air-dried and stored at room temperature while the other was frozen and stored at -20°C. B) Logistic regression of the relationship between the age of wood (years) and the WGS library preparation success in GLOR07 frozen samples (“GLOR07, frozen”) and the GLOR01-GLOR07 samples that were air-dried and stored at room temperature (“ex Dulwich, ex outliers”). The logistic regression was performed after excluding (“ex”) the samples obtained from the Dulwich Woods (“Dulwich”) core and those that contained a low amount of oak DNA (“outliers”). C) WGS library preparation outcome of samples correlated with the concentration of phenolic compounds (log of gallic acid equivalents μM) and the concentration of DNA (log ng/μl). D, E, F) Logistic regression relating the success of WGS library preparation to D) the concentration DNA as measured by fluorometric quantification (log ng/μl), E) the concentration of phenolic compounds (log of Gallic acid equivalents μM), F) the residuals of DNA concentration.
Fig 4.
DNA fragmentation and damage pattern in oak heartwood.
A-D) DNA fragments length distribution profile of (A) GLOR07, (B) GLOR03, (C) GLOR02 and (D) GLOR01 sapwood and heartwood DNA fragment length profile. We reported the fraction of total DNA fragments corresponding to each length (bp). E) Nucleotide frequency at the 3’ (-1 to -5) and 5’ (1 to 5) 5bp genomic positions of DNA reads in the core sample GLOR07. F) Misincorporation frequency at DNA read 5’- and 3’-end in the core sample GLOR07 1934–1943.
Fig 5.
WGBS is not feasible in oak heartwood.
A) Percentage of genomic bases covered by at least one WGS or WGBS DNA read generated from the sequencing of the GLOR07 and GLOR03 sapwood and heartwood samples. B-C) Number of distinct reads expected to be obtained at the increase of the total number of reads as predicted by Preseq [60]. We included the WGS (B) and WGBS (C) libraries having a number of endogenous reads compatible with the software requirement. We reported the number of reads generated by an Illumina MiSeq (21 hours run, chemistry version 3, 75 bp reads) and Illumina HiSeq2000 (8 days run, 100 bp reads).