tpc118794Supplemental

Supplemental Figures and Table

tpc118794Supplemental DS2

Supplemental Data File

tpc118794Supplemental DS4

Supplemental Data File

Inter-Species Grafting Caused Extensive and Heritable Alterations of DNA Methylation in <i>Solanaceae</i> Plants

<div><p>Background</p><p>Grafting has been extensively used to enhance the performance of horticultural crops. Since Charles Darwin coined the term “graft hybrid” meaning that asexual combination of different plant species may generate products that are genetically distinct, highly discrepant opinions exist supporting or against the concept. Recent studies have documented that grafting enables exchanges of both RNA and DNA molecules between the grafting partners, thus providing a molecular basis for grafting-induced genetic variation. DNA methylation is known as prone to alterations as a result of perturbation of internal and external conditions. Given characteristics of grafting, it is interesting to test whether the process may cause an alteration of this epigenetic marker in the grafted organismal products.</p><p>Methodology/Principal Findings</p><p>We analyzed relative global DNA methylation levels and locus-specific methylation patterns by the MSAP marker and locus-specific bisulfite-sequencing in the seed plants (wild-type controls), self- and hetero-grafted scions/rootstocks, selfed progenies of scions and their seed-plant controls, involving three <i>Solanaceae</i> species. We quantified expression of putative genes involved in establishing and/or maintaining DNA methylation by q-(RT)-PCR. We found that (1) hetero-grafting caused extensive alteration of DNA methylation patterns in a locus-specific manner, especially in scions, although relative methylation levels remain largely unaltered; (2) the altered methylation patterns in the hetero-grafting-derived scions could be inherited to sexual progenies with some sites showing further alterations or revisions; (3) hetero-grafting caused dynamic changes in steady-state transcript abundance of genes encoding for a set of enzymes functionally relevant to DNA methylation.</p><p>Conclusions/Significance</p><p>Our results demonstrate that inter-species grafting in plants could produce extensive and heritable alterations in DNA methylation. We suggest that these readily altered, yet heritable, epigenetic modifications due to interspecies hetero-grafting may shed one facet of insight into the molecular underpinnings for the still contentious concept of graft hybrid.</p></div

Real-time qRT-PCR analysis of genes encoding for enzymes involved in establishing and/or maintaining of DNA methylation.

<p>The steady-state transcript abundance of genes encoding three DNA methyltransferases, slMET1 (A), slCMT3 (B), and slDRM2 (C), two 5-methylcytosine DNA glycosylases, slDME (D) and slROS1 (E), and four proteins involved in the biogenesis of siRNAs participating in the RdDM pathway, slNRPDA (F), slAGO (G), slDRB (H), and slRDR (I) were analyzed by real-time qRT-PCR. All these genes were identified from the tomato genome (details are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061995#pone.0061995.s002" target="_blank">Table S1</a>). Reverse transcription was performed on three batches of independently isolated RNAs from young leaves of the seed-plant control (T), two independent hetero-grafted (tomato to eggplant) scion plants (eT1-2), and 12 and eight selfed progeny plants (S1) of each mother scion plant. A conserved <i>Solanaceae</i> actin gene (Genbank accession: U60481) was used as an internal control for RNA input and DNA contamination (on RNAs without reverse transcriptase). Asterisks denote statistical difference in the transcript abundance relative to that of the corresponding seed-plant control, based on t-test at <i>P</i> = 0.05 (*) and <i>P</i> = 0.01 (**) levels, respectively. Colors of asterisks refer to significant up- (blue) and down-regulation (red), respectively.</p

Altered DNA methylation patterns of 15 variant MSAP bands in the grafted tomato or eggplant scions, their predicted functional homology and their inheritance to selfed progenies.

<p>Altered DNA methylation patterns of 15 variant MSAP bands in the grafted tomato or eggplant scions, their predicted functional homology and their inheritance to selfed progenies.</p

tpc118794Supplemental DS3

Supplemental Data File

tpc118794Supplemental DS1

Supplemental Data File

Tabulated data of relative DNA methylation levels based on the MSAP analysis.

<p>Relative levels of total, CG and CHG methylation at randomly sampled 5′-CCGG sites by the MSAP marker, in three independent tomato scions hetero-grafted to eggplant (marked as eT1-3) (<b>A</b>), three independent eggplant scions hetero-grafted to tomato (marked as tE1-3) (<b>B</b>), three independent self-grafted tomato scions (marked as tT1-3) (<b>cC</b>), three independent self-grafted eggplant scions (marked as eE1-3) (<b>D</b>),and their respective seed-plant controls of tomato (T) and eggplant (E), respectively. * Values significantly lower than the values of the corresponding seed-plant controls (<i>P</i><0.05).</p

Validation of grafting-induced DNA methylation alterations and their inheritance by locus-specific bisulfite Sanger-sequencing.

<p>DNA methylation maps and collective methylation values (in percentage) for the three selected MSAP bands, unique-copy genic sequences ST4 (from tomato scion) (A and B) and SE1 (from eggplant scion) (C and D), and a low-copy retrotransposon fragment SE2 (from eggplant scion) (E and F), determined by genomic bisulfite Sanger-sequencing in the following samples: seed-plants of tomato (seed-plant T) and eggplant (seed-plant E), two independent, reciprocally hetero-grafted scion plants, tomato grafted to eggplant (eT1-2) and eggplant grafted to tomato (tE1-2), and one selfed progeny (S1) for each scion-plant of tomato (tT) and Eggplant (eE). All three types of cytosines, CG (red circles), CHG (blue circles) and CHH (green circles) were shown in the map. Filled and empty circles indicate methylated and unmethylated cytosines, respectively. The red, blue and green columns in the histograms refer to the collective methylation levels (in percentage) respectively of CG, CHG, and CHH for each sample, described above.</p