1. Chromosomes and Gene Expression
  2. Plant Biology
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Plants regenerated from tissue culture contain stable epigenome changes in rice

  1. Hume Stroud
  2. Bo Ding
  3. Stacey A Simon
  4. Suhua Feng
  5. Maria Bellizzi
  6. Matteo Pellegrini
  7. Guo-Liang Wang
  8. Blake C Meyers
  9. Steven E Jacobsen  Is a corresponding author
  1. University of California, Los Angeles, United States
  2. Ohio State University, United States
  3. Delaware Biotechnology Institute, University of Delaware, United States
  4. Howard Hughes Medical Institute, University of California, Los Angeles, United States
Research Article
Cite this article as: eLife 2013;2:e00354 doi: 10.7554/eLife.00354
6 figures, 3 tables and 1 data set

Figures

Aberrant loss of DNA methylation in regenerated rice.

(A) Genome browser views of fractional CG methylation levels. Sample numbers correspond to those listed in Table 1. Regenerated samples of the same line are grouped together in red boxes. (B) Genome coverage of identified CG hypermethylation and hypomethylation DMRs. DMRs were defined relative to sample 1 (wild type). (C) Distribution of sizes of CG hypomethylation DMRs in regenerated plants. (D) Heat map representation of hierarchical clustering based on CG methylation levels within DMRs. Rows represent all 3610 CG-DMRs identified and columns represent the samples. (E) Overlap of CG-DMRs between samples. The bottom triangle represents the percent overlap of elements listed in the x-axis with those listed in the y-axis. The upper triangle on the other hand represents the percent overlap of elements listed in the y-axis with those listed in the x-axis.

https://doi.org/10.7554/eLife.00354.004
Figure 1—source data 1

List of CG, CHG, CHH DMRs identified in this study.

Defined hypomethylation DMRs for each sample are listed.

https://doi.org/10.7554/eLife.00354.005
Stability of loss of DNA methylation over generations.

Methylation status of sample 4 (T2) DMRs in T4 and T6 generations are indicated. Loss: less than half of respective wild-type CG methylation levels. Gain: more than half of respective wild-type CG methylation levels.

https://doi.org/10.7554/eLife.00354.006
Loss of DNA methylation occurs in all three cytosine contexts.

(A) Average distributions of DNA methylation in wild type (faded) and regenerated plants (solid) were plotted over defined CG hypomethylation DMRs in the indicated samples. Flanking regions are the same lengths as the middle region. (B) Heat map of DNA methylation levels within all defined hypomethylation DMRs (CG + CHG + CHH). (C) Average distribution of smRNA-seq reads in wild type (black) and regenerated plants (red) over defined CHH hypomethylation DMRs in indicated samples. Flanking regions are the same lengths as the middle region.

https://doi.org/10.7554/eLife.00354.007
Figure 4 with 4 supplements
Loss of DNA methylation at promoters may impact gene expression.

(A) Overlap of hypomethylation DMRs with indicated genomic elements. Observed overlap (dark bars) is compared to randomized regions of similar number and size distribution as the DMRs (light bars). Gene body: transcribed region of protein coding genes. Gene promoter: TSS to 2 kb upstream of TSS. 3' downstream of gene TTS (transcription termination site): TTS to 2 kb downstream of TTS. CDS: Coding sequence. TE: Transposable element. Error bars represent standard deviation. *Significant enrichment, p<0.01. (B) Percentages of genes with CG hypomethylation DMRs near TSSs that have significantly altered expression levels (fourfold up/down regulation, FDR<0.01). Genes with zero mRNA-seq reads in both wild type and regenerated samples were removed from the analyses. An average of 11.3 genes were deregulated. (C) Genome browser views of DNA methylation and gene expression levels.

https://doi.org/10.7554/eLife.00354.009
Figure 4—source data 1

List of genes with CG hypomethylation DMRs at promoters and their expression levels.

Genes with CG hypomethylation DMRs at the promoter regions (TSS minus 2 kb to TSS) in samples 4–10 along with their normalized expression levels are listed. Also indicated are whether they were significantly up- or down-regulated based on fourfold and FDR < 0.01 cutoffs. Descriptions of genes were directly taken from the rice genome annotation project website (http://rice.plantbiology.msu.edu/).

https://doi.org/10.7554/eLife.00354.010
Figure 4—figure supplement 1
Impact of loss of DNA methylation at promoters on gene expression.

Relative expression levels of genes with CG hypomethylation DMRs near TSS. Log2 ratios between RPKM values of indicated regenerated lines and wild type (sample 2) were calculated, and data is represented as boxplots. Genes with zero mRNA-seq reads in both wild type and regenerated samples were removed from the analyses. Red lines, median; edges of boxes, 25th (bottom) and 75th (top) percentiles; error bars, minimum and maximum points within 1.5 × IQR (Interquartile range); red dots, outliers.

https://doi.org/10.7554/eLife.00354.011
Figure 4—figure supplement 2
Genome browser views of DNA methylation and gene expression levels.
https://doi.org/10.7554/eLife.00354.012
Figure 4—figure supplement 3
Significantly up-regulated genes are largely different across different lines.

Defined significantly up-regulated genes with CG hypomethylation DMRs at promoters were categorized based on the number of lines (out of seven tested) in which they were up-regulated. Gene identifiers are listed below.

https://doi.org/10.7554/eLife.00354.013
Figure 4—figure supplement 4
DNA methylation levels over upregulated TE genes in regenerated samples.

Average distributions of DNA methylation in wild type (faded lines) and regenerated lines (solid lines) over defined up-regulated TE genes in the indicated regenerated samples.

https://doi.org/10.7554/eLife.00354.014
Tissue culture step induces loss of DNA methylation.

(A) Genome coverage of identified CG hypermethylation and hypomethylation DMRs. DMRs were defined relative to sample 18 (wild type). (B) Heat map of CG methylation levels within all 1074 CG hypomethylation DMRs identified in samples 13 to 17 (callus samples and wild-type plants regenerated from callus). (C) Heat map of CG methylation levels within 241 CG hypomethylation DMRs that were observed in all tested regenerated plants. (D) Boxplot representations of (C). Red lines, median; edges of boxes, 25th (bottom) and 75th (top) percentiles; error bars, minimum and maximum points within 1.5 × IQR (Interquartile range); red dots, outliers.

https://doi.org/10.7554/eLife.00354.016
Figure 6 with 1 supplement
Tissue culture-induced CHH hypermethylation is eliminated upon regeneration.

(A) Genome browser views of DNA methylation. (B) Genome coverage of identified CHH hypermethylation and hypomethylation DMRs. Regenerated samples of the same line are grouped together in red boxes. (C) Overlap of callus CHH hypermethylation DMRs with indicated genomic elements. Observed overlap (dark bars) is compared to randomized regions of a similar number and size distribution as the DMRs (light bars). Error bars represent standard deviation.

https://doi.org/10.7554/eLife.00354.017
Figure 6—figure supplement 1
Callus induced CHH hypermethylation.

(A) Average distribution of DNA methylation over defined CHH hypermethylated regions in callus, genes, and TE genes. Flanking regions are the same lengths as the middle region. (B) Overlap between defined CHH hypermethylation DMRs of the two callus samples in this study (13 and 14).

https://doi.org/10.7554/eLife.00354.018

Tables

Table 1

BS-Seq samples analyzed in this study

https://doi.org/10.7554/eLife.00354.003
SampleDescriptionRaw readsUniquely mapping readsCoverage (X)CG error rateCHG error rateCHH error rate
1WT200323156890210057278013.51780.01760.01220.0099
2WT200720354135710437698814.02920.01070.00870.0082
3WT20111878031098430190411.33090.01580.00950.0065
4T2-PiZt-11-R22965025911871009415.95570.01390.00990.0069
5T2-PiZt-11-S26332960213647141118.34290.01010.00960.0076
6T4-PiZt-11-R27067087113105670017.61510.01170.01000.0074
7T4-PiZt-11-S25215029812846772117.26720.00960.00760.0074
8T6-PiZt-11-R23728013712196674516.39340.01050.00960.0064
9T6-Pi9-R2047526998699574211.69300.01060.00930.0050
10T6-Spin1i-1-R2154510229046823612.15970.01130.00880.0061
11T2-PiZt-839-8-R (non functional PiZt)23873028111747133215.78920.01290.00790.0056
12T2-PiZt-839-8-S (non functional PiZt)21100611910617287214.27050.01780.01290.0095
13WT Callus 12171215229614527912.92280.01850.01780.0070
14WT Callus 21992614938261764311.10450.02320.02220.0084
15WT regenerated from tissue culture 121800883511636762615.64080.01700.01550.0078
16WT regenerated from tissue culture 22252021139790514213.15930.02620.02060.0093
17WT regenerated from tissue culture 325230642810654473514.32050.01940.01600.0073
18WT2011 (replicate)25397182711814006215.87900.01720.01480.0086
  1. Number of raw sequencing reads, number of uniquely mapping reads (post-removal of identical reads), genome coverage (rice genome size = 372 Mb), and error rates are listed. DNA methylation levels of the chloroplast genome were used to estimate error rates. Samples 1–12 and samples 13–18 were prepared separately. “R” and “S” correspond to plants that either contain the transgene (R) or plants in which the transgene was segregated away (S).

Table 2

smRNA-seq samples analyzed in this study

https://doi.org/10.7554/eLife.00354.008
SampleDescriptionRaw readsUniquely mapping reads
1WT2003220306633186666
2WT2007170694982598780
3WT2011148607672399713
4T2-PiZt-11-R220248813965317
5T2-PiZt-11-S176416233127938
6T4-PiZt-11-R189994153090933
7T4-PiZt-11-S221150744258752
8T6-PiZt-11-R129951932044615
9T6-Pi9-R167005243114923
10T6-Spin1i-1-R172758132973100
  1. Number of raw sequencing reads and number of uniquely mapping reads are listed.

Table 3

mRNA-seq samples analyzed in this study

https://doi.org/10.7554/eLife.00354.015
SampleDescriptionRaw readsUniquely mapping reads
2WT20074402908929461162
3WT20113399775522657098
4T2-PiZt-11-R4255013627839598
5T2-PiZt-11-S4317376428688381
6T4-PiZt-11-R4662489135826861
7T4-PiZt-11-S3172917322667633
8T6-PiZt-11-R4662453235335627
9T6-Pi9-R3897854130623633
10T6-Spin1i-1-R4228023532485204
  1. Number of raw sequencing reads and number of uniquely mapping reads are listed.

Data availability

The following data sets were generated
  1. 1

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