IC4R010-Epigenomic-2013-23539454

Project Title

• Plants regenerated from tissue culture contain stable epigenome changes in rice

The Background of This Project

• The rice genome is DNA methylated in all three cytosine contexts (CG, CHG, CHH, where H=A, T,or C), with high levels of CG and CHG methylation and very low levels of CHH methylation (Feng et al., 2010; Zemach et al., 2010). Whole genome bisulfite sequencing (BS-seq) enables measurement of DNA methylation at single nucleotide resolution and thus allows one to distinguish DNA methylation in different cytosine contexts (Cokus et al., 2008; Lister et al., 2008).

Plant Culture & Treatment

• Wild-type rice (Oryza sativa ssp japonica cv Nipponbare) and regenerated rice lines (in Nipponbare background) were used in this study (Zhou et al., 2006; Vega-Sanchez et al., 2008). Hygromycin wasused as the selection marker in rice transformation. All the resistant plants were selfed for indicated generations (Table 1). Homozygosity was confirmed by PCR analysis of the transgene. Rice seeds were surface-sterilized and transferred to 1/2 MS medium. After germination, rice seedlings were transplanted into soil and kept in a growth chamber at 26/20°C under a 14-hr light/10-hr dark cycle. The rice plants regenerated from untransformed rice callus induced from Nipponbare seeds (WT2011)were prepared as previously described (Zhou et al., 2006; Vega-Sanchez et al., 2008). Rice leaf samples were collected at 3 weeks after transplanted into soil and the rice callus were harvested from the callus inducing media.

Research Findings

• The researchers performed deep BS-seq to map DNA methylation in nine regenerated rice lines in the Nipponbare ecotype background that were transformed by various transgenes and were at various stages of inbreeding after transformation: rice blast resistance lines PiZ-t, PiZ-t-839 (a non-functional PiZ-t), Pi9, and an RNAi line for flowering time regulator Spin1 (Zhou et al., 2006; Vega-Sanchez et al., 2008; Table 1). For the PiZ-t line, both transgenic and non-transgenic T2 and T4 plants were available by genetic segregation of the PiZ-t transgene (Table 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).'

• The researchers observed strong losses of DNA methylation at certain sites in the genome in the regenerated plants but not in wild-type plants (Figure 1A). To further characterize these sites, we defined differentially methylated regions (DMRs) in CG contexts by applying stringent thresholds (see ‘Materials and methods’). We found that all regenerated plants tested were significantly enriched with CG hypomethylation DMRs (Figure 1B). On average, we identified 1344 CG hypomethylation DMRs in the regenerated plants, whose sizes ranged from 100 to 3200 bp (Figure 1C), whereas on average we identified only eight CG hypomethylation DMRs in the inbred wild-type lines (Figure 1—source data 1).Importantly, we observed hypomethylation even in the T2/T4 non-transgenic plants in which the transgenes had been segregated away (samples 5, 7 and 12), suggesting that loss of DNA methylation is likely due to the tissue culture or transformation process, but not due to the fact that the plants contain transgenes. While loss of DNA methylation in different regenerated lines did not always occur at the same sites (Figure 1D), there were significant overlaps of hypomethylation DMRs among regenerated lines (Figure 1E). This suggests that certain sites in the genome are susceptible to loss of DNA methylation in regenerated plants.

'Figure 1. 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 Figure 1. Continued on next page'

• The researchers next investigated the stability of DNA methylation losses across generations. To test this, the researchers analyzed a line for which we had plants in T2, T4, and T6 generations (samples 4, 6, 8). 84% of sites that lost CG methylation in the T2 did not recover methylation in the T4 and T6 generations (Figure 2).

'Figure 2. 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.

Labs working on this Project

• Department of Molecular, Cell and Developmental Biology, University of California,Los Angeles, Los Angeles, United States;
• Department of Plant Pathology, Ohio State University, Columbus, United States;
• Department of Plant and Soil Sciences,Delaware Biotechnology Institute, University of Delaware, Newark, United States;
• Howard Hughes Medical Institute, University of California, Los Angeles,Los Angeles, United States.

Corresponding Author

• Steven E Jacobsen:jacobsen@ucla.edu