sRNA drives epigenetic changes in F1 hybrids

Hybrid Arabidopsis plants undergo epigenetic reprogramming producing decreased levels of 24nt siRNAs and altered patterns of DNA methylation that can affect gene expression. Driving the changes in methylation are the processes Trans Chromosomal Methylation (TCM) and Trans Chromosomal deMethylation (TCdM). In TCM/TCdM the methylation state of one allele is altered to resemble the other allele. These changes in DNA methylation and the associated changes in small RNA levels in the F1 hybrid can be maintained in subsequent generations and affect hundreds of regions in the F2 epigenome. The inheritance of these altered epigenetic states varies in F2 individuals resulting in individuals with genetically identical loci displaying different epigenetic states and gene expression profiles. At these regions the change in methylation is associated with the presence of small RNAs. Loci without any sRNA activity can have altered methylation states, suggesting that a sRNA-independent mechanism may also contribute to the altered methylation state of the F1 and F2 generations. Overall design: Arabidopsis thaliana accessions C24 and Landsberg erecta (Ler,) were used as parental lines. Reciprocal C24 x Ler, hybrids were generated by hand pollination. For all libraries immature inflorescence was used. sRNA and mRNA libraries were prepared from total RNA extracted using the Direct-zol RNA mini-prep kit. RNA was extracted from two biological replicates of pooled C24 and Landsberg erecta material. For F1 hybrids the RNA was extracted from three individual F1 plants for each reciprocal hybrids. These individual hybrid plants have been designated F1-A, F1-B, F1-C, F1-D, F1-E, F1-F. RNA was also extracted from 6 individual F2 plants designated F2-A, F2-B, F2-C, F2-D, F2-E, F2-F. One of these F2 individuals has been derived from one of the F1 individuals (i.e. F2-A is derived from F1-A). Methyl-seq libraries were prepared as described in Lister et al, 2008. 6 methyl-seq libraries were prepared from the same F2 samples used in the RNA-seq analyses (F2-A, F2-B, F2-C, F2-D, F2-E, F2-F).