Résumé
The triploid block is a major reproductive barrier that prevents the production of viableprogeny in crosses between parents of di↵ering ploidy. In Arabidopsis thaliana, triploid seedlethality is often observed in interploidy hybridizations between diploid females and tetraploidmales, which has been largely associated to the increased dosage of paternally-expressed im-printed genes (PEGs) in the endosperm. Interestingly, the paternal epigenome is also impli-cated in this process, in a way that paternal mutations in DNA methylation pathways are ableto rescue triploid seed viability. In order to identify epialleles associated to the triploid blockresponse, we have generated a population of A. thaliana plants segregating epigenetic variationinduced by somatic embryogenesis (SE). This strategy allowed the identification of a particularlystrong suppressor line where DNA hypomethylation localized to intergenic regions and trans-posable elements flanking many imprinted genes. Importantly, these di↵erentially methylatedregions (DMRs) overlapped significantly with the target sites of the DNA glycosylases DME andROS1, suggesting that triploid seed viability in SE clones is associated to the hypomethylatedstatus of these particular DMRs. To validate this hypothesis, we performed ectopic expressionof DME and ROS1 in the male germline where they are normally not expressed, and found astrong decrease in DNA methylation and triploid seed collapse when ROS1 was expressed atthe microspore stage of pollen development. These results demonstrate that DNA methylationat ROS1 targets is required for the triploid block in A. thaliana, thus opening new perspectivesfor using targeted germline epimutagenesis as a tool to facilitate the production of triploids andinterspecific hybrids with high agronomical interest.