The role of small RNAs in genomic imprinting and hybrid barriers

In this project we study the role of genomic imprinting and how this epigenetic phenomenon is regulated and plays a biological role in crosses within- and between species, that is in hybrids. We have established the mechanistic mode of imprinting for more than 50 loci in the model plant Thale cress (A.thaliana). The imprinting patterns of most genes were not affected by these mechanisms. To this end, we demonstrated that the RNA-dependent epigenetic mechanisms not substantially influence imprinting patterns. In order to follow up these findings we have used GFP-reporterlines and cell-sorting technology to isolate the expression of genes that could be uniquely expressed in specific parts of the seed. Here we have been able to identify more than 300 imprinted genes, some of which also have an unique expression patterns in the seed endosperm. These findings will be used to generate signature profiles for the different domains of the seed, making it possible to sort single-nuclei by their unique expression profiles. By using hybrid seeds from crosses between different ecotype populations of A. arenosa, we have also generated expression profiles that are used to study genomic imprinting in this species. Furthermore, we have successfully generated transgenic plants expressing GFP in the endosperm in the species A.arenosa, a close relative to A.thaliana. These plant-lines will facilitate analysis of endosperm of hybrid A.thaliana x A.arenosa seeds by cell-sorting technology. Having described genomic imprinting in both A. thaliana and A. arenosa we can compare imprinting in closely related species, but importantly also analyze how hybridization between species affect genomic imprinting.

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Fertilization in plants generates the seed, a structure with two fertilization products, the embryo, carrying the genetic makeup of the next generation, and the endosperm, a nourishing tissue. In analogy to the mammalian placenta, the endosperm is a site for the epigenetic phenomenon genomic imprinting; parent-of-origin dependent expression of genes due to epigenetic regulatory marks established in the male and female germ line. Genomic imprinting is a highly captivating example of gene regulation. The underlying epigenetic mechanisms must be able to distinguish two identical alleles and reside in the same nucleus. Imprinted expression results from differences in epigenetic marks, and the major mechanisms include maintenance DNA methylation and post-translational modifications of histones on the parental alleles. However, by far all imprinted genes can be explained by these mechanisms, and a novel type of DNA methylation guided by small, single stranded RNA emerge as a mechanistic alternative. A related and insufficiently investigated role of small RNAs is speciation. When different species interact in fertilization, hybrid seeds are generated, that usually abort due to post-zygotic barriers, traditionally explained by mis-regulation of imprinted genes. In the proposed research, small RNAs that guide epigenetic modification are hypothesized to play significant roles in imprinted gene regulation and further to act through imprinted genes or affecting other seed regulatory cascades to erect post-zygotic species barriers. To test these hypotheses we propose to use a combination of genetic dissection, transcriptomics and epigenomics. We will identify imprinted genes regulated by small RNA in the genus Arabidopsis, and further test the hypothesis that imprinted genes are evolutionary conserved between related species. Finally, we propose to experimentally demonstrate that small RNAs are involved in the establishment of post-zygotic hybrid barriers and thus speciation.