Effects of hybridization on expression of apomixis in the Ranunculus auricomus complex

In flowering plants, hybridization and polyploidy are important evolutionary processes and are often connected to changes in the reproductive system. Nevertheless, it is still not well understood how these processes trigger the alterations of reproductive pathways. The main aim of the project is to study effects of interspecific hybridization and polyploidy on embryo sac development and embryogenesis, and components of asexual reproduction via seed (apomixis) in the Ranunculus auricomus complex, a natural apomictic polyploid complex. Alterations of reproductive phenotypes and gene expression will be studied in natural allopolyploid apomicts and their sexual parental species; experimentally produced homoploid and polyploid hybrids of related sexual species will be tested with respect to alterations in the developmental pathways and possible steps towards apomictic reproduction. We further want to test the hypothesis that hybridization alters the timing of gene expression regulating reproductive pathways, which may lead to apomictic phenotypes. We will first conduct a screening of the mode of reproduction via flow cytometric seed screening, cytological and embryological microscopic investigations, and molecular progeny tests (microsatellites) to sort out candidate individuals for expression profiling. On selected individuals, sequencing of the whole transcriptome will be conducted, and microarrays will be developed for a gene expression profiling in specific reproductive tissues. This approach will allow for a direct comparison for timing and allelic variation of gene expression in sexual species and their hybrid derivates with natural apomictic plants. Results will be informative about changes in phenotype and gene expression in primary and more derived, apomictic hybrids. We expect to get insights into the constraints towards establishment of apomixis in flowering plants, which is of high relevance for both evolutionary questions and for a potential application of the apomixis technology in crops.

In flowering plants, hybridization and duplications of the genome (polyploidy) are important evolutionary processes and are often connected to changes in the reproductive system. Asexual reproduction via seed (apomixis) is a major evolutionary trait connected to polyploidy. The main aim of the project was to test the hypothesis that hybridization is a functional trigger for apomictic development. We performed crossing experiments and studied effects of interspecific hybridization and polyploidy on embryo sac development and embryogenesis, and components of apomixis in the Ranunculus auricomus complex, a natural apomictic polyploid model system. The hybrids showed components of apomixis, ie. development of somatic cells into embryo sacs, already in the first generation. Phylogenetic studies using DNA sequencing and geometric morphometrics corroborated hybrid origin of apomictic lineages. Comparative RNA sequencing studies using the Illumina technology and analysis of single nucleotide polymorphism showed that natural apomictic Ranunculus lineages originated via hybridization c. 70,000 years ago. The present lineages do not show significant genome-wide accumulation of non-synonymous substitutions, but rather a rapid divergence from each other. Thus, we disprove older hypotheses that apomixis is a dead end of evolution because of accumulation of deleterious mutations. Instead, we developed a novel theory that apomixis might be a transitional stage in the evolution of polyploid complexes and can serve as a springboard for diversification and geographical range expansion. To test this hypothesis in a broader scale, an online database was installed documenting, occurrence of apomixis in all angiosperms. Statistical evaluation of data for all angiosperms corroborated an association of apomixis to diverse and widespread plant families.We further studied genetic control mechanisms of apomixis. With the next generation sequencing data, we have developed a model system for apomixis research with >1200 annotated genes. Genes under divergent selection between sexual and apomictic taxa relate to development, meiosis and gametogenesis. Gene expression analysis of ovules on four developmental stages using microarrays revealed each over 200 genes differentially regulated in apomicts and sexuals, respectively. We conclude that apomixis is mainly due to differential expression of a whole set of genes and thus a flexible reproductive strategy. Population genetic studies and flow cytometric analyses corroborate a considerable level of facultative sexuality and genetic diversity within apomictic lineages of R. auricomus and also of other Ranunculus species. Results helped to develop a novel hypothesis that expression of sexuality is responsive to environmentally induced oxidative stress.