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Greiner, Stephan (2008): Oenothera, a unique model to study the role of plastids in speciation. Dissertation, LMU München: Fakultät für Biologie



The subject of this thesis was to develop molecular approaches appropriate to investigate speciation processes. The genus Oenothera was chosen for study, since it offers the unique possibility to exchange plastids, individual or more chromosomes and/or even entire haploid genomes (so-called Renner complexes) between species. In addition, a rich stock of information in taxonomy, cytogenetics and formal genetics is available, collected for more than a century of research. Interspecific exchange of plastids, nuclear genomes or chromosomes often leads to mis-development of the resulting hybrids. These inviable hybrids form hybridization barriers responsible for speciation. In the case of plastid and nuclear genome exchange, hybrid bleaching is frequently observed, which results from plastome-genome incompatibility (PGI) due to compartmental co-evolution. Traditional work on Oenothera was almost exclusively restricted to classical genetic and cytogenetic approaches. Subsection Oenothera, the best studied of the five subsections in the section Oenothera, was used in this work. It is comprised of three basic nuclear genomes, A, B and C, which occur in homozygous (AA, BB, CC) or stable heterozygous (AB, AC, BC) combination. In nature, the nuclear genomes are associated with five basic, genetically discernible plastid types (I - V) in distinct combinations. The following results were obtained: (i) Biochemistry with Oenothera is not trivial due to exceedingly high amounts of mucilage and tannins which adversely interfere with the isolation of macromolecules and enzymatic reactions. A basic biochemistry for the material was therefore developed initially, notably to obtain appropriate subcellular fractions, restricable, amplifyable and clonable DNA, RNA, supramolecular protein assemblies and proteins of appropriate purity. (ii) Evaluation of the PGI literature clearly indicates that PGI can form hybridization barriers according to the Dobzhansky-Muller gene pair model of speciation, even if the genes reside in different cellular compartments. (iii) Oenothera PGIs could be classified into four genetically distinct categories, which influence hybridization barriers in different ways. (iv) Co-dominant marker systems (SSLP and CAPS) were generated for both, nuclear genome and plastome. Their potential was successfully evaluated with crossing programs designed to exchange plastomes, genomes, or individual chromosomes between species. (v) The plastome markers allowed to genotype 41 subplastomes to judge inter- and intraplastome diversity and displayed molecular loci linked to the genetic behaviour of basic plastome types I - V. (vi) A single, highly polymorphic marker (M40) was sufficient to genotype 29 different Renner complexes of the basic genome types A, B and C. (vii) Markers specific for all seven Oenothera chromosomes were selected. Combined with the genetics of a partial permanent translocation heterozygote (ring of 12 chromosomes plus 1 bivalent, which behave as two distinct linkage groups) they allowed the assignment of molecular linkage group 7 to chromosome 9•8 of the classical Oenothera map. Material for the assignment of the remaining chromosomes and their arms was produced or selected so that both map types can now be fully integrated. (viii) In parallel to work on the nuclear genome, the sequences of the five basic Oenothera plastomes were completed (in cooperation). Elaborated in this thesis, due to its limited coding potential, conserved nature, and substantial knowledge about photosynthesis, plastid chromosomes provide relatively easy access to “speciation genes” and selection pressures causing speciation. (ix) Phylogenetic analysis of the sequences provided a plastome pedigree, and also an idea about the age of the subsection, i.e. back to the middle of Pleistocene, approximately 1 mya ago. This contributed to solve a long lasting question in the Oenothera literature. (x) Application of appropriate algorithms uncovered for the first time that plastomes are subject to natural selection and hence contribute to speciation. This was questioned repeatedly. (xi) A novel weighting strategy, combining classical genetic data on plastome-genome compatibility/incompatibility with molecular data and bioinformatic approaches, was applied to deduce potential plastid determinants for PGI. (xii) In a case study it could be shown that a single plastid locus contributes substantially to PGI in the interspecific hybrid AB-I, which was found to be defective in photosystem II. A plastome I-specific deletion in the bidirectional promoter region between psbB and clpP was found to be responsible for the phenotype observed. The finding is consistent with reduced levels of psbB mRNA and its product CP47 chlorophyll a apoprotein of photosystem II, with spectroscopic data and phenotype. (xiii) Available data indicate that interspecific plastome-genome hybrids represent some sort of “network mutants”. This would imply that speciation is predominantly a regulatory phenomenon. In the studied cases PGIs are is involved in the fine-tuning of regulation of photosynthesis, rather than in an adaptation of its structural components. This is considered as a major finding of this thesis.