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Abstract

Apomixis refers specifically to asexual reproduction through seed in plants. Like other modes of asexual reproduction it has received much attention from evolutionary biologists and has been subject of many studies throughout the last decades. At the same time, it attracts interest from an economic point of view, as the long-term goal to technologically induce apomixis in major crop plants offers the prospect of a potential agricultural revolution. Hence, interests have been growing in the scientific community in order to elucidate the evolution and underlying molecular genetic mechanisms of apomixis. Here I present a multifaceted approach to the problem by (1) the development of biotechnological tools in order to (2) apply molecular evolutionary methods to narrow down the possible causes and consequences of asexual reproduction in plants. In this work, representatives of two genera (Ranunculus L. and Boechera Á. LÖVE & D. LÖVE) were studied in order to advance current apomixis knowledge from different perspectives. In the framework of a microarray based transcriptomic analysis of ovules extracted from sexual and apomictic Boechera, a list of housekeeing genes (HKGs) was selected based on a stability of expression analysis subsequently conducted in different vegetative and reproductive tissues of apomictic and sexual species. Using a GeNorm algorithm, different combinations of HKGs were identified, including Ribosomal Subunit 18 (BoechRPS18), Elongation Factor Alpha 1 (BoechEfα1), Actine 2 (BoechACT2) and Ubiquitine (BoechUBQ), based on their pairwise stability in ovules, anthers, and vegetative tissue in apomictic and sexual species. These genes, specifically chosen to be reproduction mode- and tissue-specific, have subsequently been used for normalization in the experimental validation of two candidates genes related to apomixis in Boechera Next, molecular evolutionary causes and consequences of apomixis were investigated by analyzing the transcriptomic effects of asexual reproduction and its correlated traits (i.e. hybridization, polyploidy and mutation accumulation). Flower-specific RNA from sexual and apomictic species of the wild apomictic Ranunculus auricomus complex was used for high throughput transcriptome sequencing (RNAseq). The first de novo assembled transcriptome for these species was used as a reference sequence for mining Single Nucleotide Polymorphism (SNP) and insertion-deletion xii (indel) polymorphisms. The data were further used to design and manufacture a custom 3 x 1.4 million spot expression microarray. Comparative SNP analysis between apomictic and sexual individuals (specifically, two apomicts from two populations and three sexuals from three populations) corroborated the hybrid origin of apomictic Ranunculus, as proposed by Paun et al. (2006b), and could furthermore elucidate the Pleistocene origin and subsequent divergence of the apomictic individuals. In addition, sites of divergent selection were detected with the analysis of non-synonymous (dN) to synonymous (dS) substitution rates, strengthening the idea of rapid divergence in the hybrids. Finally, the custom microarray was used for the hybridization of RNA from live-microdissected ovules (four developmental stages) from the three apomictic and four sexual individuals used in the SNP analysis. The comparative stage specific transcriptome analysis was used to detect stage specific differentially expressed genes in ovules, in order to identify signatures of apomixis and to produce a list of potential candidates underlying the reproductive switch. 555 stage specific genes were found to be differentially expressed throughout ovule development, and eight genes showed a significant shift in expression pattern throughout ovule development in apomicts compared to sexuals. A further classification was conducted following the predictions made from Nogler’s extensive work in Ranunculus in which different genetic factors were proposed for the induction and penetrance of apomixis. In that light, differentially expressed homoeologous genes were classified into three categories based on their relative expression in apomicts compared to their phylogenetic sexual parent, with the final aim of classifying the number of genes potentially responsible for apomixis. In doing so, we have provided a solid base for future studies in wild (i.e with little or no genetic information available) Ranunculus species. By developing biotechnical tools for their study, identifying genes potentially involved in the establishment of apomixis, and analyzing their evolutionary history, this work presents an important step towards a more comprehensive understanding of the processes and patterns connected to apomixis in model and non-model plants, and has far-reaching potential for agricultural use.