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Identification and analysis of candidate genes involved in key steps of gametophytic apomixis

Final Activity Report Summary - IDEAGENA (Identification and analysis of candidate genes involved in key steps of gametophytic apomixis)

Reproduction is a crucial step in the life cycle of all species. In plants both sexual and asexual reproduction through seeds (apomixis) are common. Apomixis is a fascinating developmental process with a great potential for plant breeding and seed production. Key events during female sexual reproduction are: megasporogenesis involving meiosis of a megaspore mother cell (MMC) to give rise to a functional megaspore, megagametogenesis being the formation of the mature female gametophyte harboring the two gametes (the egg cell and the central cell), and double fertilisation during which female and male gametes fuse. At least three key developmental processes are altered in apomictic reproduction: meiotic reduction is omitted or altered (apomeiosis); embryogenesis is initiated autonomously (parthenogenesis); and functional endosperm is formed through various developmental adaptations (1). The genetic basis and molecular mechanism of (a)sexual reproduction are so far poorly understood, mainly because of the inaccessibility of the gametopythic cells within the ovule.

Recently, the transcriptome of the individual cells of the mature gametophyte from the sexual species Arabidopsis has been described using a microgenomics approach (2). This involves isolation of individual cells by Laser-Assisted Microdissection (LAM), RNA isolation and amplification, and GeneCHIP expression profiling. The objective of this project was to use this powerful microgenomics approach in combination with heterologous GeneCHIP hybridisations (Boechera aRNA on Arabidopsis arrays) to define the transcriptomes of cells important in key steps of apomictic reproduction, the apomictic initial cell and the egg cell of Boechera gunnnisoniana, a close relative of Arabidopsis.

To this aim the tissue embedding, the quality of the isolated RNA, and the amplification have been optimised for the cell types of interest in Boechera. Sufficient amounts of apomictic initial cells (app.4300) and gametophytic cells (app.1200) to perform transcriptome analyses have been isolated. Heterologous transcriptome analysis has been normalised using genomic Boechera DNA. Two and one replicate of GeneCHIP array hybridisations have been performed with Boechera initial cell aRNA and egg cell aRNA, respectively. The dataset will be complemented with deep sequencing using the SolidTM 4 platform (ABI) for the cell types of interest, which are already isolated.

Analysis of the transcriptome data is ongoing and identified several thousands of genes expressed in each of the cell types analysed. In addition, comparison with the expression profiles of the respective cell types in Arabidopsis indicates a large number of genes (up to app.2000) to be differentially expressed per cell type. Higher-level analysis of the data is ongoing to identify gene ontologies and pathways that are differentially represented during sexual and apomictic reproduction. In addition, the investigation of selected genes of interest for their roles in reproductive development has been initiated. The transcriptome analysis will be finalised within the next months and follow up experiments will be performed for data confirmation. The data provide important new insights in the genetic basis underlying (a)sexual reproduction.