Assessing the effects of DNA repair and homologous recombination pathways on genome integrety in plants

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As sessile organisms, plants cannot easily escape external, mutagenic risks threatening the integrity of their genomes. As the plant germline is not predetermined, acquired mutations can even be transmitted to following generations. Therefore, plants have evolved a multitude of DNA repair mechanisms for safeguarding genome stability (SGS). To analyse the individual functions of these factors on transgenerational genome integrity, we established a unique collection of mutation accumulation (MA) lines in different SGS mutant backgrounds of Arabidopsis thaliana. This includes independently grown, self-pollinated and single-seed descendent lines of 16 single and 10 double mutants that have been propagated up to generation F18. By performing whole-genome sequencing (WGS), we can specify the type and frequency of genomic changes, accumulating due to the absence of specific SGS pathways. As we expect that these changes are not only small and local, but complex rearrangements, we will not perform short read-based WGS, but long read based assembly of each of these genomes. The data will be confirmed by additional short read sequencing, which will also give us insights into sequence-based copy number estimates of repetitive elements like the 45S rDNA. As the 45S rDNA is essential for plant survival but prone to rearrangements and copy loss, we will complement the genomic data with ddPCR and FISH, for directly measuring repeat number. Moreover, we will elucidate the transcriptional response towards the genotoxic stress caused by the absence of SGS factors in the different lines. The analysis of RNAseq data will highlight the differential expression of the DNA damage response factors and unveil if transgenerational adaptation processes develop as previously proposed. In addition to changes in the genomes and transcriptomes, we expect that the accumulation of severe DNA damage in the mutant lines could also lead to defects in plant growth. We aim to analyse phenotypic effects and whether they increase over the course of multiple generations. A characterization of root growth as an indicator for replicative DNA damage will give us first insights into possible growth defects. Additionally, the effects on transgenerational fertility will be analysed in the MA lines both on the silique and seed count level as well as with a detailed cytological analysis of chromosomal aberrations. The analysis of this unique MA collection on all these levels will give us an unprecedented look into the mechanisms for SGS in plants, how different pathways ensure transgenerational genome stability and how plant genomes change in a time lapse.