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

作为固着生物，植物不能轻易逃脱外部的诱变风险，威胁其基因组的完整性。由于植物生殖系不是预先确定的，获得的突变甚至可以传递给后代。因此，植物已经进化出许多DNA修复机制来保护基因组稳定性（SGS）。为了分析这些因素对跨代基因组完整性的单独作用，我们在拟南芥不同的SGS突变体背景中建立了一个独特的突变积累（MA）系集合。这包括独立生长，自花授粉和单种子后代线的16个单一和10个双突变体已繁殖到F18代。通过进行全基因组测序（WGS），我们可以指定由于缺乏特定SGS途径而积累的基因组变化的类型和频率。由于我们预期这些变化不仅是小的和局部的，而且是复杂的重排，因此我们将不进行基于短读段的WGS，而是对这些基因组中的每一个进行基于长读段的组装。这些数据将通过额外的短读测序来确认，这也将使我们深入了解重复元件（如45S rDNA）的基于序列的拷贝数估计。由于45S rDNA对植物生存至关重要，但容易重排和拷贝丢失，我们将用ddPCR和FISH补充基因组数据，以直接测量重复数。此外，我们将阐明对遗传毒性胁迫所造成的SGS因子在不同的线的情况下的转录反应。RNAseq数据的分析将突出DNA损伤反应因子的差异表达，并揭示跨代适应过程是否如先前提出的那样发展。除了基因组和转录组的变化外，我们预计突变株系中严重DNA损伤的积累也可能导致植物生长缺陷。我们的目标是分析表型效应以及它们是否在多代过程中增加。根生长的表征作为复制DNA损伤的指标将使我们第一次洞察到可能的生长缺陷。此外，将在MA系中分析对跨代生育力的影响，包括角果和种子计数水平以及染色体畸变的详细细胞学分析。在所有这些层面上对这个独特的MA集合的分析将使我们前所未有地了解植物SGS的机制，不同的途径如何确保跨代基因组稳定性以及植物基因组如何在一段时间内发生变化。

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.