Variability of DSB repair in plant development and genome evolution, at molecular and chromosomal level

DNA双链断裂(DSB)是最复杂和最危险的DNA损伤。因此，我们进化出了几种对同源模板序列要求不同、结果精度不同的修复途径。在正在进行的项目中，我们找到了一种策略，通过细胞遗传学和从转基因靶序列中进行扩增序列测序相结合的方法来追踪大麦的所有DSB修复途径。令人惊讶的是，大多数DSB是在细胞周期的S/G2期以姐妹染色单体为模板通过互换修复的。此外，还首次在大麦中实现了DSB诱导的基因打靶。基于这些结果，我们建立了一种在目标位置同时监测NHEJ变异和HR的方法，这些变异使用不同长度的微同源性，导致恢复或转换、缺失、插入或组合突变类型。应用一种新的测序技术(PacBio)，我们现在将比较A.thaliana和大麦中相同的报告基因，以找出这两个物种在不同发育阶段修复能力的潜在差异。同时，我们将测试大基因组和小基因组之间可能存在的差异，这些差异可能通过倾向于小基因组中更多和/或更长的缺失来驱动基因组大小的进化。此外，我们打算将获得的转基因报告基因的数据与CRISPR-Cas9 RNA引导的核酸酶在拟南芥内源序列上模拟报告基因的序列排列产生的DSB进行比较(用于优先检测SSA或SDSA)。CRISPR-Cas9核酸酶诱导的DSB和CRISPR-Cas镍酶诱导的SSB对大麦修复途径光谱的影响将在同一报告基因的分子(测序)和染色体水平(染色体畸变率)和大麦内源重复序列(SCES)的水平上进行检测。此外，还将研究纯合子和半合子基因座上的DSB对不对称染色体易位起源的影响，以及这些重排在复制之前或之后发生的比例。

DNA double-strand breaks (DSBs) represent the most complex and hazardous DNA lesion. Consequently, several repair pathways with different requests on homologous template sequences, and different precision of outcome, have evolved.During the ongoing project, we found a strategy to trace all DSB repair pathways in barley by combination of cytogenetics and amplicon sequencing from transgenic target sequences. Surprisingly, the majority of DSBs were repaired during S/G2 phase of cell cycle via reciprocal exchange using the sister chromatid as a template. In addition, efficient DSB-induced gene targeting was for the first time achieved for barley. Based on these results, we established an approach to monitor at the target site simultaneously the variants of NHEJ which use various lengths of microhomology, as well as HR, resulting either in restitution or in conversion, deletion, insertion, or combined mutation types. Applying a novel sequencing technology (Pacbio), we will now compare the same reporter genes in A. thaliana and in barley to find out potential differences in repair capacity during different developmental stages in both species.At the same time we will test for possible differences between large and small genomes that might drive genome size evolution, via a bias towards more and/or longer deletions in small genomes. Furthermore, we intend to compare the data obtained for transgenic reporter genes with DSBs generated by CRISPR-Cas9 RNA-guided nucleases on Arabidopsis endogenous sequences mimicking the sequence arrangement of reporter genes (for preferential detection of either SSA or SDSA). The effect of CRISPR-Cas9 nuclease-induced DSBs and CRISPR-Cas nickase-induced SSBs on the spectrum of repair pathways will be tested at the molecular (sequencing) and the chromosomal level (SCE, chromosome aberrations) at the same reporter genes and on repetitive endogenous sequences in barley (SCEs). Additionally, the effect of DSBs at homozygous versus hemizygous loci on the origination of asymmetric chromosomal translocations will be investigated as well as the proportion at which these rearrangements occur before or after replication.