Department of Plant and Environmental Sciences, The Weizmann Institute of Science, Rehovot 7610001, Israel.
Genes (Basel). 2020 Dec 31;12(1):59. doi: 10.3390/genes12010059.
Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F hybrids from divergent parental lines, each with a different mutation at the () locus. IHSR events, namely crossover or gene conversion (GC), between the two mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F seeds were grown to test for germinal transmission of HR events. Two out of five F plants showing red sectors had their IHSR events germinally transmitted to F, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.
体细胞同源重组(HR)不如减数分裂重组那样被充分理解,并且被认为是罕见的。在之前的一项研究中,我们表明通过靶向诱导 DNA 双链断裂(DSB)可以实现同源间体细胞重组(IHSR)。在这里,我们设计了一种新的 IHSR 测定法来更深入地研究这种现象。我们利用来自不同亲本系的 F 杂种,每个系在 () 基因座上都有一个不同的突变。两个 突变等位基因(橘黄色)之间的 IHSR 事件,即交叉或基因转换(GC),可以恢复基因活性,并表现为功能获得型、野生型(红色)表型。我们的结果表明,在测试的四个内含子 DSB 靶标中,有三个靶标显示出 DSB 形成,如非同源末端连接(NHEJ)足迹所示,但只有一个靶标产生了潜在的 IHSR 事件,如橘黄色果实上的红色扇区所示。F 种子被种植以测试 HR 事件的生殖传播。在表现出红色扇区的五个 F 植物中,有两个将其 IHSR 事件生殖传递给了 F,主要是基因转换。六个独立的重组等位基因被表征:三个具有截断的转换片段,平均长度约为 1 kb。两个等位基因是通过交叉形成的,这是通过基因分型确定的,并通过全基因组测序进行了表征。我们讨论了 IHSR 如何用于未来的研究以及新型基因编辑和精确育种工具的开发。