MEIOREC_Meiotic Recombination in Plants: controlling the transition of DNA double-strand breaks to genetic crossovers

遗传变异是在减数分裂过程中通过同源重组产生的，是植物育种努力的基础，以实现确保粮食安全所需的作物快速改良。HR是由SPO11复合体形成DNA双链断裂(DSB)启动的。DSB由HR途径的组成部分处理，在那里它们被修复为交叉(CoS)或非交叉(NCO)，其中仅交换较短的DNA片段。在工厂中，大多数DSB被修复为NCO。此外，CoS的分布，特别是在谷类作物中，局限于特定的染色体区域。这些限制大大减少了在每个减数分裂过程中可能产生的遗传变异。MEIOREC调查人员的广泛研究在理解这些限制的基础以及如何解决这些限制方面取得了重大进展。然而，对控制特定基因组座位上的DSB向CO转变的因素以及如何优化这一转变的充分了解尚未阐明。我们的目标是破译DSB到CO转化过程中涉及的分子步骤，并使用模式物种评估操纵CO形成的策略，从而为随后将最有希望的作物转化为作物奠定基础。我们的研究将集中在控制植物中CO形成的三个不同阶段：(I)控制DSB形成的因素：我们将确定它们的最低要求。我们将评估MTOPVIB-CRISPR系统作为将DSB复合物靶向复合寒冷地区的基础的潜力。我们将研究使用抑制盒(DCas9)和CO抑制基因的多个短引导RNA来提高CO频率。(Ii)DSB加工、稳定的联合分子形成和CO分解：我们将通过分析MRN复合体和COM1在有效地从DSB末端移除SPO11方面的作用，来研究DSB加工和重组酶装载的早期步骤是如何联系在一起的，以及这如何影响CO/非CO的决定。我们还将验证这样的假设，即重组反应中CO的形成效率可以受到Cas9介导的MUS81/Gen1解旋酶靶向的影响。(3)染色体重塑与CO形成的关系：我们将分析染色体轴的翻译后修饰对CO形成和分布的影响。文中还将分析细线期/合线期染色体轴的广泛重塑和联会复合体与CO指定的重组中间体成熟之间的相互关系。在大麦和玉米中，染色体轴的程序性重塑和CO分布之间的联系将被进一步研究。

Genetic variation is generated through homologous recombination during meiosis and underpins plant breeding efforts to deliver the rapid improvements in crops that will be required to ensure Food Security. HR is initiated by the formation of DNA double-strand breaks (DSBs) by the SPO11 complex. DSBs are processed by components of the HR pathway where they are repaired as crossovers (COs), which recombine the homologous parental chromosomes, or non-crossovers (NCOs), where only short stretches of DNA are exchanged. In plants most DSBs are repaired as NCOs. Moreover, the distribution of COs, notably in cereal crops, is localized to particular chromosomal regions. These limitations significantly lessen the genetic variation that can be generated in each meiotic division. Extensive studies by the MEIOREC investigators have led to significant progress in understanding the basis of these limitations and how they may be addressed. Nevertheless, a full understanding of the factors that control the transition of a DSB at a particular genomic locus to a CO and how this can be optimized has not yet been elucidated. We aim to decipher the molecular steps involved in the DSB to CO transition and evaluate strategies to manipulate CO formation using model species, thereby laying the foundation for subsequent translation of the most promising into crops. Our research will focus on three different stages in the control of CO formation in plants: (i) Factors controlling DSB formation: We will determine their minimum requirements. We will evaluate the potential of a MTOPVIB-CRISPR system as the basis for targeting DSBs complexes to recombination-cold regions. We will investigate the use of a suppression cassette (dCas9) and multiple short guide RNAs of the CO suppressor genes to enhance CO frequency. (ii) DSB processing, stable joint molecule formation and CO resolution: We will address how early steps in DSB processing and recombinase loading are linked and how this impacts on the CO/non-CO decision by analysing the role of the MRN complex and COM1 in the efficient removal of SPO11 from the DSB ends that is essential for loading of the DMC1 and RAD51 recombinases. We will also test the hypothesis that efficiency of CO formation in the recombination reaction can be influenced by Cas9-mediated targeting of the MUS81/GEN1 resolvases. (iii) Relationship of chromosome remodelling and CO formation: We will analyse the influence of posttranslational modifications of the chromosome axis in relation to CO formation and distribution. The inter-relationship between extensive remodelling of the chromosome axis and synaptonemal complex at the leptotene/zygotene transition and the maturation of CO designated recombination intermediates will be also analysed. The link between the programmed remodelling of the chromosome axis and CO distribution will be further investigated in barley and maize.