Unravelling the meiotic single-cell transcriptomic atlas for the control of recombination.

大多数进行有性繁殖的生物使用一种特殊类型的细胞分裂，称为减数分裂，这对创造遗传变异和将配子中的染色体数量减半非常重要。在减数分裂过程中，大量的程序性DNA双链断裂(DSB)通过减数分裂重组途径形成并加工形成交叉，这是染色体之间遗传信息相互交换的点。交叉对于在每一代中创造新的遗传变异是必不可少的。由于驯化和密集的选择育种导致作物遗传变异的大量丧失，人们对植物减数分裂有很大的兴趣。持续改进优良品种的遗传，以缓解气候变化的挑战，将需要通过形成交叉，从野生品种中导入有益的等位基因。不幸的是，交叉主要形成在染色体末端，占基因组的不到15%，而更中心的区域，其中包含某些与农业相关的基因，如防御反应基因，在大多数主要作物中很少重组。因此，了解影响杂交模式的因素，并制定策略来重新定位作物中的杂交是及时和迫切的。在物种间和个体内观察到DSB和交叉数目的显著细胞差异。拟南芥和小麦花药含有低重组和高重组减数分裂细胞的混合物，在DSB和交换数量上变化高达70%。我们以前的研究表明，在拟南芥中，ASY1和HEI10的转录水平影响着交叉的频率和位置。因此，我们认为单核细胞的重组结果受到几个基因表达的精细平衡的影响。因此，转录组中的异质性可能是花药中观察到的低重组和高重组的小细胞的原因。然而，到目前为止，所有关于植物减数分裂的基因组研究都包括细胞池，因此无法确定导致这种变异的异质性因素。在这个项目中，我们建议在减数分裂重组的两个关键时间点(DSB形成期间的T1和交叉形成期间的T2)建立拟南芥微细胞的单细胞转录图谱，以了解从DSB形成到它们转化为交叉的转录组动力学。此外，我们将对转录高度相关的细胞进行分组，并使用已知基因的信息来推断包含超重组微核细胞的细胞群(例如，较高的HEI10转录水平对应于较高的交叉率)。然后，我们将使用这些数据来识别在重组异质性中可能起作用的基因。我们将用一组拟南芥过表达品系的特征来补充这项研究，以基于它们的转录水平寻找影响重组的基因。最后，我们将进行一个原理验证实验，以剂量敏感基因ASY1为参照，测试增加小麦减数分裂基因的表达是否可以重新定位交叉，有利于在传统育种中不易获得的区域进行重组。这些新数据将通过使用创新的方法提供影响，以了解转录组和重组异质性之间的相互关系，破译减数分裂过程中的转录组动态，并发现参与减数分裂的基因。该项目还旨在探索一条利用基因过度表达来影响小麦重组前景的新途径，这可能会为育种部门带来持久的好处。这项拟议的工作支持BBSRC的战略优先事项“前沿生物科学：理解生命规则”和“生物科学促进可持续农业和食品”。

Most organisms that reproduce sexually use a special type of cell division, called meiosis, that is important for the creation of genetic variation and halving the chromosome numbers in gametes. During meiosis, numerous programmed DNA double strand-breaks (DSBs) are formed and processed by the meiotic recombination pathway to form crossovers, which are the points of reciprocal exchange of genetic information between chromosomes. Crossovers are essential to create novel genetic variation in each generation. There is a major interest to understand meiosis in plants because domestication and intense selective breeding have led to a substantial loss of genetic variation in crops. Continued genetic improvement of elite cultivars to mitigate the challenge of climate change will require the introgression of beneficial alleles from wild varieties through the formation of crossovers. Unfortunately, crossovers are mainly formed at the end of the chromosomes, representing less than 15% of the genome, whereas more centric regions, which contain certain genes of agricultural relevance, like defence response genes, rarely recombine in most major crops. Therefore, it is both timely and imperative to understand the factors influencing crossover patterning and to create strategies to reposition crossovers in crops. Substantial cellular variation in DSB and crossover numbers is observed between species and within individuals. Arabidopsis and wheat anthers contain a mixture of hypo- and hyper-recombinant meiotic cells varying in DSB and crossover numbers by up to 70%. Our previous studies revealed that the frequency and position of the crossovers are influenced by the transcript levels of ASY1 and HEI10 in Arabidopsis. Therefore, we propose that the recombination outcome of a meiocyte is influenced by a fine balance of expression of several genes. Hence, heterogeneity in the transcriptome could be responsible for the hypo- and hyper-recombination meiocytes observed in anthers. However, all genomic studies carried out on plant meiosis have so far included pools of cells, thus preventing the identification of heterogeneous factors responsible for such variation. In this project, we propose to generate a single cell transcriptomic atlas of Arabidopsis meiocytes at two key time points of meiotic recombination (T1 during DSB formation, T2 during crossover formation) to understand the transcriptome dynamics from the formation of DSBs to their conversion into crossovers. In addition, we will group cells that are transcriptionally highly correlated and infer the cluster of cells that contains the hyper-recombinant meiocytes using information from known genes (e.g. higher HEI10 transcript level corresponds to higher crossover rate). We will then use this data to identify genes with a putative role in recombination heterogeneity. We will complement this study with the characterisation of a set of Arabidopsis over-expressing lines to find genes influencing recombination based on their transcript level. Lastly, we will perform a proof-of-principle experiment, using the dosage-sensitive gene ASY1 as a reference, to test if increasing meiotic gene expression in wheat could reposition crossovers to favour recombination in regions which are not easily accessible in conventional breeding. These new data will provide impact through the use of innovative approaches to understand the inter-relationship between transcriptome and recombination heterogeneity, decipher the transcriptome dynamics during meiosis and discover genes involved in meiosis. This project also aims to explore a novel route for impact in wheat using gene over-expression to influence the recombination landscape, which could confer lasting benefits for the breading sector. This proposed work supports BBSRC strategic priorities "Frontier bioscience: understanding the rules of life" and "Bioscience for sustainable agriculture and food".