Assembling and recombining the Arabidopsis centromeres

当细胞分裂时，染色体必须自我复制并分离到相反的细胞极。至关重要的是，每个子细胞都继承了平衡数量的染色体。这个过程是通过染色体与纺锤体微管结合来实现的。染色体通过称为着丝粒的特殊区域附着在纺锤体上。不能正确地附着在纺锤体上可能导致染色体分离缺陷，并与癌症和不孕症有关。着丝粒在确保染色体分离中的作用是细胞中一个古老而保守的功能。通常，当研究这种高度保守的过程时，所涉及的机制在不同物种之间非常相似。然而，令人惊讶的是，着丝粒的情况正好相反，与之相关的DNA序列和蛋白质是基因组中变化最快的。这种现象被称为“着丝粒悖论”。研究着丝粒的一个挑战是相关的DNA序列是高度重复的。例如，在许多物种中，着丝粒由短的（~170-180个碱基对）序列组成，它们以头尾方向串联复制多次（100 -1000秒）。这些被称为卫星阵列，在这些序列中，微管将与染色体结合。众所周知，着丝粒卫星阵列能够在物种之间快速而广泛地变化，但这些卫星阵列是如何进化和变化的却知之甚少。高度的重复使得着丝粒基本上不可能用上一代的短读DNA测序技术来研究。然而，随着包括牛津纳米孔在内的长读DNA测序技术的出现，新的机会正在出现。在本研究中，我们将首次利用长读测序技术组装模式植物拟南芥的着丝粒。我们将使用这些着丝粒的图谱来研究在生殖系中发生的重组过程如何有助于着丝粒的快速进化。这项工作的主要成果将是完成拟南芥基因组的着丝粒间隙，我们将向社区发布。除了特定的重复DNA序列外，着丝粒在细胞分裂过程中需要表观遗传标记才能发挥作用。例如，一种叫做CENH3的特殊组蛋白与着丝粒结合，对染色体附着在纺锤体微管上至关重要。此外，着丝粒经常被DNA甲基化高度修饰，尽管这种表观遗传标记在着丝粒中的功能尚不清楚。因此，在最终目标中，我们将使用长读测序来研究缺乏DNA甲基化的拟南芥突变体的着丝粒和重组。我们的工作将共同揭示着丝粒的结构以及它们如何如此迅速地进化的新见解。重要的是，在许多作物物种中，着丝粒周围的区域也被抑制以进行重组，这可能限制育种过程中的品系改进。因此，我们在这项提议中产生的知识可能为解锁靠近着丝粒的重组提供方法，以加速作物育种。

When cells divide the chromosomes must copy themselves and segregate to opposite cell poles. It is critical that each daughter cell inherits a balanced number of chromosomes. This process is achieved by the chromosomes binding to spindle microtubules. Chromosomes attach to the spindle via specialised regions called centromeres. A failure to attach to the spindle correctly can cause defects in chromosome segregation and is associated with cancer and infertility. The role of the centromeres in ensuring chromosome segregation is an ancient and deeply conserved function in cells. Typically, when such highly conserved processes are studied, the mechanisms involved are very similar between different species. However, surprisingly the opposite is true for the centromeres, and the DNA sequences and proteins associated with them are some of the fastest changing in the genome. This phenomenon is termed the 'centromere paradox'. One challenge to studying the centromeres is that the associated DNA sequences are highly repetitive. For example, in many species the centromeres consist of short (~170-180 base pairs) sequences copied many times (100s-1000s) in a tandem head-to-tail orientation. These are known as satellite arrays and it is within these sequences that the microtubules will bind to the chromosome. It is also known that the centromere satellite arrays are capable of rapid and extensive change between species, yet how these satellite arrays evolve and change is poorly understood. The very high degree of repetition has made the centromeres essentially impossible to study with the previous generation of short read DNA sequencing technologies. However, new opportunities are arising with the advent of long-read DNA sequencing technologies, including Oxford Nanopore. In this proposal we will harness long-read sequencing to assemble the centromeres of the model plant species Arabidopsis for the first time. We will use these maps of the centromeres to investigate how recombination processes occurring during the germline might contribute to the fast evolution of the centromeres. A major output from this work will be completion of the centromere gaps in the Arabidopsis genome, which we will release to the community.In addition to specific repeat DNA sequences, the centromeres are known to require epigenetic marks for their function during cell division. For example, a special histone protein called CENH3 binds to the centromeres and is critical for chromosomes to attach to the spindle microtubules. Additionally, centromeres are often highly modified by DNA methylation, although the function of this epigenetic mark in the centromeres is unknown. Therefore, in the final objective we will use long-read sequencing to investigate the centromeres and recombination in Arabidopsis mutants that lack DNA methylation. Together our work will reveal new insights into how the centromeres are structured and how they evolve so quickly. Importantly, in many crop species the regions surrounding the centromeres are also suppressed for recombination, which can limit strain improvement during breeding. The knowledge we generate in this proposal may therefore provide ways to unlock recombination close to the centromeres in order to accelerate crop breeding.