Assembling and recombining the Arabidopsis centromeres

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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.