EpiSpiX - Unlocking plant genetic diversity via epi-modification & targeted recombination.

It is vital that we continue to improve and adapt our most important crops to meet the challenges of an increasing global population and the changing climate. A major component of crop improvement is via classical crop breeding. In this approach breeders combine varieties of crops with complementary beneficial characteristics and use the natural process of recombination to recover strains that combine both sets of desirable features from the original parents. Recombination occurs between the generations when plants form sex cells (gametes) and therefore by understanding the processes that occur at this stage we will be able to breed useful strains faster and more effectively. This is important as recombination patterns can severely limit our ability to breed crop species. For example, if we consider the extremely large wheat genome (~16x larger than the human genome), recombination shows a highly skewed distribution and occurs in a minority of the DNA sequence. Despite large parts of the wheat genome being essentially silent for recombination, these regions can contain many important genes and useful variation. Therefore, these patterns can inherently limit the ability of breeders to improve our crops. As one example, the dwarfing gene RhtD1, which contributed to yield increases achieved during the Green Revolution, is located in one such non-recombining region. This has limited the ability of breeders to combine RhtD1 with other useful genes located in proximity, including important disease resistance genes. This problem is known as linkage-drag.

We are investigating the hypothesis that a major cause for suppressed recombination in these genomic regions is at the level of organisation that we term epigenetic. This concept describes organisation of the genome beyond the DNA base sequence itself. A well understood example of this is that the cytosine bases in the DNA can be modified with methyl groups and this modification can act as a type of grammar that influences how the DNA is expressed. We have previously shown that epigenetic information can have a major effect on patterns of recombination. In the proposed work we will alter epigenetic information in plant genomes and profile exactly how the recombination process changes. We will undertake this both in the model species Arabidopsis and also directly in the complex wheat genome. This will involve collaboration with the group of Pierre Sourdille (Clermont-Ferrand) who is an expert at mapping recombination in the hexaploid bread wheat genome. This proposal is also an industrial collaboration with Meiogenix who are pioneering advanced technology to direct the recombination machinery to specific locations in the genome. They key idea in this proposal is to combine these targeting technologies with manipulation of chromatin to effectively unlock recombination in silent regions of plant chromosomes. Through this work we will provide knowledge and technology that will allow variation to be accessed in breeding programmes that was previously unavailable, due to restricted distributions of recombination. These ambitious research aims capitalise on the unique knowledge and research experience of the partners and will bring novel approaches to solving the problem of recombination control.

Meiotic recombination is a fundamental feature of eukaryotic genomes that has a profound effect on genetic diversity. During meiosis homologous chromosomes pair and undergo programmed DNA double strand breaks, which can be repaired as reciprocal crossovers between the chromosomes. The frequency of meiotic recombination is highly variable within plant genomes and is typically high in gene-rich euchromatin and suppressed in repeat-rich heterochromatin. Many of our most important crops have very large genome sizes (e.g. 16 Gb in hexaploid bread wheat) and show suppression of recombination in the extensive heterochromatic regions around centromeres. Despite this, these regions contain useful genetic variation, so this can limit breeding and crop improvement. Here we propose to comprehensively map meiotic recombination in mutants with altered heterochromatin. Specifically, mutants that have reduced CG or non-CG DNA methylation, which we have shown have opposite effects on centromeric crossovers in Arabidopsis. We will generate genome-wide maps of meiotic DNA double strand breaks and crossovers in these mutants. We will also isolate wheat chromomethylase mutants with altered levels of CG and non-CG methylation and test for recombination changes using mapping populations. The industrial partner Meiogenix and the Henderson laboratory have developed technology to target recombination in plant genomes. We have fused TAL DNA binding domains to the meiotic endonuclease SPO11 and expressed these constructs from meiotic promoters. TAL-SPO11 constructs will be transformed into wild type and heterochromatin mutants and recombination tested over the centromeric regions. TAL-SPO11 constructs will also be transformed into wheat DNA methylation mutants alongside wild type to provide proof-of-principle for this approach directly in a complex crop genome. We will develop knowledge and technology to control meiotic recombination in plant genomes and unlock genetic diversity.

Breeding of natural genetic variation remains a vital tool for crop improvement. The majority of agricultural crops contain a fraction of the genetic diversity present in their wild progenitors. Reintroduction of variation in disease resistance and stress tolerance will allow higher yielding, more sustainable crops to be developed. One limitation to breeding is the skewed distribution of crossover events in crop species, including wheat, barley, maize and tomato. For example, extensive regions of the wheat genome show negligible levels of recombination that can severely limit our ability to breed with useful variation. We hypothesize that one factor limiting recombination is epigenetic modification of plant chromosomes - specifically chromatin marks such as DNA methylation that transcriptionally silence repetitive sequences close to the centromeres.

This proposal will combine the expertise and knowledge of the partners to provide a solution to this problem. First the Henderson laboratory is actively investigating the interaction between chromatin and recombination in plant genomes. Second the industrial partner Meiogenix are in a unique position globally in pioneering targeted recombination technology. This involves an extensive research & development program in several important crop species, where they are deploying targeted versions of the SPO11 endonuclease in order to manipulate recombination. Third the laboratory of Dr Pierre Sourdille (INRA, Clermont-Ferrand) is a world expert in understanding distributions of meiotic recombination in the complex hexaploid wheat genome. Through this unique combination of expertise we will develop the EpiSpiX technology necessary to unlock non-recombining regions of the wheat genome.

This proposal has three major impact objectives:

1. Demonstrate EpiSpiX proof-of-principle in Arabidopsis and secure intellectual property for translation into crop species. A major aim of the proposed work is to comprehensively map meiotic recombination in heterochromatin mutants and combine these mutants with targeted recombination technology. This will provide proof-of-principle data for the EpiSpiX approach and will generate vital knowledge that will guide deployment of targeted recombination technology in crop genomes.

2. Collaborate with Meiogenix and INRA to initiate translation of EpiSpiX technology and knowledge into crop species, primarily bread wheat. Together we will implement recombination targeting technology in wheat. In addition we will combine our constructs with wheat lines with modified DNA methylation. This will generate further proof-of-principle data that we will use to develop and manage Intellectual Property. We have an agreed strategy for IP generation and management that has been coordinated between Meiogenix, INRA, the Sourdille laboratory, the University of Cambridge research office and the Henderson laboratory.

3. Publicise to a wider audience the importance of food security and how our research specifically contribute to this. Dr Henderson and the PDRAs will directly participate in communication of our findings to academic and non-academic stakeholders, including crop breeders, farmers, wider society. This will be via presentations and discussions at the Cambridge Partnership for Plant Science, which provides a termly networking event for University researchers and industry representatives. We will also present our work at the Cambridge Science Festival and the Festival of Plants held each year at the Botanic Garden. We will also discuss presenting on the Naked Scientist podcast, which is broadcast from Cambridge to a global audience of millions. Finally, we will communicate our findings to academic beneficiaries at national and international scientific meetings and through publication in peer-reviewer journals.