Award details

HEI10: a master switch for recombination in plants

Reference	BB/S006842/1
Principal Investigator / Supervisor	Professor Ian Henderson
Co-Investigators / Co-Supervisors	Dr Sebastien Andreuzza
Institution	University of Cambridge
Department	Plant Sciences
Funding type	Research
Value (£)	619,439
Status	Current
Type	Research Grant
Start date	01/02/2019
End date	31/10/2023
Duration	57 months

Abstract

During meiosis, homologous chromosomes pair and undergo reciprocal exchange, known as crossover. Crossovers are critical for plant breeding and have a profound effect on patterns of genetic diversity. Recombination initiates from DNA double strand breaks (DSBs), a subset of which are repaired as crossovers. Using the model plant Arabidopsis thaliana we previously identified natural variation in HEI10 (a highly conserved, meiosis-specific E3 ligase) as controlling crossover levels. We also discovered that HEI10 is highly-dosage sensitive, and that higher HEI10 expression is effective at increasing crossovers genome-wide. In this project we propose to further harness the use of HEI10 as a recombination master switch, capable of increasing crossovers in plant genomes. In Objective 1 we will perform molecular genetic experiments in Arabidopsis, to define optimal HEI10 levels and the influence of gene promoters on crossover activity. We will combine HEI10 transgenic lines with mutations in the mismatch repair gene MSH2, in order to increase crossover frequency in divergent chromosome regions. In Objective 2 we will perform chromatin immunoprecipitation (ChIP-seq) to identify HEI10 binding sites throughout the genome, and relate this pattern to our maps of DSB and crossover hotspots. HEI10 binding to meiotic chromosomes is known to be dynamic. Therefore, we will use the INTACT method to purify meiotic nuclei at specific sub-stages and repeat ChIP-seq. Finally, in Objective 3, we will transform tomato with additional HEI10 transgenes, in order to increase expression level during meiosis. HEI10 transgenic tomato will be compared to empty vector controls using immunocytology and genetic mapping in order to quantify crossover numbers and identify their location throughout the genome. As crossovers are limiting for tomato breeding, this work will provide a proof-of-principle test for increased HEI10 expression acting to increase crossovers in an important crop species.

Summary

Society is facing a major challenge to improve crop yield and quality, in order to meet the needs of the growing global population. One means to address this challenge is to breed and further improve crop germ plasm. For example, disease and stress resistance traits from wild populations can be beneficial to introduce into elite varieties. In this case beneficial traits must then be introgressed over many generations to achieve an improved strain. This process of crop breeding largely relies on the trait re-assortment in offspring that occurs naturally during reproduction. Specifically, diversity is generated during a specialised cell division called meiosis, which results in production of gametes (sex cells). One mechanism that creates genetic diversity during meiosis is called crossover. Here homologous chromosomes physically pair and exchange reciprocal regions, which generates new combinations of genetic variation. However, crossover patterns along chromosomes are highly skewed in many of the most important crop species, including wheat, maize, barley and tomato. As a consequence, some regions of the genome are inaccessible for breeding, despite containing important genes with effects on agronomically important traits. When compared across plants, animals and fungi, crossover levels are typically low, with most species showing ~1-2 crossovers per chromosome per meiosis. However, there is also considerable variation in crossover levels within and between species. To explore this phenomenon we previously used Arabidopsis to identify natural variation in crossover levels. These experiments identified a major gene called HEI10 that varies between Arabidopsis populations and controls crossover levels. We also discovered that HEI10 is highly dosage sensitive. We found that by increasing the HEI10 gene copy number and expression levels we could efficiently increase total crossover levels. In the context of breeding this would allow breeders to generate more diversepopulations using smaller numbers of individuals. This would provide considerable advantages in terms of time and expense. Therefore, in the proposed work we describe a combined program of basic and applied experiments designed to refine the use of HEI10 as a master-switch for recombination, and deploy it in an important crop species (tomato). This project has three major objectives. First, we will use the model plant Arabidopsis to investigate in detail how the HEI10 gene can be used to increase recombination. We will further increase HEI10 dosage to test when saturation of the recombination response occurs. We will further vary the timing of HEI10 expression during meiosis. Additional genetic modifications will be combined with HEI10 in order to bias the pattern of crossovers in ways that would be beneficial for breeding. Second, to understand how HEI10 controls recombination at the molecular level, we will use a specialised technique to map HEI10 binding sites along the chromosomes. This will provide a high-resolution map of where HEI10 binds, which we will compare to our existing maps of recombination hotspots. Using a novel technique we will also map HEI10 binding at different stages during meiosis, in order to investigate the dynamics of its binding to the genome. This is important as HEI10 foci shows dynamic binding to chromosomes during meiosis. Third, we will transform tomato with additional HEI10 gene copies and test whether crossover increases. This is important as recombination limits tomato breeding and trait improvement, for example when introgressions are made from wild relatives. As meiosis and HEI10 are highly conserved, the outcomes of the proposed research will be readily applicable to diverse crop species. The proposed research will provide a more detailed understanding of the mechanisms of trait re-assortment during plant sexual reproduction and will lay a foundation to develop novel tools to accelerate crop breeding.

Impact Summary

Food security is an issue of critical importance both within the UK and globally. As human population size increases, more food must be harvested per area of land, using sustainable methods. The proposed work has extensive economic, societal and academic impact via application of recombination knowledge in the context of crop breeding and improvement. Our overall strategic aim in this context is to modify the number and location of crossover events in plant genomes. Via generating an increased understanding of recombination control by HEI10, the proposed work is directly relevant to achieving this strategic aim. The Henderson laboratory has completed and ongoing collaborative relationships with industrial partners in crop breeding. This includes industrial Ph.D studentships (KWS, Rijk Zwaan and Syngenta), a BBSRC Industrial Partnership Award (IPA) with Meiogenix a French biotech SME, an sLola network grant seeking to modify recombination in breadwheat involving academic and industrial partners and a Marie Curie Ph.D International Training Network, involving 11 partner laboratories across the EU and industrial partners. Through these networks we seek to develop and exploit our knowledge of recombination control, including in crop genomes. Impact Objectives 1. Generate basic knowledge relating to control of meiotic recombination, including via HEI10, and where appropriate develop and protect any arising intellectual property. 2. Use arising mechanistic knowledge of recombination within our existing relationships with plant bioscience industry to develop new technologies for crop improvement. 3. Publicise to a wider audience the importance of food security and how our research contributes to this. HEI10 represents an important example of exploitation and translation of our findings. In this case, we identified HEI10 as a major regulator of crossover numbers, which encodes a conserved meiotic ubiquitin E3 ligase (Ziolkowski 2017 Genes & Dev). Importantly, we also discovered that HEI10 is highly dosage-sensitive and introduction of additional copies of this gene more than doubled crossovers throughout the Arabidopsis chromosome arms (see also Serra et al., 2018 PNAS). Currently, a patent has been filed to protect the introduction of additional HEI10 copies to increase recombination frequency in plants (UK priority patent application GB1620641.9 co-inventor: Dr Ian Henderson). Our strategy is that this platform technology will be licenced to breeding companies to be incorporated into their crop improvement strategies and programs. Indeed, Meiogenix currently have an option to license our HEI10 patent. We anticipate that our HEI10 technology will help increase crossover numbers in plant genomes. We have filed a second patent jointly with Cold Spring Harbor Laboratory 'U.S. Provisional Application No. 62/356,957, Control of meiotic crossover in maize, Underwood CJ, Henderson IR and Martienssen RA. Filed on June 30, 2016', which seeks to use our understanding of plant heterochromatin to change the location of crossovers in plant genomes. To ensure that intellectual property resulting from the proposed work is adequately developed and protected we will work closely with the University Research Office and Cambridge Enterprise, the University's Technology Transfer Office. The major beneficiaries of our work will be: (i) Crop breeders, who will benefit from knowledge and technology that change recombination in ways that accelerate crop improvement. (ii) Farmers, who will benefit from new crops generated using knowledge of meiotic recombination, with improved yield, disease resistance and stress tolerance characteristics. (iii) Wider society, who will benefit through decreased food prices and increased food security. (iv) Scientists, who will benefit from increased basic knowledge of meiotic recombination mechanisms and chromosome biology.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Crop Science, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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