Genome-wide mapping of recombination hotspots in Arabidopsis

Harnessing natural genetic variation through breeding and genetic recombination is a cornerstone of crop improvement. During domestication crops pass through genetic bottlenecks, meaning that wild progenitor species contain abundant variation that is useful to reintroduce. One limitation to breeding is that recombination rate is highly variable along chromosomes. For example, large regions of wheat, barley and maize chromosomes are non-recombining, despite containing useful genetic variation. We are investigating the mechanistic basis of recombination control, with the aim of rationally manipulating this process to accelerate crop breeding. As the core recombination process is highly conserved between plants, our work focuses on the model species Arabidopsis thaliana, which has extensive genetic and genomic resources. Using a novel experimental technique termed pollen-typing we have recently discovered that A.thaliana chromosomes have narrow hotspot regions with a highly elevated chance of recombination. These hotspots are located in gene promoters, where open chromatin packaging allows access to the DNA, and we hypothesize that this also allows access to the recombination machinery. Hotspots in other species are controlled by a combination of chromatin structure and primary DNA sequence, and we will investigate these relationships in A.thaliana. First we will use population genetics methods to generate a genome-wide hotspot map using patterns of natural genetic polymorphism between A.thaliana populations. As recombination breaks up blocks of linked genetic variation, this effect can be used to measure recombination rate and identify hotspot locations. The A.thaliana genome is well annotated with chromatin maps and this will allow us to determine which aspects of chromosome organisation correlate with hotspot locations. In addition we will test whether hotspots associate with specific DNA sequences, to resolve the extent to which plant hotspots are genetically versus epigenetically determined. Using pollen-typing we will then experimentally validate a subset of the most active hotspots in the A.thaliana genome. To directly test the role of chromatin on hotspot activity we will repeat pollen-typing in mutant backgrounds with altered epigenetic organization. The hotspots we have already observed are located in gene promoters and we will use mutants with altered promoter chromatin to test for effects on recombination. We will also directly target chromatin modifications associated with gene silencing to hotspots and investigate whether this is sufficient to suppress recombination. Knowledge of hotspot control will provide novel insight into manipulating recombination in crop genomes. Our strategic goal is to modulate hotspot activity in crops, for example by boosting, suppressing or relocating them, and thereby facilitating breeding of high-yielding strains.

During meiosis homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). CO frequency is highly variable within genomes and this has a profound effect on patterns of genetic diversity. Importantly, crop species such as barley, wheat and maize have chromosomes with large non-recombining regions that can limit breeding of useful variation. We have discovered narrow (1-2 kb) recombination hotspots in Arabidopsis thaliana, via a novel methodology termed pollen-typing. These hotspots localize to intergenic, nucleosome-free regions in gene promoters, and are flanked by H2A.Z histone variant-containing nucleosomes. To understand hotspot patterns on a genome-wide scale we will implement the LDhat program using genetic polymorphism data from 80 Eurasian A.thaliana accessions. LDhat uses population genetics methods to estimate recombination rates from patterns of linkage disequilibrium between polymorphisms. To validate this map we will experimentally measure recombination rates using pollen-typing for a subset of the most active hotspots. Hotspots vary in the extent to which they are controlled by epigenetic information versus primary DNA sequence. Therefore, we will correlate hotspot locations with chromatin maps, for example nucleosome density, and also test for association with DNA sequence motifs. To experimentally test the role of epigenetic organisation on hotspot activity we will repeat pollen-typing in mutants with altered promoter chromatin structure, for example the H2A.Z deposition mutant arp6. As DNA methylation causes transcriptional silencing we will target this modification to hotspots using small RNAs, to test whether this is also sufficient to silence recombination activity. These experiments will functionally test the importance of chromatin organisation for hotspot recombination. A detailed understanding of hotspot control will allow us to boost, suppress or redistribute recombination during crop breeding.

ng of natural genetic variation remains a critical tool for crop improvement. The majority of crops represent 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. The key impact of our research will be via mechanistic knowledge of the recombination process that crop breeding relies upon. Recombination frequency can be highly limiting during crop breeding; for example, large parts of barley, wheat and maize chromosomes are non-recombining, despite containing abundant useful genes and variation. Through an understanding of the mechanism of recombination frequency control we aim to develop tools that will allow rational manipulation of crossover frequency and therefore facilitate breeding. Specifically, we aim to develop technology where meiotic recombination can be increased or suppressed, both locally or on a genome-wide scale. This will generate a molecular breeding 'tool-kit' and allow us to fully harness natural genetic variation existing in crop species. To explore translation of our research into crop species I collaborate with Syngenta through co-supervision of a BBSRC-CASE student, Carsten Reinhard. Carsten is investigating the relationship between recombination rate and disease resistance gene clusters, using a comparative approach between Arabidopsis thaliana and hexaploid bread wheat. To this end he has generated a >1,000 individual wheat double-haploid population with Syngenta, which we are currently genotyping over resistance gene dense regions. In addition to generating a valuable genetic resource, this project will prime us to apply the knowledge of hotspot control obtained from A.thaliana within a critically important crop species. As wheat chromosomes have large non-recombining regions, a clear need exists for technology to manipulate recombination patterns during breeding. Although cereal chromosomes are far larger than A.thaliana chromosomes, their chromatin is organized in fundamentally similar ways, meaning that our knowledge will be directly applicable between species. In the long term we aim to translate our research into enabling technologies that allow crop breeders to fully utilise natural genetic variation and achieve sustainable increases in crop productivity. We will communicate the importance of our research for crop improvement and food security to wider society via the University of Cambridge Science Festival and by publicizing research discoveries though science podcasts.