Periodic Reporting for period 4 - SHUFFLE (Recombination in large genome crop plants)
Reporting period: 2020-04-01 to 2021-01-31
During a tissue-specific phase of plant development called meiosis, recombination (or crossing-over, CO) drives the exchange of genetic materials and releases genetic diversity by creating new combinations of alleles within and among chromosomes. CO occurs in the small populations of cells that develop into the gametes (eggs and sperm). In plant breeding CO is exploited through the generation of large populations of progeny plants from which genetically improved individuals are selected. However, in some economically important crops such as barley and wheat there is an emerging problem: the rate of genetic improvement appears to have plateaued. One hypothesis to explain this is that constraints on the location of CO restricts the release of genetic diversity that is required to drive crop improvement. This is because CO in barley is restricted to the ends of chromosomes, excluding approximately two thirds of the genome from the breeding process.
If genetic gain is reduced and yields continue to plateau, then the long term societal impact will be on a human population that continues to increase while agricultural land and productivity shrinks due to environmental change. Consequently, primary food and feed commodities will become limited. While, at one level, this could result in increased food prices or a scarcity of specific products on supermarket shelves, in poor and subsistence societies it could lead to major issues such as regional famines, conflicts and mass migration.
The SHUFFLE project focuses on exploring how we can change the frequency or distribution of CO in order to release genetic diversity from currently inaccessible regions of the genome in order to improve the rate of genetic gain in crop improvement. We started out to address three critically important questions about CO in barley: Why is it restricted to the telomeric ends of chromosomes? What genes and proteins are the key players in determining this distribution? What are their roles in controlling CO? We believed that answering these questions would allow us to propose, and then evaluate, strategies for modifying the distribution of recombination in barley. In a broader context our observations may also find application in breeding other major crop plants. A short video of the project explaining the issues we address can be found here: https://www.youtube.com/watch?v=XyMhyeWMZl4
If genetic gain is reduced and yields continue to plateau, then the long term societal impact will be on a human population that continues to increase while agricultural land and productivity shrinks due to environmental change. Consequently, primary food and feed commodities will become limited. While, at one level, this could result in increased food prices or a scarcity of specific products on supermarket shelves, in poor and subsistence societies it could lead to major issues such as regional famines, conflicts and mass migration.
The SHUFFLE project focuses on exploring how we can change the frequency or distribution of CO in order to release genetic diversity from currently inaccessible regions of the genome in order to improve the rate of genetic gain in crop improvement. We started out to address three critically important questions about CO in barley: Why is it restricted to the telomeric ends of chromosomes? What genes and proteins are the key players in determining this distribution? What are their roles in controlling CO? We believed that answering these questions would allow us to propose, and then evaluate, strategies for modifying the distribution of recombination in barley. In a broader context our observations may also find application in breeding other major crop plants. A short video of the project explaining the issues we address can be found here: https://www.youtube.com/watch?v=XyMhyeWMZl4
Initially we focused on identifying mutations in the barley genome that affect meiosis. We used a potent mutagen called EMS to develop a large mutant population of GP. We then searched for lines that were semi-fertile – which is a hallmark of meiotic mutants – and identified several hundred. To screen these at the sequence level we developed a novel sequence-based using target enrichment sequencing. By characterising ALL of the semi-fertile lines we identified a large number of lesions in many of our targeted genes. We also adopted an approach called a ‘suppressor screen’ where we attempt to identify semi-fertile lines that have their fertility restored due to mutations in a different gene identifying a small number of suppressor lines. Finally used Genome Editing to produce knockout alleles of meiotic genes that had been shown previously to affect CO. We prioritised a few of the most promising mutants for detailed characterisation.
We used and high resolution immuno-cytology to investigate where, when and what the impact of the mutations we had identified were on meiosis. We also constructed populations segregating for mutant alleles and used genetic segregation analysis to compare the effect of mutant vs. wild type versions of the identified genes. We identified mutants that changed CO frequency and location. We found that some of the genes we identified had been identified previously in different species (e.g. MLH3). However, in barley we observed either subtly or radically different effects. Other mutated genes were novel. One, STICKY TELOMERES 1, is an E3 ubiquitin Ligase where mutants increased CO by >2.5 fold. In addition two lines identified from the suppressors screen restored recombination from <50% to well-above wild type levels. These were mutants in genes called FANCM and RecQL4, previously shown to restore recombination in Arabidopsis.
To discover novel proteins we also looked at the protein repertoire of meiotic cells. We developed a micro-proteomics workflow to profile the proteome individual meiotic phase barley anthers (the structure containing the meiocytes,) highlighting over 300 that changed in abundance during meiotic progression. In parallel we developed a meiotic anther RNA transcriptome using techniques that allow us to survey almost all of the RNA molecules in a cell. We identified many differentially expressed meiotic genes and assembled them in modules that showed different expression patterns during meiotic progression. These experiments have led us to focus on a family of ARGONAUTE proteins, highly specialized proteins that bind small RNAs and coordinate downstream gene-silencing events, that exhibit contrasting patterns of expression.
We studied the natural patterns of CO in barley using a combination of genetic approaches that examine CO at high resolution in natural bi- and multi-parent populations. The results show that there are ‘hotspots’ of recombination in the barley genome, just as there is a massive ‘cold spot’ across the centromeric regions. The hotspots tend to be conserved across families. We explored why the ‘cold’ regions are recombinationally inert. We observed few ancestral genomic patterns (called haplotypes), peppered with variants most likely introduced by non-crossing over or gene conversion events. To extend this work we developed a novel method for assessing CO using mutagenesis and high throughput sequencing reducing the time and expense of genetic analyses
We are now focused on exploring how our discoveries can be used in breeding.
We used and high resolution immuno-cytology to investigate where, when and what the impact of the mutations we had identified were on meiosis. We also constructed populations segregating for mutant alleles and used genetic segregation analysis to compare the effect of mutant vs. wild type versions of the identified genes. We identified mutants that changed CO frequency and location. We found that some of the genes we identified had been identified previously in different species (e.g. MLH3). However, in barley we observed either subtly or radically different effects. Other mutated genes were novel. One, STICKY TELOMERES 1, is an E3 ubiquitin Ligase where mutants increased CO by >2.5 fold. In addition two lines identified from the suppressors screen restored recombination from <50% to well-above wild type levels. These were mutants in genes called FANCM and RecQL4, previously shown to restore recombination in Arabidopsis.
To discover novel proteins we also looked at the protein repertoire of meiotic cells. We developed a micro-proteomics workflow to profile the proteome individual meiotic phase barley anthers (the structure containing the meiocytes,) highlighting over 300 that changed in abundance during meiotic progression. In parallel we developed a meiotic anther RNA transcriptome using techniques that allow us to survey almost all of the RNA molecules in a cell. We identified many differentially expressed meiotic genes and assembled them in modules that showed different expression patterns during meiotic progression. These experiments have led us to focus on a family of ARGONAUTE proteins, highly specialized proteins that bind small RNAs and coordinate downstream gene-silencing events, that exhibit contrasting patterns of expression.
We studied the natural patterns of CO in barley using a combination of genetic approaches that examine CO at high resolution in natural bi- and multi-parent populations. The results show that there are ‘hotspots’ of recombination in the barley genome, just as there is a massive ‘cold spot’ across the centromeric regions. The hotspots tend to be conserved across families. We explored why the ‘cold’ regions are recombinationally inert. We observed few ancestral genomic patterns (called haplotypes), peppered with variants most likely introduced by non-crossing over or gene conversion events. To extend this work we developed a novel method for assessing CO using mutagenesis and high throughput sequencing reducing the time and expense of genetic analyses
We are now focused on exploring how our discoveries can be used in breeding.
In the SHUFFLE project we believe we succeeded in identifying and characterising more genes involved in a single developmental process in a crop plant than any other study. We generated carefully produced and well-documented resources that are available to the research community. Our characterisation of multiple barley meiotic genes has without any doubt advanced the research field. We made unique discoveries (e.g. ST1) that are potentially significant ‘breakthroughs’. We observed gene functions that do not conform to what is published in the scientific literature and others that are considered to provide only incremental advances to knowledge. All advance the field. The major breakthrough is that we now have the genetic tools at hand to actually test whether increasing recombination can be of value in crop plant breeding and can bring the improvements that we anticipated and used as justification for the need for the research over six years ago.