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Q1- What is your goal in the project?

The goal of my laboratory is to learn more about the basic mechanisms of double strand break repair in plants and its applications for genome manipulation. More recently meiotic recombination came in focus of our interest. We are especially interested in the decision at which loci genomic changes between the parental genome are initiated and also in the extent of how much information is exchanged between the genomes. An important question we address in this project is whether we can enhance somatic and meiotic recombination to improve plant breeding.

Q2- What technologies do you use?

We are using sequence specific nucleases that are able to induce specific breaks in the genome. Moreover we are creating transgenic Arabidopsis plants that are overexpressing one or the other factor involved in recombination.

Q3- What are your main achievements so far in RECBREED?

At this point we have carried a detailed analysis of the BRCA2 genes in Arabidopsis, Mutations in the breast cancer susceptibility gene 2 (BRCA2) are correlated with hereditary breast cancer in humans. Studies revealed that mammalian BRCA2 plays crucial roles in somatic and meiotic DNA repair. Previous studies showed that also BRCA2 in Arabidopsis is involved in recombination processes. We generated an Atbrca2 double mutant and analyzed it. The Atbrca2 double mutant showed hypersensitivity against the crosslinking agent mitomycin C and displayed a substantial reduction in the somatic homologous recombination frequency. The loss of AtBRCA2 led to severe defects in male and female meiosis. Correct localization of the synaptonemal complex protein AtZYP1 and the recombinases AtRAD51 and AtDMC1 is impeded in Atbrca2 double mutants. AtBRCA2 was shown to be important for both somatic and meiotic homologous recombination. Immunolocalization proved that synapsis is disturbed in male meiotic prophase I and that the correct localization of the recombinases AtRAD51 and AtDMC1 is dependent upon AtBRCA2. Therefore we could show for the first time that BRCA2 mediates single strand invasion during homologous recombination also in seed plants.

Q4- How do you envision the impact of your work on plant breeding and biotechnology?

If we reach our goal and are able to direct recombination in a controlled manner, we for the first time in history would not only be able to decide beforehand what kind of traits will be transferred from one generation to the next but we also would be able to use the complete gene pool of the respective species for crop improvement. These gene pools document different kinds of adaptation obtained by try and error in different environments during evolution over a historical time periods. Although present - this accumulated knowledge could not be activated to major extents by classical breeding technologies. Thus plant breeding could be transformed from a basically random to a well planned directed process.


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Q1- What is your goal in the project?

The goal of my laboratory is to better understand the mechanism of homologous recombination in plants in order to enhance the rates of Gene targeting and of meiotic recombination in the model plant Arabidopsis and in tomato. In RECBREED, we propose to achieve this through the genetic manipulation of homologs of the yeast RAD52 gene. These genes, whose role is essential in homology search in yeast, were previously unknown in plants. Therefore they are an interesting gene family to study.  The second approach is to knockout or silence genes of the mismatch repair pathway in Arabidopsis and in tomato.  Our assumption, based on earlier studies, is that such genes might inhibit recombination between divergent sequences.

Q2- What technologies do you use?

We use tools of plant molecular genetics, including genetic engineering, plant tissue culture, and genetic markers.  We have developed assays, based on fluorescent seed markers, specifically to measure meiotic recombination and gene targeting in Arabidopsis, as well as DNA indel markers to measure meiotic recombination rates.

Q3- What are your main achievements so far in RECBREED?

At this point we have carried a detailed analysis of the new plant RAD52 gene family, including phylogeny, conservation, protein structure/function prediction, expression, localization and function in repair and intrachromosomal recombination.  A publication on this topic has been submitted and we are now ready for the second stage, namely the functional analysis on meiotic recombination and gene targeting.
We have optimized the expression of recombination genes in the egg cell of Arabidopsis flower in order to express genes that may promote recombination in this tissue during gene targeting.  A publication on this was recently accepted in The Plant Journal and we are ready for expressing RAD52 homologs for Gene targeting experiments.

Q4- How do you envision the impact of your work on plant breeding and biotechnology?

If successful our work may contribute to the development of gene targeting.  The proof of concept is done here in Arabidopsis but the approach should also be applicable to crops for modern plant biotechnology and breeding, for example for the targeted knockout of genes or the replacement of genes or the precise integration of transgenes in plants.  Our work on mismatch repair may facilitate the transfer of genes between wild relatives whose chromosome are divergent and under normal condition show only limited amount of pairing and recombination.  This would enable to exploit beneficial alleles from a broad range of wild relatives of our crops.

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Q1- What is your goal in the project?

We study the molecular mechanisms assuring the integrity of the structure and of the informational content of the genome. Working with the plant, Arabidopsis thaliana, our research in the RECBREED project concerns three interrelated themes:

• Studies of the mechanisms of meiotic recombination and chromosome synapsis and, in particular, the roles of the Rad51 protein family and the structure-specific endonuclease XPF/ERCC1.
• Effects of knocking-out or overexpressing these proteins on rates and patterns of recombination.
• Understanding of the complex interrelationships and regulation of DNA repair and recombination proteins and pathways in planta.

Q2- What technologies do you use?

We use tools of plant molecular genetics and cytology, including genetic engineering, plant and tissue culture, genetic markers, FISH and immunocytology. We have developed assays and biological material (eg. expression of tagged fluorescent recombination proteins; immunodetection of chromatin modification at DNA breaks and quantitative kinetic analysis of recombination at these breaks) to study recombination and DNA Repair proteins and pathways in vivo.

Q3- What are your main achievements so far in RECBREED?

Half-way through the project, we have built and confirmed proper function of tagged fusion proteins of the Rad51 recombinase family and the Xpf/Ercc1 structure-specific endonuclease proteins. "Own promoter" and constitutive overexpression promoter constructs have been built, and successfully tested in planta.  Effects on recombination in meiosis and somatic cells have been tested in knockout mutant lines for these recombination proteins and these tagged expression and overexpression constructs are now being crossed into the mutants to test for effects on rates and patterns of recombination. We have also identified a novel non-homologous recombination pathway and established the kinetics and the functional hierarchy of the four major somatic recombination pathways in the plant. Understanding and manipulation of alternative or parallel pathways of recombination will possibly be of great importance in optimising analyses of rates and patterns of homologous recombination for plant breeding and gene targeting.

Q4- How do you envision the impact of your work on plant breeding and biotechnology?

Through developement of understanding of recombination and genome maintenance processes in plants, our work aims to develop the possibility to modulate rates, patterns and types of recombination. Very much aware of the intimate relationship between recombination and genome stability, we analyze the roles of these pathways in DNA repair and chromosomal stability, as well as recombination as such.  The tools that may come from this understanding are of potentially great importance to plant breeding and biotechnology.

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Q1- What is your goal in the project?

The goal of our research within the RECBREED project is to determine the regulatory role of chromatin in meiotic recombination. It has been well documented that meiotic crossovers (COs) are suppressed in transcriptionally inactive compact, chromosomal regions encompassing centromeric and pericentromeric hypermethylated DNA. Therefore, it has been postulated that in these areas high degree of chromosome compaction, regulated in plants and mammals by DNA hypermethylation, is inhibitory to COs. Using Arabidopsis mutants depleted in DNA methylation and inbred lines with mosaic DNA methylation patterns (with methylated and demethylated chromosomal segments), we are reassessing and quantifying these effects.

Q2- What technologies do you use?

We analyse genome-wide MRR using single nucleotide polymorphisms (SNPs) as markers, which are selected such that they are distributed across all chromosomes. For determination of MRR in many individuals and across the entire genome we use high-throughput KASP genotyping (KBiosceinces, Herts, UK, http://www.kbioscience.co.uk/download/KASP.swf).

Q3- What are your main achievements so far in RECBREED?

We observed that the loss of DNA methylation had a stimulatory effect on MRR in the naturally hypomethylated euchromatic chromosome arms, while it had inhibitory outcome in heterochromatic regions encompassing centromeric and pericentromeric hypermethylated DNA. These latter observations seems counterintuitive considering the well documented suppressive effect of heterochromatin on MRR, which could have been alleviated by reduced DNA methylation, released transcriptional silencing and altered heterochromatin properties. Therefore our results reveal a surprisingly complex role of DNA methylation, which regulates the chromosomal distribution of COs and thus influences the final CO landscape.

Q4- How do you envision the impact of your work on plant breeding and biotechnology?

Identifying factors influencing meiotic crossover frequency is undoubtedly important for refining plant breeding strategies as hyper recombinant lines could be selected to facilitate breeding programs. Increased MRR in euchromatic chromosome arms could contribute to accelerate the selection for novel haplotypes, while the decreased heterochromatic recombination would be beneficial in maintaining the control of transposable elements and repeat, which constitute the major part of centromeric heterochromatin.

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Q1- What is your goal in the project?

The goal of our laboratory in the project is to develop efficient techniques with which DNA double strand breaks (DSBs) can be induced at specific sites in the genome in a controlled way.
Repair of such DSBs by the cell may lead to mutation, if this occurs by an error prone mechanism such as DNA end-joining.
In the presence of introduced DNA that shares homology with the broken region, this new DNA may be incorporated in the genome by homologous recombination, a process called gene targeting.
If formed during meiosis, such DSBs may form starting points for crossover in the chromosome.

Q2- What technologies do you use?

We apply molecular genetics and develop gene technology, but we also use cell biology including confocal microscopy and plant tissue culture.  We have developed plant vector systems based on the natural vector Agrobacterium tumefaciens for transformation, protein translocation and gene targeting.

Q3- What are your main achievements so far in RECBREED?

Genes for zinc finger nucleases with which DSBs can be induced at chosen sites in the genome of the model plant Arabidopsis thaliana have been selected. Vectors for the delivery of these genes in plants have been constructed and have been used already to study local mutation and gene targeting. Using the PPO locus we have indeed found gene targeting events. Other constructs have been provided to partner 1 to determine the effects on meiotic recombination.

Q4- How do you envision the impact of your work on plant breeding and biotechnology?

Our work should lead to improved gene targeting and targeted mutagenesis in plants. Although we use Arabidopsis thaliana as our model, we are convinced that the technology developed shall find wide application in a range of plants including crops. Applications would include the precise introduction of new transgenes, which gives much better control of expression, and the replacement of genes by alleles with higher or reduced activity (for unwanted traits).

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Q1- What is your goal in the project?

The goal of Keygene within RecBreed is to investigate the use of site specific nucleases to improve the efficiency of mutagenesis and gene targeting in the crop species tomato. Much of our knowledge on the role of plant genes has come from Arabidopsis where gene function is normally characterized by the analysis of T-DNA insertion lines which nearly always produce a null phenotype. If we want to transfer this knowledge to crop species then we must develop new methods for targeted mutagenesis to knock out gene function. Gene targeting using homologous recombination is also a very powerful tool to accurately transfer SNP’s between gene orthologs and has a direct application in many breeding programs if the efficiencies can be increased sufficiently.

Q2- What technologies do you use?

We use engineered zinc finger nucleases targeting a specific gene in tomato combined with an optimized plant tissue culture system. In addition, we have implemented an assay to score gene targeting that is reproducible and can be done in a relatively high throughput manner.

Q3- What are your main achievements so far in RECBREED?

Thus far we have further optimized our tomato tissue culture system and used this extensively in our experiments. We have been able to show that zinc finger nucleases are efficient reagents for producing tomato mutants. We have also validated our gene targeting assay and are currently assessing the gene targeting efficiency in tomato through the use of zinc finger nucleases.

Q4- How do you envision the impact of your work on plant breeding and biotechnology?

Arabidopsis is an ideal plant species to obtain proof of concept for gene function and their possible application in commercial biotechnology. However, this information then needs to be applied to a crop species, which are hampered by a lack of good tissue culture protocols and are generally a lot harder to work with than Arabidopsis. The technologies that we are testing will allow breeders to generate mutants in a fast and cost effective manner. The goal of breeding is to introduce novel genotypes from wild plant species into cultivated lines. Essentially, this involves the introduction of novel haplotypes from one plant to another and involves many generations of backcrossing. Gene targeting can be used to transfer a haplotype in a single generation and therefore represents a very powerful tool for breeders in the future.

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Q1- What is your goal in the project?

The goal of Biogemma in the project is to develop improved tools for plant transformation and for plant breeders. The first goal is to implement efficient gene targeting (GT) in maize. We aim to test in maize genes identified by other Recbreed partners that may stimulate GT. The second goal is to modify the frequency of recombination during maize meiosis, for example increased frequencies may reduce population sizes that breeders need to screen to identify favourable gene combinations and improved varieties.

Q2- What technologies do you use?

We are developing a test system to measure the frequency of GT in maize. This system is based on the reconstitution, by GT, of a selectable marker gene (nptII). Plant genetic engineering is used to introduce genes that may stimulate GT. Potential GT events are analysed using standard molecular biology techniques. Similarly our attempts to change meiotic recombination frequencies rely on the introduction of candidate genes via maize transformation.

Q3- What are your main achievements so far in RECBREED?

We have validated our GT test system by the molecular analysis of regenerated maize plants that are now resistant to the selectable marker. These plants have a reconstituted and active nptII gene.

Q4- How do you envision the impact of your work on plant breeding and biotechnology?

GT is likely to become the method of choice in the future to produce transgenic GM lines and also in crop plants to modify the genome via gene surgery. If an efficient GT system is in place for crop plants such as maize, the cost of producing genetically modified plants should be reduced. For example significantly fewer transformation events should be required if transgenes are integrated at a predefined locus which has been selected as a good site for transgene expression. It is also likely that public and authorities will prefer GM plants made via precise GT technologies, not necessarily purely on rational arguments. Precise gene replacement is not a tool that is currently readily available in crops. Efficient GT will make such gene surgery a powerful new tool for the future to cleanly replace ‘bad’ genes with improved versions.
Similarly tools to accelerate plant breeding via increasing meiotic recombination rates are not available to the breeder. Smaller population sizes will allow the breeder to screen the progeny of more genetic crosses thus improving the chances of identifying novel gene combinations, ie a new improved variety.

Thus efficient tools for developing GT and modifying recombination rates will impact plant breeding and biotechnology by aiding the creation of new improved varieties and deliver them to the marketplace more rapidly.

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