Mid-Term Report Summary - COMREC (Designed Plant Breeding by Control of Meiotic Recombination) Agriculture depends on breeding to produce crop plants that harbour high yields and are resistant to stress and pathogens. Breeders try to achieve this goal by combining desirable and eliminating unfavourable traits. Changes in genetic linkage are based on meiotic recombination. Although molecular techniques for the transfer of single traits have been developed in the last decades hardly any effort has been undertaken to control the exchange between parental genomes as such. By applying new molecular tools to control recombination the current project aims to establish a new kind of “designed” plant breeding. Thus, not only transfer or elimination of specific traits should become possible in a programmable way, but also the access to the complete gene pool of natural species. Two different levels of molecular control are addressed in the project: the induction of recombination at predefined specific sites in the genome and the regulation of the level of genome-wide exchange. For induction of recombination we apply specifically tailored artificial nucleases. At the start of the project we tried to achieve the induction of recombination using zinc finger nucleases (ZFNs). Unfortunately however, the cutting efficiency of the ZFNs applied was too low so that no significant effect on inheritance could be achieved. Therefore we adopted the transcription activator-like effector nucleases (TALENs) for the same propose, that only became available after the start of the project. Again the cutting efficiency was too low for inducing meiotic recombination in our hands. Recently, the bacterial CRISPR/Cas system was developed as tool for genome engineering. We adopted the system to induce DSB but also nicks in the genomic DNA of Arabidopsis and could already demonstrate that in somatic cells recombination is dramatically enhanced. In our hands the cutting efficiency was - as determined by deep sequencing - at least one order higher than with the other nucleases and we were also able to demonstrate that somatic mutations can be transferred through the germ line at high frequency. Experiments have already been set up to test whether the CRISPR/Cas system can also induce meiotic recombination and first results should be available till end of the year.The efficiency of mixing parental genomes during meiosis is modulated by factors involved in the resolution of recombination intermediates into gene conversions or crossovers. We were able to identify two factors, FANCM and MHF1 that suppress ectopic interactions during recombination in Arabidopsis and are thus important for the quality control of meiotic recombination. Interestingly in humans the mutation of homologues of these factors result in the severe genetic disorder Fanconi Anaemia. Moreover, we further characterized the detailed role of the RTR complex in the dissolution of recombination intermediates. In upcoming experiments we will test whether we will be able to influence the relation between crossovers and gene conversions in the respective mutated RTR backgrounds by expression of heterologous enzymes. Thus, a much more efficient mixing of parental genomes might be possible.
