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Final Report Summary - COMREC (Control of meiotic recombination: from Arabidopsis to crops)

The scientific programme of COMREC was organized into three integrated workpackages (WP2-WP4) which addressed particular factors that influence plant meiotic recombination. The WPs comprised 13 ESR projects that contribute to one or more of the WPs. Below we provide a brief overview and
summarize recent progress of the individual ESR projects.

WP2 Influence of chromatin landscape on crossover (CO) distribution (Leader Schlögelhofer UoV) ESR 1 Divya Nageswaren used a genetic screen to isolate seven mutant lines that exhibit modified patterns of recombination. Four lines with elevated levels (hcr1-4) and three with reduced recombination (lcr1-3). Backcross F2 mapping populations were developed for hcr1, hcr2, hcr3 and lcr- 3 and mutant libraries constructed based on illumina True-seq protocol. Sequencing data of hcr1, hcr2 and hcr3 were analysed using SAMtools allowing identification of the causal SNPS and putative candidate genes. hcr1 data has implicated a protein phosphatase (designated HCR1) which is linked to DNA repair. In this review period, this was confirmed by complementation of a hcr1 T-DNA insertion line. Initial epistasis studies suggest that HCR1 has an additive effect with the previously described recombination modifiers fancm and zip4. Further recent research by partner P2 (UoC) involving ESR1 in collaboration with P1/ESR7 (UoB), has shown that the recombination protein HEI10 modulates crossover frequency (Genes Dev. 2017, PMID: 28223312).

ESR2 Jason Sims (UoV) investigated what differentiates recombinationally “hot” chromosomal regions from “cold” regions. He created 3 artificial cold-spots by ectopically integrating rDNA repeat sequences into in three different loci. One of these (R4#15) was shown to modulate recombination. Recent results indicate an increase of recombination in the adjacent chromosomal region that appears to be accompanied, based on a reduction in RAD51 foci, by a reduction in recombination in the close proximity of the rDNA insert by “dragging” the genomic regions adjacent to it in the nucleolus. This suggests a new strategy for silencing recombination in specific genomic regions. We revealed meiotic DNA breaks in the NORs are not exclusively generated by SPO11 and may be a regular by-product of replication and possibly be repaired by non-homologous end-joining.

ESR3 Jihed Chouref (UoA) following extensive optimization a protocol was developed for chromatin immunoprecipitation (ChIP) protocol for mapping of recombination hotspots on tomato chromosomes. In particular, standard methods were modified to maximize recovery of input DNA which was identified
as the bottleneck in the procedure. Experiments to map MLH1 sites that mark COs were conducted and are being optimized along with those mapping binding of the recombinase DMC1. Initial results of this ChIP-seq shows that the reads of MLH1 overlap with most of the genes of the tomato genome.

WP3 Coordination of meiotic progression and crossover (CO) control (Leader Houben IPK) ESR4 Pablo Parra (UCM) investigated how polyploid plants adapt meiosis to ensure accurate chromosome segregation. Five generations of neo-tetraploid lines (lines 3151 and 3432) of A. thaliana were characterized cytologically and chiasma frequencies determined. Material has been retained from each generation in preparation fro the next stage of the project which will involve RNA seq and/or quantitative PCRs to assess possible differences in gene expression levels (genes involved in meiotic
homologous recombination) between generations in both tetraploid lines. To determine the role of key meiotic genes in the diploidisation process of polyploids in A. thaliana polyploids derived zip4, mlh3, mer3, asy1, asy3, mus81, msh5, hei10, fancm and asy1/asy3 were isolated. Recent analysis shows that in most cases the polyploids exhibit a 2-fold increase in chiamsa frequency relative to their diploid progenitors. The exceptions are fancm, which is unchanged and hei10, which shows an elevation of nearly 3.5-fold. The basis of this continues to be investigated.

ESR5 Maria Prusicki (UoH) investigated how entry into meiosis and subsequent progression is controlled focusing on the crucial role phosphorylation of meiotic proteins plays in regulating meiotic entry/progression. Having successfully developed for the first time a system for live imaging of meiosis
in A. thaliana ESR5 used time-lapse analysis to accurately quantify meiotic progression. 17 hallmark (from H0 to H16); stages were defined using a combination of 6 morphological criteria: meiocyte cell shape, nuclear position, position and shape of the nucleolus, level of chromatin condensation,
microtubule array and ploidy of the tapetum cells. This system has allowed us to gain an accurate quantitative description of meiotic progression and the methodology was used to assess the impact of mutations in key meiotic and cell-cycle genes on prophase I progression. These include the A-type cyclin
TAM1 and the recombination protein MLH3.The impact of mutations in meiotic and cell cycle genes on meiotic progression have been analysed using this novel system. The results will be published in mid- 2018. Related studies involving ESR5, who conducted the confocal microscopy analysis of the mutants used in the work, have shown that the CDKA substrate RETINOBLASTOMA RELATED1, the Arabidopsis homolog of the human tumour suppressor gene RBR controls entry into meiosis by suppression of the stem cell factor WUSCHEL. This is now published: Science, 2017, PMID: 28450583).

ESR6 Gunjita Singh (CNRS-UBP) ESR6 (CNRS-UBP) dissected the relative contribution of the RAD51 and DMC1 recombinases during meiosis. Studies of the recombinases RAD51 and DMC1 using a fluorescent seed recombination assay with markers covering different genetic intervals. have shown the latter is the primary meiotic DNA strand-exchange protein. DMC1 catalyses repair of all meiotic DNA breaks in the presence of a catalytically-dead RAD51 variant (PLoS ONE 12: e0183006–16). Previous work indicates some, albeit incomplete homolog synapsis in the absence of RAD51 and XRCC3 accompanied by the presence of many short ZYP1 fibres indicating short stretches of Synaptonemal Complex (SC). The partial synapsis is both SPO11- and DMC1-dependent and involves peri- centromeres, showing that DMC1 is able to (at least partially) drive synapsis in peri- centromeres in the absence of RAD51. During this reporting period, in an effort to better characterize this immunofluorescence and super-resolution microscopy (SIM) using ASY1, ZYP1 and CENH3 antisera were carried out in WT, rad51 and xrcc3 mutants. Although, short ZYP1 fibres at the centromeres in the mutants the rule. Nevertheless, SIM confirmed the presence of stretches of 4-chromatid fibres in xrcc3 plants suggesting some synapsis does occur. In future work the nature of the RAD51-independent partial meiotic chromosome synapsis will be analysed further.

ESR7 Marina Martinez-Garcia (UoB) investigated the influence that programmed remodelling of the chromosome axes has on CO control. Analysis of a hypomorphic topoisomerase II mutant (topii-1) and TOPII-RNAi knockdown lines have revealed that this protein is involved in the resolution of chromosome interlocks during meiotic prophase I. Recently the study has gone on to show that there are at least two mechanisms involved in interlock removal. In addition to TOPII, analysis of a nucleoporin mutant nup136 (supplied by P9, UCM), there is a requirement for chromosome movement. This work is currently under review for the Journal of Cell Biology. Also the study found that TOPII has a role during replication, loss of the protein leads to mitotic DNA breaks which affect developmental processes such as root growth. Several transgenic Arabidopsis lines expressing ASY1-eYFP proteins in which the phosphorylation sites have been mutated have been constructed and are being analysed. Initial experiments indicate that incontrast to budding yeast the conserved threonine at position 295 (T295) is not essential for protein function. However, during this reporting period site-directed mutagenesis of putative ATM/ATR kinase sites in the C-terminus of the chromosome axis protein ASY1 suggests a route to altering CO distribution.
Chiasma frequencies in six Arabidopsis ecotypes have been assessed at 20oC providing the basis for further studies to determine the impact of increased temperature on COs.

ESR8 Mateusz Zelkowski (IPK) investigated the role of the SMC5/SMC6 complex that has recently been shown to be important for meiotic recombination as well as somatic DNA repair. The objective was to study and compare SMC5/SMC6 function in Arabidopsis and in Luzula elegans, which is unusual in that it is a holocentric species Analysis of the holocentric chromosome structure of L. elegans using a sister chromatid exchange assay revealed a similar sister chromatid arrangement to that in monocentric species but with elevated levels of sister chromatid exchange (Cytogenet Genome Res. 2016, PMID: 27454585). To evaluate the function of SMC5/6 proteins different SMC5/6 T-DNA insertion lines were selected. Only heterozygous T-DNA mutants were found for NSE3 and SMC5 suggesting that their complete loss results in lethality. NSE1, NSE4A and NSE4B were found to be required for fertility, NSE1 and SMC5 for plant development, and NSE4A for normal mitosis and meiosis. Analysis of the
homozygous nse4A T-DNA mutant GK768H08 reveals defects in mitosis and meiosis, such as chromosome fragmentation, lagging chromosomes and anaphase bridges. The dynamics and localization of NSE4A was analysed using a 35S::EYFP::NSE4A fusion protein and an antibody against NSE4A. Both methods resulted in a dispersed labelling within the euchromatin in interphase nuclei. A similar labelling pattern was obtained for the subunits NSE3 and SMC5 by immunolabelling and 35S::NSE3::EYFP localization.

WP4 Manipulating meiotic recombination in crops (Leader de Jong UoW) WP4 focused on translational research in a number of key species.
ESR9 Mikel Arrieta (JHI) investigated the use of elevated temperature to modify CO distribution in barley. Use of single seed descent trays to limit growth and hence better synchronize meiosis showed that it is possible to produce a significant change in recombination through the use of a short targeted stress without some of the negative fertility effects seen in previous experiments. Next, this system was combined with an Agilent barley microarray to determine the response of meiotic genes to heat stress. Following on from investigating the effects of sustained temperature increase to 30oC. The effect of short exposure (11 days) to increased temperature and also cold-shock (4oC) was studied. This highlighted a number of genes up- and down-regulated during pre-meiotic and early meiotic stages that were the subject of further study. The results of these subsequent experiments have indicated that it is possible to produce a significant change in the distribution of recombination through the use of a short targeted heat stress without some of the negative fertility effects seen in previous experiments. Recentstudies following the release of the barley genome sequence and the development of a 50K SNP Illumina genotyping chip has allowed ESR9 to generate a considerable amount of information in the last
year of the project allowing him to delineate the physical positions of crossovers with much greater accuracy than before. The final year has also included a field trial of material generated from the heat stress experiments to investigate the effect of treatments on genetic variation observed for agronomic traits in the subsequent generations in order to establish the utility for breeding of the approach used. A parallel study in Sugar Beet has been initiated in collaboration with industrial associate partner SES VanderHave. This was intiated during a period of secondment of ESR9 to the company.

ESR 10 Adrián Gonzalo (INRA). has been shown for the first time that the FANCM gene has an anti-CO activity in a Brassica crop species as in Arabidopsis. Brassica fancm mutants thus exhibit elevated recombination. To the best of our knowledge, this is the first example of a translational biology approach to increase CO frequencies in crops. Recent studies in allopolyploid Brassica napus indicate that COs between homoeologous chromosomes (i.e. inherited from parental species) originate almost exclusively from the interference dependent (class I) pathway. In addition crossover frequency between homoeologous chromosomes is sensitive to MSH4 dosage, while CO frequency between homologues is
not. Overall the results indicate that meiotic adaptation to allopolyploidy mainly involves the class I CO pathway and could be achieved by limiting its efficiency (e.g. by decreasing gene copy number).

ESR11 Amy Whitbread (KIT) investigated how readily information obtained in Arabidopsis meiosis can be translated to tomato. The aim was to use CRISPA/Cas9 technology to specifically target key meiotic genes that modulate CO frequency in Arabidopsis. The Solanum lycopersicum genes for targeting using Cas9-mediated mutagenesis were focused on homologues of the RTR complex partners AtRMI1 and AtTOP3α, as other groups are working on similar approaches and are further progressed for example in the mutagenesis of AtFANCM. The broad knowledge of our group concerning the RTR complex in Arabidopsis and its essential and CO limiting functions in Arabidopsis meiosis, reinforce the choice of these two genes.

Bioinformatics analyses for the identification of AtRMI1 and AtTOP3α homologs were performed successfully and monoclonal peptide antibodies for both AtTOP3α and AtRMI1 proteins were produced and tested for their efficiency at localising these proteins during wild-type meiosis of both A.
thaliana and Brassica oleracea meiocytes using immunocytology techniques. An additional approach to analyzing meiotic recombination in tomato was implemented involving SPO11 and Cas9 fusion constructs. This approach involved using a catalytically inactive Cas9 (dCas9) fused to a tomato SPO11
homologous protein, to determine whether SPO11 induced DSBs can increase meiotic recombination and crossover formation. Subsequent additional crossovers will be determined utilising multi-locus KASP genotyping, using sequenced SNPs between two Micro-Tom lines obtained from Christophe Rothan (INRA). Both Micro-Tom lines were established successfully and all relevant techniques could be applied to both. The cloning of all constructs has been carried out, and were transformed into the Micro-Tom lines. Furthermore, Cas9-mediated mutagenesis of the SPO11 homologue in Micro-Tom is currently being carried out to ensure complementation of SPO11 is possible with transformation of the fusion
constructs into a knockout mutant.

ESR12 Vanesa Calvo (UoW) has been developing an approach established by UoW in conjunction with RijkZwaan (AP1). This novel “Reverse Breeding” technique based on inducing achiasmate meiosis, has a number of potentially important applications such as the generation of non-recombinant chromosome substitution lines. Virus induced gene silencing (VIGs) has been used to assess how silencing of specific recombination pathway genes may provide a method to maintain elite varieties. As a result she has shown that VIGS is a useful technique to: a) downregulate crossover recombination; b) upregulate crossover recombination ; c) omit the second meiotic division; d) Eliminate sister chromatid cohesion;
e) downregulate the expression of up to three genes simultaneously. The VIGs-based methodology is now being developed for tomato in collaboration with Rijk Zwaan. Following a successful secondment in the company, ESR12 has progressed working on crops, and she, together with the company, has
shown that VIGS-mediated alteration of recombination is feasible in crops too.

ESR13 Sevgin Demirci has investigated the effect that homo(e)ologous (closely related but not identical) sequences have on meiotic recombination and introgression. In order to access the full genetic variation that is available to breeders it is highly desirable to introgress chromosomal regions from close relatives. ESR13 harnessed bioinformatics of DNA around the recombination sites in tomato and interspecific hybrids using sequence information from S. lycopersicon x S. pimpenellifolium introgression lines and recombinant inbred lines (RILs). Building on work in the previous period a robust statistical methodology was developed. This allowed detection of hot regions of recombination showing 3-6 recombination events in 100 kb region in 52 RILs. Moreover, 20 recombination coldspots each larger than 1 Mb were identified in euchromatin regions. The cold spots indicate a candidate introgression region between two parents of RILs. Some of this work has now been published (Plant J 2017, PMID: 277274250.

Together with receiving specialist research training the COMREC ESRs completed a number of network training activities (WP1). These included a course in Bioinformatics presented by partner 11 (UoW-PRI). Workshops in advanced research techniques, grant writing and paper writing were held in conjunction with the 1st annual meeting (El Escorial, Madrid). Following the 2nd Annual meeting at IPK Gatersleben (May 2016) a Workshop on Commercial Plant Breeding was organized by the COMREC associate partners KWS and RijkZwaan. This was followed by a Workshop on Business Skills and Entrepreneurship. The first part of the workshop involved an introduction to patents and intellectual property presented by Dr Verelst, RijkZwaan and Dr Schrell (patents lawyer) IPK. Business skills and entrepreneurship were than presented and discussed by Dr Bastianelli from the biotech company Meiogenix and Mr Munoz a founder of the mobile phone and software company BQ).

The ESRs have undertaken their secondments as planned. These included the COMREC non-academic associate partners KWS (ESRs 7 and 10), SESVanderHave (ESRs 1 and 9) and Rijkzwaan (ESRs 3 and 11) which have led to new on-going translational research collaborations.

Regarding dissemination (WP5) of the ESRs have to date contributed to 8 published papers with a further 18 anticipated. Notably, these have included important publications on the control of meiotic entry (Science, 2017, PMID: 28450583) and recombination (Genes Dev. 2017, PMID: 28223312). The
ESRs have presented 87 talks and/or posters at International, local conferences and invited seminars, including the Gordon Research Conference on Meiosis (2016, USA); 21st International Chromosome Conference (Brazil); Plant and Animal Genome conference (2017 USA; with invitations for 2018) and the European Meiosis Conferences (Oxford, 2015; Croatia, 2017). ESRs 7 and 9 won awards for their oral and poster presentations respectively.

ESRs participated in 16 dissemination and outreach events to the general public including Junior Plant Scientist for the day” Thinktank Birmingham Science Museum and the Brownie and Guide Science investigator Day: Women Plant Scientists Now and Then. ESR4 has produced a project related video https://www.youtube.com/watch?v=oQVDvq6p6IQ&feature=youtu.be. Further details of all the ESRs dissemination are available at the COMREC website.

In conclusion the project has met all its planned milestones and deliverables. So far 4 ESRs have been awarded PhDs with the remainder on course to
do so in 2018. Thus COMREC has resulted in a cohort of 13 highly trained ESRs and through working alongside stakeholder breeding companies COMREC has provided them with insight into the major challenges that confront the plant breeding industry over the next 5-10 years and the necessary skillset to address them. More information about COMREC can be found at www.birmingham.ac.uk/comrec or by contacting the project manager Rachael Batchelor (r.a.batchelor@bham.ac.uk).

Reported by

THE UNIVERSITY OF BIRMINGHAM
United Kingdom

Subjects

Life Sciences
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