Arabidopsis HEAT SHOCK FACTOR BINDING PROTEIN is required to limit meiotic crossovers and HEI10 transcription.

The number of meiotic crossovers is tightly controlled and most depend on pro-crossover ZMM proteins, such as the E3 ligase HEI10. Despite the importance of HEI10 dosage for crossover formation, how HEI10 transcription is controlled remains unexplored. In a forward genetic screen using a fluorescent crossover reporter in Arabidopsis thaliana, we identify heat shock factor binding protein (HSBP) as a repressor of HEI10 transcription and crossover numbers. Using genome-wide crossover mapping and cytogenetics, we show that hsbp mutations or meiotic HSBP knockdowns increase ZMM-dependent crossovers toward the telomeres, mirroring the effects of HEI10 overexpression. Through RNA sequencing, DNA methylome, and chromatin immunoprecipitation analysis, we reveal that HSBP is required to repress HEI10 transcription by binding with heat shock factors (HSFs) at the HEI10 promoter and maintaining DNA methylation over the HEI10 5' untranslated region. Our findings provide insights into how the temperature response regulator HSBP restricts meiotic HEI10 transcription and crossover number by attenuating HSF activity.

Differentiated function and localisation of SPO11-1 and PRD3 on the chromosome axis during meiotic DSB formation in Arabidopsis thaliana.

During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis.

Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination.

During meiosis, homologous chromosomes undergo crossover recombination, which creates genetic diversity and balances homolog segregation. Despite these critical functions, crossover frequency varies extensively within and between species. Although natural crossover recombination modifier loci have been detected in plants, causal genes have remained elusive. Using natural Arabidopsis thaliana accessions, we identified two major recombination quantitative trait loci (rQTLs) that explain 56.9% of crossover variation in Col×Ler F2 populations. We mapped rQTL1 to semidominant polymorphisms in HEI10, which encodes a conserved ubiquitin E3 ligase that regulates crossovers. Null hei10 mutants are haploinsufficient, and, using genome-wide mapping and immunocytology, we show that transformation of additional HEI10 copies is sufficient to more than double euchromatic crossovers. However, heterochromatic centromeres remained recombination-suppressed. The strongest HEI10-mediated crossover increases occur in subtelomeric euchromatin, which is reminiscent of sex differences in Arabidopsis recombination. Our work reveals that HEI10 naturally limits Arabidopsis crossovers and has the potential to influence the response to selection.

Interacting Genomic Landscapes of REC8-Cohesin, Chromatin, and Meiotic Recombination in Arabidopsis.

Meiosis recombines genetic variation and influences eukaryote genome evolution. During meiosis, DNA double-strand breaks (DSBs) enter interhomolog repair to yield crossovers and noncrossovers. DSB repair occurs as replicated sister chromatids are connected to a polymerized axis. Cohesin rings containing the REC8 kleisin subunit bind sister chromatids and anchor chromosomes to the axis. Here, we report the genomic landscape of REC8 using chromatin immunoprecipitation sequencing (ChIP-seq) in Arabidopsis (Arabidopsis thaliana). REC8 associates with regions of high nucleosome occupancy in multiple chromatin states, including histone methylation at H3K4 (expressed genes), H3K27 (silent genes), and H3K9 (silent transposons). REC8 enrichment is associated with suppression of meiotic DSBs and crossovers at the chromosome and fine scales. As REC8 enrichment is greatest in transposon-dense heterochromatin, we repeated ChIP-seq in kyp suvh5 suvh6 H3K9me2 mutants. Surprisingly, REC8 enrichment is maintained in kyp suvh5 suvh6 heterochromatin and no defects in centromeric cohesion were observed. REC8 occupancy within genes anti-correlates with transcription and is reduced in COPIA transposons that reactivate expression in kyp suvh5 suvh6 Abnormal axis structures form in rec8 that recruit DSB-associated protein foci and undergo synapsis, which is followed by chromosome fragmentation. Therefore, REC8 occupancy correlates with multiple chromatin states and is required to organize meiotic chromosome architecture and interhomolog recombination.

DeepTetrad: high-throughput image analysis of meiotic tetrads by deep learning in Arabidopsis thaliana.

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate the genetic dissection of mechanisms that control meiotic recombination.

Epigenetic activation of meiotic recombination near Arabidopsis thaliana centromeres via loss of H3K9me2 and non-CG DNA methylation.

Eukaryotic centromeres contain the kinetochore, which connects chromosomes to the spindle allowing segregation. During meiosis, centromeres are suppressed for inter-homolog crossover, as recombination in these regions can cause chromosome missegregation and aneuploidy. Plant centromeres are surrounded by transposon-dense pericentromeric heterochromatin that is epigenetically silenced by histone 3 lysine 9 dimethylation (H3K9me2), and DNA methylation in CG and non-CG sequence contexts. However, the role of these chromatin modifications in control of meiotic recombination in the pericentromeres is not fully understood. Here, we show that disruption of Arabidopsis thaliana H3K9me2 and non-CG DNA methylation pathways, for example, via mutation of the H3K9 methyltransferase genes KYP/SUVH4 SUVH5 SUVH6, or the CHG DNA methyltransferase gene CMT3, increases meiotic recombination in proximity to the centromeres. Using immunocytological detection of MLH1 foci and genotyping by sequencing of recombinant plants, we observe that H3K9me2 and non-CG DNA methylation pathway mutants show increased pericentromeric crossovers. Increased pericentromeric recombination in H3K9me2/non-CG mutants occurs in hybrid and inbred backgrounds and likely involves contributions from both the interfering and noninterfering crossover repair pathways. We also show that meiotic DNA double-strand breaks (DSBs) increase in H3K9me2/non-CG mutants within the pericentromeres, via purification and sequencing of SPO11-1-oligonucleotides. Therefore, H3K9me2 and non-CG DNA methylation exert a repressive effect on both meiotic DSB and crossover formation in plant pericentromeric heterochromatin. Our results may account for selection of enhancer trap Dissociation (Ds) transposons into the CMT3 gene by recombination with proximal transposon launch-pads.

Nucleosomes and DNA methylation shape meiotic DSB frequency in Arabidopsis thaliana transposons and gene regulatory regions.

Meiotic recombination initiates from DNA double-strand breaks (DSBs) generated by SPO11 topoisomerase-like complexes. Meiotic DSB frequency varies extensively along eukaryotic chromosomes, with hotspots controlled by chromatin and DNA sequence. To map meiotic DSBs throughout a plant genome, we purified and sequenced Arabidopsis thaliana SPO11-1-oligonucleotides. SPO11-1-oligos are elevated in gene promoters, terminators, and introns, which is driven by AT-sequence richness that excludes nucleosomes and allows SPO11-1 access. A positive relationship was observed between SPO11-1-oligos and crossovers genome-wide, although fine-scale correlations were weaker. This may reflect the influence of interhomolog polymorphism on crossover formation, downstream from DSB formation. Although H3K4me3 is enriched in proximity to SPO11-1-oligo hotspots at gene 5' ends, H3K4me3 levels do not correlate with DSBs. Repetitive transposons are thought to be recombination silenced during meiosis, to prevent nonallelic interactions and genome instability. Unexpectedly, we found high SPO11-1-oligo levels in nucleosome-depleted Helitron/Pogo/Tc1/Mariner DNA transposons, whereas retrotransposons were coldspots. High SPO11-1-oligo transposons are enriched within gene regulatory regions and in proximity to immunity genes, suggesting a role as recombination enhancers. As transposon mobility in plant genomes is restricted by DNA methylation, we used the met1 DNA methyltransferase mutant to investigate the role of heterochromatin in SPO11-1-oligo distributions. Epigenetic activation of meiotic DSBs in proximity to centromeres and transposons occurred in met1 mutants, coincident with reduced nucleosome occupancy, gain of transcription, and H3K4me3. Together, our work reveals a complex relationship between chromatin and meiotic DSBs within A. thaliana genes and transposons, with significance for the diversity and evolution of plant genomes.

Interhomolog polymorphism shapes meiotic crossover within the Arabidopsis RAC1 and RPP13 disease resistance genes.

During meiosis, chromosomes undergo DNA double-strand breaks (DSBs), which can be repaired using a homologous chromosome to produce crossovers. Meiotic recombination frequency is variable along chromosomes and tends to concentrate in narrow hotspots. We mapped crossover hotspots located in the Arabidopsis thaliana RAC1 and RPP13 disease resistance genes, using varying haplotypic combinations. We observed a negative non-linear relationship between interhomolog divergence and crossover frequency within the hotspots, consistent with polymorphism locally suppressing crossover repair of DSBs. The fancm, recq4a recq4b, figl1 and msh2 mutants, or lines with increased HEI10 dosage, are known to show increased crossovers throughout the genome. Surprisingly, RAC1 crossovers were either unchanged or decreased in these genetic backgrounds, showing that chromosome location and local chromatin environment are important for regulation of crossover activity. We employed deep sequencing of crossovers to examine recombination topology within RAC1, in wild type, fancm, recq4a recq4b and fancm recq4a recq4b backgrounds. The RAC1 recombination landscape was broadly conserved in the anti-crossover mutants and showed a negative relationship with interhomolog divergence. However, crossovers at the RAC1 5'-end were relatively suppressed in recq4a recq4b backgrounds, further indicating that local context may influence recombination outcomes. Our results demonstrate the importance of interhomolog divergence in shaping recombination within plant disease resistance genes and crossover hotspots.

Heterogeneous transposable elements as silencers, enhancers and targets of meiotic recombination.

During meiosis, DNA double-strand breaks are initiated by the topoisomerase-like enzyme SPO11 and are repaired by inter-sister chromatid and inter-homologue DNA repair pathways. Genome-wide maps of initiating DNA double-strand breaks and inter-homologue repair events are now available for a number of mammalian, fungal and plant species. In mammals, PRDM9 specifies the location of meiotic recombination initiation via recognition of specific DNA sequence motifs by its C2H2 zinc finger array. In fungi and plants, meiotic recombination appears to be initiated less discriminately in accessible chromatin, including at gene promoters. Generally, meiotic crossover is suppressed in highly repetitive genomic regions that are made up of transposable elements (TEs), to prevent deleterious non-allelic homologous recombination events. However, recent and older studies have revealed intriguing relationships between meiotic recombination initiation and repair, and transposable elements. For instance, gene conversion events have been detected in maize centromeric retroelements, mouse MULE-MuDR DNA transposons undergo substantial meiotic recombination initiation, Arabidopsis Helitron TEs are among the hottest of recombination initiation hotspots, and human TE sequences can modify the crossover rate at adjacent PRDM9 motifs in cis. Here, we summarize the relationship between meiotic recombination and TEs, discuss recent insights from highly divergent eukaryotes and highlight outstanding questions in the field.

Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes.

Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.

Regulation of MicroRNA-Mediated Developmental Changes by the SWR1 Chromatin Remodeling Complex.

The ATP-dependent SWR1 chromatin remodeling complex (SWR1-C) exchanges the histone H2A-H2B dimer with the H2A.Z-H2B dimer, producing variant nucleosomes. Arabidopsis thaliana SWR1-C contributes to the active transcription of many genes, but also to the repression of genes that respond to environmental and developmental stimuli. Unlike other higher eukaryotic H2A.Z deposition mutants (which are embryonically lethal), Arabidopsis SWR1-C component mutants, including arp6, survive and display a pleiotropic developmental phenotype. However, the molecular mechanisms of early flowering, leaf serration, and the production of extra petals in arp6 have not been completely elucidated. We report here that SWR1-C is required for miRNA-mediated developmental control via transcriptional regulation. In the mutants of the components of SWR1-C such as arp6, sef, and pie1, miR156 and miR164 levels are reduced at the transcriptional level, which results in the accumulation of target mRNAs and associated morphological changes. Sequencing of small RNA libraries confirmed that many miRNAs including miR156 decreased in arp6, though some miRNAs increased. The arp6 mutation suppresses the accumulation of not only unprocessed primary miRNAs, but also miRNA-regulated mRNAs in miRNA processing mutants, hyl1 and serrate, which suggests that arp6 has a transcriptional effect on both miRNAs and their targets. We consistently detected that the arp6 mutant exhibits increased nucleosome occupancy at the tested MIR gene promoters, indicating that SWR1-C contributes to transcriptional activation via nucleosome dynamics. Our findings suggest that SWR1-C contributes to the fine control of plant development by generating a balance between miRNAs and target mRNAs at the transcriptional level.

Meiotic recombination hotspots - a comparative view.

During meiosis homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover. Meiotic recombination has a profound effect on patterns of genetic variation and is an important tool during crop breeding. Crossovers initiate from programmed DNA double-stranded breaks that are processed to form single-stranded DNA, which can invade a homologous chromosome. Strand invasion events mature into double Holliday junctions that can be resolved as crossovers. Extensive variation in the frequency of meiotic recombination occurs along chromosomes and is typically focused in narrow hotspots, observed both at the level of DNA breaks and final crossovers. We review methodologies to profile hotspots at different steps of the meiotic recombination pathway that have been used in different eukaryote species. We then discuss what these studies have revealed concerning specification of hotspot locations and activity and the contributions of both genetic and epigenetic factors. Understanding hotspots is important for interpreting patterns of genetic variation in populations and how eukaryotic genomes evolve. In addition, manipulation of hotspots will allow us to accelerate crop breeding, where meiotic recombination distributions can be limiting.

Telomere stability and development of ctc1 mutants are rescued by inhibition of EJ recombination pathways in a telomerase-dependent manner.

The telomeres of linear eukaryotic chromosomes are protected by caps consisting of evolutionarily conserved nucleoprotein complexes. Telomere dysfunction leads to recombination of chromosome ends and this can result in fusions which initiate chromosomal breakage-fusion-bridge cycles, causing genomic instability and potentially cell death or cancer. We hypothesize that in the absence of the recombination pathways implicated in these fusions, deprotected chromosome ends will instead be eroded by nucleases, also leading to the loss of genes and cell death. In this work, we set out to specifically test this hypothesis in the plant, Arabidopsis. Telomere protection in Arabidopsis implicates KU and CST and their absence leads to chromosome fusions, severe genomic instability and dramatic developmental defects. We have analysed the involvement of end-joining recombination pathways in telomere fusions and the consequences of this on genomic instability and growth. Strikingly, the absence of the multiple end-joining pathways eliminates chromosome fusion and restores normal growth and development to cst ku80 mutant plants. It is thus the chromosomal fusions, per se, which are the underlying cause of the severe developmental defects. This rescue is mediated by telomerase-dependent telomere extension, revealing a competition between telomerase and end-joining recombination proteins for access to deprotected telomeres.

Epigenetic remodeling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants.

Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes.

The FRIGIDA complex activates transcription of FLC, a strong flowering repressor in Arabidopsis, by recruiting chromatin modification factors.

The flowering of Arabidopsis thaliana winter annuals is delayed until the subsequent spring by the strong floral repressor FLOWERING LOCUS C (FLC). FRIGIDA (FRI) activates the transcription of FLC, but the molecular mechanism remains elusive. The fri mutation causes early flowering with reduced FLC expression similar to frl1, fes1, suf4, and flx, which are mutants of FLC-specific regulators. Here, we report that FRI acts as a scaffold protein interacting with FRL1, FES1, SUF4, and FLX to form a transcription activator complex (FRI-C). Each component of FRI-C has a specialized function. SUF4 binds to a cis-element of the FLC promoter, FLX and FES1 have transcriptional activation potential, and FRL1 and FES1 stabilize the complex. FRI-C recruits a general transcription factor, a TAF14 homolog, and chromatin modification factors, the SWR1 complex and SET2 homolog. Complex formation was confirmed by the immunoprecipitation of FRI-associated proteins followed by mass spectrometric analysis. Our results provide insight into how a specific transcription activator recruits chromatin modifiers to regulate a key flowering gene.

Control of meiotic crossover interference by a proteolytic chaperone network.

Meiosis is a specialized eukaryotic division that produces genetically diverse gametes for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal exchanges, called crossovers, which recombine genetic variation. Meiotic crossovers are stringently controlled with at least one obligate exchange forming per chromosome pair, while closely spaced crossovers are inhibited by interference. In Arabidopsis, crossover positions can be explained by a diffusion-mediated coarsening model, in which large, approximately evenly spaced foci of the pro-crossover E3 ligase HEI10 grow at the expense of smaller, closely spaced clusters. However, the mechanisms that control HEI10 dynamics during meiosis remain unclear. Here, through a forward genetic screen in Arabidopsis, we identified high crossover rate3 (hcr3), a dominant-negative mutant that reduces crossover interference and increases crossovers genome-wide. HCR3 encodes J3, a co-chaperone related to HSP40, which acts to target protein aggregates and biomolecular condensates to the disassembly chaperone HSP70, thereby promoting proteasomal degradation. Consistently, we show that a network of HCR3 and HSP70 chaperones facilitates proteolysis of HEI10, thereby regulating interference and the recombination landscape. These results reveal a new role for the HSP40/J3-HSP70 chaperones in regulating chromosome-wide dynamics of recombination via control of HEI10 proteolysis.

The LBD11-ROS feedback regulatory loop modulates vascular cambium proliferation and secondary growth in Arabidopsis.

Vascular cambium produces the phloem and xylem, vascular tissues that transport resources and provide mechanical support, making it an ideal target for crop improvement. However, much remains unknown about how vascular cambium proliferates. In this study, through pharmaceutical and genetic manipulation of reactive oxygen species (ROS) maxima, we demonstrate a direct link between levels of ROS and activity of LATERAL ORGAN BOUNDARIES DOMAIN 11 (LBD11) in maintaining vascular cambium activity. LBD11 activates the transcription of several key ROS metabolic genes, including PEROXIDASE 71 and RESPIRATORY BURST OXIDASE HOMOLOGS D and F, to generate local ROS maxima in cambium, which in turn enhance the proliferation of cambial cells. In a negative feedback mechanism, higher ROS levels then repress LBD11 expression and maintain the balance of cambial cell proliferation. Our findings thus reveal the role of a novel LBD11/ROS-dependent feedback regulatory system in maintaining vascular cambium-specific redox homeostasis and radial growth in plants.

DDM1-mediated gene body DNA methylation is associated with inducible activation of defense-related genes in Arabidopsis.

BACKGROUND: Plants memorize previous pathogen attacks and are "primed" to produce a faster and stronger defense response, which is critical for defense against pathogens. In plants, cytosines in transposons and gene bodies are reported to be frequently methylated. Demethylation of transposons can affect disease resistance by regulating the transcription of nearby genes during defense response, but the role of gene body methylation (GBM) in defense responses remains unclear. RESULTS: Here, we find that loss of the chromatin remodeler decrease in DNA methylation 1 (ddm1) synergistically enhances resistance to a biotrophic pathogen under mild chemical priming. DDM1 mediates gene body methylation at a subset of stress-responsive genes with distinct chromatin properties from conventional gene body methylated genes. Decreased gene body methylation in loss of ddm1 mutant is associated with hyperactivation of these gene body methylated genes. Knockout of glyoxysomal protein kinase 1 (gpk1), a hypomethylated gene in ddm1 loss-of-function mutant, impairs priming of defense response to pathogen infection in Arabidopsis. We also find that DDM1-mediated gene body methylation is prone to epigenetic variation among natural Arabidopsis populations, and GPK1 expression is hyperactivated in natural variants with demethylated GPK1. CONCLUSIONS: Based on our collective results, we propose that DDM1-mediated GBM provides a possible regulatory axis for plants to modulate the inducibility of the immune response.

High-Throughput Fluorescent Pollen Tetrad Analysis Using DeepTetrad.

Meiotic recombination initiates from ~100-200 s of programmed DNA double stranded breaks (DSBs) in plants. Meiotic DSBs can be repaired using homologous chromosomes to generate a crossover . Meiotic crossover is critical for chromosomal segregation and increasing genetic variation. The number of crossovers is limited to one and three per chromosome pair in most plant species. Genetic, epigenetic, and environmental factors control crossover frequency and distribution. Due to the limited number of crossovers it is challenging to measure crossover frequency along chromosomes. We adapted fluorescence-tagged lines (FTLs ) that contain quartet1 mutations and linked transgenes expressing dsRed, eYFP, and eCFP in pollen tetrads into the deep learning-based image analysis tool, DeepTetrad. DeepTetrad enables the measurement of crossover frequency and interference by classifying 12 types of tetrads from three-color FTLs in a high-throughput manner, using conventional microscope instruments and a Linux machine. Here, we provide detailed procedures for preparing tetrad samples, tetrad imaging, running DeepTetrad, and analysis of DeepTetrad outputs. DeepTetrad-based measurements of crossover frequency and interference ratio will accelerate the genetic dissection of meiotic crossover control.

Fast and Precise: How to Measure Meiotic Crossovers in Arabidopsis.

During meiosis, homologous chromosomes (homologs) pair and undergo genetic recombination via assembly and disassembly of the synaptonemal complex. Meiotic recombination is initiated by excess formation of DNA double-strand breaks (DSBs), among which a subset are repaired by reciprocal genetic exchange, called crossovers (COs). COs generate genetic variations across generations, profoundly affecting genetic diversity and breeding. At least one CO between homologs is essential for the first meiotic chromosome segregation, but generally only one and fewer than three inter-homolog COs occur in plants. CO frequency and distribution are biased along chromosomes, suppressed in centromeres, and controlled by pro-CO, anti-CO, and epigenetic factors. Accurate and high-throughput detection of COs is important for our understanding of CO formation and chromosome behavior. Here, we review advanced approaches that enable precise measurement of the location, frequency, and genomic landscapes of COs in plants, with a focus on Arabidopsis thaliana.