The kinase ATR controls meiotic crossover distribution at the genome scale in Arabidopsis.

Meiotic crossover, i.e., the reciprocal exchange of chromosome fragments during meiosis, is a key driver of genetic diversity. Crossover is initiated by the formation of programmed DNA double-strand breaks (DSBs). While the role of ATAXIA-TELANGIECTASIA AND RAD3-RELATED (ATR) kinase in DNA damage signaling is well-known, its impact on crossover formation remains understudied. Here, using measurements of recombination at chromosomal intervals and genome-wide crossover mapping, we showed that ATR inactivation in Arabidopsis (Arabidopsis thaliana) leads to dramatic crossover redistribution, with an increase in crossover frequency in chromosome arms and a decrease in pericentromeres. These global changes in crossover placement were not caused by alterations in DSB numbers, which we demonstrated by analyzing phosphorylated H2A.X foci in zygonema. Using the seed-typing technique, we found that hotspot usage remains mainly unchanged in atr mutants compared to wild-type individuals. Moreover, atr showed no change in the number of crossovers caused by two independent pathways, which implies no effect on crossover pathway choice. Analyses of genetic interaction indicate that while the effects of atr are independent of MMS AND UV SENSITIVE81 (MUS81), ZIPPER1 (ZYP1), FANCONI ANEMIA COMPLEMENTATION GROUP M (FANCM) and D2 (FANCD2), the underlying mechanism may be similar between ATR and FANCD2. This study extends our understanding of ATR's role in meiosis, uncovering functions in regulating crossover distribution.

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.

MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis.

During meiosis, DNA double-strand breaks undergo interhomolog repair to yield crossovers between homologous chromosomes. To investigate how interhomolog sequence polymorphism affects crossovers, we sequenced multiple recombinant populations of the model plant Arabidopsis thaliana. Crossovers were elevated in the diverse pericentromeric regions, showing a local preference for polymorphic regions. We provide evidence that crossover association with elevated diversity is mediated via the Class I crossover formation pathway, although very high levels of diversity suppress crossovers. Interhomolog polymorphism causes mismatches in recombining molecules, which can be detected by MutS homolog (MSH) mismatch repair protein heterodimers. Therefore, we mapped crossovers in a msh2 mutant, defective in mismatch recognition, using multiple hybrid backgrounds. Although total crossover numbers were unchanged in msh2 mutants, recombination was remodelled from the diverse pericentromeres towards the less-polymorphic sub-telomeric regions. Juxtaposition of megabase heterozygous and homozygous regions causes crossover remodelling towards the heterozygous regions in wild type Arabidopsis, but not in msh2 mutants. Immunostaining showed that MSH2 protein accumulates on meiotic chromosomes during prophase I, consistent with MSH2 regulating meiotic recombination. Our results reveal a pro-crossover role for MSH2 in regions of higher sequence diversity in A. thaliana.

Natural variation identifies SNI1, the SMC5/6 component, as a modifier of meiotic crossover in Arabidopsis.

The frequency and distribution of meiotic crossovers are tightly controlled; however, variation in this process can be observed both within and between species. Using crosses of two natural Arabidopsis thaliana accessions, Col and Ler, we mapped a crossover modifier locus to semidominant polymorphisms in SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), which encodes a component of the SMC5/6 complex. The sni1 mutant exhibits a modified pattern of recombination across the genome with crossovers elevated in chromosome distal regions but reduced in pericentromeres. Mutations in SNI1 result in reduced crossover interference and can partially restore the fertility of a Class I crossover pathway mutant, which suggests that the protein affects noninterfering crossover repair. Therefore, we tested genetic interactions between SNI1 and both RECQ4 and FANCM DNA helicases, which showed that additional Class II crossovers observed in the sni1 mutant are FANCM independent. Furthermore, genetic analysis of other SMC5/6 mutants confirms the observations of crossover redistribution made for SNI1 The study reveals the importance of the SMC5/6 complex in ensuring the proper progress of meiotic recombination in plants.

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.

Massive crossover elevation via combination of HEI10 and recq4a recq4b during Arabidopsis meiosis.

During meiosis, homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double-strand breaks (DSBs), which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis, competing pathways balance the repair of ∼100-200 meiotic DSBs into ∼10 crossovers per meiosis, with the excess DSBs repaired as noncrossovers. To bias DSB repair toward crossovers, we simultaneously increased dosage of the procrossover E3 ligase gene HEI10 and introduced mutations in the anticrossovers helicase genes RECQ4A and RECQ4B As HEI10 and recq4a recq4b increase interfering and noninterfering crossover pathways, respectively, they combine additively to yield a massive meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage increases crossover coincidence, which indicates an effect on interference. We also show that patterns of interhomolog polymorphism and heterochromatin drive recombination increases distally towards the subtelomeres in both HEI10 and recq4a recq4b backgrounds, while the centromeres remain crossover suppressed. These results provide a genetic framework for engineering meiotic recombination landscapes in plant genomes.

Dual Role of the Histone Variant H2A.Z in Transcriptional Regulation of Stress-Response Genes.

The influence of the histone variant H2A.Z on transcription remains a long-standing conundrum. Here, by analyzing the actin-related protein6 mutant, which is impaired in H2A.Z deposition, and by H2A.Z profiling in stress conditions, we investigated the impact of this histone variant on gene expression in Arabidopsis thaliana We demonstrate that the arp6 mutant exhibits anomalies in response to osmotic stress. Indeed, stress-responsive genes are overrepresented among those hyperactive in arp6. In wild-type plants, these genes exhibit high levels of H2A.Z in the gene body. Furthermore, we observed that in drought-responsive genes, levels of H2A.Z in the gene body correlate with transcript levels. H2A.Z occupancy, but not distribution, changes in parallel with transcriptional changes. In particular, we observed H2A.Z loss upon transcriptional activation and H2A.Z gain upon repression. These data suggest that H2A.Z has a repressive role in transcription and counteracts unwanted expression in noninductive conditions. However, reduced activity of some genes in arp6 is associated with distinct behavior of H2A.Z at their +1 nucleosome, which exemplifies the requirement of this histone for transcription. Our data support a model where H2A.Z in gene bodies has a strong repressive effect on transcription, whereas in +1 nucleosomes, it is important for maintaining the activity of some genes.

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.

Interconnections between meiotic recombination and sequence polymorphism in plant genomes.

1022 I. 1022 II. 1023 III. 1023 IV. 1025 V. 1026 1027 References 1027 SUMMARY: Meiosis is fundamental to sexual reproduction and creates genetic variation in progeny. During meiosis paired homologous chromosomes undergo recombination, which can result in reciprocal crossovers. This process can recombine independently arising mutations onto the same chromosome. Recombination locations are highly variable between meioses, although total crossover numbers are tightly regulated. In addition to the effect of meiosis on genetic variation, sequence polymorphisms between homologous chromosomes can feedback onto the recombination pathways. Here we review the major crossover pathways in plants and some of the known homeostatic mechanisms that act during meiotic recombination. We then examine how sequence polymorphisms between homologous chromosomes, that is, heterozygosity, can influence meiotic recombination pathways in cis and trans. Finally, we provide a brief perspective on the relevance of these interconnections for natural selection and adaptation in plants.

Why do plants need the ZMM crossover pathway? A snapshot of meiotic recombination from the perspective of interhomolog polymorphism.

At the heart of meiosis is crossover recombination, i.e., reciprocal exchange of chromosome fragments between parental genomes. Surprisingly, in most eukaryotes, including plants, several recombination pathways that can result in crossover event operate in parallel during meiosis. These pathways emerged independently in the course of evolution and perform separate functions, which directly translate into their roles in meiosis. The formation of one crossover per chromosome pair is required for proper chromosome segregation. This "obligate" crossover is ensured by the major crossover pathway in plants, and in many other eukaryotes, known as the ZMM pathway. The secondary pathways play important roles also in somatic cells and function mainly as repair mechanisms for DNA double-strand breaks (DSBs) not used for crossover formation. One of the consequences of the functional differences between ZMM and other DSB repair pathways is their distinct sensitivities to polymorphisms between homologous chromosomes. From a population genetics perspective, these differences may affect the maintenance of genetic variability. This might be of special importance when considering that a significant portion of plants uses inbreeding as a predominant reproductive strategy, which results in loss of interhomolog polymorphism. While we are still far from fully understanding the relationship between meiotic recombination pathways and genetic variation in populations, recent studies of crossovers in plants offer a new perspective.

MSH2 stimulates interfering and inhibits non-interfering crossovers in response to genetic polymorphism.

Meiotic crossovers can be formed through the interfering pathway, in which one crossover prevents another from forming nearby, or by an independent non-interfering pathway. In Arabidopsis, local sequence polymorphism between homologs can stimulate interfering crossovers in a MSH2-dependent manner. To understand how MSH2 regulates crossovers formed by the two pathways, we combined Arabidopsis mutants that elevate non-interfering crossovers with msh2 mutants. We demonstrate that MSH2 blocks non-interfering crossovers at polymorphic loci, which is the opposite effect to interfering crossovers. We also observe MSH2-independent crossover inhibition at highly polymorphic sites. We measure recombination along the chromosome arms in lines differing in patterns of heterozygosity and observe a MSH2-dependent crossover increase at the boundaries between heterozygous and homozygous regions. Here, we show that MSH2 is a master regulator of meiotic DSB repair in Arabidopsis, with antagonistic effects on interfering and non-interfering crossovers, which shapes the crossover landscape in relation to interhomolog polymorphism.

The effect of DNA polymorphisms and natural variation on crossover hotspot activity in Arabidopsis hybrids.

In hybrid organisms, genetically divergent homologous chromosomes pair and recombine during meiosis; however, the effect of specific types of polymorphisms on crossover is poorly understood. Here, to analyze this in Arabidopsis, we develop the seed-typing method that enables the massively parallel fine-mapping of crossovers by sequencing. We show that structural variants, observed in one of the generated intervals, do not change crossover frequency unless they are located directly within crossover hotspots. Both natural and Cas9-induced deletions result in lower hotspot activity but are not compensated by increases in immediately adjacent hotspots. To examine the effect of single nucleotide polymorphisms on crossover formation, we analyze hotspot activity in mismatch detection-deficient msh2 mutants. Surprisingly, polymorphic hotspots show reduced activity in msh2. In lines where only the hotspot-containing interval is heterozygous, crossover numbers increase above those in the inbred (homozygous). We conclude that MSH2 shapes crossover distribution by stimulating hotspot activity at polymorphic regions.

Quantifying Meiotic Crossover Recombination in Arabidopsis Lines Expressing Fluorescent Reporters in Seeds Using SeedScoring Pipeline for CellProfiler.

The number of crossovers during meiosis is relatively low, so multiple meioses need to be analyzed to accurately measure crossover frequency. In Arabidopsis, systems based on the segregation of fluorescent T-DNA reporters that are expressed in seeds (fluorescent-tagged lines, FTLs) allow for an accurate measurement of crossover frequency in specific chromosome regions. A major advantage of FTL-based experiments is the ability to analyze thousands of seeds for each biological replicate, which requires the use of automatic seed scoring. Here, we describe a protocol to computationally count the proportion of seeds that experienced a crossover event within the tested FTL interval and so measure the recombination frequency within that interval. We describe SeedScoring, a CellProfiler pipeline where the total time needed to measure crossover frequency in a single FTL line is approximately 5 min using a series of three images taken under a fluorescent stereomicroscope (3 min) and passing these images through the SeedScoring pipeline described in this protocol (2 min).

Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions.

Investigating the process of gamete formation in plants often requires the use of mutants of selected genes in various genetic backgrounds. For example, analysis of meiotic recombination based on sequencing or genotyping requires the generation of hybrids between two lines. Although T-DNA mutant collections of Arabidopsis thaliana are vast and easily accessible, they are largely confined to Col-0 background. This chapter describes how to efficiently generate knock-out mutants in different Arabidopsis accessions using CRISPR/Cas9 technology. The presented system is based on designing two single-guide RNAs (sgRNAs), which direct the Cas9 endonuclease to generate double-strand breaks at two sites, leading to genomic deletion in targeted gene. The presence of seed-expressed dsRed fluorescence cassette in the CRISPR construct facilitates preselection of genome-edited and transgene-free plants by monitoring the seed fluorescence under the epifluorescent microscope. The protocol provides the detailed information about all steps required to perform genome editing and to obtain loss-of-function mutants in different Arabidopsis accessions within merely two generations.

NuA4 and H2A.Z control environmental responses and autotrophic growth in Arabidopsis.

Nucleosomal acetyltransferase of H4 (NuA4) is an essential transcriptional coactivator in eukaryotes, but remains poorly characterized in plants. Here, we describe Arabidopsis homologs of the NuA4 scaffold proteins Enhancer of Polycomb-Like 1 (AtEPL1) and Esa1-Associated Factor 1 (AtEAF1). Loss of AtEAF1 results in inhibition of growth and chloroplast development. These effects are stronger in the Atepl1 mutant and are further enhanced by loss of Golden2-Like (GLK) transcription factors, suggesting that NuA4 activates nuclear plastid genes alongside GLK. We demonstrate that AtEPL1 is necessary for nucleosomal acetylation of histones H4 and H2A.Z by NuA4 in vitro. These chromatin marks are diminished genome-wide in Atepl1, while another active chromatin mark, H3K9 acetylation (H3K9ac), is locally enhanced. Expression of many chloroplast-related genes depends on NuA4, as they are downregulated with loss of H4ac and H2A.Zac. Finally, we demonstrate that NuA4 promotes H2A.Z deposition and by doing so prevents spurious activation of stress response genes.

Genome sequence comparison of Col and Ler lines reveals the dynamic nature of Arabidopsis chromosomes.

Large differences in plant genome sizes are mainly due to numerous events of insertions or deletions (indels). The balance between these events determines the evolutionary direction of genome changes. To address the question of what phenomena trigger these alterations, we compared the genomic sequences of two Arabidopsis thaliana lines, Columbia (Col) and Landsberg erecta (Ler). Based on the resulting alignments large indels (>100 bp) within these two genomes were analysed. There are approximately 8500 large indels accounting for the differences between the two genomes. The genetic basis of their origin was distinguished as three main categories: unequal recombination (Urec)-derived, illegitimate recombination (Illrec)-derived and transposable elements (TE)-derived. A detailed study of their distribution and size variation along chromosomes, together with a correlation analyses, allowed us to demonstrate the impact of particular recombination-based mechanisms on the plant genome evolution. The results show that unequal recombination is not efficient in the removal of TEs within the pericentromeric regions. Moreover, we discovered an unexpectedly high influence of large indels on gene evolution pointing out significant differences between the various gene families. For the first time, we present convincing evidence that somatic events do play an important role in plant genome evolution.

License to Regulate: Noncoding RNA Special Agents in Plant Meiosis and Reproduction.

The complexity of the subcellular processes that take place during meiosis requires a significant remodeling of cellular metabolism and dynamic changes in the organization of chromosomes and the cytoskeleton. Recently, investigations of meiotic transcriptomes have revealed additional noncoding RNA factors (ncRNAs) that directly or indirectly influence the course of meiosis. Plant meiosis is the point at which almost all known noncoding RNA-dependent regulatory pathways meet to influence diverse processes related to cell functioning and division. ncRNAs have been shown to prevent transposon reactivation, create germline-specific DNA methylation patterns, and affect the expression of meiosis-specific genes. They can also influence chromosome-level processes, including the stimulation of chromosome condensation, the definition of centromeric chromatin, and perhaps even the regulation of meiotic recombination. In many cases, our understanding of the mechanisms underlying these processes remains limited. In this review, we will examine how the different functions of each type of ncRNA have been adopted in plants, devoting attention to both well-studied examples and other possible functions about which we can only speculate for now. We will also briefly discuss the most important challenges in the investigation of ncRNAs in plant meiosis.

Comparative analysis of the Brassica oleracea genetic map and the Arabidopsis thaliana genome.

We further investigated genome macrosynteny for Brassica species and Arabidopsis thaliana. This work aimed at comparative map construction for B. oleracea and A. thaliana chromosomes based on 160 known A. thaliana probes: 147 expressed sequence tags (ESTs) and 13 full-length cDNA clones. Based on an in silico study of the A. thaliana genome, most of the selected ESTs (83%) represented unique or low-copy genes. We identified conserved segments by the visual inspection of comparative data with a priori assumptions, and established their significance with the LineUp algorithm. Evaluation of the number of B. oleracea gene copies per A. thaliana EST revealed a fixed upward trend. We established a segregation distortion pattern for all genetic loci, with particular consideration of the type of selection (gametic or zygotic), and discuss its possible impact on genetic map construction. Consistent with previous reports, we found evidence for numerous chromosome rearrangements and the genome fragment replication of B. oleracea that have taken place since the divergence of the two species. Also, we found that over 54% of the B. oleracea genome is covered by 24 segments conserved with the A. thaliana genome. The average conserved segment is composed of 5 loci covering 19.3 cM in the B. oleracea genetic map and 2.42 Mb in the A. thaliana physical map. We have also attempted to use a unified system of conserved blocks (previously described) to verify our results and perform a comprehensive comparison with other Brassica species.

Where to Cross Over? Defining Crossover Sites in Plants.

It is believed that recombination in meiosis serves to reshuffle genetic material from both parents to increase genetic variation in the progeny. At the same time, the number of crossovers is usually kept at a very low level. As a consequence, many organisms need to make the best possible use from the one or two crossovers that occur per chromosome in meiosis. From this perspective, the decision of where to allocate rare crossover events becomes an important issue, especially in self-pollinating plant species, which experience limited variation due to inbreeding. However, the freedom in crossover allocation is significantly limited by other, genetic and non-genetic factors, including chromatin structure. Here we summarize recent progress in our understanding of those processes with a special emphasis on plant genomes. First, we focus on factors which influence the distribution of recombination initiation sites and discuss their effects at both, the single hotspot level and at the chromosome scale. We also briefly explain the aspects of hotspot evolution and their regulation. Next, we analyze how recombination initiation sites translate into the development of crossovers and their location. Moreover, we provide an overview of the sequence polymorphism impact on crossover formation and chromosomal distribution.

Chromatin Affinity Purification (ChAP) from Arabidopsis thaliana Rosette Leaves Using in vivo Biotinylation System.

Chromatin Affinity Purification (ChAP) is widely used to study chromatin architecture and protein complexes interacting with DNA. Here we present an efficient method for ChAP from Arabidopsis thaliana rosette leaves, in which in vivo biotinylation system is used. The chromatin is digested by Micrococcal Nuclease (MNase), hence the distribution of nucleosomes is also achieved. The in vivo biotinylation system was initially developed for Drosophila melanogaster ( Mito et al., 2005 ), but the presented protocol has been developed specifically for Arabidopsis thaliana ( Sura et al., 2017 ).