DNA polymerase epsilon interacts with SUVH2/9 to repress the expression of genes associated with meiotic DSB hotspot in Arabidopsis.

Meiotic recombination is initiated by the SPORULATION 11 (SPO11)-triggered formation of double-strand breaks (DSBs) that usually occur in open chromatin with active transcriptional features in many eukaryotes. However, gene transcription at DSB sites appears to be detrimental for repair, but the regulatory mechanisms governing transcription at meiotic DSB sites are largely undefined in plants. Here, we demonstrate that the largest DNA polymerase epsilon subunit POL2A interacts with SU(VAR)3 to 9 homologs SUVH2 and SUVH9. N-SIM (structured illumination microscopy) observation shows that the colocalization of SUVH2 with the meiotic DSB marker γ-H2AX is dependent on POL2A. RNA-seq of male meiocytes demonstrates that POL2A and SUVH2 jointly repress the expression of 865 genes, which have several known characteristics associated with meiotic DSB sites. Bisulfite-seq and small RNA-seq of male meiocytes support the idea that the silencing of these genes by POL2A and SUVH2/9 is likely independent of CHH methylation or 24-nt siRNA accumulation. Moreover, pol2a suvh2 suvh9 triple mutants have more severe defects in meiotic recombination and fertility compared with either pol2a or suvh2 suvh9. Our results not only identify a epigenetic regulatory mechanism for gene silencing in male meiocytes but also reveal roles for DNA polymerase and SUVH2/9 beyond their classic functions in mitosis.

DNA polymerase epsilon binds histone H3.1-H4 and recruits MORC1 to mediate meiotic heterochromatin condensation.

Heterochromatin is essential for genomic integrity and stability in eukaryotes. The mechanisms that regulate meiotic heterochromatin formation remain largely undefined. Here, we show that the catalytic subunit (POL2A) of Arabidopsis DNA polymerase epsilon (POL ε) is required for proper formation of meiotic heterochromatin. The POL2A N terminus interacts with the GHKL adenosine triphosphatase (ATPase) MORC1 (Microrchidia 1), and POL2A is required for MORC1's localization on meiotic heterochromatin. Mutations affecting the POL2A N terminus cause aberrant morphology of meiotic heterochromatin, which is also observed in morc1. Moreover, the POL2A C-terminal zinc finger domain (ZF1) specifically binds to histone H3.1-H4 dimer or tetramer and is important for meiotic heterochromatin condensation. Interestingly, we also found similar H3.1-binding specificity for the mouse counterpart. Together, our results show that two distinct domains of POL2A, ZF1 and N terminus bind H3.1-H4 and recruit MORC1, respectively, to induce a continuous process of meiotic heterochromatin organization. These activities expand the functional repertoire of POL ε beyond its classic role in DNA replication and appear to be conserved in animals and plants.

A facile fluorescence method for the effective detection of ampicillin using antioxidant carbon dots with specific fluorescent response to ˙OH.

Monitoring methods for beta-lactam (ß-lactam) antibiotics, especially for ampicillin (AMP), with simple operation and sensitivity for realtime applications are highly required. To address this need, antioxidant carbon dots (E-CDs) with excellent fluorescence properties were synthesized using citric acid and ethylenediamine as raw materials. With a quantum yield of 81.97%, E-CDs exhibited a specific and sensitive response to ˙OH. The quenched fluorescence of E-CDs by the formed ˙OH could be restored through a competition reaction with AMP. Leveraging the signal-quenching strategy of E-CDs, H2O2, and Fe2+, a fluorescence signal-on strategy was developed using AMP as the fluorescence recovery agent for the sensitive detection of AMP. The mechanism of the quenching of E-CDs by ˙OH was attributed to the damaging effect of ˙OH on E-CDs. Under optimal conditions, the detection limit of this method for AMP was determined to be 0.38 µg mL-1. This method was successful in drug quality control and the spiked detection of AMP in lake water, milk, and sea cucumber, presenting a viable option for convenient and rapid antibiotic monitoring methods.

Regulation of interference-sensitive crossover distribution ensures crossover assurance in Arabidopsis.

During meiosis, crossovers (COs) are typically required to ensure faithful chromosomal segregation. Despite the requirement for at least one CO between each pair of chromosomes, closely spaced double COs are usually underrepresented due to a phenomenon called CO interference. Like Mus musculus and Saccharomyces cerevisiae, Arabidopsis thaliana has both interference-sensitive (Class I) and interference-insensitive (Class II) COs. However, the underlying mechanism controlling CO distribution remains largely elusive. Both AtMUS81 and AtFANCD2 promote the formation of Class II CO. Using both AtHEI10 and AtMLH1 immunostaining, two markers of Class I COs, we show that AtFANCD2 but not AtMUS81 is required for normal Class I CO distribution among chromosomes. Depleting AtFANCD2 leads to a CO distribution pattern that is intermediate between that of wild-type and a Poisson distribution. Moreover, in Atfancm, Atfigl1, and Atrmi1 mutants where increased Class II CO frequency has been reported previously, we observe Class I CO distribution patterns that are strikingly similar to Atfancd2. Surprisingly, we found that AtFANCD2 plays opposite roles in regulating CO frequency in Atfancm compared with either in Atfigl1 or Atrmi1. Together, these results reveal that although AtFANCD2, AtFANCM, AtFIGL1, and AtRMI1 regulate Class II CO frequency by distinct mechanisms, they have similar roles in controlling the distribution of Class I COs among chromosomes.

Detection of Doxycycline Using Carbon Quantum dots as Probe Based on Internal Filtering Effect.

The establishment of a convenient and effective detection method for doxycycline (DC) holds significant importance in drug monitoring and drug residue assessment. In this work, carbon quantum dots (CQDs) with excellent and stable luminescence performance (the quantum yield of CQDs was 21.8%) were synthesized by the melting method and employed as probes to monitor the fluorescence intensity variations before and after the introduction of DC. A fluorescence analytical method based on the internal filtration effect (IFE) was developed for DC determination. The mechanism of DC quenching CQDs was verified using fluorescence lifetime tests, absorption spectroscopy, and evaluation of internal filtration parameters. After optimizing experimental conditions, it was found that the DC concentration (CDC) exhibited a good linear relationship with the fluorescence quenching efficiency ((F0-F)/F0) of CQDs in the range of 5-30 µM. The fitted linear equation was Y = 0.01249*CDC+0.03625, R2 = 0.9987, and the detection limit was 2.343 µM (n = 8). This developed method has been successfully applied to accurately determine DC concentrations in both doxycycline hydrochloride tablets and human serum samples. It stands out for its simplicity, rapidity, and acceptable detection performance. Due to its advantages, this method holds great promise for application in the biomedical field for monitoring DC drug concentrations and ensuring quality control.

Meiocyte-Specific and AtSPO11-1-Dependent Small RNAs and Their Association with Meiotic Gene Expression and Recombination.

Meiotic recombination ensures accurate chromosome segregation and results in genetic diversity in sexually reproducing eukaryotes. Over the last few decades, the genetic regulation of meiotic recombination has been extensively studied in many organisms. However, the role of endogenous meiocyte-specific small RNAs (ms-sRNAs; 21-24 nucleotide [nt]) and their involvement in meiotic recombination are unclear. Here, we sequenced the total small RNA (sRNA) and messenger RNA populations from meiocytes and leaves of wild type Arabidopsis (Arabidopsis thaliana) and meiocytes of spo11-1, a mutant defective in double-strand break formation, and we discovered 2,409 ms-sRNA clusters, 1,660 of which areSPORULATION 11-1 (AtSPO11-1)-dependent. Unlike mitotic small interfering RNAs that are enriched in intergenic regions and associated with gene silencing, ms-sRNAs are significantly enriched in genic regions and exhibit a positive correlation with genes that are preferentially expressed in meiocytes (i.e. Arabidopsis SKP1-LIKE1 and RAD51), in a fashion unrelated to DNA methylation. We also found that AtSPO11-1-dependent sRNAs have distinct characteristics compared with ms-sRNAs and tend to be associated with two known types of meiotic recombination hotspot motifs (i.e. CTT-repeat and A-rich motifs). These results reveal different meiotic and mitotic sRNA landscapes and provide new insights into how sRNAs relate to gene expression in meiocytes and meiotic recombination.

Comparative transcriptomic analysis of thermally stressed Arabidopsis thaliana meiotic recombination mutants.

BACKGROUND: Meiosis is a specialized cell division that underpins sexual reproduction in most eukaryotes. During meiosis, interhomolog meiotic recombination facilitates accurate chromosome segregation and generates genetic diversity by shuffling parental alleles in the gametes. The frequency of meiotic recombination in Arabidopsis has a U-shaped curve in response to environmental temperature, and is dependent on the Type I, crossover (CO) interference-sensitive pathway. The mechanisms that modulate recombination frequency in response to temperature are not yet known. RESULTS: In this study, we compare the transcriptomes of thermally-stressed meiotic-stage anthers from msh4 and mus81 mutants that mediate the Type I and Type II meiotic recombination pathways, respectively. We show that heat stress reduces the number of expressed genes regardless of genotype. In addition, msh4 mutants have a distinct gene expression pattern compared to mus81 and wild type controls. Interestingly, ASY1, which encodes a HORMA domain protein that is a component of meiotic chromosome axes, is up-regulated in wild type and mus81 but not in msh4. In addition, SDS the meiosis-specific cyclin-like gene, DMC1 the meiosis-specific recombinase, SYN1/REC8 the meiosis-specific cohesion complex component, and SWI1 which functions in meiotic sister chromatid cohesion are up-regulated in all three genotypes. We also characterize 51 novel, previously unannotated transcripts, and show that their promoter regions are associated with A-rich meiotic recombination hotspot motifs. CONCLUSIONS: Our transcriptomic analysis of msh4 and mus81 mutants enhances our understanding of how the Type I and Type II meiotic CO pathway respond to environmental temperature stress and might provide a strategy to manipulate recombination levels in plants.

Conservation and Divergence in the Meiocyte sRNAomes of Arabidopsis, Soybean, and Cucumber.

Meiosis is a critical process for sexual reproduction. During meiosis, genetic information on homologous chromosomes is shuffled through meiotic recombination to produce gametes with novel allelic combinations. Meiosis and recombination are orchestrated by several mechanisms including regulation by small RNAs (sRNAs). Our previous work in Arabidopsis (Arabidopsis thaliana) meiocytes showed that meiocyte-specific sRNAs (ms-sRNAs) have distinct characteristics, including positive association with the coding region of genes that are transcriptionally upregulated during meiosis. Here, we characterized the ms-sRNAs in two important crops, soybean (Glycine max) and cucumber (Cucumis sativus). Ms-sRNAs in soybean have the same features as those in Arabidopsis, suggesting that they may play a conserved role in eudicots. We also investigated the profiles of microRNAs (miRNAs) and phased secondary small interfering RNAs in the meiocytes of all three species. Two conserved miRNAs, miR390 and miR167, are highly abundant in the meiocytes of all three species. In addition, we identified three novel cucumber miRNAs. Intriguingly, our data show that the previously identified phased secondary small interfering RNA pathway involving soybean-specific miR4392 is more abundant in meiocytes. These results showcase the conservation and divergence of ms-sRNAs in flowering plants, and broaden our understanding of sRNA function in crop species.

Elevated temperature increases meiotic crossover frequency via the interfering (Type I) pathway in Arabidopsis thaliana.

For most eukaryotes, sexual reproduction is a fundamental process that requires meiosis. In turn, meiosis typically depends on a reciprocal exchange of DNA between each pair of homologous chromosomes, known as a crossover (CO), to ensure proper chromosome segregation. The frequency and distribution of COs are regulated by intrinsic and extrinsic environmental factors, but much more is known about the molecular mechanisms governing the former compared to the latter. Here we show that elevated temperature induces meiotic hyper-recombination in Arabidopsis thaliana and we use genetic analysis with mutants in different recombination pathways to demonstrate that the extra COs are derived from the major Type I interference sensitive pathway. We also show that heat-induced COs are not the result of an increase in DNA double-strand breaks and that the hyper-recombinant phenotype is likely specific to thermal stress rather than a more generalized stress response. Taken together, these findings provide initial mechanistic insight into how environmental cues modulate plant meiotic recombination and may also offer practical applications.

The Largest Subunit of DNA Polymerase Delta Is Required for Normal Formation of Meiotic Type I Crossovers.

Meiotic recombination contributes to the maintenance of the association between homologous chromosomes (homologs) and ensures the accurate segregation of homologs during anaphase I, thus facilitating the redistribution of alleles among progeny. Meiotic recombination is initiated by the programmed formation of DNA double strand breaks, the repair of which requires DNA synthesis, but the role of DNA synthesis proteins during meiosis is largely unknown. Here, we hypothesized that the lagging strand-specific DNA Polymerase δ (POL δ) might be required for meiotic recombination, based on a previous analysis of DNA Replication Factor1 that suggested a role for lagging strand synthesis in meiotic recombination. In Arabidopsis (Arabidopsis thaliana), complete mutation of the catalytic subunit of POL δ, encoded by AtPOLD1, leads to embryo lethality. Therefore, we used a meiocyte-specific knockdown strategy to test this hypothesis. Reduced expression of AtPOLD1 in meiocytes caused decreased fertility and meiotic defects, including incomplete synapsis, the formation of multivalents, chromosome fragmentation, and improper segregation. Analysis of meiotic crossover (CO) frequencies showed that AtPOLD1RNAi plants had significantly fewer interference-sensitive COs than the wild type, indicating that AtPOL δ participates in type I CO formation. AtPOLD1RNAi atpol2a double mutant meiocytes displayed more severe meiotic phenotypes than those of either single mutant, suggesting that the function of AtPOLD1 and AtPOL2A is not identical in meiotic recombination. Given that POL δ is highly conserved among eukaryotes, we hypothesize that the described role of POL δ here in meiotic recombination likely exists widely in eukaryotes.

Arabidopsis RAD51, RAD51C and XRCC3 proteins form a complex and facilitate RAD51 localization on chromosomes for meiotic recombination.

Meiotic recombination is required for proper homologous chromosome segregation in plants and other eukaryotes. The eukaryotic RAD51 gene family has seven ancient paralogs with important roles in mitotic and meiotic recombination. Mutations in mammalian RAD51 homologs RAD51C and XRCC3 lead to embryonic lethality. In the model plant Arabidopsis thaliana, RAD51C and XRCC3 homologs are not essential for vegetative development but are each required for somatic and meiotic recombination, but the mechanism of RAD51C and XRCC3 in meiotic recombination is unclear. The non-lethal Arabidopsis rad51c and xrcc3 null mutants provide an opportunity to study their meiotic functions. Here, we show that AtRAD51C and AtXRCC3 are components of the RAD51-dependent meiotic recombination pathway and required for normal AtRAD51 localization on meiotic chromosomes. In addition, AtRAD51C interacts with both AtRAD51 and AtXRCC3 in vitro and in vivo, suggesting that these proteins form a complex (es). Comparison of AtRAD51 foci in meiocytes from atrad51, atrad51c, and atxrcc3 single, double and triple heterozygous mutants further supports an interaction between AtRAD51C and AtXRCC3 that enhances AtRAD51 localization. Moreover, atrad51c-/+ atxrcc3-/+ double and atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygous mutants have defects in meiotic recombination, suggesting the role of the AtRAD51C-AtXRCC3 complex in meiotic recombination is in part AtRAD51-dependent. Together, our results support a model in which direct interactions between the RAD51C-XRCC3 complex and RAD51 facilitate RAD51 localization on meiotic chromosomes and RAD51-dependent meiotic recombination. Finally, we hypothesize that maintenance of RAD51 function facilitated by the RAD51C-XRCC3 complex could be highly conserved in eukaryotes.

The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3.

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation.

Formation of interference-sensitive meiotic cross-overs requires sufficient DNA leading-strand elongation.

Meiosis halves diploid genomes to haploid and is essential for sexual reproduction in eukaryotes. Meiotic recombination ensures physical association of homologs and their subsequent accurate segregation and results in the redistribution of genetic variations among progeny. Most organisms have two classes of cross-overs (COs): interference-sensitive (type I) and -insensitive (type II) COs. DNA synthesis is essential for meiotic recombination, but whether DNA synthesis has a role in differentiating meiotic CO pathways is unknown. Here, we show that Arabidopsis POL2A, the homolog of the yeast DNA polymerase-ε (a leading-strand DNA polymerase), is required for plant fertility and meiosis. Mutations in POL2A cause reduced fertility and meiotic defects, including abnormal chromosome association, improper chromosome segregation, and fragmentation. Observation of prophase I cell distribution suggests that pol2a mutants likely delay progression of meiotic recombination. In addition, the residual COs in pol2a have reduced CO interference, and the double mutant of pol2a with mus81, which affects type II COs, displayed more severe defects than either single mutant, indicating that POL2A functions in the type I pathway. We hypothesize that sufficient leading-strand DNA elongation promotes formation of some type I COs. Given that meiotic recombination and DNA synthesis are conserved in divergent eukaryotes, this study and our previous study suggest a novel role for DNA synthesis in the differentiation of meiotic recombination pathways.

The DNA replication factor RFC1 is required for interference-sensitive meiotic crossovers in Arabidopsis thaliana.

During meiotic recombination, induced double-strand breaks (DSBs) are processed into crossovers (COs) and non-COs (NCO); the former are required for proper chromosome segregation and fertility. DNA synthesis is essential in current models of meiotic recombination pathways and includes only leading strand DNA synthesis, but few genes crucial for DNA synthesis have been tested genetically for their functions in meiosis. Furthermore, lagging strand synthesis has been assumed to be unnecessary. Here we show that the Arabidopsis thaliana DNA replication factor C1 (RFC1) important for lagging strand synthesis is necessary for fertility, meiotic bivalent formation, and homolog segregation. Loss of meiotic RFC1 function caused abnormal meiotic chromosome association and other cytological defects; genetic analyses with other meiotic mutations indicate that RFC1 acts in the MSH4-dependent interference-sensitive pathway for CO formation. In a rfc1 mutant, residual pollen viability is MUS81-dependent and COs exhibit essentially no interference, indicating that these COs form via the MUS81-dependent interference-insensitive pathway. We hypothesize that lagging strand DNA synthesis is important for the formation of double Holliday junctions, but not alternative recombination intermediates. That RFC1 is found in divergent eukaryotes suggests a previously unrecognized and highly conserved role for DNA synthesis in discriminating between recombination pathways.

Stefin B alleviates the gouty arthritis in mice by inducing the M2 polarization of macrophages.

The present study aims to explore the therapeutic effect of Stefin B on gouty arthritis (GA) and the polarization of macrophages in mice. Stefin B-overexpressed or knockdown M0 macrophages were constructed. The GA model was established in mice by injecting 25 mg/mL MSU, followed by a single injecting of Stefin B-overexpressing adenovirus vector (GA model + Stefin B OE) or an empty vector (GA model + Stefin B OE NC). Stefin B was found lowly expressed in M1 macrophages. CD206 was markedly upregulated and IL-10 release was signally increased in Stefin B-overexpressed macrophages. In gouty arthritis mice, marked redness and swelling were observed in the ankle joint. Dramatical infiltration of inflammatory cells was observed in the GA model and GA model + Stefin B OE NC groups, which was suppressed in the Stefin B OE group. Increased proportion of F4/80+CD86+ cells observed in GA mice was markedly repressed by Stefin B overexpression, accompanied by the declined level of Caspase-1 and IL-17. Collectively, Stefin B alleviated the GA in mice by inducing the M2 polarization of macrophages.

Risk of deep vein thrombosis (DVT) in lower extremity after total knee arthroplasty (TKA) in patients over 60 years old.

PURPOSE: There is a significant risk of DVT after TKA. We aim to evaluate the potential risk factors for postoperative DVT in the lower extremities in TKA patients over 60 years of age and provide a reference for the effective prevention of DVT. METHODS: This retrospective study included patients older than 60 who underwent TKA surgery in our hospital from May 2015 to May 2022 and compared and analyzed patients' personal characteristics and clinical data with or without postoperative DVT. Logistic regression analysis was performed to determine the potential risk factors for DVT after TKA. The sensitivity and specificity of each risk factor in the diagnosis of DVT were compared by the ROC curve, and the value of this model in the diagnosis of DVT was further investigated using a multivariable combined diagnosis ROC curve model. RESULTS: A total of 661 patients over 60 who underwent TKA were included. Preoperative Hematocrit (HCT), platelet count, anesthesia mode, postoperative D-dimer, ESR, diabetes mellitus, and other aspects of the DVT group and non-DVT group were statistically significant after TKA (P < 0.05). Multivariate logistics regression analysis showed that preoperative HCT, anesthesia mode, and diabetes were independent risk factors for DVT in patients over 60 years old after TKA. Compared with the univariate ROC model, the multivariable combined ROC curve analysis model has a higher diagnostic value for the diagnosis of DVT. CONCLUSION: DVT is common in patients over 60 years of age after TKA, and there is a multivariable influence on its pathogenesis. For patients over 60 with diabetes, neuraxial anesthesia is recommended for patients with high preoperative HCT levels, which may reduce the incidence of postoperative DVT.

Isolation of Meiocytes and Cytological Analyses of Male Meiotic Chromosomes in Soybean, Lettuce, and Maize.

Meiosis is a specialized cell division that halves the number of chromosomes following a single round of DNA replication, thus leading to the generation of haploid gametes. It is essential for sexual reproduction in eukaryotes. Over the past several decades, with the well-developed molecular and cytogenetic methods, there have been great advances in understanding meiosis in plants such as Arabidopsis thaliana and maize, providing excellent references to study meiosis in other plants. A chapter in the previous edition described molecular cytological methods for studying Arabidopsis meiosis in detail. In this chapter, we focus on methods for studying meiosis in soybean (Glycine max), lettuce (Lactuca sativa), and maize (Zea mays). Moreover, we include the method that was recently developed for examination of epigenetic modifications, such as DNA methylation and histone modifications on meiotic chromosomes in plants.

Effectively synthesized functional Si-doped carbon dots with the applications in tyrosinase detection and lysosomal imaging.

There has been significant interest in the preparation and versatile applications of carbon dots (CDs) due to their immense potential value in sensors and imaging. In this work, silicon-doped green carbon dots (Si-CDs) with high quantum yield and rich epoxypropyl were effectively synthesized. Given the clinical diagnostic importance of abnormal levels of tyrosinase (TYR), sensitive detection of TYR is significant for clinical research. A fluorescence signal-off strategy with Si-CDs as probe was constructed to determine TYR based on the oxidation of dopamine by TYR. The detection ranges of this method were 0.01-1.5 and 10-30 U/mL with the detection limit of 0.0046 U/mL, the lower limit of quantification (LLOQ) was 0.01 U/mL, and TYR was successfully and accurately monitored in human serum. Additionally, due to the role of lysosomes in cellular regulatory processes, including TYR levels and fluorescence stability characteristics of Si-CDs in acidic conditions, it was envisaged to use Si-CDs as probe to establish real-time monitoring of lysosomes. According to fluorescence colocation analysis, Si-CDs had intrinsic lysosomal targeting ability to HepG2 and L-02 (with Pearson correlation coefficients were 0.90 and 0.91, respectively). The targeting of Si-CDs to lysosomes was due to the acidophilic effect of the epoxypropyl on its surface.

Evaluation of Crossover Number, Distribution, and Interference Using Cytological Assays in Arabidopsis.

Meiosis involves the replication of nuclear chromosomes in a parent cell, followed by two successive nuclear divisions to produce haploid spores, which differentiate into the gametophyte generations that produce the egg and sperm in plants. Meiotic recombination or crossover (CO) is a hallmark of meiosis that allows shuffling of genetic information between homologous chromosomes (homologs), thereby giving rise to genetically diverse progeny cells and, ultimately, individuals in the progeny; this opens vast opportunities for genetic differentiation and hence speciation. Meiotic COs also ensure the formation of bivalents and fidelity of their equal segregation. Therefore, mechanisms that regulate meiotic recombination have been extensively studied in multiple species. Several approaches have been developed to observe or estimate the frequency of CO, in which CO can be visualized and analyzed cytologically by estimating the number of chiasma (plural chiasmata), a position where non-sister chromatids exchange genetic material between homologs. Furthermore, one CO event might influence the occurrence of another one nearby, along a chromosome; this is known as CO interference. Over the past decades, visualizing CO events and measuring CO interference have contributed greatly to our understanding of the regulatory mechanisms of meiotic recombination. Here, we describe protocols to estimate the number of chiasmata and CO interference in Arabidopsis using cytological methods involving chromosome spreads and immunostaining. Specifically, we describe how chromosome spreads can be used to estimate the number of chiasmata based on the conformations of metaphase I bivalents and provide a revised acid-based quick immunostaining assay that permits high-throughput and quantitative digital estimation of the relative distance between adjacent interference-sensitive CO foci at diakinesis. These methods can be easily established or modified, if necessary, for studying meiotic recombination in other plants and crops. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Estimation of the number of chiasmata per nucleus based on metaphase I bivalent conformations Basic Protocol 2: A chromosome spread-based immunostaining method for relative distance analysis of adjacent interference-sensitive CO foci at diakinesis in Arabidopsis meiocytes.