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.

Crossover-active regions of the wheat genome are distinguished by DMC1, the chromosome axis, H3K27me3, and signatures of adaptation.

The hexaploid bread wheat genome comprises over 16 gigabases of sequence across 21 chromosomes. Meiotic crossovers are highly polarized along the chromosomes, with elevation in the gene-dense distal regions and suppression in the Gypsy retrotransposon-dense centromere-proximal regions. We profiled the genomic landscapes of the meiotic recombinase DMC1 and the chromosome axis protein ASY1 in wheat and investigated their relationships with crossovers, chromatin state, and genetic diversity. DMC1 and ASY1 chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed strong co-enrichment in the distal, crossover-active regions of the wheat chromosomes. Distal ChIP-seq enrichment is consistent with spatiotemporally biased cytological immunolocalization of DMC1 and ASY1 close to the telomeres during meiotic prophase I. DMC1 and ASY1 ChIP-seq peaks show significant overlap with genes and transposable elements in the Mariner and Mutator superfamilies. However, DMC1 and ASY1 ChIP-seq peaks were detected along the length of each chromosome, including in low-crossover regions. At the fine scale, crossover elevation at DMC1 and ASY1 peaks and genes correlates with enrichment of the Polycomb histone modification H3K27me3. This indicates a role for facultative heterochromatin, coincident with high DMC1 and ASY1, in promoting crossovers in wheat and is reflected in distalized H3K27me3 enrichment observed via ChIP-seq and immunocytology. Genes with elevated crossover rates and high DMC1 and ASY1 ChIP-seq signals are overrepresented for defense-response and immunity annotations, have higher sequence polymorphism, and exhibit signatures of selection. Our findings are consistent with meiotic recombination promoting genetic diversity, shaping host-pathogen co-evolution, and accelerating adaptation by increasing the efficiency of selection.

FIGL1 prevents aberrant chromosome associations and fragmentation and limits crossovers in polyploid wheat meiosis.

Meiotic crossovers (COs) generate genetic diversity and are crucial for viable gamete production. Plant COs are typically limited to 1-3 per chromosome pair, constraining the development of improved varieties, which in wheat is exacerbated by an extreme distal localisation bias. Advances in wheat genomics and related technologies provide new opportunities to investigate, and possibly modify, recombination in this important crop species. Here, we investigate the disruption of FIGL1 in tetraploid and hexaploid wheat as a potential strategy for modifying CO frequency/position. We analysed figl1 mutants and virus-induced gene silencing lines cytogenetically. Genetic mapping was performed in the hexaploid. FIGL1 prevents abnormal meiotic chromosome associations/fragmentation in both ploidies. It suppresses class II COs in the tetraploid such that CO/chiasma frequency increased 2.1-fold in a figl1 msh5 quadruple mutant compared with a msh5 double mutant. It does not appear to affect class I COs based on HEI10 foci counts in a hexaploid figl1 triple mutant. Genetic mapping in the triple mutant suggested no significant overall increase in total recombination across examined intervals but revealed large increases in specific individual intervals. Notably, the tetraploid figl1 double mutant was sterile but the hexaploid triple mutant was moderately fertile, indicating potential utility for wheat breeding.

Identification, characterization, and rescue of CRISPR/Cas9 generated wheat SPO11-1 mutants.

Increasing crop yields through plant breeding is time consuming and laborious, with the generation of novel combinations of alleles being limited by chromosomal linkage blocks and linkage-drag. Meiotic recombination is essential to create novel genetic variation via the reshuffling of parental alleles. The exchange of genetic information between homologous chromosomes occurs at crossover (CO) sites but CO frequency is often low and unevenly distributed. This bias creates the problem of linkage-drag in recombination 'cold' regions, where undesirable variation remains linked to useful traits. In plants, programmed meiosis-specific DNA double-strand breaks, catalysed by the SPO11 complex, initiate the recombination pathway, although only ~5% result in the formation of COs. To study the role of SPO11-1 in wheat meiosis, and as a prelude to manipulation, we used CRISPR/Cas9 to generate edits in all three SPO11-1 homoeologues of hexaploid wheat. Characterization of progeny lines shows plants deficient in all six SPO11-1 copies fail to undergo chromosome synapsis, lack COs and are sterile. In contrast, lines carrying a single copy of any one of the three wild-type homoeologues are phenotypically indistinguishable from unedited plants both in terms of vegetative growth and fertility. However, cytogenetic analysis of the edited plants suggests that homoeologues differ in their ability to generate COs and in the dynamics of synapsis. In addition, we show that the transformation of wheat mutants carrying six edited copies of SPO11-1 with the TaSPO11-1B gene, restores synapsis, CO formation, and fertility and hence opens a route to modifying recombination in this agronomically important crop.

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.

Factors underlying restricted crossover localization in barley meiosis.

Meiotic recombination results in the formation of cytological structures known as chiasmata at the sites of genetic crossovers (COs). The formation of at least one chiasma/CO between homologous chromosome pairs is essential for accurate chromosome segregation at the first meiotic division as well as for generating genetic variation. Although DNA double-strand breaks, which initiate recombination, are widely distributed along the chromosomes, this is not necessarily reflected in the chiasma distribution. In many species there is a tendency for chiasmata to be distributed in favored regions along the chromosomes, whereas in others, such as barley and some other grasses, chiasma localization is extremely pronounced. Localization of chiasma to the distal regions of barley chromosomes restricts the genetic variation available to breeders. Studies reviewed herein are beginning to provide an explanation for chiasma localization in barley. Moreover, they suggest a potential route to manipulating chiasma distribution that could be of value to plant breeders.

Unravelling mechanisms that govern meiotic crossover formation in wheat.

Wheat is a major cereal crop that possesses a large allopolyploid genome formed through hybridisation of tetraploid and diploid progenitors. During meiosis, crossovers (COs) are constrained in number to 1-3 per chromosome pair that are predominantly located towards the chromosome ends. This reduces the probability of advantageous traits recombining onto the same chromosome, thus limiting breeding. Therefore, understanding the underlying factors controlling meiotic recombination may provide strategies to unlock the genetic potential in wheat. In this mini-review, we will discuss the factors associated with restricted CO formation in wheat, such as timing of meiotic events, chromatin organisation, pre-meiotic DNA replication and dosage of CO genes, as a means to modulate recombination.

Meiotic chromosome axis remodelling is critical for meiotic recombination in Brassica rapa.

Meiosis generates genetic variation through homologous recombination (HR) that is harnessed during breeding. HR occurs in the context of meiotic chromosome axes and the synaptonemal complex. To study the role of axis remodelling in crossover (CO) formation in a crop species, we characterized mutants of the axis-associated protein ASY1 and the axis-remodelling protein PCH2 in Brassica rapa. asy1 plants form meiotic chromosome axes that fail to synapse. CO formation is almost abolished, and residual chiasmata are proportionally enriched in terminal chromosome regions, particularly in the nucleolar organizing region (NOR)-carrying chromosome arm. pch2 plants show impaired ASY1 loading and remodelling, consequently achieving only partial synapsis, which leads to reduced CO formation and loss of the obligatory CO. PCH2-independent chiasmata are proportionally enriched towards distal chromosome regions. Similarly, in Arabidopsis pch2, COs are increased towards telomeric regions at the expense of (peri-) centromeric COs compared with the wild type. Taken together, in B. rapa, axis formation and remodelling are critical for meiotic fidelity including synapsis and CO formation, and in asy1 and pch2 CO distributions are altered. While asy1 plants are sterile, pch2 plants are semi-sterile and thus PCH2 could be an interesting target for breeding programmes.

Affinity proteomics reveals extensive phosphorylation of the Brassica chromosome axis protein ASY1 and a network of associated proteins at prophase I of meiosis.

During meiosis, the formation of crossovers (COs) generates genetic variation and provides physical links that are essential for accurate chromosome segregation. COs occur in the context of a proteinaceous chromosome axis. The transcriptomes and proteomes of anthers and meiocytes comprise several thousand genes and proteins, but because of the level of complexity relatively few have been functionally characterized. Our understanding of the physical and functional interactions between meiotic proteins is also limited. Here we use affinity proteomics to analyse the proteins that are associated with the meiotic chromosome axis protein, ASY1, in Brassica oleracea anthers and meiocytes. We show that during prophase I ASY1 and its interacting partner, ASY3, are extensively phosphorylated, and we precisely assign phosphorylation sites. We identify 589 proteins that co-immunoprecipitate with ASY1. These correspond to 492 Arabidopsis orthologues, over 90% of which form a coherent protein-protein interaction (PPI) network containing known and candidate meiotic proteins, including proteins more usually associated with other cellular processes such as DNA replication and proteolysis. Mutant analysis confirms that affinity proteomics is a viable strategy for revealing previously unknown meiotic proteins, and we show how the PPI network can be used to prioritise candidates for analysis. Finally, we identify another axis-associated protein with a role in meiotic recombination. Data are available via ProteomeXchange with identifier PXD006042.

Identification of ASYNAPTIC4, a Component of the Meiotic Chromosome Axis.

During the leptotene stage of prophase I of meiosis, chromatids become organized into a linear looped array via a protein axis that forms along the loop bases. Establishment of the axis is essential for the subsequent synapsis of the homologous chromosome pairs and the progression of recombination to form genetic crossovers. Here, we describe ASYNAPTIC4 (ASY4), a meiotic axis protein in Arabidopsis (Arabidopsis thaliana). ASY4 is a small coiled-coil protein that exhibits limited sequence similarity with the carboxyl-terminal region of the axis protein ASY3. We used enhanced yellow fluorescent protein-tagged ASY4 to show that ASY4 localizes to the chromosome axis throughout prophase I. Bimolecular fluorescence complementation revealed that ASY4 interacts with ASY1 and ASY3, and yeast two-hybrid analysis confirmed a direct interaction between ASY4 and ASY3. Mutants lacking full-length ASY4 exhibited defective axis formation and were unable to complete synapsis. Although the initiation of recombination appeared to be unaffected in the asy4 mutant, the number of crossovers was reduced significantly, and crossovers tended to group in the distal parts of the chromosomes. We conclude that ASY4 is required for normal axis and crossover formation. Furthermore, our data suggest that ASY3/ASY4 are the functional homologs of the mammalian SYCP2/SYCP3 axial components.

Arabidopsis PCH2 Mediates Meiotic Chromosome Remodeling and Maturation of Crossovers.

Meiotic chromosomes are organized into linear looped chromatin arrays by a protein axis localized along the loop-bases. Programmed remodelling of the axis occurs during prophase I of meiosis. Structured illumination microscopy (SIM) has revealed dynamic changes in the chromosome axis in Arabidopsis thaliana and Brassica oleracea. We show that the axis associated protein ASY1 is depleted during zygotene concomitant with synaptonemal complex (SC) formation. Study of an Atpch2 mutant demonstrates this requires the conserved AAA+ ATPase, PCH2, which localizes to the sites of axis remodelling. Loss of PCH2 leads to a failure to deplete ASY1 from the axes and compromizes SC polymerisation. Immunolocalization of recombination proteins in Atpch2 indicates that recombination initiation and CO designation during early prophase I occur normally. Evidence suggests that CO interference is initially functional in the mutant but there is a defect in CO maturation following designation. This leads to a reduction in COs and a failure to form COs between some homologous chromosome pairs leading to univalent chromosomes at metaphase I. Genetic analysis reveals that CO distribution is also affected in some chromosome regions. Together these data indicate that the axis remodelling defect in Atpch2 disrupts normal patterned formation of COs.

Absence of SUN1 and SUN2 proteins in Arabidopsis thaliana leads to a delay in meiotic progression and defects in synapsis and recombination.

The movement of chromosomes during meiosis involves location of their telomeres at the inner surface of the nuclear envelope. Sad1/UNC-84 (SUN) domain proteins are inner nuclear envelope proteins that are part of complexes linking cytoskeletal elements with the nucleoskeleton, connecting telomeres to the force-generating mechanism in the cytoplasm. These proteins play a conserved role in chromosome dynamics in eukaryotes. Homologues of SUN domain proteins have been identified in several plant species. In Arabidopsis thaliana, two proteins that interact with each other, named AtSUN1 and AtSUN2, have been identified. Immunolocalization using antibodies against AtSUN1 and AtSUN2 proteins revealed that they were associated with the nuclear envelope during meiotic prophase I. Analysis of the double mutant Atsun1-1 Atsun2-2 has revealed severe meiotic defects, namely a delay in the progression of meiosis, absence of full synapsis, the presence of unresolved interlock-like structures, and a reduction in the mean cell chiasma frequency. We propose that in Arabidopsis thaliana, overlapping functions of SUN1 and SUN2 ensure normal meiotic recombination and synapsis.

The condensin complexes play distinct roles to ensure normal chromosome morphogenesis during meiotic division in Arabidopsis.

Meiosis is a specialized cell division essential for sexual reproduction. During meiosis the chromosomes are highly organized, and correct chromosome architecture is required for faithful segregation of chromosomes at anaphase I and II. Condensin is involved in chromosome organization during meiotic and mitotic cell divisions. Three condensin subunits, AtSMC4 and the condensin I and II specific subunits AtCAP-D2 and AtCAP-D3, respectively, have been studied for their role in meiosis. This has revealed that both the condensin I and condensin II complexes are required to maintain normal structural integrity of the meiotic chromosomes during the two nuclear divisions. Their roles appear functionally distinct in that condensin I is required to maintain normal compaction of the centromeric repeats and 45S rDNA, whereas loss of condensin II was associated with extensive interchromosome connections at metaphase I. Depletion of condensin is also associated with a slight reduction in crossover formation, suggesting a role during meiotic prophase I.

Identification of the pollen self-incompatibility determinant in Papaver rhoeas.

Higher plants produce seed through pollination, using specific interactions between pollen and pistil. Self-incompatibility is an important mechanism used in many species to prevent inbreeding; it is controlled by a multi-allelic S locus. 'Self' (incompatible) pollen is discriminated from 'non-self' (compatible) pollen by interaction of pollen and pistil S locus components, and is subsequently inhibited. In Papaver rhoeas, the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca(2+)-dependent signalling network, resulting in pollen inhibition and programmed cell death. Here we have cloned three alleles of a highly polymorphic pollen-expressed gene, PrpS (Papaver rhoeas pollen S), from Papaver and provide evidence that this encodes the pollen S locus determinant. PrpS is a single-copy gene linked to the pistil S gene (currently called S, but referred to hereafter as PrsS for Papaver rhoeas stigma S determinant). Sequence analysis indicates that PrsS and PrpS are equally ancient and probably co-evolved. PrpS encodes a novel approximately 20-kDa protein. Consistent with predictions that it is a transmembrane protein, PrpS is associated with the plasma membrane. We show that a predicted extracellular loop segment of PrpS interacts with PrsS and, using PrpS antisense oligonucleotides, we demonstrate that PrpS is involved in S-specific inhibition of incompatible pollen. Identification of PrpS represents a major advance in our understanding of the Papaver self-incompatibility system. As a novel cell-cell recognition determinant it contributes to the available information concerning the origins and evolution of cell-cell recognition systems involved in discrimination between self and non-self, which also include histocompatibility systems in primitive chordates and vertebrates.

Inter-homolog crossing-over and synapsis in Arabidopsis meiosis are dependent on the chromosome axis protein AtASY3.

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation.

Replication protein A (AtRPA1a) is required for class I crossover formation but is dispensable for meiotic DNA break repair.

Replication protein A (RPA) is involved in many aspects of DNA metabolism including meiotic recombination. Many species possess a single RPA1 gene but Arabidopsis possesses five RPA1 paralogues. This feature has enabled us to gain further insight into the meiotic role of RPA1. Proteomic analysis implicated one of the AtRPA1 family (AtRPA1a) in meiosis. Immunofluorescence studies confirmed that AtRPA1a is associated with meiotic chromosomes from leptotene through to early pachytene. Analysis of an Atrpa1a mutant revealed that AtRPA1a is not essential at early stages in the recombination pathway. DNA double-strand breaks are repaired in Atrpa1a, but the mutant is defective in the formation of crossovers, exhibiting a 60% reduction in chiasma frequency. Consistent with this, localization of recombination proteins AtRAD51 and AtMSH4 appears normal, whereas the numbers of AtMLH1 and AtMLH3 foci at pachytene are significantly reduced. This suggests that the defect in Atrpa1a is manifested at the stage of second-end capture. Analysis of Atrpa1a/Atmsh4 and Atrpa1a/Atmlh3 double mutants indicates that loss of AtRPA1a predominantly affects the formation of class I, interference-dependent crossovers.

The RecQ helicase AtRECQ4A is required to remove inter-chromosomal telomeric connections that arise during meiotic recombination in Arabidopsis.

RecQ helicases are a conserved group of proteins with a role in the maintenance of genome integrity. In Saccharomyces cerevisiae (budding yeast), meiotic recombination is increased in the absence of the RecQ helicase Sgs1. Here we investigated the potential meiotic role of the Sgs1 homologue AtRECQ4A and the closely related AtRECQ4B. Both proteins have been shown to function during recombination in somatic cells, but so far their meiotic role has not been investigated. Both AtRECQ4A and AtRECQ4B were expressed in reproductive tissues. Although immunolocalization studies showed that AtRECQ4A associates with recombination intermediates, we found no evidence that its loss or that of AtRECQ4B had a significant effect on meiotic cross-overs, suggesting functional redundancy with other RECQ family members. Nevertheless, pollen viability decreased in Atrecq4A, resulting in a reduction in fertility, although this was not the case in Atrecq4B. Cytological analysis revealed chromatin bridges between the telomeres of non-homologous chromosomes in Atrecq4A at metaphase I, in some instances accompanied by chromosome fragmentation at anaphase I. The bridges required telomeric repeats and were dependent on meiotic recombination. Immunolocalization confirmed the association of AtRECQ4A with the telomeres during prophase I, which we propose enables dissolution of recombination-dependent telomeric associations. Thus, this study has identified a hitherto unknown role for a member of the RECQ helicase family during meiosis that contributes to the maintenance of chromosome integrity. As telomere structure is generally conserved, it seems likely that these associations may arise during meiosis in other species, where they must also be removed.

Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen.

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible ('self') pollen. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network resulting in pollen tube inhibition and programmed cell death. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.

Cytogenetic Techniques for Analyzing Meiosis in Hexaploid Bread Wheat.

This chapter describes several cytogenetic procedures developed for investigating meiotic recombination in pollen mother cells (PMCs) of hexaploid wheat (Triticum aestivum) using standard fluorescence microscopy. Two basic methods are used to prepare slides for microscopy. In the cytological technique, wheat anthers are excised, fixed and used to prepare chromosome spreads which can be visualized following the application of a fluorescent DNA stain. In the immunocytological technique, fresh anthers are used to prepare chromosome spreads for analyzing the localization of meiotic proteins by applying specific antibodies followed by fluorescently tagged secondary antibodies. Both methods can be combined with the use of DNA probes to label specific chromosome regions such as telomeres, centromeres, and rDNA sequences in a procedure known as fluorescence in situ hybridisation (FISH). In addition, the cytological technique can be used in conjunction with S-phase incorporation of the DNA base analog, 5-bromo-2'-deoxyuridine (BrdU), and a modified immunolocalization procedure for a convenient meiotic time course assay. Although these protocols were developed for T. aestivum cv. Cadenza, they are directly applicable to other varieties and we have used them successfully for several other hexaploid cultivars and the tetraploid Triticum turgidum cv. Kronos.