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Selected examples of bivalent 4 at metaphase I showing different patterns of chiasma distribution. The locations of 45S and 5S rDNA sequences in the short arm are marked by green and red signals (FISH), respectively. (A) Ring bivalent with three chiasmata. (B, C) Bivalents with two chiasmata. (D–F) Three rod bivalents showing a single chiasma in the long arm located in an interstitial (D), subdistal (E), and distal (F) region. (G) A rod bivalent with a single distal chiasma in the short arm. Bars represent 2 l m. A diagrammatic representation of the corresponding bivalents illustrating the position of chiasmata is displayed at the bottom of the figure.
Source publication
Information concerning natural variation either in chiasma frequency or in the genetic basis of any such variation is a valuable
tool to characterize phenotypic traits and their genetic control. Here meiotic recombination frequencies are analysed in nine
geographically and ecologically diverse accessions of Arabidopsis thaliana, and a comparative s...
Contexts in source publication
Context 1
... one chiasma, it tended to occupy subdistal and interstitial positions, whereas, when two chiasmata were present, one was far from the other, occupying more proximal and distal locations. Thus, the operation of positive interference can be reasonably inferred. Different examples of these patterns of chiasma distribution in bivalent 4 are shown in Fig. 2. It must be noted that short arms appeared associated via their distal NORs (45S signals). The presence of an unlabelled chroma- tin strip running continuously between the homologous chromosomes, flanked by 45S signals, confirmed the chias- matic nature of these associations (Fig. 2C). In those cases in which the 45S signal formed a ...
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... these patterns of chiasma distribution in bivalent 4 are shown in Fig. 2. It must be noted that short arms appeared associated via their distal NORs (45S signals). The presence of an unlabelled chroma- tin strip running continuously between the homologous chromosomes, flanked by 45S signals, confirmed the chias- matic nature of these associations (Fig. 2C). In those cases in which the 45S signal formed a solid continuous block, it was reasoned that these are also chiasmatic bonds but the diagnostic unlabelled strip is invisible due to the plane of viewing ( Fig. 2A, B, ...
Context 3
... continuously between the homologous chromosomes, flanked by 45S signals, confirmed the chias- matic nature of these associations (Fig. 2C). In those cases in which the 45S signal formed a solid continuous block, it was reasoned that these are also chiasmatic bonds but the diagnostic unlabelled strip is invisible due to the plane of viewing ( Fig. 2A, B, ...
Citations
... As a consequence, studying crossover frequency along the chromosome is challenging and requires the analysis of many meiotic events. In plants, crossover numbers are commonly assessed by cytological analyses, for instance by counting chiasmata on metaphase I chromosome spreads, or by counting foci of crossover-associated proteins, for example HEI10 or MLH1 [2][3][4][5][6]. However, those techniques require specialized expertise and can be challenging for plants with small chromosomes such as Arabidopsis. ...
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).Key wordsMeiosisCrossoverRecombinationFluorescent-Tagged Lines (FTLs)Traffic Lines
SeedScoring
CellProfilerArabidopsis
... COs between homologous pairs in Arabidopsis have been visualized and scored as chiasmata at metaphase I using a simple combination of acid spreading, DAPI (4,6-diamidino-2-phenylindole) staining, and epifluorescence ( Fig. 2A) (López et al., 2012;Moran et al., 2001;Sanchez-Moran et al., 2002). Bivalents are highly compacted at metaphase I and aligned at the metaphase I plate ( Figs. 2A and 2B). ...
... Fluorescence in situ hybridization (FISH), also known as chromosome painting, uses fluorescent-labeled chromosome-specific nucleic acid probes to identify chromosomes and their aberrations (Cremer et al., 1988;Pinkel et al., 1988). In Arabidopsis, FISH staining of 5S and 45S rDNA regions enables the identification of individual chromosomes, and the number of chiasmata can be counted based on bivalent shape (López et al., 2012;Moran et al., 2001;Sanchez-Moran et al., 2002). Painting whole chromosomes by FISH has led to the investigation of meiotic pairing, chromosome rearrangements, and even CO sites in crop species (Albert et al., 2019;Zhao et al., 2019). ...
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.
... Cytological methods rely on microscopy to identify the chromosome features related to crossover formation in addition to axis formation, DSB formation, and synapsis [9]. The morphology of bivalent chromosomes and chiasmata when applied with DAPI staining provides evidence of the number and position of crossovers [10,11]. Immunohistochemistry of MLH1 foci at late pachytene, diplotene, or diakinesis can identify the number and position of Class I crossovers [12,13]. ...
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.Key wordsMeiosisCrossoverTetrad analysisQRT1FTLsDeepTetrad
... Metaphase I nuclei are also analyzed to determine the number of chiasmata formed in a meiotic nucleus. Individual chromosomes are identified by fluorescence in situ hybridization (FISH) staining for the 5S and 45S rDNA repeats (see section "Widefield Epifluorescence Microscopy") and the number of chiasmata per chromosome arm is deduced from bivalent shape (Sanchez Moran et al., 2001;Lopez et al., 2012). ...
... The FISH technique has been widely used within the meiotic plant community and is an essential tool to determine the chiasma frequency on individual chromosomes. To this end, bivalents are unequivocally identified by FISH labeling of the 5S and 45S rDNA regions and the number of chiasmata per chromosome arm is deduced from bivalent shape (Sanchez Moran et al., 2001;Lopez et al., 2012;Armstrong, 2013;Kurzbauer et al., 2018Kurzbauer et al., , 2021. Finally, single-molecule RNA-FISH has become increasingly popular to analyze transcription in plant tissues. ...
Visualization of meiotic chromosomes and the proteins involved in meiotic recombination have become essential to study meiosis in many systems including the model plant Arabidopsis thaliana. Recent advances in super-resolution technologies changed how microscopic images are acquired and analyzed. New technologies enable observation of cells and nuclei at a nanometer scale and hold great promise to the field since they allow observing complex meiotic molecular processes with unprecedented detail. Here, we provide an overview of classical and advanced sample preparation and microscopy techniques with an updated Arabidopsis meiotic atlas based on super-resolution microscopy. We review different techniques, focusing on stimulated emission depletion (STED) nanoscopy, to offer researchers guidance for selecting the optimal protocol and equipment to address their scientific question.
... Fixation, spread preparation, FISH, and chiasma counts were performed as described previously (Sanchez Moran et al., 2001;Lopez et al., 2012;Armstrong, 2013;Kurzbauer et al., 2018), except that locked nucleic acid (LNA) probes were used (5S rDNA: 5 0 -TYE563-CAAGCACGCTTAACTGCGGAG TTCTGAT-3 0 ; 45S rDNA: 5 0 -TYE655-GGTCCGAGGATTTGT CGACCAG-3 0 ; both produced by Exiqon). Imaging was performed with a conventional fluorescence microscope (Zeiss Axioplan) and appropriate filters. ...
Meiosis is a specialized cell division that gives rise to genetically distinct gametic cells. Meiosis relies on the tightly controlled formation of DNA double-strand breaks (DSBs) and their repair via homologous recombination for correct chromosome segregation. Like all forms of DNA damage, meiotic DSBs are potentially harmful and their formation activates an elaborate response to inhibit excessive DNA break formation and ensure successful repair. Previous studies established the protein kinase ATM as a DSB sensor and meiotic regulator in several organisms. Here we show that Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover (CO) formation and synaptonemal complex (SC) organization, all vital for the successful completion of meiosis. We developed a single-molecule approach to quantify meiotic breaks and determined that ATM is essential to limit the number of meiotic DSBs. Local and genome-wide recombination screens showed that ATM restricts the number of interference-insensitive COs, while super-resolution STED nanoscopy of meiotic chromosomes revealed that the kinase affects chromatin loop size and SC length and width. Our study extends our understanding of how ATM functions during plant meiosis and establishes it as an integral factor of the meiotic program.
... It is well-known that genetic variation among varieties can lead to variation in chromosome-pairing behaviour and both global and local CO frequencies in various species (Esch et al. 2007). These include rose (Bourke et al. 2017), Arabidopsis (Sanchez-Moran et al. 2002López et al. 2012), maize (Bauer et al. 2013) and potato (Swaminathan 1954), and also animals, including mice (Dumont et al. 2009). However, the extent of variation in chiasma frequency in modern commercial S. tuberosum germplasm is largely unknown. ...
Naturally occurring autopolyploid species, such as the autotetraploid potato Solanum tuberosum, face a variety of challenges during meiosis. These include proper pairing, recombination and correct segregation of multiple homologous chromosomes, which can form complex multivalent configurations at metaphase I, and in turn alter allelic segregation ratios through double reduction. Here, we present a reference map of meiotic stages in diploid and tetraploid S. tuberosum using fluorescence in situ hybridisation (FISH) to differentiate individual meiotic chromosomes 1 and 2. A diploid-like behaviour at metaphase I involving bivalent configurations was predominant in all three tetraploid varieties. The crossover frequency per bivalent was significantly reduced in the tetraploids compared with a diploid variety, which likely indicates meiotic adaptation to the autotetraploid state. Nevertheless, bivalents were accompanied by a substantial frequency of multivalents, which varied by variety and by chromosome (7–48%). We identified possible sites of synaptic partner switching, leading to multivalent formation, and found potential defects in the polymerisation and/or maintenance of the synaptonemal complex in tetraploids. These findings demonstrate the rise of S. tuberosum as a model for autotetraploid meiotic recombination research and highlight constraints on meiotic chromosome configurations and chiasma frequencies as an important feature of an evolved autotetraploid meiosis.
... The absence of the obligate number of COs can be deleterious and lead to production of aneuploid gametes (Ritz et al., 2017). In two acrocentric Arabidopsis chromosomes (chromosomes 2 and 4), the mean chiasma frequency is higher in the long than in the short arm (López et al., 2012). This difference was also observed for chromosome 4 by analyzing the CO rate variation after single-nucleotide polymorphism genotyping. ...
Centromere position may change despite conserved chromosomal collinearity. Centromere repositioning and evolutionary new centromeres (ENC) were frequently encountered during vertebrate genome evolution, but only rarely observed in plants. The largest crucifer tribe Arabideae (c. 550 species; Brassicaceae, the mustard family) diversified into several well-defined subclades in the virtual absence of chromosome number variation. BAC-based comparative chromosome painting uncovered a constancy of genome structures among 10 analyzed genomes representing seven Arabideae subclades classified as four genera (Arabis, Aubrieta, Draba and Pseudoturritis). Interestingly, the intra-tribal diversification was marked by the high frequency of ENCs on five out of the eight homeologous chromosomes in the crown-group genera, but not in the most ancestral Pseudoturritis genome. From the 32 documented ENCs, at least 26 originated independently including four ENCs recurrently formed at the same position in not closely related species. While chromosomal localization of ENCs does not reflect the phylogenetic position of the Arabideae subclades, centromere seeding was usually confined to long chromosome arms, transforming acrocentric chromosomes to (sub)metacentric ones. Centromere repositioning is proposed as the key mechanism differentiating overall conserved homeologous chromosomes across the crown-group Arabideae subclades. The evolutionary significance of centromere repositioning is discussed in the context of possible adaptive effects on recombination and epigenetic regulation of gene expression.
... In the present case, 38.41% of PMCs showed quadrivalent formation at diakinesis and M I. Occurrence of quadrivalents in the wild diploid individuals seems to be the result of the reciprocal translocations (Mahama and Palmer 2003). Lopez et al. (2012), while studying the chiasma frequency in different ecotypes of Arabidopsis thaliana, suggested that depending upon the type of segregation, translocations significantly affect the pollen fertility. During adjacent type 1 and type 2 cases of segregation, homozygotes and translocated chromosomes go to one pole, producing unstable or non-viable gametes. ...
Cytological investigations have been carried out in one wild accession of Anemone rupicola Cambess. collected from the cold deserts of Ladakh division of Jammu & Kashmir India. The chromosome count of 2n=16 was observed and is the first chromosome record for the species from India. Meiotic analysis showed structural heterozygosity for reciprocal translocations as indicated by the presence of multiple associations of four chromosomes. Detailed meiotic analysis showed Pollen Mother Cells (PMCs) with laggards (32.87%) and chromatin bridges (21.91%) at anaphases I, II (A I, II)/telophases I, II (T I, T II), leading to abnormal microsporogenesis. Consequently, abnormal sporads such as triads (37.14%) and polyads (28.57%) were formed. Structural heterozygosis and other associated abnormal meiotic irregularities in the species showed considerable pollen sterility (32.20%) which could be attributed to the presence of ring/chain shaped multivalent in PMCs.
... Intra-species variability in the recombination rate observed for plants and animals (Dvor ak and McGuire, 1981;Lynn et al., 2004;Esch et al., 2007;Pradillo et al., 2012;Bauer et al., 2013;Dreissig et al., 2015;Ziolkowski et al., 2017) was shown to be affected by both genes controlling meiotic division and the distribution of local genomic features and/or chromatin structure (Akhunov et al., 2003;Yandeau-Nelson et al., 2006;Liu et al., 2009;Wijnker et al., 2013;Rodgers-Melnick et al., 2015;Shilo et al., 2015;Melamed-bessudo et al., 2016). Meiotically induced double-stranded breaks (DSBs) are repaired through pathways resulting in either crossover (CO) or non-crossover events, which can be detected when the interaction of the DSB region occurs between non-sister chromatids (Mercier et al., 2015). ...
Recombination affects the fate of alleles in populations by imposing constraints on the reshuffling of genetic information. Understanding the genetic basis of these constraints is critical for manipulating the recombination process to improve the resolution of genetic mapping, and reducing the negative effects of linkage drag and deleterious genetic load in breeding. Using sequence‐based genotyping of a wheat NAM population of 2,100 recombinant inbred lines created by crossing 29 diverse lines, we mapped QTL affecting the distribution and frequency of 102,000 crossovers (CO). Genome‐wide recombination rate variation was mostly defined by rare alleles with small effects together explaining up to 48.6% of variation. The majority of QTL were additive and showed predominantly trans‐acting effects. The QTL affecting the proximal COs also acted additively without increasing the frequency of distal COs. We showed that the regions with decreased recombination carry more SNPs with possible deleterious effects than the regions with a high recombination rate. Thus, our study offers insights into the genetic basis of recombination rate variation in wheat and its effect on the distribution of deleterious SNPs across the genome. The identified trans‐acting additive QTL can be utilized to manipulate CO frequency and distribution in the large polyploid wheat genome opening the possibility to improve the efficiency of gene pyramiding and reducing the deleterious genetic load in the low recombining pericentromeric regions of chromosomes.
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... Such an adverse effect of structural heterozygotes on pollen sterility has been reported in many plant species, Chrysanthemum zawadskii (Kim et al. 2008), Artemisia parviflora , A. absinthium (Malik et al. 2010), Astragalus chlorostachys (Rana et al. 2012), Achillea millefolium (Singhal et al. 2014), Tanacetum artemisioides (Singhal et al. 2016) and Anthoxanthum odoratum (Singhal and Kumari 2017). Lopez et al. (2012) suggested that depending upon the type of orientation and subsequent segregations, the translocation significantly affect the pollen fertility. We have also been noticed that frequency of meiocytes depicting the reciprocal translocations is directly correlated with the amount of pollen fertility in this observation (Table 1). ...
Male meiotic studies have been performed on three wild accessions of Arthraxon hispidus scored from Parvati valley and Solang valley of Kullu district in Himachal Pradesh. The accessions shared the same diploid chromosome number of 2n=30 and added a variable dysploid chromosome report for the species against the earlier reports of 2n=18, 36, 38, 40. Multiple chromosomal associations due to structural heterozygosity for reciprocal translocations involving four to six chromosomes seem to have increased the chiasma frequency in meiocytes. Owing to adjacent type orientation of multiple chromosomal associations coupled with some meiotic irregularities, the studied accessions also related with some pollen sterility.
