Bivalent Formation Research Articles

The Essential Function of SETDB1 in Homologous Chromosome Pairing and Synapsis during Meiosis

SETDB1 is a histone-lysine N-methyltransferase critical for germline development. However, its function in early meiotic prophase I remains unknown. Here, we report that Setdb1 null spermatocytes display aberrant centromere clustering during leptotene, bouquet formation during zygotene, and subsequent failure in pairing and synapsis of homologous chromosomes, as well as compromised meiotic silencing of unsynapsed chromatin, which leads to meiotic arrest before pachytene and apoptosis of spermatocytes. H3K9me3 is enriched in centromeric or pericentromeric regions and is present in many sites throughout the genome, with a subset changed in the Setdb1 mutant. These observations indicate that SETDB1-mediated H3K9me3 is essential for the bivalent formation in early meiosis. Transcriptome analysis reveals the function of SETDB1 in repressing transposons and transposon-proximal genes and in regulating meiotic and somatic lineage genes. These findings highlight a mechanism in which SETDB1-mediated H3K9me3 during early meiosis ensures the formation of homologous bivalents and survival of spermatocytes.

Assessment of the roles of SPO11-2 and SPO11-4 in meiosis in rice using CRISPR/Cas9 mutagenesis.

In Arabidopsis, chromosomal double-strand breaks at meiosis are presumably catalyzed by two distinct SPO11 transesterases, AtSPO11-1 and AtSPO11-2, together with M-TOPVIB. To clarify the roles of the SPO11 paralogs in rice, we used CRISPR/Cas9 mutagenesis to produce null biallelic mutants in OsSPO11-1, OsSPO11-2, and OsSPO11-4. Similar to Osspo11-1, biallelic mutations in the first exon of OsSPO11-2 led to complete panicle sterility. Conversely, all Osspo11-4 biallelic mutants were fertile. To generate segregating Osspo11-2 mutant lines, we developed a strategy based on dual intron targeting. Similar to Osspo11-1, the pollen mother cells of Osspo11-2 progeny plants showed an absence of bivalent formation at metaphase I, aberrant segregation of homologous chromosomes, and formation of non-viable tetrads. In contrast, the chromosome behavior in Osspo11-4 male meiocytes was indistinguishable from that in the wild type. While similar numbers of OsDMC1 foci were revealed by immunostaining in wild-type and Osspo11-4 prophase pollen mother cells (114 and 101, respectively), a surprisingly high number (85) of foci was observed in the sterile Osspo11-2 mutant, indicative of a divergent function between OsSPO11-1 and OsSPO11-2. This study demonstrates that whereas OsSPO11-1 and OsSPO11-2 are the likely orthologs of AtSPO11-1 and AtSPO11-2, OsSPO11-4 has no major role in wild-type rice meiosis.

Parthenogenesis as a Solution to Hybrid Sterility: The Mechanistic Basis of Meiotic Distortions in Clonal and Sterile Hybrids.

Hybrid sterility is a hallmark of speciation, but the underlying molecular mechanisms remain poorly understood. Here, we report that speciation may regularly proceed through a stage at which gene flow is completely interrupted, but hybrid sterility occurs only in male hybrids whereas female hybrids reproduce asexually. We analyzed gametogenic pathways in hybrids between the fish species Cobitis elongatoides and C. taenia, and revealed that male hybrids were sterile owing to extensive asynapsis and crossover reduction among heterospecific chromosomal pairs in their gametes, which was subsequently followed by apoptosis. We found that polyploidization allowed pairing between homologous chromosomes and therefore partially rescued the bivalent formation and crossover rates in triploid hybrid males. However, it was not sufficient to overcome sterility. In contrast, both diploid and triploid hybrid females exhibited premeiotic genome endoreplication, thereby ensuring proper bivalent formation between identical chromosomal copies. This endoreplication ultimately restored female fertility but it simultaneously resulted in the obligate production of clonal gametes, preventing any interspecific gene flow. In conclusion, we demonstrate that the emergence of asexuality can remedy hybrid sterility in a sex-specific manner and contributes to the speciation process.

SPO11.2 is essential for programmed double‐strand break formation during meiosis in bread wheat (Triticum aestivum L.)

Meiotic recombination is initiated by formation of DNA double-strand breaks (DSBs). This involves a protein complex that includes in plants the two similar proteins, SPO11-1 and SPO11-2. We analysed the sequences of SPO11-2 in hexaploid bread wheat (Triticum aestivum), as well as in its diploid and tetraploid progenitors. We investigated its role during meiosis using single, double and triple mutants. The three homoeologous SPO11-2 copies of hexaploid wheat exhibit high nucleotide and amino acid similarities with those of the diploids, tetraploids and Arabidopsis. Interestingly, however, two nucleotides deleted in exon-2 of the A copy lead to a premature stop codon and suggest that it encodes a non-functional protein. Remarkably, the mutation was absent from the diploid A-relative Triticum urartu, but present in the tetraploid Triticum dicoccoides and in different wheat cultivars indicating that the mutation occurred after the first polyploidy event and has since been conserved. We further show that triple mutants with all three copies (A, B, D) inactivated are sterile. Cytological analyses of these mutants show synapsis defects, accompanied by severe reductions in bivalent formation and numbers of DMC1 foci, thus confirming the essential role of TaSPO11-2 in meiotic recombination in wheat. In accordance with its 2-nucleotide deletion in exon-2, double mutants for which only the A copy remained are also sterile. Notwithstanding, some DMC1 foci remain visible in this mutant, suggesting a residual activity of the A copy, albeit not sufficient to restore fertility.

Dynamic localization of SPO11-1 and conformational changes of meiotic axial elements during recombination initiation of maize meiosis.

Meiotic double-strand breaks (DSBs) are generated by the evolutionarily conserved SPO11 complex in the context of chromatin loops that are organized along axial elements (AEs) of chromosomes. However, how DSBs are formed with respect to chromosome axes and the SPO11 complex remains unclear in plants. Here, we confirm that DSB and bivalent formation are defective in maize spo11-1 mutants. Super-resolution microscopy demonstrates dynamic localization of SPO11-1 during recombination initiation, with variable numbers of SPO11-1 foci being distributed in nuclei but similar numbers of SPO11-1 foci being found on AEs. Notably, cytological analysis of spo11-1 meiocytes revealed an aberrant AE structure. At leptotene, AEs of wild-type and spo11-1 meiocytes were similarly curly and discontinuous. However, during early zygotene, wild-type AEs become uniform and exhibit shortened axes, whereas the elongated and curly AEs persisted in spo11-1 mutants, suggesting that loss of SPO11-1 compromised AE structural maturation. Our results reveal an interesting relationship between SPO11-1 loading onto AEs and the conformational remodeling of AEs during recombination initiation.

How to Perform an Accurate Analysis of Metaphase I Chromosome Configurations in Autopolyploids of Arabidopsis thaliana.

During meiosis, accurate segregation of chromosomes requires the formation of bivalents at metaphase I. In autopolyploids, there are more than two copies of each chromosome with the same chance to form chiasmata at meiosis. This leads to the formation of multivalent configurations in which chiasma quantification is rather complicated. Here, we present an improved cytological protocol, including fluorescence in situ hybridization, to obtain high quality spreads of metaphase I chromosomes from Arabidopsis thaliana autotetraploids. This method allows an accurate analysis of the different meiotic configurations and enables the assessment of the number of chiasmata formed by each tetrasome (group of four homologs).

Comparative meiosis and cytogenomic analysis in euploid and aneuploid hybrids of Urochloa P. Beauv.

The genus Urochloa includes most of the important grasses and hybrids currently used as pastures in the tropical regions. Cytogenetic analyzes have identified some aneuploid hybrids that provide new perspectives for genetic breeding. The objective was to analyze the meiotic behavior in euploid (2n = 4x = 36) and aneuploid (2n = 4x = 36 + 2) hybrids of U. ruziziensis x U. decumbens and U. ruziziensis x U. brizantha. Later, the chromosomes and respective genomes involved in pairing configurations and abnormalities were identified through GISH, with an emphasis on tracking the behavior of the additional chromosomes in the aneuploid hybrid U. ruziziensis x U. decumbens (B1B2B2B2 genomes). The aneuploid U. ruziziensis x U. decumbens shows a higher frequency of univalents, reduction of bivalents, and higher index of irregularities compared with the euploid hybrid. For the aneuploid U. ruziziensis x U. brizantha, there was a reduction in the frequency of univalents, an increase in bivalent and trivalent rates and a lower frequency of abnormalities when compared with the euploid hybrid. The rates of meiotic abnormalities and pairing configurations are parental genotype-dependent and influenced by trisomy. The chromosomes of the B1 and B2 genomes of the aneuploid hybrid (U. ruziziensis x U. decumbens) are involved in the formation of univalents, bivalents, and multivalents in inter-, intra- and inter-intragenomic pairings. In general, the segregation times of chromosomes of the genomes are different, since the chromosomes of the B1 genome segregate more slowly.

70. PREIMPLANTATION GENETIC TESTING FOR YQH-AUTOSOME TRANSLOCATION CARRIERS: ANALYSIS OF GAMETE SEGREGATION

Introduction Y-autosome translocations occur in approximately 1:2000 in population and represent special group of reciprocal exchanges. Due to a balanced nature they do not influence on phenotype in general. However, Y-autosome translocation can dislocate sex bivalent formation and chromosome synapsis in the first meiotic division prophase that led to gamete aneuploidy or pachytene arrest. More often Y-derived DNA, predominantly Yq12 band, is translocated onto the short arm of acrocentric chromosomes, mainly chromosomes 15 and 22 due to a strong correlation in their satellite DNA sequences. In women inheritance of such derivative acrocentric chromosomes could be associated with the risk of ovarian malignancy, but the particular mechanism of this pathological effect is not entirely clear since the Yq12 band consists of tandemly organized satellite DNA sequences. It can be associated with the transcription of satellite DNAwhich occurs during carcinogenesis. Material & methods Two families with Yq12/15p and one – with Yq12/22p translocations were analyzed during IVF cycle with PGT in International Centre for Reproductive Medicine. The mean age of men and women was 36±2.8 and 34.25±1.4 years respectively. Karyotyping analysis was performed using GTG banding technique on metaphase chromosomes from peripheral blood lymphocytes. Fluorescence in situ hybridization (FISH) was used to clarify the breakpoint on chromosomes and confirm the chromosomes-specificity of the probes. Additionally, seven chromosomes (13, 14, 15 or 22, 16, 18, 21, X), which are not involved in rearrangement, were analyzed. Results FISH with chromosome specific probes was performed on blastomere from cleavage-stage embryo. A total of 42 blastomeres were analyzed. In 43% of the blastomeres «adjacent-1» segregationwith derivate autosome and X or Y chromosomes was revealed. Segregation 3:1 was observed in 10% of the cells, with no predominant inheritance of the derivative autosome. In 21% of cells the type of chromosome segregation could not be determined due to mosaicismor polyploidy. 45% of the cells were aneuploid for autosomes, which are not involved in the translocation, which may be connected with interchromosomal effect. Conclusions Carriers of Yq12/15p or Yq12/22p translocations have a predominant inheritance of the derivative autosome, which can be associated with the disruption ofsex bivalent segregation. This research was supported by Russian Foundation for Basic Research (grant # 18-34-00279 ).

Interspecies hybridization is not the end: the lesson from asexual fishes from European spined loaches complex (Cobitis)

Interspecies hybridization is not the end: the lesson from asexual fishes from European spined loaches complex (Cobitis)

Cytomorphological Comparison in Natural Intraspecific Cytotypes (2<i>x</i>, 4<i>x</i>) in <i>Brachyactis pubescens</i> from Northwest Himalayas, India

Intraspecific euploid cytotypes (2n=18, 36) were found in the wild plants of Brachyactis pubescens in North West Himalayas from the hills of Ladakh division of Jammu and Kashmir and Solang Valley, Kullu district, Himachal Pradesh. The plants with each ploidy level showed regular bivalent formation, equal segregation of chromosomes during anaphases and normal sporads. The 2x plants were found growing at higher elevations in the Kashmir Himalayas and adjoining hills of Afghanistan while the 4x plants grow at lower elevations in the Solang Valley, Himachal Pradesh. The 4x and 2x plants could be differentiated in various morphometric parameters. The 4x plants were noticed to be robust and much taller and show gigantism in leaf and floral characters besides depicting the increase in the size of trichomes, stomata and pollen grains. Analysis of meiotic chromosome pairing in the tetraploid taxa indicates towards its allopolyploid nature.

Revisiting Meiosis in Sugarcane: Chromosomal Irregularities and the Prevalence of Bivalent Configurations.

Traditional sugarcane cultivars (Saccharum officinarum) proved highly susceptible to diseases, and this led breeders to progress to interspecific crosses resulting in disease resistance. A backcrossing program to S. officinarum was then required to boost sucrose content. Clonal selection across generations and incorporation of other germplasm into cultivated backgrounds established the (narrow) genetic base of modern cultivars (Saccharum spp.), which have a man-made genome. The genome complexity has inspired several molecular studies that have elucidated aspects of sugarcane genome constitution, architecture, and cytogenetics. However, there is a critical shortage of information on chromosome behavior throughout meiosis in modern cultivars. In this study, we examined the microsporogenesis of a contemporary variety, providing a detailed analysis of the meiotic process and chromosome association at diakinesis, using FISH with centromeric probes. Chromosomal abnormalities were documented by examining high quality preparations of pollen mother cells (700 in total). Approximately 70% of the cells showed abnormalities, such as metaphase chromosomes not lined up at the plate, lagging chromosomes and chromosomal bridges, and tetrad cells with micronuclei. Some dyads with asynchronous behavior were also observed. Due to the hybrid composition of the sugarcane genome, we suggest that bivalent incomplete pairing may occur in the first prophase leading to univalency. The presence of rod bivalents showing the lagging tendency is consistent with a reduction in chiasma frequency. Finally, the presence of chromatin bridges indicates the indirect occurrence of chromosomal inversions, although chromosome fragments were not clearly recognized. Possible reasons for such meiotic abnormalities and the large prevalence of bivalent formation are discussed.

The fate of ribosomal RNA genes in spontaneous polyploid dogrose hybrids [Rosa L. sect. Caninae (DC.) Ser.] exhibiting non-symmetrical meiosis.

Dogroses represent an exceptional system for studying the effects of genome doubling and hybridization: their asymmetrical meiosis enables recombination in bi-parentally inherited chromosomes but prevents it in maternally inherited ones. We employed fluorescent insitu hybridization, genome skimming, amplicon sequencing of genomic and cDNA as well as conventional cloning of nuclear ribosomal DNA in two phylogenetically distinct pentaploid (2n=5x=35) species, Rosa canina and Rosainodora, and their naturally occurring reciprocal hybrids, Rosa dumalis (5x) and Rosa agrestis (5x, 6x). Both progenitor species differed in composition, meiotic behaviour and expression of rDNA loci: R.canina (five 18S and 5-8 5S loci) was dominated by the Canina ribotypes, but R.inodora (four 18S loci and 7-8 5S loci) by the Rubiginosa ribotype. The co-localized 5S/18S loci occurred on either bivalent-forming (R.canina) or univalent-forming (R.inodora) chromosomes. Ribosomal DNA loci were additively inherited; however, the Canina ribotypes were dominantly expressed, even in genotypes with relatively low copy number of these genes. Moreover, we observed rDNA homogenization towards the paternally transmitted Canina ribotype in 6x R.agrestis. The here-observed variation in arrangement and composition of rDNA types between R.canina and R.inodora suggests the involvement of different genomes in bivalent formation. This results supports the hypothesis that the asymmetrical meiosis arose at least twice by independent ancient hybridization events.

Meiotic Recombination: Mixing It Up in Plants.

Meiosis halves diploid chromosome numbers to haploid levels that are essential for sexual reproduction in most eukaryotes. Meiotic recombination ensures the formation of bivalents between homologous chromosomes (homologs) and their subsequent proper segregation. It also results in genetic diversity among progeny that influences evolutionary responses to selection. Moreover, crop breeding depends upon the action of meiotic recombination to rearrange elite traits between parental chromosomes. An understanding of the molecular mechanisms that drive meiotic recombination is important for both fundamental research and practical applications. This review emphasizes advances made during the past 5 years, primarily in Arabidopsis and rice, by summarizing newly characterized genes and proteins and examining the regulatory mechanisms that modulate their action.

Hybrids of Passiflora: P. gardneri versus P. gibertii, confirmation of paternity, morphological and cytogenetic characterization

Two new varieties of interspecific hybrids of Passiflora have been developed from the cross between P. gardneri versus P. gibertii, both registered under the Passiflora Society International. Twelve putative hybrids were analyzed. Hybridization was confirmed using RAPD and SSR markers. Primer UBC11 (5′-CCGGCCTTAC-3′) generated informative bands. Primer SSR Pe75 has amplified species-specific fragments and a heterozygote status was observed with two parent bands 300 and 350 bp. The molecular markers generated have been analyzed for the presence or absence of specific informative bands. Based on the morphological characterization, we have identified two hybrid varieties: P. ‘Gabriela’ and P. ‘Bella’. P. ‘Gabriela’ produced flowers in bluish tones, bluish petals on the adaxial and abaxial faces, light blue sepals on the adaxial and light green on the abaxial faces, corona with the base of filaments in intense lilac color and white apex. P. ‘Bella’ produced flowers in lilac tones, intense lilac petals on the adaxial and abaxial faces, dark lilac sepals with whitish edges on the adaxial and light green on the abaxial faces, corona with the base of filaments in intense lilac color and white apex. The cytogenetic analysis verified that the hybrids have the same chromosomal number as the parents (2n = 18); the formation of bivalents between the homeologous chromosomes (n = 9) was observad, leading to regular meiosis, which allows the sexual reproduction and use of these hybrids in breeding programs.

Microscopic Characterization of a Transposon-Induced Male-Sterile, Female-Sterile Mutant in Glycine max L.

Premise of research. A male-sterile, female-sterile mutant was discovered in a w4-m mutable line of Glycine max L. The mechanism of its sterility was not well understood. Therefore, different cytological and microscopic techniques were undertaken to better understand the process of mutant phenotype development. Molecular research indicated that mer3 was responsible for the sterility.Methodology. Macro images were collected of whole plants, flowers, anthers, pods, and ovules. Chromosome spreads from anthers at various meiotic stages were examined. Confocal scanning laser microscopy using optical sectioning was utilized on whole anthers and ovules at various developmental stages. Whole mature anthers and isolated pollen images were collected and studied with SEM.Pivotal results. In observations of the mutant, male cell development was found to begin normally and then digresses at metaphase I of meiosis, when abnormal segregation of chromosomes with reduced bivalent formation was observed. It was the abnorma...

Cytogenetic analysis of five recommended and released Philippine varieties of sugarcane (Saccharum officinarumL.) and their parentals

AbstractMeiotic chromosomal behavior of five recommended and released Philippine varieties of sugarcane and their parents, namely VMC 95-09 (Q 90 × Unidentified), PSR 97-45 (VMC 74-292 × CP 72-1312), PSR 00-343 (VMC 84-947 × Unidentified), PSR 00-161 (VMC 86-550 × VMC 87-599), and PSR 00-34 (VMC 84-194 × PHIL 89-43) were characterized and analyzed. Samples were collected from the tassel source of Philippine Sugar Research Institute Foundation Inc. (PHILSURIN) located in Negros Occidental, Philippines. Gametic number for each variety was determined at diakinesis and at least 50 cells per meiotic stage was analyzed. All varieties exhibited chromosome mosaicism at diakinesis with n = 40–60. Chromosome clumping was observed at diakinesis of Q 90 (68%), CP 72-1312 (35%), and PSR 97-45 (22%) pollen mother cells. High frequency of bivalent formation (68–100%) was observed, both in parents and hybrids, indicating that chromosome pairing is autosyndetic. Irregularities at meiosis were also observed at high frequen...

BIME2, a novel gene required for interhomolog meiotic recombination in the protist model organism Tetrahymena

Meiotic recombination is initiated by DNA double-strand breaks (DSBs). Most DSBs are converted into nonreciprocal exchanges (gene conversions) or crossovers (COs) between sister chromatids. Only a minority of DSBs are processed toward interhomolog COs, the precursors of the chiasmata that connect homologous chromosomes. Dmc1, the meiosis-specific paralog of the universal recombination protein Rad51, is required for interhomolog COs; in its absence, univalents are primarily formed. Here, we report a ciliate-specific novel meiotic gene, BIME2, which also promotes interhomolog crossing over. In the bime2Δ mutant, DSBs are formed and repaired normally, but bivalent formation is strongly reduced. Bime2 protein forms foci on chromatin during meiotic prophase, and chromatin localization of Bime2 and Dmc1 is largely interdependent. Bime2 distantly resembles budding yeast Rdh54/Tid1 and the vertebrate Rad54B helicases and may have similar functions in promoting or stabilizing Dmc1 nucleoprotein filaments.

Bivalent Formation 1, a plant-conserved gene, encodes an OmpH/coiled-coil motif-containing protein required for meiotic recombination in rice.

Meiosis is essential for eukaryotic sexual reproduction and plant fertility. In comparison with over 80 meiotic genes identified in Arabidopsis, there are only ~30 meiotic genes characterized in rice (Oryza sativa L.). Many genes involved in the regulation of meiotic progression remain to be determined. In this study, we identified a sterile rice mutant and cloned a new meiotic gene, OsBVF1 (Bivalent Formation 1) by map-based cloning. Molecular genetics and cytological approaches were carried out to address the function of OsBVF1 in meiosis. Phylogenetic analyses were used to study the evolution of OsBVF1 and its homologs in plant species. Here we showed that the bvf1 male meiocytes were defective in formation of meiotic double strand break, thereby resulting in a failure of bivalent formation in diakinesis and unequal chromosome segregation in anaphase I. The causal gene, OsBVF1, encodes a unique OmpH/coiled-coil motif-containing protein and its homologs are highly conserved in the plant kingdom and seem to be a single-copy gene in the majority of plant species. Our study demonstrates that OsBVF1 is a novel plant-conserved factor involved in meiotic recombination in rice, providing a new insight into understanding of meiotic progression regulation.

SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes.

SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre-driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females.

Meiotic behaviour and B chromosomes in Argentine populations ofChrysolaena verbascifolia(Vernonieae: Asteraceae)

Fil: Via Do Pico, Gisela Mariel. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Nordeste. Instituto de Botanica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botanica del Nordeste; Argentina