Homologous Chromosome Pairing Research Articles

Cytogenetic characterization of EPSPS gene amplification in glyphosate-resistant Hordeum glaucum and Bromus diandrus from Australia.

As a result of extensive selection, two polyploid grass weeds, Hordeum glaucum (northern barley grass; 2n = 4x = 28) and Bromus diandrus (ripgut brome; 2n = 8x = 56), have evolved resistance to glyphosate, in Australia. Previous research suggested amplification of 5-enolpyruvylshikimate-3-Phosphate synthase (EPSPS) gene confers resistance in these two weed species. The objective of this research was to investigate the genomic organization of the EPSPS gene in these two species through molecular cytogenetic analyses of fluorescence in situ hybridization (FISH) to understand possible mechanism of amplification of this gene. EPSPS copy number of H. glaucum and B. diandrus plants was estimated via quantitative polymerase chain reaction. The susceptible plants of both species had one copy of EPSPS, whereas the resistant plants of H. glaucum and B. diandrus had 14-17 and 16-32 copies, respectively. FISH analysis of glyphosate-susceptible (Hg-RWS) H. glaucum, revealed four faint signals of the EPSPS gene in two pairs of homologous chromosomes, at the telomeric region. The glyphosate-resistant H. glaucum (Hg-YP1) also showed amplification of EPSPS gene at telomeric regions in two pairs of homologous chromosomes, but the signals were brighter and appeared as cluster of EPSPS genes. Similarly, the glyphosate-susceptible B. diandrus (Bd-S) plants showed faint signals of EPSPS gene on two homologous chromosomes, at the telomeric position. However, samples of two glyphosate-resistant, B. diandrus, Bd-SA988 and Bd-Vic showed much brighter hybridization signals of EPSPS gene, located at the telomere on two homologous chromosomes, suggesting an increase in EPSPS gene copies at this position. Overall, unequal crossover during meiosis may have triggered the initial EPSPS gene duplication sparking the evolution of glyphosate resistance.

Impact of interactions between Su(Hw)-dependent insulators on the transvection effect in <I>Drosophila melanogaster</i>

Transvection is a phenomenon of interallelic communication in which enhancers can activate a specific promoter located on a homologous chromosome. Insulators play a significant role in ensuring functional interactions between enhancers and promoters. In the presented work, we created the model where two or three copies of the insulator are located next to enhancers and promoters localized on homologous chromosomes. Using the Su(Hw) insulator as model, we showed that the functional interaction between a pair of insulators promotes enhancer-promoter trans interactions. The interaction between the three insulators, on the contrary, can lead to the formation of chromatin loops that sterically hinder the full enhancer-promoter interaction. The results of the work suggest the participation of insulators in the regulation of homologous chromosome pairing and in communication between distant genomic loci.

Proteins and noncoding RNAs that promote homologous chromosome recognition and pairing in fission yeast meiosis undergo condensate formation invitro.

Pairing of homologous chromosomes during meiosis is crucial for successful sexual reproduction. Previous studies have shown that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 locus and plays a key role in mediating robust pairing during meiosis. Several RNA-binding proteins accumulate at the sme2 and other lncRNA gene loci in conjunction with the lncRNAs transcribed from these loci. These lncRNA-protein complexes form condensates that exhibit phase separation properties on chromosomes and are necessary for robust pairing of homologous chromosomes. To further understand the mechanisms by which phase separation affects homologous chromosome pairing, we conducted an invitro phase separation assay with the sme2 RNA-associated proteins (Smps) and RNAs. Our findings reveal that one of the Smps, Seb1, forms condensates resembling phase separation; the observed number and size of these condensates increase upon the addition of another Smp, Rhn1, and purified RNAs. Additionally, we have found that RNAs protect Smp condensates from treatment with 1,6-hexanediol. The Smp condensates containing different types of RNA display distinct FRAP profiles, and the Smp condensates containing the same type of RNA tend to fuse together more readily than those containing different types of RNAs. Collectively, these results indicate that the specific RNA species within condensates modulate their physical properties, potentially enabling the formation of regional RNA-Smp condensates with distinct characteristics that facilitate homologous chromosome pairing.

G6PC3 is involved in spermatogenesis by maintaining meiotic sex chromosome inactivation.

Meiosis, a process unique to germ cells, involves formation and repair of double-stranded nicks in DNA, pairing and segregation of homologous chromosomes, which ultimately achieves recombination of homologous chromosomes. Genetic abnormalities resulted from defects in meiosis are leading causes of infertility in humans. Meiotic sex chromosome inactivation (MSCI) plays a crucial role in the development of male germ cells in mammals, yet its underlying mechanisms remain poorly understood. In this study, we illustrate the predominant presence of a protein known as glucose 6 phosphatase catalyzed 3 (G6PC3) in pachytene spermatocytes, with a high concentration in the sex body (XY body), suggesting its significant involvement in male germ cell development. By employing CRISPR-Cas9 technology, we generate mice deficient in the G6pc3 gene, resulting in complete meiotic arrest at the pachytene stage in spermatocytes and are completely sterile. Additionally, we observe abnormal XY body formation and impaired MSCI in G6pc3-knockout spermatocytes. These findings underscore G6pc3 as a new essential regulator that is essential for meiotic progression. G6PC3 is involved in spermatocyte during male spermatogenesis development by the maintenance of meiosis chromosome silencing.

A haplotype-resolved genome assembly of Coptis teeta, an endangered plant of significant medicinal value

Coptis teeta Wall. (Ranunculaceae), an endangered plant species of significant medicinal value, predominantly undergoes clonal propagation, potentially compromising the species’ evolutionary potential and ultimately increase its risk of extinction. In this study, we successfully assembled two sets of haploid genomes (Hap1 and Hap2) for C. teeta, comprising nine homologous chromosome pairs, by employing Illumina and PacBio sequencing technologies. The genome annotation identified a total of 43,979 and 46,311 protein-coding genes in Hap1 and in Hap2, and most of them were functionally annotated. The high-quality reference genome will serve as an indispensable genomic resource for conservation and comprehensive exploitation of this endangered species. Between the two haploid genomes, numerous structural alterations were detected within the nine homologous chromosome pairs, potentially resulting in aberrant synapsis and irregular chromosomal segregation and thus contributing to the sustained preservation of clonal propagation in C. teeta. The findings offer new perspective for elucidating the genetic mechanism underlying the compromised sexual reproductive capacity of C. teeta, thereby facilitating its enhancement though molecular breeding and genetic improvement.

Kinesin-1-like protein PSS1 is essential for full-length homologous pairing and synapsis in rice meiosis.

The pairing and synapsis of homologous chromosomes are crucial for their correct segregation during meiosis. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex can recruit kinesin protein at the nuclear envelope, affecting telomere bouquet formation and homologous pairing. Kinesin-1-like protein Pollen Semi-Sterility1 (PSS1) plays a pivotal role in male meiotic chromosomal behavior and is essential for fertility in rice. However, its exact role in meiosis, especially as kinesin involved in homologous pairing and synapsis, has not been fully elucidated. Here, we generated three pss1 mutants by genome editing technology to dissect PSS1 biological functions in meiosis. The pss1 mutants exhibit alterations in the radial microtubule organization at pachytene and manifest a deficiency in telomere clustering, which is critical for full-length homologous pairing. We reveal that PSS1 serves as a key mediator between chromosomes and cytoskeleton, thereby regulating microtubule organization and transmitting the force to nuclei to facilitate homologous chromosome pairing and synapsis in meiosis.

A low-cost dpMIG-seq method for elucidating complex inheritance in polysomic crops: a case study in tetraploid blueberry.

Next-generation sequencing (NGS) library construction often requires high-quality DNA extraction, precise adjustment of DNA concentration, and restriction enzyme digestion to reduce genome complexity, which results in increased time and cost in sample preparation and processing. To address these challenges, a PCR-based method for rapid NGS library preparation, named dpMIG-seq, has been developed and proven effective for high-throughput genotyping. However, the application of dpMIG-seq has been limited to diploid and polyploid species with disomic inheritance. In this study, we obtained genome-wide single nucleotide polymorphism (SNP) markers for tetraploid blueberry to evaluate genotyping and downstream analysis outcomes. Comparison of genotyping qualities inferred across samples with different DNA concentrations and multiple bioinformatics approaches revealed high accuracy and reproducibility of dpMIG-seq-based genotyping, with Pearson's correlation coefficients between replicates in the range of 0.91 to 0.98. Furthermore, we demonstrated that dpMIG-seq enables accurate genotyping of samples with low DNA concentrations. Subsequently, we applied dpMIG-seq to a tetraploid F1 population to examine the inheritance probability of parental alleles. Pairing configuration analysis supported the random meiotic pairing of homologous chromosomes on a genome-wide level. On the other hand, preferential pairing was observed on chr-11, suggesting that there may be an exception to the random pairing. Genotypic data suggested quadrivalent formation within the population, although the frequency of quadrivalent formation varied by chromosome and cultivar. Collectively, the results confirmed applicability of dpMIG-seq for allele dosage genotyping and are expected to catalyze the adoption of this cost-effective and rapid genotyping technology in polyploid studies.

Transcriptomic analysis of meiotic genes during the mitosis-to-meiosis transition in Drosophila females.

Germline cells produce gametes, which are specialized cells essential for sexual reproduction. Germline cells first amplify through several rounds of mitosis before switching to the meiotic program, which requires specific sets of proteins for DNA recombination, chromosome pairing, and segregation. Surprisingly, we previously found that some proteins of the synaptonemal complex, a prophase I meiotic structure, are already expressed and required in the mitotic region of Drosophila females. Here, to assess if additional meiotic genes were expressed earlier than expected, we isolated mitotic and meiotic cell populations to compare their RNA content. Our transcriptomic analysis reveals that all known meiosis I genes are already expressed in the mitotic region; however, only some of them are translated. As a case study, we focused on mei-W68, the Drosophila homolog of Spo11, to assess its expression at both the mRNA and protein levels and used different mutant alleles to assay for a premeiotic function. We could not detect any functional role for Mei-W68 during homologous chromosome pairing in dividing germ cells. Our study paves the way for further functional analysis of meiotic genes expressed in the mitotic region.

CDK phosphorylation of Sfr1 downregulates Rad51 function in late-meiotic homolog invasions.

Meiosis is the developmental program that generates gametes. To produce healthy gametes, meiotic recombination creates reciprocal exchanges between each pair of homologous chromosomes that facilitate faithful chromosome segregation. Using fission yeast and biochemical, genetic, and cytological approaches, we have studied the role of CDK (cyclin-dependent kinase) in the control of Swi5-Sfr1, a Rad51-recombinase auxiliary factor involved in homolog invasion during recombination. We show that Sfr1 is a CDK target, and its phosphorylation downregulates Swi5-Sfr1 function in the meiotic prophase. Expression of a phospho-mimetic sfr1-7D mutant inhibits Rad51 binding, its robust chromosome loading, and subsequently decreases interhomolog recombination. On the other hand, the non-phosphorylatable sfr1-7A mutant alters Rad51 dynamics at late prophase, and exacerbates chromatin segregation defects and Rad51 retention observed in dbl2 deletion mutants when combined with them. We propose Sfr1 phospho-inhibition as a novel cell-cycle-dependent mechanism, which ensures timely resolution of recombination intermediates and successful chromosome distribution into the gametes. Furthermore, the N-terminal disordered part of Sfr1, an evolutionarily conserved feature, serves as a regulatory platform coordinating this phospho-regulation, protein localization and stability, with several CDK sites and regulatory sequences being conserved.

Identification of the Polo-like kinase substrate required for homologous synapsis in C. elegans.

The synaptonemal complex (SC) is a zipper-like protein structure that aligns homologous chromosome pairs and regulates recombination during meiosis. Despite its conserved appearance and function, how synapsis occurs between chromosome axes remains elusive. Here, we demonstrate that Polo-like kinases (PLKs) phosphorylate a single conserved residue in the disordered C-terminal tails of two paralogous SC subunits, SYP-5 and SYP-6, to establish an electrostatic interface between the SC central region and chromosome axes in C. elegans . While SYP-5/6 phosphorylation is dispensable for the ability of SC proteins to self-assemble, local phosphorylation by PLKs at the pairing center is crucial for SC elongation between homologous chromosome axes. Additionally, SYP-5/6 phosphorylation is essential for asymmetric SC disassembly and proper PLK-2 localization after crossover designation, which drives chromosome remodeling required for homolog separation during meiosis I. This work identifies a key regulatory mechanism by which localized PLK activity mediates the SC-axis interaction through phosphorylation of SYP-5/6, coupling synapsis initiation to homolog pairing.

Distinct roles of spindle checkpoint proteins in meiosis

Gametes are produced via meiosis, a specialized cell division associated with frequent errors that cause birth defects and infertility. Uniquely in meiosis I, homologous chromosomes segregate to opposite poles, usually requiring their linkage by chiasmata, the products of crossover recombination.1 The spindle checkpoint delays cell-cycle progression until all chromosomes are properly attached to microtubules,2 but the steps leading to the capture and alignment of chromosomes on the meiosis I spindle remain poorly understood. In budding yeast meiosis I, Mad2 and Mad3BUBR1 are equally important for spindle checkpoint delay, but biorientation of homologs on the meiosis I spindle requires Mad2, but not Mad3BUBR1.3,4 Here we reveal the distinct functions of Mad2 and Mad3BUBR1 in meiosis I chromosome segregation. Mad2 promotes the prophase to metaphase I transition, while Mad3BUBR1 associates with the TOGL1 domain of Stu1CLASP, a conserved plus-end microtubule protein that is important for chromosome capture onto the spindle. Homologous chromosome pairs that are proficient in crossover formation but fail to biorient rely on Mad3BUBR1-Stu1CLASP to ensure their efficient attachment to microtubules and segregation during meiosis I. Furthermore, we show that Mad3BUBR1-Stu1CLASP are essential to rescue the segregation of mini-chromosomes lacking crossovers. Our findings define a new pathway ensuring microtubule-dependent chromosome capture and demonstrate that spindle checkpoint proteins safeguard the fidelity of chromosome segregation both by actively promoting chromosome alignment and by delaying cell-cycle progression until this has occurred.

Enhanced recombination empowers the detection and mapping of Quantitative Trait Loci

Modern plant breeding, such as genomic selection and gene editing, is based on the knowledge of the genetic architecture of desired traits. Quantitative trait loci (QTL) analysis, which combines high throughput phenotyping and genotyping of segregating populations, is a powerful tool to identify these genetic determinants and to decipher the underlying mechanisms. However, meiotic recombination, which shuffles genetic information between generations, is limited: Typically only one to two exchange points, called crossovers, occur between a pair of homologous chromosomes. Here we test the effect on QTL analysis of boosting recombination, by mutating the anti-crossover factors RECQ4 and FIGL1 in Arabidopsis thaliana full hybrids and lines in which a single chromosome is hybrid. We show that increasing recombination ~6-fold empowers the detection and resolution of QTLs, reaching the gene scale with only a few hundred plants. Further, enhanced recombination unmasks some secondary QTLs undetected under normal recombination. These results show the benefits of enhanced recombination to decipher the genetic bases of traits.

SCC3 is an axial element essential for homologous chromosome pairing and synapsis

Cohesin is a multi-subunit protein that plays a pivotal role in holding sister chromatids together during cell division. Sister chromatid cohesion 3 (SCC3), constituents of cohesin complex, is highly conserved from yeast to mammals. Since the deletion of individual cohesin subunit always causes lethality, it is difficult to dissect its biological function in both mitosis and meiosis. Here, we obtained scc3 weak mutants using CRISPR-Cas9 system to explore its function during rice mitosis and meiosis. The scc3 weak mutants displayed obvious vegetative defects and complete sterility, underscoring the essential roles of SCC3 in both mitosis and meiosis. SCC3 is localized on chromatin from interphase to prometaphase in mitosis. However, in meiosis, SCC3 acts as an axial element during early prophase I and subsequently situates onto centromeric regions following the disassembly of the synaptonemal complex. The loading of SCC3 onto meiotic chromosomes depends on REC8. scc3 shows severe defects in homologous pairing and synapsis. Consequently, SCC3 functions as an axial element that is essential for maintaining homologous chromosome pairing and synapsis during meiosis.

SCC3 is an axial element essential for homologous chromosome pairing and synapsis.

Cohesin is a multi-subunit protein that plays a pivotal role in holding sister chromatids together during cell division. Sister chromatid cohesion 3 (SCC3), constituents of cohesin complex, is highly conserved from yeast to mammals. Since the deletion of individual cohesin subunit always causes lethality, it is difficult to dissect its biological function in both mitosis and meiosis. Here, we obtained scc3 weak mutants using CRISPR-Cas9 system to explore its function during rice mitosis and meiosis. The scc3 weak mutants displayed obvious vegetative defects and complete sterility, underscoring the essential roles of SCC3 in both mitosis and meiosis. SCC3 is localized on chromatin from interphase to prometaphase in mitosis. However, in meiosis, SCC3 acts as an axial element during early prophase I and subsequently situates onto centromeric regions following the disassembly of the synaptonemal complex. The loading of SCC3 onto meiotic chromosomes depends on REC8. scc3 shows severe defects in homologous pairing and synapsis. Consequently, SCC3 functions as an axial element that is essential for maintaining homologous chromosome pairing and synapsis during meiosis.

Mapping of Repetitive Sequences in Brachyhypopomus brevirostris (Hypopomidae, Gymnotiformes) from the Brazilian Amazon.

Brachyhypopomus (Hypopomidae, Gymnotiformes) is a monophyletic genus consisting of 28 formally described species. Karyotypic data are available for 12 species. The same karyotype is described for two species (B. brevirostris and B. hamiltoni), as well as different karyotypes for the same species from distinct locations (B. brevirostris). In this context, B. brevirostris may constitute a cryptic species complex. Thus, in the present study, we analyzed the karyotype of B. brevirostris, from Santarém, Pará, and Tefé, Amazonas, using classical cytogenetics (conventional staining and C-banding) and molecular techniques (fluorescence in situ hybridization using 18S rDNA, 5S rDNA, U2 snRNA, and telomeric probes). The results show that samples from both locations present 2n = 38, with all chromosomes being acrocentric (FC = 38a). In both populations, 18S rDNA sequences are present on only one pair of homologous chromosomes and telomeric sequences occur only at the ends of the chromosomes. In the Tefé sample, the 5S rDNA occurs in two pairs, and the U2 snRNA in three pairs. These results are the first descriptions of these sequences for B. brevirostris samples from the Tefé locality, as well as the first karyotypic description for the Santarém locality. Future cytotaxonomic studies of this genus can benefit from these results.

Impact of Interactions between Su(Hw)-Dependent Insulators on the Transvection Effect in Drosophila melanogaster.

Transvection is a phenomenon of interallelic communication in which enhancers can activate a specific promoter located on a homologous chromosome. Insulators play a significant role in ensuring functional interactions between enhancers and promoters. In the presented work, we created a model where two or three copies of the insulator are located next to enhancers and promoters localized on homologous chromosomes. Using the Su(Hw) insulator as a model, we showed that the functional interaction between a pair of insulators promotes enhancer-promoter trans-interactions. The interaction between the three insulators, on the contrary, can lead to the formation of chromatin loops that sterically hinder the full enhancer-promoter interaction. The results of the work suggest the participation of insulators in the regulation of homologous chromosome pairing and in communication between distant genomic loci.

Agent-based modeling of nuclear chromosome ensembles identifies determinants of homolog pairing during meiosis.

During meiosis, pairing of homologous chromosomes (homologs) ensures the formation of haploid gametes from diploid precursor cells, a prerequisite for sexual reproduction. Pairing during meiotic prophase I facilitates crossover recombination and homolog segregation during the ensuing reductional cell division. Mechanisms that ensure stable homolog alignment in the presence of an excess of non-homologous chromosomes have remained elusive, but rapid chromosome movements appear to play a role in the process. Apart from homolog attraction, provided by early intermediates of homologous recombination, dissociation of non-homologous associations also appears to contribute to homolog pairing, as suggested by the detection of stable non-homologous chromosome associations in pairing-defective mutants. Here, we have developed an agent-based model for homolog pairing derived from the dynamics of a naturally occurring chromosome ensemble. The model simulates unidirectional chromosome movements, as well as collision dynamics determined by attractive and repulsive forces arising from close-range physical interactions. Chromosome number and size as well as movement velocity and repulsive forces are identified as key factors in the kinetics and efficiency of homologous pairing in addition to homolog attraction. Dissociation of interactions between non-homologous chromosomes may contribute to pairing by crowding homologs into a limited nuclear area thus creating preconditions for close-range homolog attraction. Incorporating natural chromosome lengths, the model accurately recapitulates efficiency and kinetics of homolog pairing observed for wild-type and mutant meiosis in budding yeast, and can be adapted to nuclear dimensions and chromosome sets of other organisms.

Homologous chromosome pairing starts at the ends.

Homologous chromosome pairing starts at the ends.

Prenatal ultrasound findings and clinical outcomes of uniparental disomy: a retrospective study

BackgroundUniparental disomy is the inheritance of a homologous chromosome pair or part of homologous chromosomes from only one parent. However, the clinical significance of uniparental disomy and the difference among the prognosis of involvement of different chromosomes remain unclear.ObjectiveTo assess the associated prenatal ultrasound presentations and clinical outcomes of uniparental disomy on different chromosomes and to analyze the relationship between prenatal ultrasound markers and clinical outcomes.Study designWe retrospectively analyzed data from fetuses with uniparental disomy diagnosed using chromosome microarray analysis with the Affymetrix CytoScan HD array at our institution between January 2013 and September 2022. The relationship between prenatal ultrasound findings, the involved chromosome(s), and clinical outcomes was evaluated.ResultsDuring the study period, 36 fetuses with uniparental disomy were diagnosed, and two cases were excluded for non-available postnatal data. Finally, 34 fetuses were included in our study, of which 30 (88.2%) had uniparental disomy occurring on a single chromosome, while four (11.8%) were identified with uniparental disomy on different chromosomes. The most frequently involved chromosomes were chromosomes 16, X and 2, which presented in 8 (23.5%), 5 (14.7%) and 4 (11.8%), respectively. Prenatal ultrasound abnormalities were detected in 21 fetuses, with the most common category being multiple abnormalities (12 (57.1%)). Fetal growth restriction was identified in 14 (41.2%) fetuses, all of which coexisted with other abnormal findings. The rate of adverse perinatal outcomes in patients with uniparental disomy and fetal abnormalities was significantly higher than those without abnormalities (76.2% versus 15.4%, P = 0.002). The incidence of fetal or neonatal death was significantly higher in fetuses with fetal growth restriction than those without (85.7% versus 30.0%, P = 0.004).ConclusionsThe prognosis of fetuses with uniparental disomy combined with fetal abnormalities, especially fetal growth restriction, was much poorer than those without.

Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes.

Programmed DNA double-strand break (DSB) formation is a crucial feature of meiosis in most organisms. DSBs initiate recombination-mediated linking of homologous chromosomes, which enables correct chromosome segregation in meiosis. DSBs are generated on chromosome axes by heterooligomeric focal clusters of DSB-factors. Whereas DNA-driven protein condensation is thought to assemble the DSB-machinery, its targeting to chromosome axes is poorly understood. We uncover in mice that efficient biogenesis of DSB-machinery clusters requires seeding by axial IHO1 platforms. Both IHO1 phosphorylation and formation of axial IHO1 platforms are diminished by chemical inhibition of DBF4-dependent kinase (DDK), suggesting that DDK contributes to the control of the axial DSB-machinery. Furthermore, we show that axial IHO1 platforms are based on an interaction between IHO1 and the chromosomal axis component HORMAD1. IHO1-HORMAD1-mediated seeding of the DSB-machinery on axes ensures sufficiency of DSBs for efficient pairing of homologous chromosomes. Without IHO1-HORMAD1 interaction, residual DSBs depend on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters. Thus, recombination initiation is ensured by complementary pathways that differentially support seeding and growth of DSB-machinery clusters, thereby synergistically enabling DSB-machinery condensation on chromosomal axes.