Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination

During meiosis, homologous chromosomes undergo crossover recombination, which creates genetic diversity and balances homolog segregation. Despite these critical functions, crossover frequency varies extensively within and between species. Although natural crossover recombination modifier loci have been detected in plants, causal genes have remained elusive. Using natural Arabidopsis thaliana accessions, we identified two major recombination quantitative trait loci (rQTLs) that explain 56.9% of crossover variation in ColxLer F2 populations. We mapped rQTL1 to semidominant polymorphisms in HEI10, which encodes a conserved ubiquitin E3 ligase that regulates crossovers. Null hei10 mutants are haploinsufficient, and, using genome-wide mapping and immunocytology, we show that transformation of additional HEI10 copies is sufficient to more than double euchromatic crossovers. However, heterochromatic centromeres remained recombination-suppressed. The strongest HEI10-mediated crossover increases occur in subtelomeric euchromatin, which is reminiscent of sex differences in Arabidopsis recombination. Our work reveals that HEI10 naturally limits Arabidopsis crossovers and has the potential to influence the response to selection

The genetic and epigenetic landscape of the Arabidopsis centromeres.

Centromeres attach chromosomes to spindle microtubules during cell division and, despite this conserved role, show paradoxically rapid evolution and are typified by complex repeats. We used longread sequencing to generate the Col-CEN Arabidopsis thaliana genome assembly that resolves all five centromeres. The centromeres consist of megabase-scale tandemly repeated satellite arrays, which support CENH3 occupancy and are densely DNA methylated, with satellite variants private to each chromosome. CENH3 preferentially occupies satellites that show least divergence and occur in higherorder repeats. The centromeres are invaded by ATHILA retrotransposons, which disrupt genetic and epigenetic organization. Centromeric crossover recombination is suppressed, yet low levels of meiotic DSBs occur that are regulated by DNA methylation. We propose that Arabidopsis centromeres are evolving via cycles of satellite homogenization and retrotransposon-driven diversification.BBSRC grants BB/S006842/1, BB/S020012/1 and BB/V003984/1

Cellular Communication through Light

Information transfer is a fundamental of life. A few studies have reported that cells use photons (from an endogenous source) as information carriers. This study finds that cells can have an influence on other cells even when separated with a glass barrier, thereby disabling molecule diffusion through the cell-containing medium. As there is still very little known about the potential of photons for intercellular communication this study is designed to test for non-molecule-based triggering of two fundamental properties of life: cell division and energy uptake. The study was performed with a cellular organism, the ciliate Paramecium caudatum. Mutual exposure of cell populations occurred under conditions of darkness and separation with cuvettes (vials) allowing photon but not molecule transfer. The cell populations were separated either with glass allowing photon transmission from 340 nm to longer waves, or quartz being transmittable from 150 nm, i.e. from UV-light to longer waves. Even through glass, the cells affected cell division and energy uptake in neighboring cell populations. Depending on the cuvette material and the number of cells involved, these effects were positive or negative. Also, while paired populations with lower growth rates grew uncorrelated, growth of the better growing populations was correlated. As there were significant differences when separating the populations with glass or quartz, it is suggested that the cell populations use two (or more) frequencies for cellular information transfer, which influences at least energy uptake, cell division rate and growth correlation. Altogether the study strongly supports a cellular communication system, which is different from a molecule-receptor-based system and hints that photon-triggering is a fine tuning principle in cell chemistry

A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing

Background:We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates. Results:Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations. Conclusions:We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation.Miriam Schreiber, Abdellah Barakate, Nicola Uzrek, Malcolm Macaulay, Adeline Sourdille, Jenny Morris, Pete E. Hedley, Luke Ramsay and Robbie Waug

Education needs of nurses in thrombosis and hemostasis: An international, mixed-methods study

Background: The need for a more integrated, multidisciplinary approach to care for individuals with bleeding or clotting disorders has been highlighted in recent years. Evidence-based education adapted to nurses' needs is essential for a successful evolution. However, limited data currently exist on the clinical challenges nurses face in this specialty area. Objectives: Identify barriers and challenges faced by specialty nurses, and determine possible causes, to develop appropriate educational interventions. Methods: A mixed-methods study, combining qualitative (semi-structured interviews) and quantitative (online survey) data was conducted on the challenges experienced by hemostasis nurses in nine countries (Argentina, Australia, Canada, China, France, Germany, Spain, the UK, and the US), and deployed in five languages (English, French, German, Mandarin, and Spanish). Qualitative data were analyzed using thematic analysis. Quantitative data were analyzed using frequency tables, chi-squares and standard deviations. Results: Participants (n = 234) included nurses (n = 212; n = 22 qualitative; n = 190 quantitative); and patients receiving care for bleeding or clotting conditions or their caretakers (n = 22 qualitative phase only). Through triangulated data analysis, six challenging areas emerged: (a) Understanding of von Willebrand disease (VWD); (b) Anticoagulant safety profile in specific patients; (c) Understanding the treatment of patients with inhibitors; (d) Patient risk assessments; (e) Individualization of care and communication with patients; and (f) Accessing and implementing relevant professional education. Conclusions: This needs assessment provides a comprehensive illustration of the current challenges faced by nurses in the field of bleeding and clotting disorders, and indicates where gaps in skills, knowledge or confidence would benefit from nurse-specific educational programming

MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis

During meiosis, DNA double-strand breaks undergo interhomolog repair to yield crossovers between homologous chromosomes. To investigate how interhomolog sequence polymorphism affects crossovers, we sequenced multiple recombinant populations of the model plant Arabidopsis thaliana. Crossovers were elevated in the diverse pericentromeric regions, showing a local preference for polymorphic regions. We provide evidence that crossover association with elevated diversity is mediated via the Class I crossover formation pathway, although very high levels of diversity suppress crossovers. Interhomolog polymorphism causes mismatches in recombining molecules, which can be detected by MutS homolog (MSH) mismatch repair protein heterodimers. Therefore, we mapped crossovers in a msh2 mutant, defective in mismatch recognition, using multiple hybrid backgrounds. Although total crossover numbers were unchanged in msh2 mutants, recombination was remodelled from the diverse pericentromeres towards the less-polymorphic sub-telomeric regions. Juxtaposition of megabase heterozygous and homozygous regions causes crossover remodelling towards the heterozygous regions in wild type Arabidopsis, but not in msh2 mutants. Immunostaining showed that MSH2 protein accumulates on meiotic chromosomes during prophase I, consistent with MSH2 regulating meiotic recombination. Our results reveal a pro-crossover role for MSH2 in regions of higher sequence diversity in A. thaliana

Control of meiotic crossover interference by a proteolytic chaperone network

Meiosis is a specialized eukaryotic division that produces genetically diverse gametes for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal exchanges, called crossovers, which recombine genetic variation. Meiotic crossovers are stringently controlled with at least one obligate exchange forming per chromosome pair, while closely spaced crossovers are inhibited by interference. In Arabidopsis, crossover positions can be explained by a diffusion-mediated coarsening model, in which large, approximately evenly spaced foci of the pro-crossover E3 ligase HEI10 grow at the expense of smaller, closely spaced clusters. However, the mechanisms that control HEI10 dynamics during meiosis remain unclear. Here, through a forward genetic screen in Arabidopsis, we identified high crossover rate3 (hcr3), a dominant-negative mutant that reduces crossover interference and increases crossovers genome-wide. HCR3 encodes J3, a co-chaperone related to HSP40, which acts to target protein aggregates and biomolecular condensates to the disassembly chaperone HSP70, thereby promoting proteasomal degradation. Consistently, we show that a network of HCR3 and HSP70 chaperones facilitates proteolysis of HEI10, thereby regulating interference and the recombination landscape. These results reveal a new role for the HSP40/J3-HSP70 chaperones in regulating chromosome-wide dynamics of recombination via control of HEI10 proteolysis