Crossover Events Research Articles

Examining the Effects of Temperature on Recombination in Wheat.

Meiotic recombination plays a crucial role in the generation of new varieties. The effectiveness of recombination is limited by the distribution of crossover events, which in wheat and many other crops is skewed toward the distal regions of the chromosomes. Whole-genome sequencing of wheat has revealed that there are numerous important genes in the pericentromeric regions, which are inaccessible to manipulation due to the lack of crossover events. Studies in barley have shown that the distribution of recombination events can be shifted toward the centromeres by increasing temperature during meiosis. Here we present an analysis of the effects of temperature on the distribution and frequency of recombination events in wheat. Our data show that although increased temperature during meiosis does cause an inward shift in recombination distribution for some chromosomes, its overall utility is limited, with many genes remaining highly linked.

Heteronuclear Iron(III)-Schiff Base Complexes with the Hexacyanidocobaltate(III) Anion: On the Quest To Understand the Governing Factors of Spin Crossover.

Two heteronuclear compounds (1 and 2) containing three ferric centers linked in facial-like mode with the magnetically silent hexacyanidocobaltate(III) anion were prepared and studied. The structural investigation revealed that both compounds are tetranuclear complexes with molecular formulas of [{Fe(L1)NC}3Co(CN)3]·2CH3OH·2.5CH3CN (1) and [{Fe(L2)NC}3Co(CN)3]·2H2O·1CH3OH (2). The magnetic properties of both complexes are controlled by the molecular design of the corresponding pentadentate Schiff base anions L12- and L22-. While compound 2 with a symmetric ligand prepared from salicylaldehyde shows high-spin state properties, compound 1 containing the asymmetric ligand with naphthalene units either is low-spin in its solvated form or shows a gradual but hysteretic spin crossover event when desolvated. The magnetic behavior was analyzed with respect to the Ising-like model and spin Hamiltonian, respectively, and the results were confronted with ab initio calculations. Additionally, the influence of structural features, lattice solvent molecules, the distribution of electronic terms, and active orbitals on the spin state properties of reported complexes is discussed.

Minimum anesthetic concentration of isoflurane and sparing effect of midazolam in Quaker parrots (Myiopsitta monachus)

ObjectiveTo determine the effects of midazolam on the minimum anesthetic concentration (MAC) reduction of end-tidal isoflurane concentration (Fe′Iso) measured using an electrical stimulus in Quaker parrots (Myiopsitta monachus). Study designRandomized crossover experimental study. AnimalsA group of six adult Quaker parrots, weighing 98–124 g. MethodsBirds were anesthetized with isoflurane in oxygen delivered by mask, then tracheally intubated and mechanically ventilated. Three treatments were applied with a 4 day interval between anesthetic events. Each anesthetized bird was administered midazolam (1 mg kg−1; treatment MID1), midazolam (2 mg kg−1; treatment MID2) or electrolyte solution (control) intramuscularly. The treatments were administered using a replicated Latin square design and the observers were blinded. Based on a pilot bird, the starting Fe′Iso was 1.8%. After equilibration for 10 minutes, a supramaximal stimulus was delivered using an electrical current (20 V and 50 Hz for 10 ms) and birds were observed for non-reflex movement. The Fe′Iso was titrated by 0.1% until a crossover event was observed. The MAC was estimated using logistic regression. ResultsThe MAC of isoflurane (MACISO) was estimated at 2.52% [95% confidence interval (CI), 2.19–2.85] with a range of 1.85–2.65%. MACISO in MID1 was 2.04% (95% CI, 1.71–2.37) and in MID2 was 1.81% (95% CI, 1.48–2.14); reductions in MACISO from control of 19% (p = 0.001) and 28% (p < 0.001), respectively. Heart rate, temperature, sex and anesthetic time were not different among treatments. ConclusionsMidazolam (1–2 mg kg−1) intramuscularly resulted in a significant isoflurane-sparing effect in response to a noxious stimulus in Quaker parrots without observable adverse effects. Clinical relevanceMidazolam can be used as part of a balanced anesthetic approach using isoflurane in Quaker parrots, and potentially in other psittacine species.

Bosonic and magnonic magnon dispersions

The analytical crossover events in the magnon dispersion relations are discussed. As the available experimental data show, only a few types of magnon dispersion relations are observed for all spin- and lattice-structures. When the magnon spectrum exhibits a finite energy gap at q = 0 the dispersion consists of two q-sections with different functions of wave vector. Such analytical changes indicate that two mechanisms dominate the dispersion relation alternately. For magnon energies of larger than the gap, the dispersion exhibits over a finite q-range a single qx power function of wave vector. Within the experimental error limits, the exponent x is a rational number and is independent of the spin structure, i.e. universal, but depends on whether the spin quantum number is integer or half-integer. As we now know, this is the typical indication that the qx function is determined by the bosons of the continuous magnetic medium. The qx function holds up to the crossover to a sine-function of wave vector for antiferromagnets but to a sine-function squared for ferromagnets. One therefore has to distinguish between the bosonic part of the magnon dispersion relation, at small q-values, and the magnonic part at large q-values. As is well known, sine functions of wave vector are the dispersion of the linear spin chain. Since the linear spin chain is not ordered at any finite temperature it follows that the observed magnons are not indicative of a long-range magnetic order. From the fact that no principal change of the magnon dispersions occurs upon crossing the magnetic ordering temperature it follows that the exchange interactions are not involved directly in the magnetic ordering process. The long-range ordered system is the boson field. At the critical temperature, the boson field orders. Ordering of the boson field is associated with the formation of domains and with the emergence of a magnon gap. In the ordered state, propagation of the bosons is restricted to the few different domain axes. The dimensionality of the ordered boson field can be recognized from the number of the in-equivalent domain orientations. For all lattice structures the boson field within each magnetic domain is perfectly one-dimensional, and aligns all spins parallel. This is the origin of the linear chain dispersion. The mass-less bosons are, however, not visible for neutrons. If the magnon-boson interaction is strong, the q-range of the sine functions is small. It then proves necessary to add a phenomenological phase shift in the argument of the sine function. As a consequence, magnon dispersions cannot be understood considering exchange interactions alone.

Tetrasomy 21 pter→q21.3 due to an extra +dic(21;21)mat in a severely psychomotor-retarded female patient without Down syndrome phenotype.

Complete or partial tetrasomy 21 has been reported only in rare cases. We report a Japanese female patient with tetrasomy 21 due to an extra chromosome derived from chromosome 21 (Chr21). The patient had severe psychomotor retardation without Down syndrome (DS) phenotype; she showed short stature, microcephaly, round face, cleft lip and palate, and other dysmorphic features. The chromosome analyses for the patient detected an extra dicentric Chr21 consisting of two partial Chr21 copies fused together within their long arms. Her karyotype was revealed to be 47,XX,+dic(21;21). Allelic ratios of heterozygous SNPs observed in the patient indicated the maternal origin of the extra Chr21. Copy number and structural variant analyses using whole genome sequencing data indicated that the distal breakpoint of the dicentric Chr21 was located within 21q21.3 and that the extra Chr21 did not simply consist of inverted duplications of the pter→q21.3 region, but likely contained multiple partial deletions, duplications, and inversions within it. Fluorescence in situ hybridization results were consistent with the karyotype and genomic analyses. The patient's lack of DS phenotype turned out to be due to the normal copy number of the DS critical region (21q22.13-22.3). A possible molecular mechanism leading to the complex genomic rearrangements in the tetrasomic region consists mainly of breakage-fusion-bridge cycles with an unequal crossing-over event.

An ancestral variant causing type I xanthinuria in Turkmen and Arab families is predicted to prevail in the Afro-Asian stone-forming belt.

Classical xanthinuria is a rare autosomal recessive metabolic disorder characterized by lack of xanthine dehydrogenase activity that often manifests as xanthine urolithiasis and risk of drug toxicity. Variants in the XDH or HMCS gene underlie classical xanthinuria type I and type II, respectively. Here we present two Israeli Arab families affected by type I xanthinuria in whom a c.2164A>T (Lys722Ter) variant in the XDH gene, previously reported in a Turkish family of Turkmen origin, was identified. Analysis of polymorphic markers surrounding the variant site revealed common haplotypes spanning 0.6 Mbp shared by all three, and 1.7 Mbp shared by two of the studied families. By applying Bayesian methods to a simple model of crossover events through generations in the chromosomes carrying the variant, the most recent common ancestor of these families was found to be 179 (95% credible limit 70) generations old. The estimated antiquity of the variant, the historical genealogy of the affected families and the history and present day dispersion of their people strongly suggest prevalence of this variant in the Afro‐Asian stone‐forming belt. As far as we are aware, this is a first report of an ancient variant causing xanthinuria with potential wide geographical dispersion.

Polymorphisms in the human PRDM9 gene may lead to meiotic arrest and azoospermia

PRDM9 is responsible for directing the location of programmed double strand breaks and subsequent crossover events between homologous chromosomes during meiosis in human gametes. Prdm9 is also essential for meiosis and fertility in mice, with PRDM9 knockout males displaying complete azoospermia. PRDM9 contains a rapidly evolving DNA binding zinc finger array that is coded by a ∼84 nucleotide repeating unit mini-satellite sequence. The human A-type allele, which accounts for ∼90% of the alleles in the human population, contains 13 repeating units. We sought to develop a s strategy to effectively genotype this mini-satellite with PacBio sequencing and to determine whether PRDM9 variation, including mini-satellite length polymorphisms, is associated with infertility in the human male. Observational study. Using a normospermic human control, a two-step polymerase chain reaction (PCR) protocol was established to successfully amplify the PRDM9 zinc finger array mini-satellite and sequence it using both Sanger and PacBio next generation sequencing, which has the advantage of circular consensus sequencing and long reads. We next amplified PRDM9 from the genomic DNA of 48 azoospermic men and 5 controls. The samples were visually analyzed with gel electrophoresis, and potential mutant alleles that had an atypical band appearance were compared to the wild type. PacBio sequencing results from the normospermic control were found to be an identical match to the known human A allele, confirming our ability to effectively genotype the mini-satellite array in a single PacBio run. Gel electrophoresis of PCR amplified PRDM9 alleles from azoospermic men identified 6 potential mutant variants of distinct mini-satellite lengths differing from the A-allele. We have developed a protocol to effectively genotype the human PRDM9 zinc finger array mini-satellite to evaluate a potential etiology of azoospermia in the infertile human male. We found 6 potential PRDM9 alleles differing from the known A-allele in a small (n=48) azoospermia cohort. Current/ongoing research includes applying our PacBio sequencing protocol to genotype all 48 azoospermic men in comparison to a control group and evaluate if there is an association between PRDM9 mini-satellite repeat length, polymorphisms, or de novo mutations and azoospermia.

A high-resolution map of non-crossover events reveals impacts of genetic diversity on mammalian meiotic recombination

During meiotic recombination, homologue-templated repair of programmed DNA double-strand breaks (DSBs) produces relatively few crossovers and many difficult-to-detect non-crossovers. By intercrossing two diverged mouse subspecies over five generations and deep-sequencing 119 offspring, we detect thousands of crossover and non-crossover events genome-wide with unprecedented power and spatial resolution. We find that both crossovers and non-crossovers are strongly depleted at DSB hotspots where the DSB-positioning protein PRDM9 fails to bind to the unbroken homologous chromosome, revealing that PRDM9 also functions to promote homologue-templated repair. Our results show that complex non-crossovers are much rarer in mice than humans, consistent with complex events arising from accumulated non-programmed DNA damage. Unexpectedly, we also find that GC-biased gene conversion is restricted to non-crossover tracts containing only one mismatch. These results demonstrate that local genetic diversity profoundly alters meiotic repair pathway decisions via at least two distinct mechanisms, impacting genome evolution and Prdm9-related hybrid infertility.

Assessment by Extended-Coverage Next-Generation Sequencing Typing of DPA1 and DPB1 Mismatches in Siblings Matching at HLA-A, -B, -C, -DRB1, and -DQ Loci.

Allogeneic hematopoietic stem cell transplant from an HLA matched sibling donor is usually the preferable choice. The use of next-generation sequencing (NGS) for HLA typing in clinical practice provides broader coverage and higher resolution of HLA genes. We evaluated the frequency of DPB1 crossing-over events among patients and potential related donors typed with NGS. From July 2016 to January 2018, 593 patients and 2385 siblings were typed. We evaluated sibling matching status in 546 patients, and 44.8% of these patients had siblings that matched at HLA-A, -B, -C, -DRB1, and -DQB1 loci. In 306 patient-HLA matched sibling pairs, we found 6 pairs (1.96%) with 1 DPB1 mismatch, and 5 of these pairs included an additional mismatch in DPA1. No additional mismatches were observed at the low expression loci. Using the T cell epitope algorithm, 4 of these DP mismatches were classified as permissive, 1 as nonpermissive in the host-versus-graft direction, and 1 as nonpermissive in the graft-versus-host direction. The frequency of DPB1 and DPA1 mismatches is low, and their impact in related donor transplants is not well established. Although DP typing in related transplants goes beyond guidelines, it is especially relevant for sensitized patients. NGS-based HLA typing provides full gene coverage, and its use in clinical practice can enable better donor selection.

28. KARYOMAPPING USING WHOLE GENOME SEQUENCING DATA: PRECLINICAL VALIDATION OF A PERSONALISED APPROACH TO PGT-M

Introduction Karyomapping utilises microarray technology to track informative single nucleotide polymorphisms (SNPs) and assign haplotypes to embryos without the need for direct mutation detection (Handyside et al. 2010). Although suitable for most couples undergoing PGT-M, the use of a fixed set of SNPs may provide inadequate information in certain genetic regions or in consanguineous couples (Konstantinidis et al. 2015). Whole genome sequencing (WGS) has the potential to individualise informative SNP detection and perform concurrent direct mutation detection. However, no studies have evaluated the potential for application of the karyomapping algorithm to WGS data. Material and Methods The Platinum Genomes dataset ( https://sapac.illumina.com/platinumgenomes.html ) was analysed as a model for the application of karyomapping to WGS data. Variant call format (VCF) files were analysed according to the principles of karyomapping to create a map of informative biallelic SNPs in each grandparental haplotype. A model of embryo biopsy was created by diluting DNA from one of the family members (NA12878) to 30pg and whole genome amplification was performed with the Repli-G single cell kit (Qiagen, Hilden, Germany) as a model of trophectoderm biopsy. Whole genome sequencing was performed in triplicate on a high output NextSeq 2500 run (Illumina, California, USA) at average 15x coverage and the number of informative SNPs detected compared to the karyomapping SNP microarray. Comparison of variant calling methods was performed using the Genome Analysis Toolkit v3.4 (Broad Institute, Massachusetts, USA) and DeepVariant (Google, California, USA). Results Analysis of WGS data from the Platinum Genomes family revealed a total of 2,338,158 informative SNPs distributed evenly between grandparental haplotypes, of which 85,053 (3.6%) would have been detectable using the karyomapping SNP array. Analysis of the karyomapping window around common genes requiring PGT-M demonstrated an average 32x increase in the number of targets available for analysis in a PGT-M cycle. Analysis of 15x coverage WGS data in a model of embryo biopsy demonstrated an average 279,851 informative SNPs detected using standard variant calling and an average 285,107 using AI-based variant calling. The median distance between informative SNPs was reduced from 26,782 nucleotides to 1,212 nucleotides when comparing WGS-based karyomapping to SNP array-based karyomapping. Conclusions This study provides preclinical validation for the application of the karyomapping algorithm to WGS data. The markedly increased number of informative SNPs will allow successful feasibility studies for all couples presenting for PGT-M. The decreased distance between informative SNPs detected in a model embryo biopsy will improve diagnostic accuracy, particularly in detecting small double crossover events and reducing the number of cycles in which inadequate informative markers have been detected in the embryo sample. Finally, the use of WGS for diagnostic purposes, especially for rare and undiagnosed genetic disease, will allow the creation of a high resolution digital karyomap for couples with an affected child and improve the transition to IVF and PGT-M.

Multi-centre evaluation of a comprehensive preimplantation genetic test through haplotyping-by-sequencing.

Can reduced representation genome sequencing offer an alternative to single nucleotide polymorphism (SNP) arrays as a generic and genome-wide approach for comprehensive preimplantation genetic testing for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR) in human embryo biopsy samples? Reduced representation genome sequencing, with OnePGT, offers a generic, next-generation sequencing-based approach for automated haplotyping and copy-number assessment, both combined or independently, in human single blastomere and trophectoderm samples. Genome-wide haplotyping strategies, such as karyomapping and haplarithmisis, have paved the way for comprehensive PGT, i.e. leveraging PGT-M, PGT-A and PGT-SR in a single workflow. These methods are based upon SNP array technology. This multi-centre verification study evaluated the concordance of PGT results for a total of 225 embryos, including 189 originally tested for a monogenic disorder and 36 tested for a translocation. Concordance for whole chromosome aneuploidies was also evaluated where whole genome copy-number reference data were available. Data analysts were kept blind to the results from the reference PGT method. Leftover blastomere/trophectoderm whole genome amplified (WGA) material was used, or secondary trophectoderm biopsies were WGA. A reduced representation library from WGA DNA together with bulk DNA from phasing references was processed across two study sites with the Agilent OnePGT solution. Libraries were sequenced on an Illumina NextSeq500 system, and data were analysed with Agilent Alissa OnePGT software. The embedded PGT-M pipeline utilises the principles of haplarithmisis to deduce haplotype inheritance whereas both the PGT-A and PGT-SR pipelines are based upon read-count analysis in order to evaluate embryonic ploidy. Concordance analysis was performed for both analysis strategies against the reference PGT method. PGT-M analysis was performed on 189 samples. For nine samples, the data quality was too poor to analyse further, and for 20 samples, no result could be obtained mainly due to biological limitations of the haplotyping approach, such as co-localisation of meiotic crossover events and nullisomy for the chromosome of interest. For the remaining 160 samples, 100% concordance was obtained between OnePGT and the reference PGT-M method. Equally for PGT-SR, 100% concordance for all 36 embryos tested was demonstrated. Moreover, with embryos originally analysed for PGT-M or PGT-SR for which genome-wide copy-number reference data were available, 100% concordance was shown for whole chromosome copy-number calls (PGT-A). Inherent to haplotyping methodologies, processing of additional family members is still required. Biological limitations caused inconclusive results in 10% of cases. Employment of OnePGT for PGT-M, PGT-SR, PGT-A or combined as comprehensive PGT offers a scalable platform, which is inherently generic and thereby, eliminates the need for family-specific design and optimisation. It can be considered as both an improvement and complement to the current methodologies for PGT. Agilent Technologies, the KU Leuven (C1/018 to J.R.V. and T.V.) and the Horizon 2020 WIDENLIFE (692065 to J.R.V. and T.V). H.M. is supported by the Research Foundation Flanders (FWO, 11A7119N). M.Z.E, J.R.V. and T.V. are co-inventors on patent applications: ZL910050-PCT/EP2011/060211- WO/2011/157846 'Methods for haplotyping single cells' and ZL913096-PCT/EP2014/068315 'Haplotyping and copy-number typing using polymorphic variant allelic frequencies'. T.V. and J.R.V. are co-inventors on patent application: ZL912076-PCT/EP2013/070858 'High-throughput genotyping by sequencing'. Haplarithmisis ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies') has been licensed to Agilent Technologies. The following patents are pending for OnePGT: US2016275239, AU2014345516, CA2928013, CN105874081, EP3066213 and WO2015067796. OnePGT is a registered trademark. D.L., J.T. and R.L.R. report personal fees during the conduct of the study and outside the submitted work from Agilent Technologies. S.H. and K.O.F. report personal fees and other during the conduct of the study and outside the submitted work from Agilent Technologies. J.A. reports personal fees and other during the conduct of the study from Agilent Technologies and personal fees from Agilent Technologies and UZ Leuven outside the submitted work. B.D. reports grants from IWT/VLAIO, personal fees during the conduct of the study from Agilent Technologies and personal fees and other outside the submitted work from Agilent Technologies. In addition, B.D. has a patent 20160275239 - Genetic Analysis Method pending. The remaining authors have no conflicts of interest.

Sample-size dependent temperature dependence of the spontaneous magnetization

It is shown that the precise temperature dependence of the spontaneous magnetization depends on the domain structure and, additionally, on the macroscopic dimensions of the sample. In the multi-domain zero-field ground state of cubic ferromagnets (or antiferromagnets) with half-integer spin, thermal decrease of the spontaneous magnetization is by universal T2 function up to crossover to critical power function. Power functions of temperature that hold over a large temperature range and are independent of spin structure are the typical indication that the spin dynamics is controlled by a boson field. For the magnets with half-integer spin, the T2 function means a three-dimensional boson field. In the mono-domain state prepared by ferromagnetic saturation, all moments are oriented parallel to the field axis. For this one-dimensional spin configuration the boson field can be anticipated to be one-dimensional and the spontaneous magnetization should decrease according to universal T5/2 function (for S = half-integer). However, only for sufficiently large samples the T5/2 function is confirmed. For smaller samples successive crossover events in the sequence T2 → T3/2 → T5/2 can occur for increasing temperature. Occurrence of boson fields with a higher dimensionality (T2, T3/2) in the state of ferromagnetic saturation can be explained by scattering processes of the bosons at the inner surface of the sample. When the diameter of the sample is less than the mean free path of the bosons, all bosons will be reflected (or scattered) at the inner surface of the sample and the resulting boson field can become three-dimensional (T2 universality class). A one-dimensional spin orientation due to the applied demagnetization field but a three-dimensional boson field that controls spin dynamics is possible only for the T2 universality class because there is nearly no interaction between boson field and spins (magnons) in this universality class. With increasing temperature the mean free path of the bosons decreases and only a fraction of the bosons gets scattered at the inner surface of the sample. Crossover to T3/2 function and, eventually, to T5/2 function then can occur. A similar situation as for the polarized ferromagnets is observed in the axial-symmetric zero-field ground state of the hcp ferromagnet cobalt. Only for sufficiently large samples scattering of the bosons at the inner surface of the sample is of no importance and the intrinsic T5/2 function, typical for parallel alignment of all spins, shows up. The mean free path of the bosons can be estimated to be of the order of a few mm for T → 0.

Crossover recombination and synapsis are linked by adjacent regions within the N terminus of the Zip1 synaptonemal complex protein.

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes. Most meiotic recombination intermediates that give rise to interhomolog crossovers are embedded within a hallmark chromosomal structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known structural components of the budding yeast SC, the Zip1 protein is unique for its independent role in promoting crossover recombination; Zip1 is specifically required for the large subset of crossovers that also rely on the meiosis-specific MutSγ complex. Here we report that adjacent regions within Zip1’s N terminus encompass its crossover and synapsis functions. We previously showed that deletion of Zip1 residues 21–163 abolishes tripartite SC assembly and prevents robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSγ crossover recombination. We find the reciprocal phenotype when Zip1 residues 2–9 or 10–14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSγ crossovers are strongly diminished. Interestingly, Zip1 residues 2–9 or 2–14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSγ crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15–20 does not detectably prevent Zip3’s localization at Zip1 polycomplex and supports some MutSγ crossing over but prevents normal SC assembly and Ecm11 SUMOylation. Our results highlight distinct N terminal regions that are differentially critical for Zip1’s roles in crossing over and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors within close proximity of one another.

MRLR: unraveling high-resolution meiotic recombination by linked reads.

Meiotic recombination facilitates the transmission of exchanged genetic material between homologous chromosomes and plays a crucial role in increasing the genetic variations in eukaryotic organisms. In humans, thousands of crossover events have been identified by genotyping related family members. However, most of these crossover regions span tens to hundreds of kb, which is not sufficient resolution to accurately identify the crossover breakpoints in a typical trio family. We have developed MRLR, a software using 10X linked reads to identify crossover events at a high resolution. By reconstructing the gamete genome, MRLR only requires a trio family dataset and can efficiently discover the crossover events. Using MRLR, we revealed a fine-scale pattern of crossover regions in six human families. From the two closest heterozygous alleles around the crossovers, we determined that MRLR achieved a median resolution 4.5kb. This method can delineate a genome-wide landscape of crossover events at a precise scale, which is important for both functional and genomic features analysis of meiotic recombination. MRLR is freely available at https://github.com/ChongLab/MRLR, implemented in Perl. Supplementary data are available at Bioinformatics online.

Use of the counter selectable marker PheS* for genome engineering in Staphylococcus aureus.

The gold standard method for the creation of gene deletions in Staphylococcus aureus is homologous recombination using allelic exchange plasmids with a temperature-sensitive origin of replication. A knockout vector that contains regions of homology is first integrated into the chromosome of S. aureus by a single crossover event selected for at high temperatures (non-permissive for plasmid replication) and antibiotic selection. Next, the second crossover event is encouraged by growth without antibiotic selection at low temperature, leading at a certain frequency to the excision of the plasmid and the deletion of the gene of interest. To detect or encourage plasmid loss, either a beta-galactosidase screening method or, more typically, a counterselection step is used. We present here the adaptation of the counter-selectable marker pheS*, coding for a mutated subunit of the phenylalanine tRNA synthetase, for use in S. aureus. The PheS* protein variant allows for the incorporation of the toxic phenylalanine amino acid analogue para-chlorophenylalanine (PCPA) into proteins and the addition of 20-40 mM PCPA to rich media leads to drastic growth reduction for S. aureus and supplementing chemically defined medium with 2.5-5 mM PCPA leads to complete growth inhibition. Using the new allelic exchange plasmid pIMAY*, we delete the magnesium transporter gene mgtE in S. aureus USA300 LAC* (SAUSA300_0910/SAUSA300_RS04895) and RN4220 (SAOUHSC_00945) and demonstrate that cobalt toxicity in S. aureus is mainly mediated by the presence of MgtE. This new plasmid will aid the efficient and easy creation of gene knockouts in S. aureus.

Suppression of template switching in postreplication gaps via Uup and RadD proteins

We are investigating the role of two bacterial proteins, RadD and Uup, in suppressing genomic stability. Uup is a UvrA‐like ABC family ATPase and RadD is a RecQ‐like SF2 family ATPase. Both of these proteins bind to branched DNAs. More specifically, RadD suppresses crossover events in the cell. The proteins resolve branched DNA intermediates that have arisen from template switching in post replication gaps. When a replication fork comes upon a lesion in the DNA, and a gap forms, the cell attempts to fill this gap and repair the DNA. One way of doing this is through template switching, a RecA‐independent process that creates recombination‐like branched DNA intermediates. These are the DNA intermediates at which RadD and Uup function. When genomic repeats exist in the chromosome, template switching sometimes results in mispairing that produces genomic deletions. Under normal growth conditions, with repeats in the chromosome, the deletion of Uup and RadD leads to the generation of chromosomal dimers that are not readily resolved. This experiment brings light to the functions of both proteins and recognizes the importance of genomic stability for cell viability. The results indicate that template switching happens often and that Uup and RadD play important roles in suppressing genomic stability.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Counterselectable Sucrose Sensitivity Marker Permits Efficient and Flexible Mutagenesis in Streptococcus agalactiae.

Streptococcus agalactiae (group B Streptococcus [GBS]) is a cause of severe infections, particularly during the newborn period. While methods exist for generating chromosomal mutations in GBS, they are cumbersome and inefficient and present significant challenges if the goal is to study subtle mutations, such as single-base-pair polymorphisms. To address this problem, we have developed an efficient and flexible GBS mutagenesis protocol based on sucrose counterselection against levansucrase (SacB) expressed from a temperature-selective shuttle vector. GBS containing the SacB expression cassette demonstrates lethal sensitivity to supplemental sucrose whether the plasmid DNA is replicating outside of the chromosome or has been integrated during a crossover event. Transmission electron microscopy shows that SacB-mediated lethal sucrose sensitivity results from the accumulation of inclusion bodies that eventually lead to complete degradation of normal cellular architecture and subsequent lysis. We used this new mutagenesis technique to generate an in-frame, allelic exchange knockout of the GBS sortase gene srtA, demonstrating that >99% of colonies that emerge from our protocol had the expected knockout phenotype and that among a subset tested by sequencing, 100% had the correct genotype. We also generated barcoded nonsense mutations in the cylE gene in two GBS strains, showing that the approach can be used to make small, precise chromosomal mutations.IMPORTANCE The ability to generate chromosomal mutations is fundamental to microbiology. Historically, however, GBS pathogenesis research has been made challenging by the relative genetic intractability of the organism. Generating a single knockout in GBS using traditional techniques can take many months, with highly variable success rates. Furthermore, traditional methods do not offer a straightforward way to generate single-base-pair polymorphisms or other subtle changes, especially to noncoding regions of the chromosome. We have developed a new sucrose counterselection-based method that permits rapid, efficient, and flexible GBS mutagenesis. Our technique requires no additional equipment beyond what is needed for traditional approaches. We believe that it will catalyze rapid advances in GBS genetics research by significantly easing the path to generating mutants.

Sequencing and the single sperm

Recombination During meiosis, homologous chromosomes undergo doublestrand breaks in DNA that can cross over, shuffling genetic material. However, not every double-strand break resolves in a crossover event. Hinch et al. wanted to determine the rules governing DNA recombination. They developed a method to sequence individual mouse sperm and applied it to mice carrying two different alleles of a protein involved in mammalian crossovers. A high-resolution genetic map revealed the relationships between the distribution of crossovers, proteins involved in recombination, and specific factors determining whether a double-strand break becomes a crossover. Science , this issue p. [eaau8861][1] [1]: /lookup/doi/10.1126/science.aau8861

Polymorphism in the short arm of 1R rye chromosomes in wheat lines with 1RS.1BL translocation and 1R(1B) substitution from different sources

Aim. The short arm of 1R rye (Secale cereale L.) chromosome is widely used in the breeding of bread wheat (Triticum aestivum L.), in particular 1RS.1BL, to introsgress genes of resistance to leaf (Lr26), stem (Sr31), striped (Yr9) rusts, as well as powdery mildew (Pm8); 1RS.1AL carries Gb2/Gb6 resistance genes to the wheat aphid (Schizaphis graminum Rondani), powdery mildew (Pm17), and the Cmc4 resistance gene to the Aceria tosichella Koifer mite, which is a vector for spreading of wheat mosaic virus. The aim of the research is to reveal molecular genetic polymorphisms of short arm rye 1RS chromosomes of different origins in bread wheat lines with 1RS.1BL translocation or 1R(1B) substitution from different sources. Methods. Genetic polymorphism of lines was analyzed by using PCR with a number of rye and wheat microsatellite markers. Results. It was shown that the CWXs line has a recombinant 1RS arm that contains the chromosomes parts of 1RS of the parental lines H242/97-2 and H273/97, due to crossover event, which led to the recombination of marked loci. Conclusions. Molecular genetic polymorphism has been reviled in 1RS.1BL translocations and 1R substituted rye chromosomes of different origins in H242/97-2, CWXs, H273/97, PavonMA1, Salmon lines, as there are different alleles present at loci: Xscm9, Xtsm422, Xgwm752, Xgwm18, Taglgap. Keywords: polymorphism, 1RS.1BL translocation, PCR analysis, microsatellites markers.

Single gametophyte sequencing reveals that crossover events differ between sexes in maize

Meiotic crossover (CO) plays a key role in producing gametophytes and generating genetic variation. The patterns of CO production differ inter- and intra-species, as well as between sexes. However, sex-specific patterns of CO production have not been accurately profiled independently of genetic backgrounds in maize. Here, we develop a method to isolate single female gametophyte for genomes sequencing in maize. We show that more COs are observed in male (19.3 per microspore) than in female (12.4 per embryo sac). Based on Beam-Film model, the more designated class I and II COs are identified in male than in female. In addition, CO maturation inefficiency (CMI) is detected in some genetic backgrounds, suggesting that maize may be an ideal model for dissecting CMI. This research provides insights toward understanding the molecular mechanism of CO production between sexes and may help to improve maize breeding efficiency through paternal selection.