The construction of a high-density consensus genetic map for soybean based on SNP markers derived from genotyping-by-sequencing.

Genetic linkage maps are used to localize markers on the genome based on the recombination frequency. Most often, these maps are based on the segregation observed within a single biparental population of limited size (n < 300) where relatively few recombination events are sampled and in which some genomic regions are monomorphic because both parents carry the same alleles. Together, these two limitations affect both the resolution and extent of genome coverage of such maps. Consensus genetic maps overcome the limitations of individual genetic maps by merging the information from multiple segregating populations derived from a greater diversity of parental combinations, thus increasing the number of recombination events and reducing the number of monomorphic regions. The aim of this study was to construct a high-density consensus genetic map for single nucleotide polymorphism (SNP) markers obtained through a genotyping-by-sequencing (GBS) approach. Individual genetic maps were generated from six F4:5 mapping populations (n = 278-365), totaling 1857 individuals. The six linkage maps were then merged to produce a consensus map comprising a total of 16 311 mapped SNPs that jointly cover 99.5% of the soybean genome with only two gaps larger than 10 cM. Compared to previous soybean consensus maps, it offers a more extensive and uniform coverage.

A high-resolution consensus linkage map for barley based on GBS-derived genotypes.

As genotyping-by-sequencing (GBS) is widely used in barley genetic studies, the translation of the physical position of GBS-derived SNPs into accurate genetic positions has become relevant. The main aim of this study was to develop a high-resolution consensus linkage map based on GBS-derived SNPs. The construction of this integrated map involved 11 bi-parental populations composed of 3743 segregating progenies. We adopted a uniform set of SNP-calling and filtering conditions to identify 50 875 distinct SNPs segregating in at least one population. These SNPs were grouped into 18 580 non-redundant SNPs (bins). The resulting consensus linkage map spanned 1050.1 cM, providing an average density of 17.7 bins and 48.4 SNPs per cM. The consensus map is characterized by the absence of large intervals devoid of marker coverage (significant gaps), the largest interval between bins was only 3.7 cM and the mean distance between adjacent bins was 0.06 cM. This high-resolution linkage map will contribute to several applications in genomic research, such as providing useful information on the recombination landscape for QTLs/genes identified via GWAS or ensuring a uniform distribution of SNPs when developing low-cost genotyping tools offering a limited number of markers.

FKBP25 participates in DNA double-strand break repair.

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis-trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25's catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination.

Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.

A linear-amplification VDJ-seq technique for quantification of immunoglobulin and T cell receptor diversity.

The V(D)J recombination is essential for generating a highly diverse repertoire of antigen receptors expressed on T and B lymphocytes. Here, we developed a linear-amplification VDJ-seq technique for quantifying V(D)J recombination of antigen receptor genes. This technique takes advantage of linear amplification using in vitro transcription and reverse transcription to avoid bias generated by the PCR amplification of low copy number of target DNA. The unrearranged alleles are removed by in vitro cleavage with the CRISPR-Cas9 system. The linear-amplification VDJ-seq assay was applied in quantification of the Vκ-Jκ recombination of the mouse Igκ gene with Jκ capture primers. The Jκ genes were detected in 95.86% of clean reads with more than half containing the Vκ gene, indicating high specificity of capturing and amplification. We also applied this approach to quantify the usage of Jα within the Trav12 gene family of the Tcra gene.

Sex solves Haldane's dilemma.

The cumulative reproductive cost of multi-locus selection has been considered to be a potentially limiting factor on the rate of adaptive evolution. In this paper, we show that Haldane's arguments for the accumulation of reproductive costs over multiple loci are valid only for a clonally reproducing population of asexual genotypes. We show that a sexually reproducing population avoids this accumulation of costs. Thus, sex removes a perceived reproductive constraint on the rate of adaptive evolution. The significance of our results is twofold. First, the results demonstrate that adaptation based on multiple genes-such as selection acting on the standing genetic variation-does not entail a huge reproductive cost as suggested by Haldane, provided of course that the population is reproducing sexually. Second, this reduction in the cost of natural selection provides a simple biological explanation for the advantage of sex. Specifically, Haldane's calculations illustrate the evolutionary disadvantage of asexuality; sexual reproduction frees the population from this disadvantage.

Limited evidence of fungicide-driven triazole-resistant Aspergillus fumigatus in Hamilton, Canada.

Aspergillus fumigatus is a ubiquitous opportunistic fungal pathogen that can cause aspergillosis in humans. Over the last decade there have been increasing global reports of treatment failure due to triazole resistance. An emerging hypothesis states that agricultural triazole fungicide use causes clinical triazole resistance. Here we test this hypothesis in Hamilton, Ontario, Canada, by examining a total of 195 agricultural, urban, and clinical isolates using 9 highly polymorphic microsatellite markers. For each isolate, the in vitro susceptibilities to itraconazole and voriconazole, 2 triazole drugs commonly used in the management of patients, were also determined. Our analyses suggested frequent gene flow among the agricultural, urban environmental, and clinical populations of A. fumigatus and found evidence for widespread sexual recombination within and among the different populations. Interestingly, all 195 isolates analyzed in this study were susceptible to both triazoles tested. However, compared with the urban population, agricultural and clinical populations showed significantly reduced susceptibility to itraconazole and voriconazole, consistent with ecological niche-specific selective pressures on A. fumigatus populations in Hamilton. Frequent gene flow and genetic recombination among these populations suggest greater attention should be paid to monitor A. fumigatus populations in Hamilton and other similar jurisdictions.

Microsatellites mapping for non-model species with chromosomal rearrangement: a case study in the frog Quasipaa boulengeri (Anura: Dicroglossidae).

Gene mapping is an important resource for understanding the evolution of genes and cytogenetics. Model species with a known genetic map or genome sequence allow for the selection of genetic markers on a desired chromosome, while it is hard to locate these markers on chromosomes of non-model species without such references. A frog species, Quasipaa boulengeri, shows chromosomal rearrangement polymorphisms, making itself a fascinating model for chromosomal speciation mediated by suppressed recombination. However, no markers have been located on its rearranged chromosomes. We present a complete protocol to map microsatellites based on mechanical microdissection and chromosome amplification techniques. Following this protocol, we mapped 71 microsatellites of Q. boulengeri at the chromosome level. In total, eight loci were assigned to rearranged chromosomes, and the other 63 loci might attach to other chromosomes. These microsatellites could be used to compare the gene flow and verify the chromosomal suppressed recombination hypothesis in Q. boulengeri. This integrated protocol could be effectively used to map genes to chromosomes for non-model species.

Novel insights into RAD51 activity and regulation during homologous recombination and DNA replication.

In this review we focus on new insights that challenge our understanding of homologous recombination (HR) and Rad51 regulation. Recent advances using high-resolution microscopy and single molecule techniques have broadened our knowledge of Rad51 filament formation and strand invasion at double-strand break (DSB) sites and at replication forks, which are one of most physiologically relevant forms of HR from yeast to humans. Rad51 filament formation and strand invasion is regulated by many mediator proteins such as the Rad51 paralogues and the Shu complex, consisting of a Shu2/SWS1 family member and additional Rad51 paralogues. Importantly, a novel RAD51 paralogue was discovered in Caenorhabditis elegans, and its in vitro characterization has demonstrated a new function for the worm RAD51 paralogues during HR. Conservation of the human RAD51 paralogues function during HR and repair of replicative damage demonstrate how the RAD51 mediators play a critical role in human health and genomic integrity. Together, these new findings provide a framework for understanding RAD51 and its mediators in DNA repair during multiple cellular contexts.

Meiotic recombination is suppressed near the histone-defined border of euchromatin and heterochromatin on chromosome 2L of Drosophila melanogaster.

In Drosophila melanogaster, the borders between pericentric heterochromatin and euchromatin on the major chromosome arms have been defined in various ways, including chromatin-specific histone modifications, the binding patterns of heterochromatin-enriched chromosomal proteins, and various cytogenetic techniques. Elucidation of the genetic properties that independently define the different chromatin states associated with heterochromatin and euchromatin should help refine the boundary. Since meiotic recombination is present in euchromatin, but absent in heterochromatin, it constitutes a key genetic property that can be observed transitioning between chromatin states. Using P element insertion lines marked with a su(Hw) insulated mini-white gene, meiotic recombination was found to transition in a region consistent with the H3K9me2 transition observed in ovaries.

Male recombination in Brazilian populations of Drosophila ananassae.

With few exceptions, spontaneous crossing over does not normally occur in male Drosophila. Drosophila ananassae males show considerable amounts of crossing over. In wild males of D. ananassae from Asian (2008) and Brazilian populations (1986 and 2007) variable frequencies of meiotic crossing over, estimated from chiasmata counts, suggested the existence of factors controlling male crossing over in these populations. To corroborate for such prediction, we present data on spontaneous recombination in F1 males of D. ananassae heterozygous for chromosomes of the same Brazilian populations (1986) and marker chromosomes using three testers stocks. Mean recombination value was low, although high variability existed between individual frequencies. Recombination frequencies between lines in each tester stock were not significantly different, excepting when the 3ple-px and 3ple-cy testers were compared (p < 0.05). These two testers differ in respect to the regional distribution of crossovers. The occurrence of recombination in chromosomes 2 and 3 in F1 males tested with e(65) se; bri ru was not related, suggesting they are under independent genetic control. Our data are consistent with proposed genetic factors controlling male crossing over in the tester stocks and to the presence of enhancers and suppressors of male crossing over segregating in the Brazilian populations (1986).

Gene flow, recombination, and positive selection in Stenotrophomonas maltophilia: mechanisms underlying the diversity of the widespread opportunistic pathogen.

Stenotrophomonas maltophilia is a global multidrug-resistant human opportunistic pathogen in clinical environments. Stenotrophomonas maltophilia is also ubiquitous in aqueous environments, soil, and plants. Various molecular typing methods have revealed that S. maltophilia exhibits high levels of phenotypic and genotypic diversity. However, information regarding the genomic diversity within S. maltophilia and the corresponding genetic mechanisms resulting in said diversity remain scarce. The genome sequences of 17 S. maltophilia strains were selected to investigate the mechanisms contributing to genetic diversity at the genome level. The core and large pan-genomes of the species were first estimated, resulting in a large, open pan-genome. A species phylogeny was also reconstructed based on 344 orthologous genes with one copy per genome, and the contribution of four evolutionary mechanisms to the species genome diversity was quantified: 15%-35% of the genes showed evidence for recombination, 0%-25% of the genes in one genome were likely gained, 0%-44% of the genes in some genomes were likely lost, and less than 0.3% of the genes in a genome were under positive selection pressures. We observed that, among the four main mechanisms, homologous recombination plays a key role in maintaining diversity in S. maltophilia. In this study, we provide an overview of evolution in S. maltophilia to provide a better understanding of its evolutionary dynamics and its relationship with genome diversity.

Tyramide-FISH mapping of single genes for development of an integrated recombination and cytogenetic map of chromosome 5 of Allium cepa.

Chromosome 5 of onion carries major quantitative trait loci (QTL) that control dry-matter content, pungency and storability of bulbs, amounts and types of epicuticular waxes, and resistances to abiotic factors, all of which are of interest to breeders. SNPs, SSRs, and RFLPs in expressed regions of the onion genome have been genetically mapped, and we used these clones and sequences from the NCBI database to develop DNA probes for in situ hybridization to integrate the genetic and physical maps of onion chromosome 5. We produced genomic amplicons from expressed regions of the onion genome that carried both exons and introns in order to increase the hybridization specificity of the probes and to enlarge the target DNA sizes. Tyramide-FISH technique was used to increase the detection sensitivity of relatively short target DNA regions, which range from 950 to 2100 bp. Through the integration of genetic and chromosomal maps, we were able to estimate the distribution of recombination events along onion chromosome 5. We demonstrated the efficiency of chromosomal in situ mapping of exon-intron genomic clones for the extremely large genome of onion.

[Current post-surgical treatment strategies in first-line ovarian cancer]. / Stratégies thérapeutiques post-chirurgicales actuelles dans les cancers de l'ovaire en première ligne de traitement.

Although the management of epithelial ovarian cancer has evolved significantly over the past few years, it remains a public health issue, as most patients are diagnosed at an advanced stage and relapse after first line treatment. Chemotherapy remains the standard adjuvant treatment for International Federation of Gynecology and Obstetrics (FIGO) stage I and II tumors, with some exceptions. For FIGO stage III/IV tumors, carboplatin- and paclitaxel-based chemotherapy are the standard of care, in combination with targeted therapies, especially bevacizumab and/or poly-(ADP-ribose) polymerase inhibitors, that have become a key milestone of first-line treatment. Our decision making for the maintenance therapy is based on the FIGO stage, tumor histology, timing of surgery (i.e. primary or interval debulking surgery), residual tumor, response to chemotherapy, BRCA mutation and homologous recombination (HR) status.

[Homologous recombination deficiency in epithelial ovarian cancers: from molecular characterization to patient journey]. / Déficit de la recombinaison homologue dans les cancers épithéliaux de l'ovaire : de la caractérisation moléculaire au parcours des patientes.

High-grade serous ovarian carcinoma (HGSOC), the most frequent and aggressive form of epithelial ovarian cancer is characterized in half of cases by homologous recombination deficiency (HRD). This molecular alteration is defined by distinct causes and consequences. The main and most characterized cause is the presence of an alteration affecting BRCA1 and BRCA2 genes. Regarding consequences, a specific genomic instability leads to increased sensitivity to platinum salts and poly (ADP-ribose) polymerase (PARPi) inhibitors. This latter point enabled the advent of PARPi in first and second line maintenance. As such, the initial and rapid evaluation of HRD status with molecular tests is a key step in the management of HGSOC. Until recently, the range of tests offered proved to be very limited and suffered from technical and medical limitations. This has recently led to the development and validation of alternatives, including academic ones. This "state of the art" review will bring a synthesis concerning the assessment of HRD status in HGSOCs. After a brief introduction of HRD (including main causes and consequences) and of its predictive value regarding PARPi, we will discuss the limitations of current molecular tests and the existing alternatives. Finally, we will contextualize this to the situation in France, with special consideration concerning the positioning and the financial coverage of these tests, with the perspective of optimizing patient management .

[Homologous recombination deficiency and PARP inhibitors in therapeutics]. / Défauts de la recombinaison homologue et inhibiteurs de PARP en thérapeutique.

PARP inhibitors are effective in different types of tumors such as ovarian, breast, prostate and pancreatic cancer. Many studies are in progress and may lead to prescription evolution. PARP inhibitors prescription is almost reserved to patients with a constitutional BRCA mutation or a somatic BRCA alteration or a tumor with a deficiency in homologous recombination. Nowadays, the diagnosis of homologous recombination deficit, HRD, is possible with the prescription of a myChoice CDx (Myriad) test. PARP inhibitors are studied in association with chemotherapy and targeted therapies but also with radiotherapy and with immune checkpoint inhibitors. Access to PARP inhibitors is challenged with the emergence of resistance mechanism. Various trials are now studying the possibility of reversing these resistance mechanisms.

Diagnostic histologique et moléculaire des cancers de l'ovaire - recommandations pour la pratique clinique Saint-Paul 2021: Histological and molecular diagnosis of ovarian.

Oncogenetic testing is now part of standard management in high grade ovarian cancer, including at least mutational status of BRCA1/BRCA2 genes. If necessary, tumor genetic testing is followed by constitutional testing to either confirm the constitutional origin of variants identified in BRCA1/2 genes or detect variants in other predisposition genes. The whole process including prescription of tumoral testing, retrieval of analysis report and communication of results must be formalized, as well as information on possible consequences of the results for the patient and her family. Tumor material must meet criteria of size and cellularity to allow high-quality analysis. These samples are processed during the preanalytical phase with two major steps : time of cold ischemia and fixation. Only pathogenic (Class V) and likely pathogenic (Class IV) variants shown in tumor tissue are mentioned in the report. Currently, only BRCA1 and BRCA2 genes are routinely studied but, in the future, analysis will be extended to other genes involved in homologous recombination repair. In patients without BRCA mutation, other biomarkers reflecting sensitivity to PARP inhibitors, such as HRD scores (homologous recombination deficiency) that appeared recently, will have to be implemented in routine practice in order to better select patients for these treatments and choose optimal therapy.

[DNA repair as a therapeutic target]. / Nouvelles perspectives dans le ciblage thérapeutique de la réparation de l'ADN.

The transmission of an intact and stable genetic code at each cell division relies on different DNA repair systems. Germline mutations of some of these genes cause cancer predisposition, whereas somatic mutations are frequently found in various cancer types, generating genomic instability. As a consequence, cancer cell becomes more susceptible to additional DNA damage. Pharmacological inhibition of DNA repair pathways exploits this frailty: it triggers more damages than cancer cell can tolerate, finally leading to apoptosis. The success of PARP (poly-ADP-ribose polymerase) inhibitors in BRCA1/2-mutated ovarian cancer shows the clinical relevance of this strategy. Herein, we explain the functioning of different DNA-repair pathways, describe the implicated proteins, and their close relation with cell-cycle checkpoints. We focus on novel therapeutic agents targeting DNA repair, their clinical results, and discuss challenges of combination therapies.

Meiotic recombination mechanisms.

Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries.