Genetic dissection of the tissue-specific roles of type III effectors and phytotoxins in the pathogenicity of Pseudomonas syringae pv. syringae to cherry.

When compared with other phylogroups (PGs) of the Pseudomonas syringae species complex, P. syringae pv. syringae (Pss) strains within PG2 have a reduced repertoire of type III effectors (T3Es) but produce several phytotoxins. Effectors within the cherry pathogen Pss 9644 were grouped based on their frequency in strains from Prunus as the conserved effector locus (CEL) common to most P. syringae pathogens; a core of effectors common to PG2; a set of PRUNUS effectors common to cherry pathogens; and a FLEXIBLE set of T3Es. Pss 9644 also contains gene clusters for biosynthesis of toxins syringomycin, syringopeptin and syringolin A. After confirmation of virulence gene expression, mutants with a sequential series of T3E and toxin deletions were pathogenicity tested on wood, leaves and fruits of sweet cherry (Prunus avium) and leaves of ornamental cherry (Prunus incisa). The toxins had a key role in disease development in fruits but were less important in leaves and wood. An effectorless mutant retained some pathogenicity to fruit but not wood or leaves. Striking redundancy was observed amongst effector groups. The CEL effectors have important roles during the early stages of leaf infection and possibly acted synergistically with toxins in all tissues. Deletion of separate groups of T3Es had more effect in P. incisa than in P. avium. Mixed inocula were used to complement the toxin mutations in trans and indicated that strain mixtures may be important in the field. Our results highlight the niche-specific role of toxins in P. avium tissues and the complexity of effector redundancy in the pathogen Pss 9644.

Nanoscale imaging of Fe-rich inclusions in single-crystal zircon using X-ray ptycho-tomography.

We apply X-ray ptycho-tomography to perform high-resolution, non-destructive, three-dimensional (3D) imaging of Fe-rich inclusions in paleomagnetically relevant materials (zircon single crystals from the Bishop Tuff ignimbrite). Correlative imaging using quantum diamond magnetic microscopy combined with X-ray fluorescence mapping was used to locate regions containing potential ferromagnetic remanence carriers. Ptycho-tomographic reconstructions with voxel sizes 85 nm and 21 nm were achievable across a field-of-view > 80 µm; voxel sizes as small as 5 nm were achievable over a limited field-of-view using local ptycho-tomography. Fe-rich inclusions 300 nm in size were clearly resolved. We estimate that particles as small as 100 nm-approaching single-domain threshold for magnetite-could be resolvable using this "dual-mode" methodology. Fe-rich inclusions (likely magnetite) are closely associated with apatite inclusions that have no visible connection to the exterior surface of the zircon (e.g., via intersecting cracks). There is no evidence of radiation damage, alteration, recrystallisation or deformation in the host zircon or apatite that could provide alternative pathways for Fe infiltration, indicating that magnetite and apatite grew separately as primary phases in the magma, that magnetite adhered to the surfaces of the apatite, and that the magnetite-coated apatite was then encapsulated as primary inclusions within the growing zircon. Rarer examples of Fe-rich inclusions entirely encapsulated by zircon are also observed. These observations support the presence of primary inclusions in relatively young and pristine zircon crystals. Combining magnetic and tomography results we deduce the presence of magnetic carriers that are in the optimal size range for carrying strong and stable paleomagnetic signals but that remain below the detection limits of even the highest-resolution X-ray tomography reconstructions. We recommend the use of focused ion beam nanotomography and/or correlative transmission electron microscopy to directly confirm the presence of primary magnetite in the sub 300 nm range as a necessary step in targeted paleomagnetic workflows.

Micromagnetic calculation of the magnetite magnetosomal morphology control of magnetism in magnetotactic bacteria.

Magnetotactic bacteria (MTB), which precisely bio-synthesize magnetosomes of magnetite or greigite nanoparticles, have attracted broad interdisciplinary interests in microbiology, magnetic materials, biotechnology and geobiology. Previous experimental and numerical investigations demonstrate a close link among MTB species, magnetosome crystal habits, and magnetic characteristics, but quantitative constraints are currently lacking. In this study, we build three-dimensional finite-element micromagnetic models of intact magnetosome chains in common MTB species and corresponding collapsed chains. Realistic numerical microstructures were constructed for the three typical biogenic magnetite crystal forms-cuboctahedron, prism and bullet. Our calculations reveal characteristic magnetic properties associated with specific magnetite crystal forms and MTB species. Cuboctahedron and bullet crystals show distinct low coercivity (less than 30 mT) and high coercivity (greater than 50 mT) clusters, respectively. Prismatic crystals have a broad range of hysteresis parameters that are strongly controlled by chain structure. This magnetic property clustering, combined with magnetic unmixing methods and electron microscopy observations, can fingerprint biogenic magnetite components in geological and environmental samples. The passive magnetic orientation efficiency of various magnetosome chains was calculated. Some bullet-shaped magnetosome chains have higher magnetic moments than those with cuboctahedron and prism magnetosomes, which may enable larger MTB cells to overcome viscous resistance for efficient magnetic navigation.

Indian Ocean glacial deoxygenation and respired carbon accumulation during mid-late Quaternary ice ages.

Reconstructions of ocean oxygenation are critical for understanding the role of respired carbon storage in regulating atmospheric CO2. Independent sediment redox proxies are essential to assess such reconstructions. Here, we present a long magnetofossil record from the eastern Indian Ocean in which we observe coeval magnetic hardening and enrichment of larger, more elongated, and less oxidized magnetofossils during glacials compared to interglacials over the last ~900 ka. Our multi-proxy records of redox-sensitive magnetofossils, trace element concentrations, and benthic foraminiferal Δδ13C consistently suggest a recurrence of lower O2 in the glacial Indian Ocean over the last 21 marine isotope stages, as has been reported for the Atlantic and Pacific across the last glaciation. Consistent multi-proxy documentation of this repeated oxygen decline strongly supports the hypothesis that increased Indian Ocean glacial carbon storage played a significant role in atmospheric CO2 cycling and climate change over recent glacial/interglacial timescales.

Chromosome-scale genome sequence assemblies of the 'Autumn Bliss' and 'Malling Jewel' cultivars of the highly heterozygous red raspberry (Rubus idaeus L.) derived from long-read Oxford Nanopore sequence data.

Red raspberry (Rubus idaeus L.) is an economically valuable soft-fruit species with a relatively small (~300 Mb) but highly heterozygous diploid (2n = 2x = 14) genome. Chromosome-scale genome sequences are a vital tool in unravelling the genetic complexity controlling traits of interest in crop plants such as red raspberry, as well as for functional genomics, evolutionary studies, and pan-genomics diversity studies. In this study, we developed genome sequences of a primocane fruiting variety ('Autumn Bliss') and a floricane variety ('Malling Jewel'). The use of long-read Oxford Nanopore Technologies sequencing data yielded long read lengths that permitted well resolved genome sequences for the two cultivars to be assembled. The de novo assemblies of 'Malling Jewel' and 'Autumn Bliss' contained 79 and 136 contigs respectively, and 263.0 Mb of the 'Autumn Bliss' and 265.5 Mb of the 'Malling Jewel' assembly could be anchored unambiguously to a previously published red raspberry genome sequence of the cultivar 'Anitra'. Single copy ortholog analysis (BUSCO) revealed high levels of completeness in both genomes sequenced, with 97.4% of sequences identified in 'Autumn Bliss' and 97.7% in 'Malling Jewel'. The density of repetitive sequence contained in the 'Autumn Bliss' and 'Malling Jewel' assemblies was significantly higher than in the previously published assembly and centromeric and telomeric regions were identified in both assemblies. A total of 42,823 protein coding regions were identified in the 'Autumn Bliss' assembly, whilst 43,027 were identified in the 'Malling Jewel' assembly. These chromosome-scale genome sequences represent an excellent genomics resource for red raspberry, particularly around the highly repetitive centromeric and telomeric regions of the genome that are less complete in the previously published 'Anitra' genome sequence.

Direct age constraints on the magnetism of Jack Hills zircon.

A potential record of Earth's magnetic field going back 4.2 billion years (Ga) ago is carried by magnetite inclusions in zircon grains from the Jack Hills. This magnetite may be secondary in nature, however, meaning that the magnetic record is much younger than the zircon crystallization age. Here, we use atom probe tomography to show that Pb-bearing nanoclusters in magnetite-bearing Jack Hills zircons formed during two discrete events at 3.4 and <2 Ga. The older population of clusters contains no detectable Fe, whereas roughly half of the younger population of clusters is Fe bearing. This result shows that the Fe required to form secondary magnetite entered the zircon sometime after 3.4 Ga and that remobilization of Pb and Fe during an annealing event occurred more than 1 Ga after deposition of the Jack Hills sediment at 3 Ga. The ability to date Fe mobility linked to secondary magnetite formation provides new possibilities to improve our knowledge of the Archean geodynamo.

Comparative Genomic Analysis of 31 Phytophthora Genomes Reveals Genome Plasticity and Horizontal Gene Transfer.

Phytophthora species are oomycete plant pathogens that cause great economic and ecological impacts. The Phytophthora genus includes over 180 known species, infecting a wide range of plant hosts, including crops, trees, and ornamentals. We sequenced the genomes of 31 individual Phytophthora species and 24 individual transcriptomes to study genetic relationships across the genus. De novo genome assemblies revealed variation in genome sizes, numbers of predicted genes, and in repetitive element content across the Phytophthora genus. A genus-wide comparison evaluated orthologous groups of genes. Predicted effector gene counts varied across Phytophthora species by effector family, genome size, and plant host range. Predicted numbers of apoplastic effectors increased as the host range of Phytophthora species increased. Predicted numbers of cytoplasmic effectors also increased with host range but leveled off or decreased in Phytophthora species that have enormous host ranges. With extensive sequencing across the Phytophthora genus, we now have the genomic resources to evaluate horizontal gene transfer events across the oomycetes. Using a machine-learning approach to identify horizontally transferred genes with bacterial or fungal origin, we identified 44 candidates over 36 Phytophthora species genomes. Phylogenetic reconstruction indicates that the transfers of most of these 44 candidates happened in parallel to major advances in the evolution of the oomycetes and Phytophthora spp. We conclude that the 31 genomes presented here are essential for investigating genus-wide genomic associations in genus Phytophthora. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genomic and functional analysis of phage-mediated horizontal gene transfer in Pseudomonas syringae on the plant surface.

Many strains of Pseudomonas colonise plant surfaces, including the cherry canker pathogens, Pseudomonas syringae pathovars syringae and morsprunorum. We have examined the genomic diversity of P. syringae in the cherry phyllosphere and focused on the role of prophages in transfer of genes encoding Type 3 secreted effector (T3SE) proteins contributing to the evolution of virulence. Phylogenomic analysis was carried out on epiphytic pseudomonads in the UK orchards. Significant differences in epiphytic populations occurred between regions. Nonpathogenic strains were found to contain reservoirs of T3SE genes. Members of P. syringae phylogroups 4 and 10 were identified for the first time from Prunus. Using bioinformatics, we explored the presence of the gene encoding T3SE HopAR1 within related prophage sequences in diverse P. syringae strains including cherry epiphytes and pathogens. Results indicated that horizontal gene transfer (HGT) of this effector between phylogroups may have involved phage. Prophages containing hopAR1 were demonstrated to excise, circularise and transfer the gene on the leaf surface. The phyllosphere provides a dynamic environment for prophage-mediated gene exchange and the potential for the emergence of new more virulent pathotypes. Our results suggest that genome-based epidemiological surveillance of environmental populations will allow the timely application of control measures to prevent damaging diseases.

Identification of novel genetic regions associated with resistance to European canker in apple.

BACKGROUND: European canker, caused by the fungal pathogen Neonectria ditissima, is an economically damaging disease in apple producing regions of the world - especially in areas with moderate temperatures and high rainfall. The pathogen has a wide host range of hardwood perennial species, causing trunk cankers, dieback and branch lesions in its hosts. Although apple scion germplasm carrying partial resistance to the disease has been described, little is still known of the genetic basis for this quantitative resistance. RESULTS: Resistance to Neonectria ditissima was studied in a multiparental population of apple scions using several phenotyping methods. The studied population consists of individuals from multiple families connected through a common pedigree. The degree of disease of each individual in the population was assessed in three experiments: artificial inoculations of detached dormant shoots, potted trees in a glasshouse and in a replicated field experiment. The genetic basis of the differences in disease was studied using a pedigree-based analysis (PBA). Three quantitative trait loci (QTL), on linkage groups (LG) 6, 8 and 10 were identified in more than one of the phenotyping strategies. An additional four QTL, on LG 2, 5, 15 and 16 were only identified in the field experiment. The QTL on LG2 and 16 were further validated in a biparental population. QTL effect sizes were small to moderate with 4.3 to 19% of variance explained by a single QTL. A subsequent analysis of QTL haplotypes revealed a dynamic response to this disease, in which the estimated effect of a haplotype varied over the field time-points. CONCLUSIONS: This study describes the first identified QTL associated with resistance to N. ditissima in apple scion germplasm. The results from this study show that QTL present in germplasm commonly used in apple breeding have a low to medium effect on resistance to N. ditissima. Hence, multiple QTL will need to be considered to improve resistance through breeding.

Identifying resistance in wild and ornamental cherry towards bacterial canker caused by Pseudomonas syringae.

Bacterial canker is a major disease of stone fruits and is a critical limiting factor to sweet cherry (Prunus avium) production worldwide. One important strategy for disease control is the development of resistant varieties. Partial varietal resistance in sweet cherry is discernible using shoot or whole tree inoculations; however, these quantitative differences in resistance are not evident in detached leaf assays. To identify novel sources of resistance to canker, we used a rapid leaf pathogenicity test to screen a range of wild cherry, ornamental Prunus species and sweet cherry × ornamental cherry hybrids with the canker pathogens, Pseudomonas syringae pvs syringae, morsprunorum races 1 and 2, and avii. Several Prunus accessions exhibited limited symptom development following inoculation with each of the pathogens, and this resistance extended to 16 P. syringae strains pathogenic on sweet cherry and plum. Resistance was associated with reduced bacterial multiplication after inoculation, a phenotype similar to that of commercial sweet cherry towards nonhost strains of P. syringae. Progeny resulting from a cross of a resistant ornamental species Prunus incisa with susceptible sweet cherry (P. avium) exhibited resistance indicating it is an inherited trait. Identification of accessions with resistance to the major bacterial canker pathogens is the first step towards characterizing the underlying genetic mechanisms of resistance and introducing these traits into commercial germplasm.

CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12.

BACKGROUND: Gene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in various filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™). RESULTS: We present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0-40% of isolates. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum. CONCLUSIONS: Using an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum for use as a research tool, for understanding the controls of secondary metabolism and hyphal development and validating prototypes of strains produced using traditional methods for strain improvement.

Genomic Informed Breeding Strategies for Strawberry Yield and Fruit Quality Traits.

Over the last two centuries, breeders have drastically modified the fruit quality of strawberries through artificial selection. However, there remains significant variation in quality across germplasm with scope for further improvements to be made. We reported extensive phenotyping of fruit quality and yield traits in a multi-parental strawberry population to allow genomic prediction and quantitative trait nucleotide (QTN) identification, thereby enabling the description of genetic architecture to inform the efficacy of implementing advanced breeding strategies. A negative relationship (r = -0.21) between total soluble sugar content and class one yield was identified, indicating a trade-off between these two essential traits. This result highlighted an established dilemma for strawberry breeders and a need to uncouple the relationship, particularly under June-bearing, protected production systems comparable to this study. A large effect of quantitative trait nucleotide was associated with perceived acidity and pH whereas multiple loci were associated with firmness. Therefore, we recommended the implementation of both marker assisted selection (MAS) and genomic prediction to capture the observed variation respectively. Furthermore, we identified a large effect locus associated with a 10% increase in the number of class one fruit and a further 10 QTN which, when combined, are associated with a 27% increase in the number of marketable strawberries. Ultimately, our results suggested that the best method to improve strawberry yield is through selecting parental lines based upon the number of marketable fruits produced per plant. Not only were strawberry number metrics less influenced by environmental fluctuations, but they had a larger additive genetic component when compared with mass traits. As such, selecting using "number" traits should lead to faster genetic gain.

The Genome of the CTG(Ser1) Yeast Scheffersomyces stipitis Is Plastic.

Microorganisms need to adapt to environmental changes, and genome plasticity can lead to rapid adaptation to hostile environments by increasing genetic diversity. Here, we investigate genome plasticity in the CTG(Ser1) yeast Scheffersomyces stipitis, an organism with an enormous potential for second-generation biofuel production. We demonstrate that S. stipitis has an intrinsically plastic genome and that different S. stipitis isolates have genomes with distinct chromosome organizations. Real-time evolution experiments show that S. stipitis genome plasticity is common and rapid since extensive genomic changes with fitness benefits are detected following in vitro evolution experiments. Hybrid MinION Nanopore and Illumina genome sequencing identify retrotransposons as major drivers of genome diversity. Indeed, the number and position of retrotransposons are different in different S. stipitis isolates, and retrotransposon-rich regions of the genome are sites of chromosome rearrangements. Our findings provide important insights into the adaptation strategies of the CTG(Ser1) yeast clade and have critical implications in the development of second-generation biofuels. These data highlight that genome plasticity is an essential factor for developing sustainable S. stipitis platforms for second-generation biofuels production. IMPORTANCE Genomes contain genes encoding the information needed to build the organism and allow it to grow and develop. Genomes are described as stable structures where genes have specific positions within a chromosome. Changes in gene dosage and position are viewed as harmful. However, it is becoming increasingly clear that genome plasticity can benefit microbial organisms that need to adapt rapidly to environmental changes. Mechanisms of genome plasticity are still poorly understood. This study focuses on Scheffersomyces stipitis, a yeast that holds great potential for second-generation biofuel production generated from forestry and agriculture waste. We demonstrate that S. stipitis chromosomes are easily reshuffled and that chromosome reshuffling is linked to adaptation to hostile environments. Genome sequencing demonstrates that mobile genetic elements, called transposons, mediate S. stipitis genome reshuffling. These data highlight that understanding genome plasticity is important for developing sustainable S. stipitis platforms for second-generation biofuels production.

Transposon Mutagenesis of Pseudomonas syringae Pathovars syringae and morsprunorum to Identify Genes Involved in Bacterial Canker Disease of Cherry.

Bacterial canker of Prunus, affecting economically important stone fruit crops including cherry, peach, apricot and plum, is caused by the plant pathogen Pseudomonas syringae (P.s.). Strains from two pathovars-P.s. pv. syringae (Pss) and P.s. pv. morsprunorum race 1 (PsmR1) and 2 (PsmR2)-in three phylogenetically distant clades have convergently evolved to infect Prunus. The bacteria enter woody tissues through wounds and leaf scars, causing black necrotic cankers. Symptoms are also produced on blossom, fruit and leaves. Little is known about the mechanisms P.s. uses to colonise tree hosts such as Prunus. Here, we created transposon (Tn) mutant libraries in one strain of P.s. from each of the three clades and screened the mutants on immature cherry fruit to look for changes in virulence. Mutants (242) with either reduced or enhanced virulence were detected and further characterised by in vitro screens for biofilm formation, swarming ability, and pathogenicity on leaves and cut shoots. In total, 18 genes affecting virulence were selected, and these were involved in diverse functions including motility, type III secretion, membrane transport, amino acid synthesis, DNA repair and primary metabolism. Interestingly, mutation of the effector gene, hopAU1, led to an increase in virulence of Psm R2.

Comparative Analysis of Host-Associated Variation in Phytophthora cactorum.

Phytophthora cactorum is often described as a generalist pathogen, with isolates causing disease in a range of plant species. It is the causative agent of two diseases in the cultivated strawberry, crown rot (CR; causing whole plant collapse) and leather rot (LR; affecting the fruit). In the cultivated apple, P. cactorum causes girdling bark rots on the scion (collar rot) and rootstock (crown rot), as well as necrosis of the fine root system (root rot) and fruit rots. We investigated evidence for host specialisation within P. cactorum through comparative genomic analysis of 18 isolates. Whole genome phylogenetic analysis provided genomic support for discrete lineages within P. cactorum, with well-supported non-recombining clades for strawberry CR and apple infecting isolates specialised to strawberry crowns and apple tissue. Isolates of strawberry CR are genetically similar globally, while there is more diversity in apple-infecting isolates. We sought to identify the genetic basis of host specialisation, demonstrating gain and loss of effector complements within the P. cactorum phylogeny, representing putative determinants of host boundaries. Transcriptomic analysis highlighted that those effectors found to be specific to a single host or expanded in the strawberry lineage are amongst those most highly expressed during infection of strawberry and give a wider insight into the key effectors active during strawberry infection. Many effectors that had homologues in other Phytophthoras that have been characterised as avirulence genes were present but not expressed in our tested isolate. Our results highlight several RxLR-containing effectors that warrant further investigation to determine whether they are indeed virulence factors and host-specificity determinants for strawberry and apple. Furthermore, additional work is required to determine whether these effectors are suitable targets to focus attention on for future resistance breeding efforts.

Identification and Expression of Secreted In Xylem Pathogenicity Genes in Fusarium oxysporum f. sp. pisi.

Fusarium oxysporum is a soilborne fungal plant pathogen responsible for causing disease in many economically important crops with "special forms" (formae speciales) adapted to infect specific plant hosts. F. oxysporum f. sp. pisi (FOP) is the causal agent of Fusarium wilt disease of pea. It has been reported in every country where peas are grown commercially. Disease is generally controlled using resistant cultivars possessing single major gene resistance and therefore there is a constant risk of breakdown. The main aim of this work was to characterise F. oxysporum isolates collected from diseased peas in the United Kingdom as well as FOP isolates obtained from other researchers representing different races through sequencing of a housekeeping gene and the presence of Secreted In Xylem (SIX) genes, which have previously been associated with pathogenicity in other F. oxysporum f. spp. F. oxysporum isolates from diseased United Kingdom pea plants possessed none or just one or two known SIX genes with no consistent pattern of presence/absence, leading to the conclusion that they were foot-rot causing isolates rather than FOP. In contrast, FOP isolates had different complements of SIX genes with all those identified as race 1 containing SIX1, SIX6, SIX7, SIX9, SIX10, SIX11, SIX12, and SIX14. FOP isolates that were identified as belonging to race 2 through testing on differential pea cultivars, contained either SIX1, SIX6, SIX9, SIX13, SIX14 or SIX1, SIX6, SIX13. Significant upregulation of SIX genes was also observed in planta over the early stages of infection by different FOP races in pea roots. Race specific SIX gene profiling may therefore provide potential targets for molecular identification of FOP races but further research is needed to determine whether variation in complement of SIX genes in FOP race 2 isolates results in differences in virulence across a broader set of pea differential cultivars.

Micromagnetic Tomography for Paleomagnetism and Rock-Magnetism.

Our understanding of the past behavior of the geomagnetic field arises from magnetic signals stored in geological materials, e.g., (volcanic) rocks. Bulk rock samples, however, often contain magnetic grains that differ in chemistry, size, and shape; some of them record the Earth's magnetic field well, others are unreliable. The presence of a small amount of adverse behaved magnetic grains in a sample may already obscure important information on the past state of the geomagnetic field. Recently it was shown that it is possible to determine magnetizations of individual grains in a sample by combining X-ray computed tomography and magnetic surface scanning measurements. Here we establish this new Micromagnetic Tomography (MMT) technique and make it suitable for use with different magnetic scanning techniques, and for both synthetic and natural samples. We acquired reliable magnetic directions by selecting subsets of grains in a synthetic sample, and we obtained rock-magnetic information of individual grains in a volcanic sample. This illustrates that MMT opens up entirely new venues of paleomagnetic and rock-magnetic research. MMT's unique ability to determine the magnetization of individual grains in a nondestructive way allows for a systematic analysis of how geological materials record and retain information on the past state of the Earth's magnetic field. Moreover, by interpreting only the contributions of known magnetically well-behaved grains in a sample, MMT has the potential to unlock paleomagnetic information from even the most complex, crucial, or valuable recorders that current methods are unable to recover.

Kinship and social organization in Copper Age Europe. A cross-disciplinary analysis of archaeology, DNA, isotopes, and anthropology from two Bell Beaker cemeteries.

We present a high-resolution cross-disciplinary analysis of kinship structure and social institutions in two Late Copper Age Bell Beaker culture cemeteries of South Germany containing 24 and 18 burials, of which 34 provided genetic information. By combining archaeological, anthropological, genetic and isotopic evidence we are able to document the internal kinship and residency structure of the cemeteries and the socially organizing principles of these local communities. The buried individuals represent four to six generations of two family groups, one nuclear family at the Alburg cemetery, and one seemingly more extended at Irlbach. While likely monogamous, they practiced exogamy, as six out of eight non-locals are women. Maternal genetic diversity is high with 23 different mitochondrial haplotypes from 34 individuals, whereas all males belong to one single Y-chromosome haplogroup without any detectable contribution from Y-chromosomes typical of the farmers who had been the sole inhabitants of the region hundreds of years before. This provides evidence for the society being patrilocal, perhaps as a way of protecting property among the male line, while in-marriage from many different places secured social and political networks and prevented inbreeding. We also find evidence that the communities practiced selection for which of their children (aged 0-14 years) received a proper burial, as buried juveniles were in all but one case boys, suggesting the priority of young males in the cemeteries. This is plausibly linked to the exchange of foster children as part of an expansionist kinship system which is well attested from later Indo-European-speaking cultural groups.

No Evidence That Homologs of Key Circadian Clock Genes Direct Circadian Programs of Development or mRNA Abundance in Verticillium dahliae.

Many organisms harbor circadian clocks that promote their adaptation to the rhythmic environment. While a broad knowledge of the molecular mechanism of circadian clocks has been gained through the fungal model Neurospora crassa, little is known about circadian clocks in other fungi. N. crassa belongs to the same class as many important plant pathogens including the vascular wilt fungus Verticillium dahliae. We identified homologs of N. crassa clock proteins in V. dahliae, which showed high conservation in key protein domains. However, no evidence for an endogenous, free-running and entrainable rhythm was observed in the daily formation of conidia and microsclerotia. In N. crassa the frequency (frq) gene encodes a central clock protein expressed rhythmically and in response to light. In contrast, expression of Vdfrq is not light-regulated. Temporal gene expression profiling over 48 h in constant darkness and temperature revealed no circadian expression of key clock genes. Furthermore, RNA-seq over a 24 h time-course revealed no robust oscillations of clock-associated transcripts in constant darkness. Comparison of gene expression between wild-type V. dahliae and a ΔVdfrq mutant showed that genes involved in metabolism, transport and redox processes are mis-regulated in the absence of Vdfrq. In addition, VdΔfrq mutants display growth defects and reduced pathogenicity in a strain dependent manner. Our data indicate that if a circadian clock exists in Verticillium, it is based on alternative mechanisms such as post-transcriptional interactions of VdFRQ and the WC proteins or the components of a FRQ-less oscillator. Alternatively, it could be that whilst the original functions of the clock proteins have been maintained, in this species the interactions that generate robust rhythmicity have been lost or are only triggered when specific environmental conditions are met. The presence of conserved clock genes in genomes should not be taken as definitive evidence of circadian function.

Helium ion microscope - secondary ion mass spectrometry for geological materials.

The helium ion microscope (HIM) is a focussed ion beam instrument with unprecedented spatial resolution for secondary electron imaging but has traditionally lacked microanalytical capabilities. With the addition of the secondary ion mass spectrometry (SIMS) attachment, the capabilities of the instrument have expanded to microanalysis of isotopes from Li up to hundreds of atomic mass units, effectively opening up the analysis of all natural and geological systems. However, the instrument has thus far been underutilised by the geosciences community, due in no small part to a lack of a thorough understanding of the quantitative capabilities of the instrument. Li represents an ideal element for an exploration of the instrument as a tool for geological samples, due to its importance for economic geology and a green economy, and the difficult nature of observing Li with traditional microanalytical techniques. Also Li represents a "best-case" scenario for isotopic measurements. Here we present details of sample preparation, instrument sensitivity, theoretical, and measured detection limits for both elemental and isotopic analysis as well as practicalities for geological sample analyses of Li alongside a discussion of potential geological use cases of the HIM-SIMS instrument.