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788 results for “genotypic data”
Phased genotype data for FILET's analysis of introgression in D. simulans and D. sechellia
<p>This dataset contains all genotype files prepared for Schrider et al.'s analysis of introgression between D. simulans and D. sechellia. For more information see https://www.biorxiv.org/content/early/2017/09/25/170670</p>
Genotype data of 14 nuclear microsatellite loci for 18 Quercus chenii populations in China
<p>This dataset includes genotype data of 14 nuclear microsatellite loci for 419 individuals of Quercus chenii from 18 natural populations in China.</p>
Data for: Akhund-Zade et al The effect of environmental enrichment on behavioral variability depends on genotype, behavior, and type of enrichment
<p>Data and analysis scripts for the manuscript entitled "The effect of environmental enrichment on behavioral variability depends on genotype, behavior, and type of enrichment" by Jamilla Akhund-Zade et al. </p>
Data for "Differential effects of Daphnia genotype composition on spatial environmental heterogeneity in experimental metacommunities"
Open the record for dataset details and reuse information.
Data from: Independent and interactive effects of plant genotype and environment on plant traits and insect herbivore performance: a meta-analysis with Salicaceae
1. Ecological research has increasingly highlighted the importance of intraspecific variation in shaping the structure and function of communities and ecosystems. Indeed, the effects of intraspecific variation can match or exceed those of interspecific variation. Previous reviews of intraspecific variation in plant traits across heterogeneous environments have focused primarily on mean phenotypic effects. We propose that a richer and fuller understanding of the ecological causes and consequences of intraspecific variation would be provided by partitioning trait variance into its subcomponents (genetic, environment, genotype by environment interaction). 2. We used a meta-analysis of 352 sets of genetic, environment, and genotype by environment (GxE) variation estimates from 72 studies of Salicaceae to compare these sources of variation across plant traits (growth, foliar nitrogen, defense compounds), insect herbivore performance metrics (e.g., survival, growth, fecundity), and environmental conditions (e.g., soil nutrients, water, defoliation). 3. Our findings revealed that variation in levels of defense compounds (both condensed tannins and salicinoids) and insect herbivore performance were primarily genetically determined, while variation in plant growth and foliar nitrogen were more environmentally determined. 4. Plasticity in plant growth, foliar nitrogen levels, and insect herbivore performance varied substantially across different sites (year x location), and nutrient, water, and carbon dioxide environments. Plasticity was lowest for chemical defense traits and all traits in contrasting ozone and defoliation environments. 5. Our quantitative review also revealed several gaps in the literature, including a need for surveying more mature plants (>2 years-old), a wider variety of insect herbivore species (e.g., leaf-modifiers, specialist insects), and underrepresented environmental treatments (e.g., competition, defoliation, disease, light, water). This work will help to assess how the patterns within this meta-analysis may or may not be confined within particular parameters (e.g., plant maturity). 6. Findings from this analysis further highlight the importance of and patterns within intraspecific variation in shaping the evolvability and plasticity of traits and in governing plant-insect interactions.
Data from: Fine-scale temporal analysis of genotype-dependent mortality at settlement in the Pacific oyster Crassostrea gigas
Settlement and metamorphosis mark a critical transition in the life cycle of marine invertebrates, during which substantial mortality occurs in both field and laboratory settings. Previous pair-crossing experiments with the Pacific oyster Crassostrea gigas have revealed significant selective or genotype-dependent mortality around the metamorphic transition, but the fine-scale nature and timing of this mortality is not known, particularly whether it occurs before, during or after metamorphosis. In this laboratory study, microsatellite marker segregation ratios were followed daily throughout the settlement and metamorphosis of an F2 cross of the Pacific oyster to examine the fine-scale patterns of genotype dependent mortality at this transition and whether settlement timing (early vs. late) might be under genetic control and affect inference of genotype dependent mortality. Settlement occurred over nine days (day 18 to day 27 post-fertilization) with 68% of individuals settling either early (day 19) or late (day 24). Tracking the survival of spat for 40 days after initial settlement revealed almost no mortality and thus no appreciable genetic mortality. Temporal genetic analysis revealed that 3/11 loci exhibited genotype dependent mortality around the metamorphic transition, one of which (Cg205) was followed throughout settlement and metamorphosis. Alternative temporal patterns of strong selection against each homozygous genotype at Cg205 revealed possible defects in both the competency pathway (inability to initiate metamorphosis) and the morphogenesis pathway (mortality during the metamorphic transition). Quantitative trait locus (QTL) mapping of settlement timing identified three individual and one epistatic QTL with significant genetic effects on this trait (29% of the variance explained in total); however, two of these loci were linked to markers exhibiting selective mortality at metamorphosis, potentially confounding their apparent association with settlement timing. Overall, the results of this study highlight the complex nature of mortality and behavior during settlement and metamorphosis in oysters and suggest that endogenous sources of mortality at settlement may play an important role in the recruitment dynamics of oysters and possibly other broadcast spawning marine invertebrates.
Data from: A set of plastid loci for use in multiplex fragment length genotyping for intraspecific variation in Pinus (Pinaceae)
Premise of the study: Recently released Pinus plastome sequences support characterization of 15 plastid Simple Sequence Repeat (ptSSR) loci originally published for P. contorta and P. thunbergii. This allows selection of loci for single-tube PCR multiplexed genotyping in any subsection of the genus. Methods: Unique placement of primers and primer conservation across the genus were investigated, and a set of six loci were selected for single-tube multiplexing. We compare interspecific variation between ptSSRs and nucleotide sequences of ycf1 then test intraspecific variation for ptSSRs using 911 samples in the P. ponderosa species complex. Results: The ptSSR loci contain mononucleotide and complex repeats with additional length variation in flanking regions. They are not located in hypervariable regions and most primers are conserved across the genus. A single PCR per sample multiplexed for six loci yielded 45 alleles in 911 samples. Discussion: The protocol allows efficient genotyping of many samples. The ptSSR loci are too variable for Pinus phylogenies but are useful for the study of genetic structure within and among populations. The multiplex method could easily be extended to other plant groups by choosing primers for ptSSR loci in a plastome alignment for the target group.
Data from: Skin swabbing of amphibian larvae yields sufficient DNA for efficient sequencing and reliable microsatellite genotyping
Skin swabbing, a minimally invasive DNA sampling method recently developed on adult amphibians, was tested on larvae of fire salamanders (Salamandra salamandra). The quality and quantity of the sampled DNA was evaluated by (i) measuring DNA concentration in DNA extracts, (ii) sequencing part of the mtDNA cytochrome b gene (692 bp) and (iii) genotyping eight polymorphic nuclear microsatellite loci. The multiple-tubes approach was used for calculating allelic dropout (ADO) and false allele (FA) rates to evaluate the reliability of the genotypes. DNA extracts from tissue samples of road-killed individuals were included in the study as positive controls. Our results showed that skin swabs of fire salamander larvae can provide DNA in sufficient quantity and quality, as sequencing was successful and no allelic dropouts or false alleles were detected. This method, tested for the first time on amphibian larvae, has proven to be an efficient and reliable alternative to the controversial tail fin clipping procedure.
Data from: Genotyping-in-Thousands by sequencing (GT-seq) panel development and application to minimally-invasive DNA samples to support studies in molecular ecology
Minimally-invasive sampling (MIS) is widespread in wildlife studies; however, its utility for massively parallel DNA sequencing (MPS) is limited. Poor sample quality and contamination by exogenous DNA can make MIS challenging to use with modern genotyping-by-sequencing approaches, which have been traditionally developed for high-quality DNA sources. Given that MIS is often more appropriate in many contexts, there is a need to make such samples practical for harnessing MPS. Here, we test the ability for Genotyping-in-Thousands by sequencing (GT-seq), a multiplex amplicon sequencing approach, to effectively genotype minimally-invasive cloacal DNA samples collected from the Western Rattlesnake (Crotalus oreganus), a threatened species in British Columbia, Canada. As there was no previous genetic information for this species, an optimized panel of 362 SNPs was selected for use with GT-seq from a de novo restriction-site associated DNA sequencing (RADseq) assembly. Comparisons of genotypes generated within and among RADseq and GT-seq for the same individuals found low rates of genotyping error (GT-seq: 0.50%; RADseq: 0.80%) and discordance (2.57%), the latter likely due to the different genotype calling models employed. GT-seq mean genotype discordance between blood and cloacal swab samples collected from the same individuals was also minimal (1.37%). Estimates of population diversity parameters were similar across GT-seq and RADseq datasets, as were inferred patterns of population structure. Overall, GT-seq can be effectively applied to low quality DNA samples, minimizing the inefficiencies presented by exogenous DNA typically found in minimally-invasive samples and continuing the expansion of molecular ecology and conservation genetics in the genomics era.
Data from: Persistence of resident and transplanted genotypes of the undomesticated yeast, Saccharomyces paradoxus in forest soil
One might expect yeasts in soil to be highly dispersed via water or insects, forming ephemeral, genetically heterogeneous populations subject to competition and environmental stochasticity. Here, we report persistence of genotypes of the yeast Saccharomyces paradoxus in space and time. Within 1 km2 in a mixed hardwood forest on scales from centimeters to tens of meters, we detect persistence over 3 years of native genotypes, identified by SNPs genome-wide, of the wild yeast, Saccharomyces paradoxus around Quercus rubra and Q. alba. Yeasts were recovered by enrichment in ethanol-containing medium, which measures only presence or absence, not abundance. Additional transplantation experiments employed strains marked with spontaneous defects in the URA3 gene, which also confer resistance to 5-Fluoroorotic acid (5FOA). Plating soil suspensions from transplant sites on 5FOA medium permitted one-step quantification of yeast colony-forming units, with no interference from other unmarked yeasts or microorganisms. After an initial steep decrease in abundance, the yeast densities fluctuated over time, increasing in association with rainfall and decreasing in association with drought. After 18 months, the transplanted yeasts remain in place on the nine sites. In vitro transplantation experiments into non-sterile soil in petri dishes showed similar patterns of persistence and response to moisture and drought. To determine whether S. cerevisiae, not previously recovered from soils regionally, can persist in our cold-climate sites, we transplanted marked S. cerevisiae alone and in mixture with S. paradoxus in fall, 2017. Five months on, S. cerevisiae persist to the same extent as S. paradoxus.
Data from: Analysis-ready datasets for insecticide resistance phenotype and genotype frequency in African malaria vectors
The impact of insecticide resistance in malaria vectors is poorly understood and quantified. Here a series of geospatial datasets for insecticide resistance in malaria vectors are provided so that trends in resistance in time and space can be quantified and the impact of resistance found in wild populations on malaria transmission in Africa can be assessed. Data are also provided for common genetic markers of resistance to support analyses of whether these genetic data can improve the ability to monitor resistance in low resource settings. Specifically, data have been collated and geopositioned for the prevalence of insecticide resistance, as measured by standard bioassays, in representative samples of individual species or species complexes. Data are provided for the Anopheles gambiae species complex, the Anopheles funestus subgroup, and for nine individual vector species. In addition, allele frequencies for known resistance associated markers in the Voltage-gated sodium channel (Vgsc) are provided. In total, eight analysis-ready, standardised, geopositioned datasets encompassing over 20,000 African mosquito collections between 1957 and 2017 are provided.
Data from: Spartina alterniflora genotypic identity affects plant and consumer responses in an experimental marsh community
1. Competition and herbivory are ubiquitous processes known to interactively shape plant performance, distribution and community assembly. Likewise, plant genetic variation and associated trait differences can impact both plant-plant and plant-herbivore interactions individually, yet few studies have explored these interactions simultaneously. 2. Salt marsh communities are an ideal system to study these questions, as they are dominated along the Atlantic and Gulf coasts of the United States by a foundation plant species, Spartina alterniflora, with high levels of genetically-based trait variation. Furthermore, consumer pressure and plant competitors are known to influence both the distribution and production of Spartina. We manipulated Spartina genetic identity, neighbor identity (needlerush, Juncus roemerianus), and consumer presence (snail, Littoraria irrorata) in a one-year field experiment in St. Joseph Bay, FL to test how the strength and direction of neighbor and consumer interactions vary by plant genotype. 3. Consumer effects on Spartina were generally stronger and more variable than those of the plant neighborhood, and these effects were generally consistent across Spartina genotypes. However, genotype-specific variation in morphology, phenology, and palatability significantly influenced both consumer and neighbor responses to Spartina: the consumer Littoraria was more likely to climb palatable Spartina genotypes, and neighboring Juncus had shorter stems in the presence of highly productive Spartina genotypes. 4. Synthesis. Our results add to the growing list of examples highlighting the role of intraspecific variation in species interactions, and suggest that variation in these interactions may promote the maintenance of plant genetic variation and community composition.
Data from: Investigating the production of sexual resting structures in a plant pathogen reveals unexpected self-fertility and genotype-by-environment effects
The sexual stage of pathogens governs recombination patterns and often also provides means of surviving the off-season. Despite its importance for evolutionary potential and between-season epidemiology, sexual systems have not been carefully investigated for many important pathogens, and what generates variation in successful sexual reproduction of pathogens remains unexplored. We surveyed the sexually produced resting structures (chasmothecia) across 86 natural populations of fungal pathogen Podosphaera plantaginis (Ascomycota) naturally infecting Plantago lanceolata in the Åland archipelago, southwest of Finland. For this pathosystem, these resting structures are a key life-history stage, as more than half of the local pathogen populations go extinct every winter. We uncovered substantial variation in the level of chasmothecia produced among populations, ranging from complete absence to presence on all infected leaves. We found that chasmothecia developed within clonal isolates (single strain cultures). Additionally, these clonal isolates all contained both MAT1-1-1 and MAT1-2-1 genes that characterize mating-types in Ascomycetes. Hence, contrary to expectations, we conclude that this species is capable of haploid selfing. In controlled inoculations we discovered that pathogen genotypes varied in their tendency to produce chasmothecia. Production of chasmothecia was also affected by ambient temperature (E), and by the interaction between temperature and pathogen genotype (G × E). These G, E and G × E effects found both at a European scale, as well as within Åland, may partly explain the high variability observed among populations in chasmothecia levels. Consequently, they may be key drivers of the evolutionary potential and epidemiology of this highly dynamic pathosystem.
Data from: Genome-wide genotyping uncovers genetic profiles and history of the Russian cattle breeds
One of the most economically important areas within the Russian agricultural sector is dairy and beef cattle farming contributing about $11 billion to the Russian economy annually. Trade connections, selection and breeding have resulted in the establishment of a number of breeds that are presumably adapted to local climatic conditions. Little however is known about the ancestry and history of Russian native cattle. To address this question, we genotyped 274 individuals from 18 breeds bred in Russia and compared them to 135 additional breeds from around the world that had been genotyped previously. Our results suggest a shared ancestry between most of the Russian cattle and European taurine breeds, apart from a few breeds that shared ancestry with the Asian taurines. The Yakut cattle, belonging to the latter group, was found to be the most diverged breed in the whole combined dataset according to structure results. Haplotype sharing further suggests that the Russian cattle can be divided into four major clusters reflecting ancestral relations with other breeds. Herein, we therefore shed light on to the history of Russian cattle and identified closely related breeds to those from Russia. Our results will facilitate future research on detecting signatures of selection in cattle genomes and eventually inform future genetics-assisted livestock breeding programs in Russia and in other countries.
Data from: A rapid and cost-effective quantitative microsatellite genotyping protocol to estimate intraspecific competition in protist microcosm experiments
High levels of intra-specific variation are commonly observed in natural microbial populations, yet the consequences of this variation for ecological and evolutionary processes remains poorly understood. Protists are excellent experimental models for investigating fundamental and applied questions in ecology and evolution, but studying intra-specific variation remains a challenge due to a lack of molecular resources to aid in quantifying and distinguishing strains during experiments. Here we present a molecular method, quantitative microsatellite genotyping, to accurately quantify strain specific frequencies from microcosm experiments of the marine flagellate Oxyrrhis marina, both between many pairs of strains and between strains in a multi-strain mixture. We find that for pairs of strains the method is effective for relative frequencies as low as 0.02 and with around 99% accuracy. The method is able to quantify four strains reasonably well, though less accurately than for pairs (range 92%-97% accuracy). This makes accessible a cheap and easy to implement method for quantifying strain (or allele) frequencies, and is suitable for use in a broad range of single celled eukaryotes (Protists) where copy number should correlate well with number of individuals (i.e. cells). This opens up the possibility of examining the role of intra-specific variation using experimental protist microcosms.
Data from: Linking genotype to phenotype in a changing ocean: inferring the genomic architecture of a blue mussel stress response with genome-wide association
A key component to understanding the evolutionary response to a changing climate is linking underlying genetic variation to phenotypic variation in stress response. Here we use a genome-wide association approach (GWAS) to understand the genetic architecture of calcification rates under simulated climate stress. We take advantage of the genomic gradient across the blue mussel hybrid zone (Mytilus edulis and Mytilus trossulus) in the Gulf of Maine (GOM) to link genetic variation with variance in calcification rates in response to simulated climate change. Falling calcium carbonate saturation states are predicted to negatively impact many marine organisms that build calcium carbonate shells - like blue mussels. We sampled wild mussels and measured net calcification phenotypes after exposing mussels to a "climate change" common garden, where we raised temperature 3°C, decreased pH by 0.2 units, and limited food supply by filtering out planktonic particles > 5 μm, compared to ambient GOM conditions in the summer. This climate change exposure greatly increased phenotypic variation in net calcification rates compared to ambient conditions. We then used regression models to link the phenotypic variation with over 170,000 single nucleotide polymorphism loci (SNPs) generated by genotype by sequencing to identify genomic locations associated with calcification phenotype, and estimate heritability and architecture of the trait. We identified at least one of potentially 2-10 genomic regions responsible for 30% of the phenotypic variation in calcification rates that are potential targets of natural selection by climate change. Our simulations suggest a power of 13.7% with our study's average effective sample size of 118 individuals and rare alleles, but a power of > 90% when effective sample size is 900.
Data from: Genotyping-by-sequencing reveals genomic homogeneity among overwintering Pacific Dunlin (Calidris alpina pacifica) aggregations along the Pacific coast of North America
Information on how migratory populations are genetically structured during the overwintering season of the annual cycle can improve our understanding of the strength of migratory connectivity and help identify populations as units for management. Here, we use a genotype-by-sequencing approach to investigate whether population genetic structure exists among overwintering aggregations of the Pacific Dunlin subspecies (Calidris alpina pacifica) sampled at two spatial scales (i.e. within and among overwintering sites) in the eastern Pacific Flyway. Genome-wide analyses of 874 single nucleotide polymorphisms across 80 sampled individuals revealed no evidence for genetic differentiation among aggregations overwintering at three locations within the Fraser River Estuary (FRE) of British Columbia. Similarly, comparisons of aggregations in the FRE and those overwintering in southern sites in California and Mexico indicated no genetic segregation between northern and southern overwintering areas. These results suggest that Pacific Dunlin residing within the FRE, Sacramento Valley (California) and Guerrero Negro (Mexico) are genetically homogeneous, with no evident genetic structure between sampled sites or regions across the overwintering range. Despite no evidence for differentiation among aggregations, we identified a significant effect of geographical distance between sites on the distribution of individual genotypes in a redundancy analysis; however, a small proportion of the total genotypic variance (R2 = 0.036, P = 0.011) was explained by the combined effect of latitude and longitude, suggesting weak genomic patterns of isolation-by-distance that are consistent with chain-like migratory connectivity between breeding and overwintering areas. Our study represents the first genome-scale investigation of population structure for a Dunlin subspecies and provides essential baseline estimates of genomic diversity and differentiation within the Pacific Dunlin.
Data from: High-throughput SNP genotyping of historical and modern samples of five bird species via sequence capture of ultraconserved elements
Sample availability limits population genetics research on many species, especially taxa from regions with high diversity. However, many such species are well represented in museum collections assembled before the molecular era. Development of techniques to recover genetic data from these invaluable specimens will benefit biodiversity science. Using a mixture of freshly preserved and historical tissue samples, and a sequence capture probe set targeting >5000 loci, we produced high-confidence genotype calls on thousands of single nucleotide polymorphisms (SNPs) in each of five South-East Asian bird species and their close relatives (N = 27–43). On average, 66.2% of the reads mapped to the pseudo-reference genome of each species. Of these mapped reads, an average of 52.7% was identified as PCR or optical duplicates. We achieved deeper effective sequencing for historical samples (122.7×) compared to modern samples (23.5×). The number of nucleotide sites with at least 8× sequencing depth was high, with averages ranging from 0.89 × 106 bp (Arachnothera, modern samples) to 1.98 × 106 bp (Stachyris, modern samples). Linear regression revealed that the amount of sequence data obtained from each historical sample (represented by per cent of the pseudo-reference genome recovered with ≥8× sequencing depth) was positively and significantly (P ≤ 0.013) related to how recently the sample was collected. We observed characteristic post-mortem damage in the DNA of historical samples. However, we were able to reduce the error rate significantly by truncating ends of reads during read mapping (local alignment) and conducting stringent SNP and genotype filtering.
Genotyping-by-sequencing data for a Haitian sorghum breeding program
<p>Rapid environmental change can lead to extinction of populations or evolutionary rescue via genetic adaptation. In the past several years, smallholder and commercial cultivation of sorghum (Sorghum bicolor), a global cereal and forage crop, has been threatened by a global outbreak of an aggressive new biotype of sugarcane aphid (SCA; Melanaphis sacchari). Here we characterized genomic signatures of adaptation in a Haitian sorghum breeding population, which had been recently founded from admixed global germplasm, extensively intercrossed, and subjected to intense selection under SCA infestation. We conducted evolutionary population genomics analyses of 296 post-selection Haitian lines compared to 767 global accessions at 159,683 single nucleotide polymorphisms. Despite intense selection, the Haitian population retains high nucleotide diversity through much of the genome due to diverse founders and an intercrossing strategy. A genome-wide fixation (FST) scan and geographic analyses suggests that adaptation to SCA in Haiti is conferred by a globally-rare East African allele of RMES1, which has also spread to other breeding programs in Africa, Asia, and the Americas. De novo genome sequencing data for SCA resistant and susceptible lines revealed putative causative variants at RMES1. Convenient low-cost markers were developed from the RMES1 selective sweep and successfully predicted resistance in independent U.S. × African breeding lines and eight U.S. commercial and public breeding programs, demonstrating the global relevance of the findings. Together, the findings highlight the potential of evolutionary genomics to develop adaptive trait breeding technology and the value of global germplasm exchange to facilitate evolutionary rescue.</p>
Genotyping-by-Sequencing data of weedy and domesticated Brassica rapa L.
<p>The study of domestication contributes to our knowledge of evolution and crop genetic resources. Human selection has shaped wild <em>Brassica rapa</em> into diverse turnip, leafy, and oilseed crops. Despite its worldwide economic importance and potential as a model for understanding diversification under domestication, insights into the number of domestication events and initial crop(s) domesticated in <em>B. rapa</em> have been limited due to a lack of clarity about the wild or feral status of conspecific non-crop relatives. To address this gap and reconstruct the domestication history of <em>B. rapa</em>, we analyzed 68,468 genotyping-by-sequencing-derived SNPs for 416 samples in the largest diversity panel of domesticated and weedy <em>B. rapa</em> to date. To further understand the center of origin, we modeled the potential range of wild <em>B. rapa</em> during the mid-Holocene. Our analyses of genetic diversity across <em>B. rapa</em> morphotypes suggest that non-crop samples from the Caucasus, Siberia, and Italy may be truly wild, while those occurring in the Americas and much of Europe are feral. Clustering, tree-based analyses, and parameterized demographic inference further indicate that turnips were likely the first crop type domesticated, from which leafy types in East Asia and Europe were selected from distinct lineages. These findings clarify the domestication history and nature of wild crop genetic resources for <em>B. rapa</em>, which provides the first step toward investigating cases of possible parallel selection, the domestication and feralization syndrome, and novel germplasm for <em>Brassica</em> crop improvement.</p>
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Allen Brain Atlas
Allen Brain Atlas is an Allen Institute collection of brain map atlases, datasets, APIs, and analysis tools covering mouse, human, and non-human primate brain resources.
Annotated Behaviour and Observability Dataset (ABODe)
ABODe is a University of Edinburgh DataShare dataset for behavior classification in group-housed mice using home-cage video, identities, bounding boxes, ground-plate positions, and annotator labels.
DANDI Archive for NWB datasets
DANDI is a BRAIN Initiative archive for publishing and sharing neurophysiology data, including electrophysiology, optophysiology, and behavioral data packaged as NWB and related standards.
International Brain Laboratory public data
The International Brain Laboratory public data releases expose standardized mouse decision-making experiments, including Neuropixels recordings, widefield calcium imaging, behavior, and session metadata accessed through the ONE API.
OpenNeuro
OpenNeuro is a free, open platform for sharing neuroimaging datasets, with public search, dataset pages, and download paths for web, S3, DataLad, and the OpenNeuro CLI.