Clonal Genotypes Research Articles

Horizontal acquisition of Symbiodiniaceae in the Anemonia viridis (Cnidaria, Anthozoa) species complex.

All metazoans are in fact holobionts, resulting from the association of several organisms, and organismal adaptation is then due to the composite response of this association to the environment. Deciphering the mechanisms of symbiont acquisition in a holobiont is therefore essential to understanding the extent of its adaptive capacities. In cnidarians, some species acquire their photosynthetic symbionts directly from their parents (vertical transmission) but may also acquire symbionts from the environment (horizontal acquisition) at the adult stage. The Mediterranean snakelocks sea anemone, Anemonia viridis (Forskål, 1775), passes down symbionts from one generation to the next by vertical transmission, but the capacity for such horizontal acquisition is still unexplored. To unravel the flexibility of the association between the different host lineages identified in A.viridis and its Symbiodiniaceae, we genotyped both the animal hosts and their symbiont communities in members of host clones in five different locations in the North Western Mediterranean Sea. The composition of within-host-symbiont populations was more dependent on the geographical origin of the hosts than their membership to a given lineage or even to a given clone. Additionally, similarities in host-symbiont communities were greater among genets (i.e. among different clones) than among ramets (i.e. among members of the same given clonal genotype). Taken together, our results demonstrate that A.viridis may form associations with a range of symbiotic dinoflagellates and suggest a capacity for horizontal acquisition. A mixed-mode transmission strategy in A.viridis, as we posit here, may help explain the large phenotypic plasticity that characterizes this anemone.

DNA Content Variation and SNP Diversity Within a Single Population of Asexual Snails.

A growing body of research suggests that many clonal populations maintain genetic diversity even without occasional sexual reproduction. The purpose of our study was to document variation in single-nucleotide polymorphism (SNP) diversity, DNA content, and pathogen susceptibility in clonal lineages of the New Zealand freshwater snail, Potamopyrgus antipodarum. We studied snails that were collected from multiple field sites around a single lake (Lake Alexandrina), as well as isofemale clonal lineages that had been isolated and maintained in the laboratory. We used the kompetitive allele specific PCR (KASP) method to genotype our samples at 46 nuclear SNP sites, and we used flow cytometry to estimate DNA content. We found high levels of SNP diversity, both in our field samples and in our clonal laboratory lines. We also found evidence of high variation in DNA content among clones, even among clones with identical genotypes across all SNP sites. Controlled pathogen exposures of the laboratory populations revealed variation in susceptibility among distinct clonal genotypes, which was independent of DNA content. Taken together, these results show high levels of diversity among asexual snails, especially for DNA content, and they suggest rapid genome evolution in asexuals.

Hybridization, polyploidy and clonality influence geographic patterns of diversity and salt tolerance in the model halophyte seashore paspalum (Paspalum vaginatum).

In plant species, variation in levels of clonality, ploidy and interspecific hybridization can interact to influence geographic patterns of genetic diversity. These factors commonly vary in plants that specialize on saline habitats (halophytes) and may play a role in how they adapt to salinity variation across their range. One such halophyte is the turfgrass and emerging genomic model system seashore paspalum (Paspalum vaginatum Swartz). To investigate how clonal propagation, ploidy variation, and interspecific hybridization vary across ecotypes and local salinity levels in wild P.vaginatum, we employed genotyping-by-sequencing, cpDNA sequencing and flow cytometry in 218 accessions representing>170 wild collections from throughout the coastal southern United States plus USDA germplasm. We found that the two morphologically distinct ecotypes of P.vaginatum differ in their adaptive strategies. The fine-textured ecotype is diploid and appears to reproduce in the wild both sexually and by clonal propagation; in contrast, the coarse-textured ecotype consists largely of clonally-propagating triploid and diploid genotypes. The coarse-textured ecotype appears to be derived from hybridization between fine-textured P.vaginatum and an unidentified Paspalum species. These clonally propagating hybrid genotypes are more broadly distributed than clonal fine-textured genotypes and may represent a transition to a more generalist adaptive strategy. Additionally, the triploid genotypes vary in whether they carry one or two copies of the P.vaginatum subgenome, indicating multiple evolutionary origins. This variation in subgenome composition shows associations with local ocean salinity levels across the sampled populations and may play a role in local adaptation.

Isolation, Genotyping, and Mouse Virulence Characterization of Toxoplasma gondii From Free Ranging Iberian Pigs.

The present study aimed to isolate and perform molecular and phenotypic characterization of Toxoplasma gondii strains infecting Iberian pigs bred under semi-free conditions and destined for human consumption. Blood and heart tissue samples from 361 fattening pigs from 10 various herds selected in the main areas of Iberian pig production were collected at a slaughterhouse; the sera were tested for anti-T. gondii antibodies using a commercial indirect ELISA kit, and a mouse bioassay was carried out using heart muscle of seropositive individual representatives from each geographical location. Seventy-nine (21.9%) of the 361 animals tested positive for anti-T. gondii antibodies according to the serology test. Fifteen samples of myocardial tissue were subjected to bioassay and 5 isolates (TgPigSp1 to TgPigSp5) were obtained. The isolates were characterized by using 11 PCR-RFLP genetic markers; three isolates had a ToxoDB #3 genotype (3/5) and two isolates had a ToxoDB #2 genotype (2/5). The TgPigSp1 and TgPigSp4 isolates were selected for virulence in mice characterization as instances of each different RFLP-genotype found. The TgPigSp1 isolate (#2 genotype) was virulent in mice with notable cumulative mortality (87.5%) and morbidity rates (100%); the TgPigSp4 (#3) was nonvirulent and triggered mild clinical signs in 42.1% of seropositive mice. Infection dynamics and organ distribution of both isolates were analyzed; the data revealed significant differences, including substantially higher parasite load in the lung during the acute phase of infection, in mice infected with TgPigSp1 than in the case of TgPigSp4 (median parasite load 7.6 vs. 0 zoites/mg, respectively; p < 0.05). Furthermore, degrees of severity of detected histopathological lesions appeared to be related to higher parasite burdens. Taking into account the unexpectedly high mortality rate and parasite load associated with the clonal genotype III, which is traditionally considered nonvirulent in mice, the need for further investigation and characterization of the T. gondii strains circulating in any host in Europe is emphasized.

Multi-locus sequence analyses reveal a clonal L. borgpetersenii genotype in a heterogeneous invasive Rattus spp. community across the City of Johannesburg, South Africa

BackgroundRattus spp. are frequently implicated as key reservoir hosts for leptospirosis, one of the most common, but neglected, bacterial zoonoses in the world. Although leptospirosis is predicted to be a significant public health threat in Africa, studies from the continent are limited.MethodsRattus spp. (n = 171) were sampled (January–May 2016) across the City of Johannesburg, South Africa’s largest inland metropole. Rattus spp. genetic diversity was evaluated by full length (1140 bp) cyt b sequencing of 42 samples. For comparison, a further 12 Rattus norvegicus samples collected in Cape Town, South Africa’s largest coastal metropole, were also genotyped. Leptospira infections were identified and genotyped using real-time PCR and multi-locus (lfb1, secY and lipL41) DNA sequencing.ResultsFive R. norvegicus haplotypes were identified across Johannesburg, four of which have not previously been detected in South Africa, and one in Cape Town. Across Johannesburg we identified a Leptospira spp. infection prevalence of 44% (75/171) and noted significant differences in the prevalence between administrative regions within the metropole. Multi-locus sequence analyses identified a clonal genotype consistent with L. borgpetersenii serogroup Javanica (serovar Ceylonica).DiscussionThe prevalence of infection identified in this study is amongst the highest detected in Rattus spp. in similar contexts across Africa. Despite the complex invasion history suggested by the heterogeneity in R. norvegicus haplotypes identified in Johannesburg, a single L. borgpetersenii genotype was identified in all infected rodents. The lack of L. interrogans in a rodent community dominated by R. norvegicus is notable, given the widely recognised host-pathogen association between these species and evidence for L. interrogans infection in R. norvegicus in Cape Town. It is likely that environmental conditions (cold, dry winters) in Johannesburg may limit the transmission of L. interrogans. Spatial heterogeneity in prevalence suggest that local factors, such as land use, influence disease risk in the metropole.ConclusionsIn South Africa, as in other African countries, leptospirosis is likely underdiagnosed. The high prevalence of infection in urban rodents in Johannesburg suggest that further work is urgently needed to understand the potential public health risk posed by this neglected zoonotic pathogen.Graphical

Base Editing in Human Cells to Produce Single-Nucleotide-Variant Clonal Cell Lines.

Base-editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome-editing methods that use DNA double-strand breaks, such as zinc finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) system. This allows the generation of single-nucleotide-variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time- and resource-effective manner. These single-nucleotide-variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype-to-phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base-editing constructs, transfect adherent cells, quantify base-editing efficiencies in bulk, and generate single-nucleotide-variant clonal cell lines. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Design and production of plasmids for base-editing experiments Basic Protocol 2: Transfection of adherent cells and harvesting of genomic DNA Basic Protocol 3: Genotyping of harvested cells using Sanger sequencing Alternate Protocol 1: Next-generation sequencing to quantify base editing Basic Protocol 4: Single-cell isolation of base-edited cells using FACS Alternate Protocol 2: Single-cell isolation of base-edited cells using dilution plating Basic Protocol 5: Clonal expansion to generate isogenic cell lines and genotyping of clones.

Genetic structure of Metarhizium species in western USA: Finite populations composed of divergent clonal lineages with limited evidence for recent recombination

Globally distributed, soil associated Metarhizium species used in insect biological control are evidently facultatively sexual and obligately outcrossing, yet sexual morphs have not been observed for most species and corroboration that they recombine in nature remains limited. Community-wide genetic diversity of Metarhizium species among 480 soil isolates from 14 states of western USA was investigated to assess the contributions of clonality and recombination in determining each species’ population structure. Seven species, varying greater than 100-fold in relative abundance, were identified by phylogenetic analysis of 5′ EF1-α (5TEF), including M. robertsii (n = 372), M. guizhouense (n = 37), M. brunneum (n = 37), M. lepidiotae (n = 14), M. pemphigi (n = 11), M. anisopliae (n = 7) and M. pingshaense (n = 2). Analyses of composite multilocus genotypes integrating 5TEF sequences, multilocus microsatellites and mating type idiomorphs conducted on a subset of 239 isolates revealed that all species populations display pronounced clonal structure. Following clone-correction procedures to remove redundant clonal genotypes and collapse clonal lineages, each species’ population sample was determined to be composed of a dozen or fewer genetically unique individuals. Thus, the Metarhizium community inhabiting western USA is conservatively estimated to comprise as few as 34 distinct genetic individuals, with a single, geographically ubiquitous clonal lineage of M. robertsii constituting 45% of total isolates. M. robertsii was the only population determined to be in linkage equilibrium. However, the high proportion of private alleles differentiating most M. robertsii clonal lineages argues against contemporary panmixia, thus the recombination signal detected may be historical. Nevertheless, within M. robertsii, M. brunneum and M. guizhouense there are closely related genotypes of opposite mating type, which suggests that if recombination is contemporary, it likely occurs between closely related individuals. The restricted number of genetic individuals observed throughout western North American Metarhizium species may signify that these represent peripheral populations descended from limited numbers of founders among which there has been little recombination relative to the extent of clone expansion and within-clone genetic divergence.

Impact of biased sex ratio on the genetic diversity, structure, and differentiation ofPopulus nigra(European black poplar)

Effective population size is a crucial concept of conservation biology. It is reduced by biased sex ratio, consequently causing loss of genetic variation. To evaluate genetic diversity related to gender, and investigate the possible effects of biased sex ratio, we analyzed available microsatellite DNA markers from 120 samples of Populus nigra L. (European black poplar) originating from five geographical regions in Turkey. Using 12 microsatellite markers, we detected 60 clones of the same genotype, out of 120 trees. The clone genotype was observed both in males and females, which might suggest that P. nigra deviates from dioecism. Three genetic clusters were detected, two of which possibly correspond to commercially available trees. Overall allelic richness was found to be similar for both genders, whereas heterozygosity was slightly higher in males. Additionally, a simulation software prototype was developed to see the effects of sex ratio on diversity and allele frequency trends in future generations, given the available molecular data. Results showed that if biased sex ratio persists, allele loss and fixation might occur in a higher rate, causing loss of variation.

The Climate-Driven Genetic Diversity Has a Higher Impact on the Population Structure of Plasmopara viticola Than the Production System or QoI Fungicide Sensitivity in Subtropical Brazil.

Downy mildew, caused by Plasmopara viticola, is the main disease affecting vineyards in subtropical Brazil. Here, we collected 94 P. viticola isolates from four organic and conventional vineyards in the two main grape-growing states of Brazil to evaluate the sensitivity to the quinone outside inhibitor (QoI) azoxystrobin by pheno- and genotyping assays. The impact of location, production system and sensitivity to QoI fungicides on the population genetics and structure of P. viticola was determined using 10 microsatellite markers. Cytochrome b sequencing revealed that 28 and 100% of the isolates from vineyards under organic and conventional management carried the G143A mutation, respectively. The G143A mutation was associated with high levels of azoxystrobin resistance. Three out of the 94 isolates analyzed carried the M125I alteration, not previously described in P. viticola, which was associated with a five-fold reduction in azoxystrobin sensitivity compared to wild-type isolates. Haplotype network analysis based on cytochrome b gene sequences suggested that the Brazilian populations are more closely related to the European than the North American population. A total of six haplotypes were identified, with two of them carrying the G143A mutation. Microsatellite analysis revealed high allelic and genotypic variation among the four populations. Population differentiation analyses indicated that state of origin directly influences the population biology of P. viticola, while production system and QoI sensitivity have little effect. Great genetic diversity, sexual reproduction and high levels of admixture were observed in Rio Grande do Sul State. In contrast, populations in São Paulo State were dominated by a few clonal genotypes, and no admixed genotype was detected between the two genetic pools identified in the state. This study raises the hypothesis that winter weather conditions influence the overwinter survival strategy with profound effects in the population biology of P. viticola.

New generation surface synthetic of alfalfa sowing (Medicago sativa L.)

Aim. Evaluation of starting material on a complex of economically valuable features and creation with its participation of a variety-synthetics of alfalfa sowing. Methods. Field, laboratory. Results. The results of research and creation of a synthetic variety of alfalfa of sowing Radoslav at the Institute of forages and agriculture of Podillya of NAAS are presented. Listed on the State Register of Plant Varieties Eligible for Distribution in Ukraine (hereinafter – the Register of Plant Varieties of Ukraine) (Certificate of State Registration No. 15196001 of March 14, 2019, Patent for the Plant Variety of Radoslaw No. 190654 of May 5, 2019). When evaluating fodder and seed productivity in the research fields of the institute, the preference for the Radoslav variety to the Sinyukh standard was established. Extensive testing of the suitability of the variety for propagation (SPD) in 5 research points of the Polesia zone and 4 ˗ of the Forest-Steppe Institute of Plant Variety Examination confirmed higher rates of fodder and seed productivity and growth rate of the specified variety compared to the conventional standard. Conclusions. In order to enhance the efficiency of the breeding process aimed at the creation of synthetic varieties, clonal genotypes should be used, which, along with the complex of economically valuable traits, are distinguished by a high overall combining ability. It is recommended to use synthetic alfalfa of sowing Radoslaw in clean and mixed crops with other perennial herbs for production of high-quality forages in the soil-climatic zones of Polissya and Forest-steppe.&#x0D; Keywords: Medicago sativa L., clone, synthetic variety, dry matter, seeds.

Early induction of orthotropic shoots in Coffea canephora

ABSTRACT In Coffea canephora, the number of orthotropic shoots is directly related to plant yield. The objective of this study was to evaluate the vegetative and yield performance of Coffea canephora, from the botanical variety Conilon, under different management systems for early emission of orthotropic shoots. The experiment was carried out in Ouro Preto do Oeste, Rondonia, between November 2011 and May 2014. Three types of management techniques were tested for early induction of orthotropic shoots in coffee trees (T1 = bending of the orthotropic shoot; T2 = apical pruning of the orthotropic shoot, T3 = free growing). The experiment was conducted in a randomized block design with 20 replicates. Each block consisted of a clonal genotype (clone) from the Embrapa Coffee Breeding Program. The techniques pruning and apical pruning were efficient to induce early growth of orthotropic shoots in Coffea canephora, resulting in higher yield in the first commercial bean production.

Detection of Toxoplasma gondii in retail meat samples in Scotland

Toxoplasma gondii is a globally important zoonotic parasite ranked as one of the most significant causes of disease burden among the major foodborne pathogens. Consumption of undercooked meat is a well-known risk factor for infection so the aim of this study was to investigate the presence of T. gondii in meat samples from retail outlets in Scotland. In Sampling Period 1, 300 meat samples (39 beef, 21 chicken, 87 lamb, 71 pork and 82 venison) were purchased from butchers', farmers' markets, farm shops and supermarkets, and in Sampling Period 2, 67 pure venison samples only were purchased from farmers' markets, farm shops and supermarkets. DNA was extracted and screened for T. gondii using a quantitative PCR targeting the 529 bp repeat element, and any positive samples were genotyped using PCR-RFLP targeting 10 markers. Meat juice was screened for T. gondii antibodies using a commercial ELISA or modified agglutination assay. Toxoplasma gondii DNA was detected in 0/39 (0%) beef samples, 1/21 (4.8%) chicken samples, 6/87 (6.9%) lamb samples, 3/71 (4.2%) pork samples and 29/82 (35.4%; Sampling Period 1) and 19/67 (28.4%; Sampling Period 2) venison samples. Partial PCR-RFLP genotyping revealed both clonal and non-clonal genotypes. Antibodies to T. gondii were detected in the meat juice of 2/38 (5.3%) beef samples, 3/21 (14.3%) chicken samples, 14/85 (16.5%) lamb samples, 2/68 (2.9%) pork samples and 11/78 (14.1%; Sampling Period 1) and 8/50 (16%; Sampling Period 2) venison samples. This is the first study to report the presence of T. gondii in retail meat products in Scotland and has highlighted venison as a potentially high risk meat. Further work is required to determine viability of parasites in this particular meat product.

A hidden Markov modeling approach for identifying tumor subclones in next-generation sequencing studies.

Allele-specific copy number alteration (ASCNA) analysis is for identifying copy number abnormalities in tumor cells. Unlike normal cells, tumor cells are heterogeneous as a combination of dominant and minor subclones with distinct copy number profiles. Estimating the clonal proportion and identifying mainclone and subclone genotypes across the genome are important for understanding tumor progression. Several ASCNA tools have recently been developed, but they have been limited to the identification of subclone regions, and not the genotype of subclones. In this article, we propose subHMM, a hidden Markov model-based approach that estimates both subclone region and region-specific subclone genotype and clonal proportion. We specify a hidden state variable representing the conglomeration of clonal genotype and subclone status. We propose a two-step algorithm for parameter estimation, where in the first step, a standard hidden Markov model with this conglomerated state variable is fit. Then, in the second step, region-specific estimates of the clonal proportions are obtained by maximizing region-specific pseudo-likelihoods. We apply subHMM to study renal cell carcinoma datasets in The Cancer Genome Atlas. In addition, we conduct simulation studies that show the good performance of the proposed approach. The R source code is available online at https://dceg.cancer.gov/tools/analysis/subhmm. Expectation-Maximization algorithm; Forward-backward algorithm; Somatic copy number alteration; Tumor subclones.

Population structure and genetic diversity of Trichomonas vaginalis clinical isolates in Australia and Ghana

Population genetic studies of Trichomonas vaginalis have detected high genetic diversity associated with phenotypic differences in clinical presentations. In this study, microscopy and next generation-multi-locus sequence typing (NG-MLST) were used to identify and genetically characterise T. vaginalis isolates from patients in Australia and Ghana. Seventy-one polymorphic nucleotide sites, 36 different alleles, 48 sequence types, 24 of which were novel, were identified among 178 isolates, revealing a geneticallly diverse T. vaginalis population. Polymorphism was found at most loci, clustering genotypes into eight groups among both Australian and Ghanaian isolates, although there was some variation between countries. The number of alleles for each locus ranged from two to nine. Study results confirmed geographic expansion and diversity of the T. vaginalis population. Two-type populations in almost equal frequencies and a third unassigned group were identified in this study. Linkage disequilibrium was observed, suggesting T. vaginalis population is highly clonal. Multillocus disequilibrium was observed even when analysing clades separately, as well as widespread clonal genotypes, suggesting that there is no evidence of recent recombination. A more comprehensive study to assess the extent of genetic diversity and population structure of T. vaginalis and their potential impact on varied pathology observed among infected individuals is recommended.

Parentage analyses identify local dispersal events and sibling aggregations in a natural population of Millepora hydrocorals, a free‐spawning marine invertebrate

Dispersal is a critical process for the persistence and productivity of marine populations. For many reef species, there is increasing evidence that local demography and self-recruitment have major consequences on their genetic diversity and adaptation to environmental change. Yet empirical data of dispersal patterns in reef-building species remain scarce. Here, we document the first genetic estimates of self-recruitment and dispersal distances in a free-spawning marine invertebrate, the hydrocoral Millepora cf. platyphylla. Using twelve microsatellite markers, we gathered genotypic information from 3,160 georeferenced colonies collected over 27,000m2 of a single reef in three adjacent habitats in Moorea, French Polynesia; the mid slope, upper slope, and back reef. Although the adult population was predominantly clonal (85% were clones), our parentage analysis revealed a moderate self-recruitment rate with a minimum of 8% of sexual propagules produced locally. Assigned offspring often settled at <10m from their parents and dispersal events decrease with increasing geographic distance. There were no discrepancies between the dispersal distances of offspring assigned to parents belonging to clonal versus nonclonal genotypes. Interhabitat dispersal events via cross-reef transport were also detected for sexual and asexual propagules. Sibship analysis showed that full siblings recruit nearby on the reef (more than 40% settled at <30m), resulting in sibling aggregations. Our findings highlight the importance of self-recruitment together with clonality in stabilizing population dynamics, which may ultimately enhance local sustainability and resilience to disturbance.

RobustClone: a robust PCA method for tumor clone and evolution inference from single-cell sequencing data.

Single-cell sequencing (SCS) data provide unprecedented insights into intratumoral heterogeneity. With SCS, we can better characterize clonal genotypes and reconstruct phylogenetic relationships of tumor cells/clones. However, SCS data are often error-prone, making their computational analysis challenging. To infer the clonal evolution in tumor from the error-prone SCS data, we developed an efficient computational framework, termed RobustClone. It recovers the true genotypes of subclones based on the extended robust principal component analysis, a low-rank matrix decomposition method, and reconstructs the subclonal evolutionary tree. RobustClone is a model-free method, which can be applied to both single-cell single nucleotide variation (scSNV) and single-cell copy-number variation (scCNV) data. It is efficient and scalable to large-scale datasets. We conducted a set of systematic evaluations on simulated datasets and demonstrated that RobustClone outperforms state-of-the-art methods in large-scale data both in accuracy and efficiency. We further validated RobustClone on two scSNV and two scCNV datasets and demonstrated that RobustClone could recover genotype matrix and infer the subclonal evolution tree accurately under various scenarios. In particular, RobustClone revealed the spatial progression patterns of subclonal evolution on the large-scale 10X Genomics scCNV breast cancer dataset. RobustClone software is available at https://github.com/ucasdp/RobustClone. lwan@amss.ac.cn or maliang@ioz.ac.cn. Supplementary data are available at Bioinformatics online.

Production of cuttings and nutrient export by Coffea canéfora in different periods in the Southwestern Amazon

ABSTRACT Cutting is the main vegetative propagation method used for the production of Coffea canephora plantlets. In this method, parent plants are conducted in a specific field, called clonal garden, which must be managed to produce clonal cuttings. The objective of this study was to evaluate the production of cuttings and the export of nutrients by Coffea canephora in different periods in the Southwestern Amazon. The experiment was carried out in a 6 × 3 split-plot scheme (repeated measures in time), consisting of the combination of six clonal Coffea canephora genotypes (plot) and three cutting collection periods (subplots). The genotypes were: C-057, C-088, C-089, C-125, C-130 and C-160; and the evaluation sampling times were January/2017, May/2017 and September/2017. The applied design was a completely randomized design with four repetitions. The dry season, from May to September, promotes lower orthotropic stem dry mass accumulation by Coffea canephora, but allows obtaining a larger number of viable cuttings for the production of clonal plantlets. Nutrient accumulation by orthotropic stems for cutting production follows the following order: N &gt; K &gt; Ca &gt; Mg &gt; S &gt; P &gt; Mn &gt; Fe &gt; Zn &gt; Cu. Nutrient export by the cuttings follows a descending order: K &gt; N &gt; Ca &gt; Mg &gt; P &gt; S &gt; Mn &gt; Fe &gt; Zn &gt; Cu.

Power and pitfalls of computational methods for inferring clone phylogenies and mutation orders from bulk sequencing data

Tumors harbor extensive genetic heterogeneity in the form of distinct clone genotypes that arise over time and across different tissues and regions in cancer. Many computational methods produce clone phylogenies from population bulk sequencing data collected from multiple tumor samples from a patient. These clone phylogenies are used to infer mutation order and clone origins during tumor progression, rendering the selection of the appropriate clonal deconvolution method critical. Surprisingly, absolute and relative accuracies of these methods in correctly inferring clone phylogenies are yet to consistently assessed. Therefore, we evaluated the performance of seven computational methods. The accuracy of the reconstructed mutation order and inferred clone groupings varied extensively among methods. All the tested methods showed limited ability to identify ancestral clone sequences present in tumor samples correctly. The presence of copy number alterations, the occurrence of multiple seeding events among tumor sites during metastatic tumor evolution, and extensive intermixture of cancer cells among tumors hindered the detection of clones and the inference of clone phylogenies for all methods tested. Overall, CloneFinder, MACHINA, and LICHeE showed the highest overall accuracy, but none of the methods performed well for all simulated datasets. So, we present guidelines for selecting methods for data analysis.

Nickel and copper accumulate at low concentrations in cacao beans cotyledons and do not affect the health of chocolate consumers

Aim of study: Nickel (Ni) and Copper (Cu) are essential metals for the growth and development of plants. In view of the above, the aim of this work was to quantify and correlate Ni and Cu concentrations in the leaf and the parts of the fruit [pod husk, pulp, tegument (seed coating) and cotyledons] of clonal cacao genotype PH 16.Area of study: Cacao genotypes were collected from adult plants grown on farms located in three different climatic regions of southern Bahia, Brazil.Material and methods: Plant material was collected in four plots of twenty farms, located under different edaphic and topographic conditions. They were subjected to chemical analysis and later to statistical analyses.Main results: There was high variability of Ni and Cu concentrations in all evaluated plant materials. Leaf, pulp, and tegument were the plant materials that accumulated more Ni. On the other hand, the greatest accumulation of Cu occurred in the tegument and in the pod husk, while in the cotyledons there was little accumulation of these metals. The concentrations of Ni were influenced by the three climatic regions, a fact not observed for Cu, except at the leaf level. There was interdependence between the accumulation of Ni in the leaves and in the different parts of the fruit, a fact not observed for Cu.Research highlights: Since Ni and Cu accumulated in low concentrations in the cacao beans cotyledons, raw material for the manufacture of chocolate and other food products, these metallic elements do not affect the consumers' health.

Genotypic diversity and genotype identity of resident species drive community composition

Abstract Aims Species-rich plant communities are more resistant to invasions. In the past decade it was demonstrated that genetic variation also has many ecological effects. In our study we aimed to test whether the patterns of response to the genetic diversity of a resident species differ between colonizing species of different growth forms and whether the response is affected by soil nutrients. Methods We established experimental stands of a common grass, Festuca rubra, harbouring three levels of genetic diversity (1, 6 or 18 clonal genotypes, referred to as genotypic diversity) under two soil nutrient levels. In the fourth year after the stands were established, we sowed a mixture of four colonizers into the stands: a stoloniferous legume (Trifolium repens), a broad-leaf tussock grass (Anthoxanthum odoratum), a large-rosette forb (Plantago lanceolata) and a small-rosette forb (Campanula rotundifolia). We observed species establishment and growth over 3 years. We tested whether colonization success depended on genotypic diversity, specific Festuca genotypes, soil nutrients and colonizer growth form. Important Findings The colonization success and biomass of the colonizers were significantly affected by the genotypic diversity and the genotype identity of the resident clonal grass. The response, however, differed between the colonizers. The strongest response to the genotypic diversity of the resident species was observed in the tussock grass with a growth form and architecture similar to the resident species. The large-rosette species responded in early stages of growth whereas the stoloniferous legume did not respond at all. The intraspecific genotypic diversity and genotype identity of the resident species play an important role in the assembly of plant communities.