Identification and Characterization of Reduced Epicuticular Wax Mutants in Rice

AbstractEpicuticular wax forms the outermost protective barrier of the aerial surfaces of land plants, working in concert with other components of the plant cuticle to prevent uncontrolled loss of water and to provide protection against an array of external environmental stress. In this study, chemically-mutagenized populations of rice (Oryza sativa L.) derived from approximately 4 750 M2 families were screened for adhesion of water droplets resulting in a wet leaf/glossy (wlg) phenotype. Mutants were identified in 11 independently-derived M2 families. Scanning electron microscopy analysis confirmed the association of the wlg phenotype with changes in the epicuticular wax crystals of these plants. The phenotypes of five mutants (7-17A, 26.1, 524.2, 680.2, and 843.1) were confirmed to be the result of single recessive gene mutation. Evaluation of mutants from 3 (6-1A, 7-17A, and 11-39A) of 11 M2 families revealed significant reductions (> 50%) in surface wax content and increases in cuticle membrane permeability

Discovery of chemically induced mutations in rice by TILLING

BACKGROUND: Rice is both a food source for a majority of the world's population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic tools. While these can be powerful, a non-transgenic, unbiased targeted mutagenesis method that can generate a range of allele types would add considerably to the analysis of the rice genome. TILLING (Targeting Induced Local Lesions in Genomes), a general reverse genetic technique that combines traditional mutagenesis with high throughput methods for mutation discovery, is such a method. RESULTS: To apply TILLING to rice, we developed two mutagenized rice populations. One population was developed by treatment with the chemical mutagen ethyl methanesulphonate (EMS), and the other with a combination of sodium azide plus methyl-nitrosourea (Az-MNU). To find induced mutations, target regions of 0.7–1.5 kilobases were PCR amplified using gene specific primers labeled with fluorescent dyes. Heteroduplexes were formed through denaturation and annealing of PCR products, mismatches digested with a crude preparation of CEL I nuclease and cleaved fragments visualized using denaturing polyacrylamide gel electrophoresis. In 10 target genes screened, we identified 27 nucleotide changes in the EMS-treated population and 30 in the Az-MNU population. CONCLUSION: We estimate that the density of induced mutations is two- to threefold higher than previously reported rice populations (about 1/300 kb). By comparison to other plants used in public TILLING services, we conclude that the populations described here would be suitable for use in a large scale TILLING project

Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing

<p>Abstract</p> <p>Background</p> <p>The availability of low cost sequencing has spurred its application to discovery and typing of variation, including variation induced by mutagenesis. Mutation discovery is challenging as it requires a substantial amount of sequencing and analysis to detect very rare changes and distinguish them from noise. Also challenging are the cases when the organism of interest has not been sequenced or is highly divergent from the reference.</p> <p>Results</p> <p>We describe the development of a simple method for reduced representation sequencing. Input DNA was digested with a single restriction enzyme and ligated to Y adapters modified to contain a sequence barcode and to provide a compatible overhang for ligation. We demonstrated the efficiency of this method at SNP discovery using rice and arabidopsis. To test its suitability for the discovery of very rare SNP, one control and three mutagenized rice individuals (1, 5 and 10 mM sodium azide) were used to prepare genomic libraries for Illumina sequencers by ligating barcoded adapters to <it>NlaIII </it>restriction sites. For genome-dependent discovery 15-30 million of 80 base reads per individual were aligned to the reference sequence achieving individual sequencing coverage from 7 to 15×. We identified high-confidence base changes by comparing sequences across individuals and identified instances consistent with mutations, i.e. changes that were found in a single treated individual and were solely GC to AT transitions. For genome-independent discovery 70-mers were extracted from the sequence of the control individual and single-copy sequence was identified by comparing the 70-mers across samples to evaluate copy number and variation. This <it>de novo </it>"genome" was used to align the reads and identify mutations as above. Covering approximately 1/5 of the 380 Mb genome of rice we detected mutation densities ranging from 0.6 to 4 per Mb of diploid DNA depending on the mutagenic treatment.</p> <p>Conclusions</p> <p>The combination of a simple and cost-effective library construction method, with Illumina sequencing, and the use of a bioinformatic pipeline allows practical SNP discovery regardless of whether a genomic reference is available.</p

Wilson Loops, Geometric Transitions and Bubbling Calabi-Yau's

Motivated by recent developments in the AdS/CFT correspondence, we provide several alternative bulk descriptions of an arbitrary Wilson loop operator in Chern-Simons theory. Wilson loop operators in Chern-Simons theory can be given a description in terms of a configuration of branes or alternatively anti-branes in the resolved conifold geometry. The representation of the Wilson loop is encoded in the holonomy of the gauge field living on the dual brane configuration. By letting the branes undergo a new type of geometric transition, we argue that each Wilson loop operator can also be described by a bubbling Calabi-Yau geometry, whose topology encodes the representation of the Wilson loop. These Calabi-Yau manifolds provide a novel representation of knot invariants. For the unknot we confirm these identifications to all orders in the genus expansion.Comment: 26 pages; v.2 typos corrected, explanations clarified; v.3 typos corrected, reference adde

Efficient Genome-Wide Detection and Cataloging of EMS-Induced Mutations Using Exome Capture and Next-Generation Sequencing

Chemical mutagenesis efficiently generates phenotypic variation in otherwise homogeneous genetic backgrounds, enabling functional analysis of genes. Advances in mutation detection have brought the utility of induced mutant populations on par with those produced by insertional mutagenesis, but systematic cataloguing of mutations would further increase their utility. We examined the suitability of multiplexed global exome capture and sequencing coupled with custom-developed bioinformatics tools to identify mutations in well-characterized mutant populations of rice (Oryza sativa) and wheat (Triticum aestivum). In rice, we identified ∼18,000 induced mutations from 72 independent M2 individuals. Functional evaluation indicated the recovery of potentially deleterious mutations for >2600 genes. We further observed that specific sequence and cytosine methylation patterns surrounding the targeted guanine residues strongly affect their probability to be alkylated by ethyl methanesulfonate. Application of these methods to six independent M2 lines of tetraploid wheat demonstrated that our bioinformatics pipeline is applicable to polyploids. In conclusion, we provide a method for developing large-scale induced mutation resources with relatively small investments that is applicable to resource-poor organisms. Furthermore, our results demonstrate that large libraries of sequenced mutations can be readily generated, providing enhanced opportunities to study gene function and assess the effect of sequence and chromatin context on mutations

Explicitly modelling microtopography in permafrost landscapes in a land surface model (JULES vn5.4_microtopography)

Microtopography can be a key driver of heterogeneity in the ground thermal and hydrological regime of permafrost landscapes. In turn, this heterogeneity can influence plant communities, methane fluxes, and the initiation of abrupt thaw processes. Here we have implemented a two-tile representation of microtopography in JULES (the Joint UK Land Environment Simulator), where tiles are representative of repeating patterns of elevation difference. Tiles are coupled by lateral flows of water, heat, and redistribution of snow, and a surface water store is added to represent ponding. Simulations are performed of two Siberian polygon sites, (Samoylov and Kytalyk) and two Scandinavian palsa sites (Stordalen and Iškoras). The model represents the observed differences between greater snow depth in hollows vs. raised areas well. The model also improves soil moisture for hollows vs. the non-tiled configuration (“standard JULES”) though the raised tile remains drier than observed. The modelled differences in snow depths and soil moisture between tiles result in the lower tile soil temperatures being warmer for palsa sites, as in reality. However, when comparing the soil temperatures for July at 20 cm depth, the difference in temperature between tiles, or “temperature splitting”, is smaller than observed (3.2 vs. 5.5 ∘C). Polygons display small (0.2 ∘C) to zero temperature splitting, in agreement with observations. Consequently, methane fluxes are near identical (+0 % to 9 %) to those for standard JULES for polygons, although they can be greater than standard JULES for palsa sites (+10 % to 49 %). Through a sensitivity analysis we quantify the relative importance of model processes with respect to soil moisture and temperatures, identifying which parameters result in the greatest uncertainty in modelled temperature. Varying the palsa elevation between 0.5 and 3 m has little effect on modelled soil temperatures, showing that using only two tiles can still be a valid representation of sites with a range of palsa elevations. Mire saturation is heavily dependent on landscape-scale drainage. Lateral conductive fluxes, while small, reduce the temperature splitting by ∼ 1 ∘C and correspond to the order of observed lateral degradation rates in peat plateau regions, indicating possible application in an area-based thaw model

Discriminatory performance of adiponectin and leptin in the identification of impaired glucose tolerance:The Guangzhou Biobank Cohort Study - Cardiovascular Disease Subcohort

BackgroundTo evaluate the additional discriminatory performance of adiponectin, leptin, and their ratio in the identification of impaired glucose tolerance (IGT) in men and women without diabetes on top of conventional risk factors.Methods & resultsA total of 698 subjects underwent an oral glucose tolerance test (oGTT) and adipocytokine measurements. A comprehensive stepwise selection procedure was performed, followed by c-statistics and integrated discrimination improvement (IDI) analysis. In males, adiponectin levels were significantly lower in the IGT group compared to the non-IGT group (Whitney U test, p ConclusionsIn Chinese individuals without diabetes, no significant evidence for the potential discriminatory value of adiponectin, leptin or their ratio in the identification of IGT on top of conventional risk factors was observed

Nursing heroism in the 21st Century'

Abstract Background The Vivian Bullwinkel Oration honours the life and work of an extraordinary nurse. Given her story and that of her World War II colleagues, the topic of nursing heroism in the 21st century could not be more germane. Discussion Is heroism a legitimate part of nursing, or are nurses simply 'just doing their job' even when facing extreme personal danger? In this paper I explore the place and relevance of heroism in contemporary nursing. I propose that nursing heroism deserves a broader appreciation and that within the term lie many hidden, 'unsung' or 'unrecorded' heroisms. I also challenge the critiques of heroism that would condemn it as part of a 'militarisation' of nursing. Finally, I argue that nursing needs to be more open in celebrating our heroes and the transformative power of nursing achievements. Summary The language of heroism may sound quaint by 21st Century standards but nursing heroism is alive and well in the best of our contemporary nursing ethos and practice.</p

Operational Earthquake Forecasting: State of Knowledge and Guidelines for Utilization

Following the 2009 L'Aquila earthquake, the Dipartimento della Protezione Civile Italiana (DPC), appointed an International Commission on Earthquake Forecasting for Civil Protection (ICEF) to report on the current state of knowledge of short-term prediction and forecasting of tectonic earthquakes and indicate guidelines for utilization of possible forerunners of large earthquakes to drive civil protection actions, including the use of probabilistic seismic hazard analysis in the wake of a large earthquake. The ICEF reviewed research on earthquake prediction and forecasting, drawing from developments in seismically active regions worldwide. A prediction is defined as a deterministic statement that a future earthquake will or will not occur in a particular geographic region, time window, and magnitude range, whereas a forecast gives a probability (greater than zero but less than one) that such an event will occur. Earthquake predictability, the degree to which the future occurrence of earthquakes can be determined from the observable behavior of earthquake systems, is poorly understood. This lack of understanding is reflected in the inability to reliably predict large earthquakes in seismically active regions on short time scales. Most proposed prediction methods rely on the concept of a diagnostic precursor; i.e., some kind of signal observable before earthquakes that indicates with high probability the location, time, and magnitude of an impending event. Precursor methods reviewed here include changes in strain rates, seismic wave speeds, and electrical conductivity; variations of radon concentrations in groundwater, soil, and air; fluctuations in groundwater levels; electromagnetic variations near and above Earth's surface; thermal anomalies; anomalous animal behavior; and seismicity patterns. The search for diagnostic precursors has not yet produced a successful short-term prediction scheme. Therefore, this report focuses on operational earthquake forecasting as the principle means for gathering and disseminating authoritative information about time-dependent seismic hazards to help communities prepare for potentially destructive earthquakes. On short time scales of days and weeks, earthquake sequences show clustering in space and time, as indicated by the aftershocks triggered by large events. Statistical descriptions of clustering explain many features observed in seismicity catalogs, and they can be used to construct forecasts that indicate how earthquake probabilities change over the short term. Properly applied, short-term forecasts have operational utility; for example, in anticipating aftershocks that follow large earthquakes. Although the value of long-term forecasts for ensuring seismic safety is clear, the interpretation of short-term forecasts is problematic, because earthquake probabilities may vary over orders of magnitude but typically remain low in an absolute sense (&amp;lt; 1% per day). Translating such low-probability forecasts into effective decision-making is a difficult challenge. Reports on the current utilization operational forecasting in earthquake risk management were compiled for six countries with high seismic risk: China, Greece, Italy, Japan, Russia, United States. Long-term models are currently the most important forecasting tools for civil protection against earthquake damage, because they guide earthquake safety provisions of building codes, performance-based seismic design, and other risk-reducing engineering practices, such as retrofitting to correct design flaws in older buildings. Short-term forecasting of aftershocks is practiced by several countries among those surveyed, but operational earthquake forecasting has not been fully implemented (i.e., regularly updated and on a national scale) in any of them. Based on the experience accumulated in seismically active regions, the ICEF has provided to DPC a set of recommendations on the utilization of operational forecasting in Italy, which may also be useful in other countries. The public should be provided with open sources of information about the short-term probabilities of future earthquakes that are authoritative, scientific, consistent, and timely. Advisories should be based on operationally qualified, regularly updated seismicity forecasting systems that have been rigorously reviewed and updated by experts in the creation, delivery, and utility of earthquake information. The quality of all operational models should be evaluated for reliability and skill by retrospective testing, and they should be under continuous prospective testing against established long-term forecasts and alternative time-dependent models. Alert procedures should be standardized to facilitate decisions at different levels of government and among the public. Earthquake probability thresholds should be established to guide alert levels based on objective analysis of costs and benefits, as well as the less tangible aspects of value-of-information, such as gains in psychological preparedness and resilience. The principles of effective public communication established by social science research should be applied to the delivery of seismic hazard information

Elliptic flow from two- and four-particle correlations in Au + Au collisions at sqrt{s_{NN}} = 130 GeV

Elliptic flow holds much promise for studying the early-time thermalization attained in ultrarelativistic nuclear collisions. Flow measurements also provide a means of distinguishing between hydrodynamic models and calculations which approach the low density (dilute gas) limit. Among the effects that can complicate the interpretation of elliptic flow measurements are azimuthal correlations that are unrelated to the reaction plane (non-flow correlations). Using data for Au + Au collisions at sqrt{s_{NN}} = 130 GeV from the STAR TPC, it is found that four-particle correlation analyses can reliably separate flow and non-flow correlation signals. The latter account for on average about 15% of the observed second-harmonic azimuthal correlation, with the largest relative contribution for the most peripheral and the most central collisions. The results are also corrected for the effect of flow variations within centrality bins. This effect is negligible for all but the most central bin, where the correction to the elliptic flow is about a factor of two. A simple new method for two-particle flow analysis based on scalar products is described. An analysis based on the distribution of the magnitude of the flow vector is also described.Comment: minor text change