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Exome resequencing and GWAS for growth, ecophysiology, and chemical and metabolomic composition of wood of Populus trichocarpa
Fernando P. Guerra, Haktan Suren, Jason Holliday, James H. Richards, and 7 more
https://doi.org/10.21203/rs.2.9589/v2
published 20 Nov, 2019
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Background: Populus trichocarpa is an important forest tree species for the generation of lignocellulosic ethanol. Understanding the genomic basis of biomass production and chemical composition of wood is fundamental in supporting genetic improvement programs. Considerable variation has been observed in this species for complex traits related to growth, phenology, ecophysiology and wood chemistry. Those traits are influenced by both polygenic control and environmental effects, and their genome architecture and regulation are only partially understood. Genome wide association studies (GWAS) represent an approach to advance that aim using thousands of single nucleotide polymorphisms (SNPs). Genotyping using exome capture methodologies represent an efficient approach to identify specific functional regions of genomes underlying phenotypic variation. Results: We identified 813K SNPs, which were utilized for genotyping 461 P. trichocarpa clones, representing 101 provenances collected from Oregon and Washington, and established in California. A GWAS performed on 20 traits, considering single SNP-marker tests identified a variable number of significant SNPs (p-value < 6.1479E-8) in association with diameter, height, leaf carbon and nitrogen contents, and δ 15 N. The number of significant SNPs ranged from 2 to 220 per trait. Additionally, multiple-marker analyses by sliding-windows tests detected between 6 and 192 significant windows for the analyzed traits. The significant SNPs resided within genes that encode proteins belonging to different functional classes as such protein synthesis, energy/metabolism and DNA/RNA metabolism, among others. Conclusion: SNP-markers within genes associated with traits of importance for biomass production were detected. They contribute to characterize the genomic architecture of P. trichocarpa biomass required to support the development and application of marker breeding technologies.
Epigenetics & Genomics
sequence capture
wood metabolome
Populus species and their hybrids are suitable feedstocks for second-generation biofuel production due to their rapid growth rates and favorable cell wall chemistry [1, 2]. In particular, the model species Populus trichocarpa Torr. & A. Gray (black cottonwood), native to western North America, has been used in breeding for generating commercial cultivars [3]. Biomass yield and chemical quality of P. trichocarpa cultivars, as well as their improvement, depend on multiple biological and environmental factors [4]. Considerable phenotypic and genetic variation has been observed in P. trichocarpa for complex traits related to growth, phenology, morphology, ecophysiology and wood chemistry [5-10]. These phenotypes include diameter and height [11, 12], bud set and flush [6, 13, 14], leaf morphology [15], water-use efficiency (WUE) [16, 17], secondary xylem composition [18] and wood metabolome [5]. This sort of traits has been also correlated with environmental variables such as latitude, daylength and temperature [5, 6, 14-16, 19].
Association analyses based on SNPs have been applied in recent years to identify polymorphisms controlling variation in complex traits of interest for biofuel production in Populus species [9, 15, 18-21]. Different approaches (candidate gene or GWAS) as well as genotyping platforms have been used, with single SNP-markers accounting for, in general, a low percentage of the phenotypic variation (1-8 %) in studied traits. These results support the polygenic nature and complexity of inheritance patterns and justifies increasing efforts to elucidate the genomic basis controlling those phenotypes.
Among "next-generation" sequencing alternatives, genome complexity reduction by sequence capture, or targeted sequencing, represents an efficient approach to performing genome wide analysis [22]. This method restricts attention only to specific genome regions (both genic and intergenic) of interest for molecular breeding as well as investigations into the diversity, population structure and demographic history of unstructured natural populations among others [23]. This approach has advantage of being quick, simple, and requires relatively small amount of input DNA [24]. Furthermore, compared with alternatives such as whole genome sequencing, it is reduced in terms of non-pertinent repetitive sequences, allows multiplexing of more samples for a given sequencing space, identifies functional molecular markers, provides high coverage for identification of low frequency sequence variants, and can circumvent problems arising from the presence of paralogous genes derived from duplication or polyploidization events [24]. This is particularly important for Populus species, which have experienced a whole-genome duplication event [25]. It was demonstrated by the application of an exome capture approach for analyzing the genomic architecture of clinal variation in P. trichocarpa [26].
In the present study, we employ sequence capture for genotyping and performing a GWAS in a P. trichocarpa population of 461 clones from 101 provenances collected from the Pacific Northwest (Oregon and Washington) in the United States. In an previous study [5], representatives of these clones were established in a clonal trial in California and characterized, both by traditional field measurements and high-throughput phenotyping, in describing a suite of traits involved in biomass production and wood chemical composition. Now, we coupled these phenotypic measures with specific exome capture-based genotyping to identify SNPs underlying observed trait variation. The association population was generated with germplasm collected from the southern part of the P. trichocarpa range in North America, and it was established and evaluated (at age two) in a trial located significantly to the south than that range. That represents a particular environmental/experimental condition, useful to determine, for example, the effects of geographic relocation on the P. trichocarpa performance. Understanding genetic variation at a genome-wide scale is fundamental for developing genome-based breeding technologies suitable for supporting the development of genetically improved plantations for bioethanol production.
Association population, growth conditions and phenotypic characterization
The association population was comprised of a set of 461 P. trichocarpa clones. These represented 101 provenances, within 14 river systems located west of the Cascade Mountains in Oregon and Washington between 48°54' N latitude (Nooksack River, Whatcom County, Washington) and 43°47' N latitude (Middle Fork, Willamette River, Lane County, Oregon) collected by GreenWood Resources [18]. A clonal trial was established at the University of California, Davis, California (38°32'42" N, 121°47'42" W) for phenotypic measurements and sample collection. The experiment was located on a silt loam soil (Entisol, Yolo series). Plants were produced from rooted containerized cuttings and established in April 2009, in an array of 1.83 x 1.83 m, following a randomized block design, with three blocks and one ramet per clone per block. Blocks were considered to control a north–south variation in the trial, associated with non-homogenous soil conditions. From 2009 to 2011, plants were irrigated once a week from June to September each year. No fertilizer was applied during the study.
The clonal trial was already characterized by Guerra et al. [5], in terms of traits dealing with growth, spring bud phenology, ecophysiology, and the chemical composition and metabolome of wood. Table 1 summarizes main statistics and clonal repeatability (heritability) estimates for those traits. A brief description about their measurements is indicated in the following subsections. Individual broad-sense heritability (H2i ) was estimated using formula (1) [5]: (See Formula 1 in the Supplementary Files)
where σ2g , σ2c , σ2fxb and σ2e represent the variance due the genetic cluster, clone within cluster, cluster x block and residual, respectively, and σ2ee' is the covariance between residuals of the same clone in two blocks.
Data used in the phenotypic characterization is available at the TreeGenes platform (http://dendrome.ucdavis.edu/treegenes/) under the accession number TGDR050.
Diameter, height, volume and spring-bud phenology measurements
Three growth parameters (diameter at breast height [DBH], total height [h] and volume index [Vol]), at age two, were measured in October 2010, as described by Guerra et al. [5]. In particular, Vol was estimated as Vol= π (DBH/ 2)2× h [13]. Additionally, days to bud flush (DBF), were recorded every three days from March to April 2011, as indicated previously [5].
Chemical composition and metabolome of wood
Wood cores collected from tree stems (0.3 m above ground level), at age three, in September 2011, were utilized for analyzing the chemical composition and metabolome of wood [5]. The content of 5 and 6-carbon sugars and lignin, as well as the syringyl:guaiacyl monolignol ratio (S:G) were determined from wood cores by high-throughput pyrolysis molecular beam mass spectrometry (pyMBMS), at the National Renewable Energy Laboratory (Golden, CO, USA). Simultaneously, wood metabolites were quantified from another set of wood cores by gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS), at the West Coast Metabolomics Center, at UCDavis. Methodological details have been described elsewhere [5]. For association analysis, five metabolites were selected. These corresponded to those with the highest estimates of heritabilities according to Guerra et al. [5]: 4-hydroxybenzoic acid (HbA; ), galactinol (Gal; ), adenosine (Ade; ), galactonic acid (GAc; ), and alpha tocopherol (Toc; ). These estimates involved a significant genetic variation across the analyzed genotypes, indicating their importance on wood composition variation. They were selected to maximize the power for detecting significant associations.
Ecophysiology traits
Morphological and ecophysiological characteristics are determinants of biomass productivity [4]. For that reason, independent leaf samples were obtained from the top of each tree and used for stable isotope analyses and leaf area estimation, respectively. This sort of measurements was carried considering their relationship with tree physiology and they are easy to collect (compared to physiological traits). Sampled leaves collected in August 2011 were processed to determine carbon (C) and nitrogen (N) concentrations and C and N stable isotope compositions by continuous flow isotope ratio mass spectrometry, at the UCDavis Stable Isotope Facility, as described elsewhere [5]. In this stage, the 461 clones were sampled. Additionally, a complimentary second subset of leaves, collected in August 2012, was utilized for measuring leaf area and dry biomass to estimate the specific leaf area (SLA) and N content per SLA ratio (NArea), according to Easlon et al. [27]. In this case, the subset of leaves was obtained from 177 clones, including three replicates per clone (one per block). These clones were chosen including those with extreme geographical origins and minimal and maximal volume indices. Thus, from these measurements, a set of variables were generated, including: C and N content, C:N ratio, carbon isotope discrimination (Δ), δ15N, SLA, and NArea.
DNA isolation
Additional young leaves were collected for DNA isolation. Circle punches were obtained and deposited in plastic vials, along with silica gel packets, until desiccation. This sort of leaf samples is compatible with automated grinding and pipetting systems. DNA isolation was performed using the Qiagen DNeasyPlant Mini Kit (Qiagen, Inc, Valencia, California, USA), according manufacturer's instructions. Quality was assessed with spectrophotometer (NanoDrop, Thermo Scientific). Acceptable extractions had a 260/280 ratio of 1.7-2.0 with a minimum concentration of 20 ng/μl.
Phytozome version 7.0 annotation and assembly files for P. trichocarpa (corresponding to assembly version 2.0 of the black cottonwood genome) were used to design oligonucleotide baits, complementary to short genomic regions that targeted exons, promoter, and intergenic control regions, as described by Zhou et al.[23]. A total of 230,720 baits of 120 bp were designed using SureSelect eArray software (Agilent Technologies, Santa Clara, California, USA). The baits targeted more than 39,000 of the 40,668 annotated protein-coding transcripts of the P. trichocarpa genome. As the cumulative length of the predicted exons exceeded the available baits, following bait design in eArray, we looped through the gene list, selecting one bait for each gene at each pass, until the maximum number of baits (i.e. 230,720) was reached. In addition to exons, baits were included in the design for genes with an annotated 5'-UTR targeting the 240 bp upstream, as well as 1000 baits targeting intergenic regions to be used as selectively neutral control regions. These control regions were selected at random from non-repetitive intergenic intervals at least 1000 bp from any gene model. This strategy of bait design has demonstrated previously that capture efficiency has not been significantly impacted by the presence of paralogous genes [25]. After design, a custom biotinylated RNA bait library was synthesized. Library preparation and target enrichment were performed following the Agilent SureSelectXT protocol (Version B). Briefly, 3.0 μg of poplar genomic DNA was sheared on a ultrasonicator (Covaris S220) at the Virginia Bioinformatics Institute (VBI), followed by end repair, 3'-end adenylation, adaptor ligation, and amplification. Agencourt AMPure XP beads were used to purify the libraries following each step, and library quality was assessed using an Agilent Bioanalyzer 2100 instrument, with Agilent DNA 1000 chips. Samples were randomly assigned one of 96 available index sequences and subsequently randomly assigned to groups of 16, each of which corresponding to a HiSeq sequencing lane. The prepared libraries were hybridized to the RNA baits in solution at 65 °C in an Eppendorf Mastercycler PCR machine (Eppendorf, Hamburg, Germany), and subsequently purified on magnetic beads. The multiplexed libraries were sequenced using an Illumina HiSeq 2500 System in a 2x100 paired-end format at VBI. Sequences of the prepared libraries are available at the Sequence Read Archive (http://www. ncbi.nlm.nih.gov/Traces/sra/) under the accession number SRA058855.
Data analysis and SNP calling
Short reads from poplar samples were pre-filtered using a collection of scripts in Biopieces (https://github.com/maasha/biopieces; version 2.0). First, interleaved pair-ended sequences were filtered based on the Illumina filter flag, and subsequently trimmed of adapters and bases with quality < 35. Following trimming, very short reads were eliminated (length < 35) to prevent ambiguous alignments. Lastly, reads having poor local quality scores (score < 25, window size = 5) were removed from the analysis. The short reads were aligned to the Populus trichocarpa (version 3.0) reference genome with the Burrows-Wheeler Aligner mem algorithm [28]. Resulting alignment files (in Sequence Alignment/Map, SAM, format) were converted via SAMtools v3.1 [29] to their binary versions (Binary Alignment/Map, BAM) for variant calling. Prior to SNP calling, duplicate reads were identified and removed using Picard software (version 2.6; https://broadinstitute.github.io/picard/command-line-overview.html) and the Genome Analysis Toolkit v3.x (GATK; https://software.broadinstitute.org/gatk/), with MarkDuplicates and DuplicateReadFilter functions, respectively [30]. Indels were realigned using the GATK IndelRealigner function. The HaplotypeCaller algorithm of GATK was then used to call SNPs (options: min_base_quality_score > 9, and standard_min_confidence_threshold_for_calling > 29) [31]. Variant calling was performed on individual chromosomes and scaffolds to reduce the run time. After merging all variant calling format (VCF) files, the VariantsToTable tool of GATK was used to produce SNP tables for downstream analysis. SNPs with a minor allele frequency < 5% and departure from Hardy-Weinberg equilibrium were excluded from the analyses. Similarly, SNPs that were missing data across more than 5% individuals (clones) were also removed from following stages. After these filtering steps, from 5.1 million identified SNPs, a final set comprised of 813,280 SNPs was utilized for GWAS.
GWAS analyses
The distributions of the different traits were checked for departures from normality. Logarithmic transformations were applied to normalize the variables Vol, DBF, C and N concentration, C/N ratio, SLA, NArea, lignin, C5-sugars and C6-sugars. Outlier observations were excluded from tests. Clonal means were adjusted by Best Linear Unbiased Predictor, using Proc MIXED in the software SAS v9.2 (SAS Institute, Cary, NC, USA), in order to correct a significant block effect observed across the trial area. Prediction model included the factors clone and block, considered as random and fixed effects, respectively. The GWAS was based on Mixed Linear Models (MLM), implemented in the software GCTA v1.25 [32] (http://cnsgenomics.com/software/gcta/index.html). SNP data were first converted to PLINK format using TASSEL v3.0 [33] (http://www.maizegenetics.net/tassel), and then converted to binary PLINK (bed file) using PLINK v1.9 command line tool [34]. The genetic relatedness (kinship) was determined by the genetic relationship matrix (GRM) option at the GCTA's GRM module, based on identical-by-descent estimates [35]. The population structure matrix (Q) was estimated by a classical multidimensional scaling [36], also called Principal Coordinates Analysis, using the cmdscale function in the package Stats in R [37]. With the marker (M), kinship (K), population structure (Q) and predictor variables, we ran an "M+K+Q" linear mixed model to identify significant associations with traits. Marker and Q were assumed fixed effects, whereas K represented a random effect. The goodness of fit including (or not) Q was assessed for each trait by the Bayesian information criterion (BIC), utilizing the package Stats in R [37]. Associations were adjusted for multiple testing, using the Bonferroni correction, in the package Stats in R [37]. The threshold for p-values was 6.1479E-8 at significant level of 5% after Bonferroni correction (0.05/813,280). We additionally used the Random Forest method to estimate the percentage variance explained by the top SNPs for each trait (SNP contribution), by using the randomForest package in R [38]. The SNP contribution standard deviation (SCSD) was also calculated by the same package. We set the number of trees (ntree) grown as 2,000 and averaged 50 repeats per trait in our estimates.
Multiple-SNP testing for each trait was carried out using an overlapping sliding-window analysis on association results, based on the number of the significant associations (p-value < 0.00001) for 10 k window size with 1 k slide, using a custom script. Empirical p-values for each slide were estimated assuming the significant slides follow a Poisson distribution [39]. To this end, Poisson tests were carried out by comparing the rate of total significant SNPs and the total number of SNPs with the similar rate for the tested window using the probability formula below. (See Formula 2 in the Supplementary Files)
Where lambda (ℷ) is the average number of significant SNPs, e is the constant Euler's number and k is the average number of significant SNPs for the window being tested. Empirical p-values were recorded for each window per trait and then were adjusted by Bonferroni (0.05/number of tested windows) and reported for each trait. Finally, p-values were visualized using a Manhattan plot, highlighting only the significant slides.
Linkage disequilibrium
Linkage disequilibrium (LD) was estimated among pairwise combinations of SNPs per chromosome. It was expressed in terms of the squared correlation of allele frequencies r2. The r2 value between pairs of SNP markers, within each chromosome, was estimated using TASSEL 5.2 [33], utilizing the option of sliding window (120 SNPs per window). To assess the extent of LD, the decay of LD within physical distance (base pairs) between SNPs, within each chromosome, was evaluated by nonlinear regression analysis of r2 values [40]. Analysis was performed applying the NLIN procedure in SAS 9.4.
Gene models, annotations and expression data
Gene models and gene ontologies were obtained from the Phytozome platform version 12 (Populus trichocarpa v 3.0) and Quick GO site (http://www.ebi.ac.uk/QuickGO-Beta/), respectively.
We used GWAS to identify DNA polymorphisms associated with biomass production and wood chemical composition in P. trichocarpa, which determine its potential as feedstock for lignocellulosic ethanol. This approach complements our previous phenotypic characterization of the same association population [5] by identifying SNPs underlying traits of growth, ecophysiology and wood quality, the primary traits targeted for the development of genetically improved clones suitable for dedicated biomass and bioenergy plantations. An approach based on sequence capture allowed us to detect genotype-phenotype associations across the P. trichocarpa gene exome.
The association population used in this study consisted of 461 clones (from 101 provenances), comprising part of the natural distribution range of P. trichocarpa in the Pacific Northwest of the United States. In a previous study [5], we observed significant phenotypic and genetic variation for growth, spring bud phenology, water use efficiency, C and N assimilation, as well as lignocellulosic components and metabolome of wood (Table 1). Similarly, clonal repeatability, represented in terms of individual heritability estimates, also varied among the traits. We hypothesized from this information that multiple polymorphic loci across the genome should be detected in association with phenotypes, and particularly, those with high heritability should reveal a large number of significant SNP-markers.
The processes of exome sequencing and genotyping identified 5.1 million SNPs across the P. trichocarpa genome in the association population, and after filtering, a set of 813,280 SNPs was used for association analyses (Table 2). The number of selected SNPs was proportional to chromosome size, ranging from 29,287 to 100,299 SNPs, for chromosomes 9 and 1, respectively (Table 2, Fig. 1a). Considering the full genome length, an average of one SNP every 482 bp (Table 2) was included in the analyses. Taking advantage of the full genome assembly, genotyping methodologies such as those based on sequence capture can target entire exons or genes across the genome, avoiding bias arising by a priori selection of candidate loci [23, 25]. In comparison to similar preceding studies that used SNP array platforms [6, 18, 19, 41], the number of SNPs in our analyses represent an increase in the power of applied genomic scanning. However, this amount is lower than the utilized by approaches based on whole-genome sequencing developed recently [7, 15].
Intra-chromosomal linkage disequilibrium
The extent of linkage disequilibrium (LD) was analyzed across each chromosome. On average, the LD over physical distance decayed below r2 0.2 at 26.9 kbp. A representative example, for Chromosome 12, is depicted in Fig. 1 b. The complete set of chromosomes with its LD is included in Fig. S1. The decay varied depending on specific chromosomes, with the most rapid decay observed on chromosomes 7 and 15 (r2 0.2 at 18.9 kbp) and the slowest decay on chromosome 11 (r2 0.2 at 51.6 kbp). Genome-wide LD decay exhibited different extents among chromosomes (Table 2). LD decay to r2<0.2 was observed on average at 26.9 kbp. High variation of LD across the genome (among and within chromosomes) has been reported for this species [23]. The estimated extent of LD decay predicted in our study is higher than the observed by Wegrzyn et al. [18] (r2 0.2 at ~0.5 kbp) and Wang et al.[42] (r2 0.2 at ~8 kbp) for P. trichocarpa. Distinct methodologies, number of markers, population sizes, genetic origins and standard errors among the studies may account for the different findings. Compared with other tree species extent of LD estimated in this study is similar to species belonging to Fraxinus [43], Prunus [44] and Eucalyptus [45] genus.
Single SNP-marker associations
Significant associations (p-value < 6.1479E-8) were identified for DBH, h, leaf C and N content, and δ15N. Figure 2 (a and c) depicts the number of associations detected per chromosome for a selected set of traits. A detailed list for each trait is provided in Table S1. Similarly, Manhattan plots for each phenotype are included in Fig. S2.
In general, and consistently with chromosome length, the highest numbers of significant associations were observed for chromosomes 1 and 5. The lowest number of associations was observed for chromosome 16. The proportion of significant SNPs of the total analyzed, ranged from 0.02 ‰ to 0.50 ‰ for leaf C content on chromosome 10, along with δ15N on chromosomes 6 and 10, and leaf N content on chromosome 5 (Table S1b), respectively. In the case of growth traits, 2 and 148 associations were detected for DBH and h, respectively. Within the ecophysiological traits, the number of significant associations ranged from 12 to 220 for C content and leaf N-content, respectively. For traits related to the chemical composition of wood, associated SNP-markers were over the significance cutoff (p-value < 6.1479E-8). Similarly, in the case of wood metabolites, considering a selected subset of those with the top five highest heritability estimates, no significant associations meeting the adjusted p-value were identified for Adenosine (Ade), Hydroxybenzoic Acid (HbA), Galactinol (Gal), Galactonic Acid (GAc) and Alpha tocopherol (Toc). The proportion of phenotypic variation accounted for the cumulative effect of significantly associated SNPs was 0.2 %, 1.1 %, 0.1 %, 0.7 % and 0.7 % for DBH, h, leaf C content, leaf N content, and δ15N, respectively.
Significant single nucleotide polymorphisms associated with phenotype were identified mostly in exonic regions. SNPs are part of genes encoding proteins belonging to the functional classes: Protein Synthesis/Modification (54.5 %), DNA/RNA Metabolism (27.3 %), Energy/Metabolism (9.1 %) and Signal transduction (9.1 %) (Fig. 3a). A list with these SNPs and genes is given in Table S3. An example for the Protein Synthesis/Modification category was a gene encoding a Periodic Tryptophan Protein 1 (Potri.007G019500), which was associated with height, and leaf N and δ15N. Among genes related with proteins involved in DNA/RNA Metabolism, one for a helicase senataxin (without gene model in Phytozome) was significant for height and leaf N. For genes in the Energy/Metabolism functional class, a representative was one (Potri.015G119700) encoding a Domain of unknown function (PGG), which was associated with DBH. For the Signal transduction class, the gene encoding a Rop Guanine Nucleotide Exchange Factor 1 (Potri.009G140100) was significant for height and leaf N.
Considering the applied significance threshold with Bonferroni correction (p-value < 6.1479E-8), GWAS performed on single-SNPs was successful in identifying polymorphisms associated with growth traits (DBH and h), leaf C and N-contents, as well as stable isotope parameters (δ15N) (Fig 2, Table S1). For traits related to spring bud phenology (DBF), wood chemical components (C5 and C6 sugars, lignin) and wood metabolites (GAc, Gal and HbA) significant associations at p-value< 0.0001 were detected, but they did not reach the adjusted threshold. The presence or lack of significant SNPs for these traits appears to be independent of heritability estimates for each. For some traits with moderate to high H2i (e.g. S:G ratio or DBF), GWAS did not detect single-SNP associations. On the other hand, for traits with low to moderate H2i (e.g. leaf C-content and δ15N) a relatively higher number of SNPs were identified. Similar situations were observed for phenology traits in previous studies with P. trichocarpa [19]. On average for all traits with significant associations ~ 1 % of phenotypic variation was accounted for by the cumulative effect of significant SNPs. The influence of multiple SNPs associated with phenotypes is particularly interesting in the context of the development of models for genomic selection, where large numbers of markers are utilized to predict the genetic merit of individuals [46]. Differences among traits in terms of the number of significant SNP-markers suggest the differential effect of both the variable number of SNPs influencing each trait and the individual impact of some SNPs. In that sense, some individual SNPs could have a such low effect size that none reach statistical significance. Furthermore, the apparent lack of correspondence between estimates of H2i and the phenotypic variance collectively accounted for by SNPs, could be explained by non-additive effects (e.g epistasis, GxE effect) or epigenetic factors acting on some traits. These types of effects are usually underestimated because MLM utilized for GWAS only suppose additive interactions [19]. Finally, another factor influencing the number of significant associated SNPs (and their effect on phenotypes) deals with the complexity of analyzing thousands of single markers across the genome. Stringent thresholds for controlling type I error are required for p-value adjustment in GWAS, given the correlated nature of markers along a chromosome [47]. For example, it has been suggested that the general applicability of the traditional false discovery ratio (FDR) [48] may suffer from several problems when applied to association analysis of a single trait [49]. In that sense, we utilized the Bonferroni correction to define the significance threshold. Thus, in spite of significant associations were detected at p-value < 0.00001 (and even lesser) in traits such as Vol, DBF, lignin or GAc, they did not reach the adjusted p-value threshold and were considered non-significant.
Sliding window analyses
The multiple-marker analysis by sliding-window allowed us to identify genomic regions containing different sets of SNPs jointly associated with each trait. Figure 4a depicts a representative Manhattan plot with the significant windows identified for leaf δ15N. Manhattan plots for other traits are included in Fig. S3. A variable number of windows per chromosome were detected among the phenotypes (Fig. 2 b and d). The total number of significant windows ranged from 6 for HbA, to 192 for N content (Table S2). For most traits, the main contributions were observed by chromosomes 8 and 1. However, for traits such as DBF, C:N, δ15N, and Toc, the most relevant chromosomes in terms of the number of significant windows included to 6, 4, 5 and 10, respectively. The multiple-SNP approach applied by sliding window analysis has been proposed as a robust alternative for identifying clustered significant patterns of SNPs, that are associated with complex traits, in a chromosomal context in humans and plants [39, 50-52]. In our study, significant windows identified a series of SNP clusters which were coincident with coding regions of multiple genes (Table S4). The graphical relationship between SNPs identified by single-marker associations and the detection by sliding window analysis is depicted in Fig.4, where the highlighted window (Fig.4 a) contains 14 significant SNPs belonging to the XRN4 gene (Fig.4 b). Additionally, information coming from both detection approaches allowed us to define genome zones with high LD, significantly associated with phenotypic variation, revealing the presence of phenotypically-relevant haplotypes (Fig. 4c). Although more evidence will be necessary, haplotype blocks defined by this way could be indicative of polymorphic regions with pleiotropic effects.
Considering the top three most significant windows across all chromosomes and traits, the most represented functional classes were Protein with Unknown Function (21.2 %), Energy/Metabolism (21.1 %), Protein Synthesis/Modification (19.2 %), and Transcription (15.4 %) (Fig. 3b). A list with the windows and genes included in these classes are given in Table S4. Some of the detected genes encoding proteins with roles in Protein Synthesis/Modification were those expressing Similar to Threonyl-tRNA Synthetase (Potri.008G145600), Interleukin-1 Receptor-Associated Kinase 4 (Potri.008G145900) and Leucine Rich Repeat (LRR1) (Potri.005G015700) associated with wood C5-sugars, C6-sugars and height, respectively. An example of the genes dealing with proteins belonging to the Energy/Metabolism class were Exostosin Heparan Sulfate Glycosyltransferase-Related (Potri.010G197900) and Similar to Aldehyde Dehydrogenase 1 Precursor (Potri.012G078700), associated with δ15N and DBF, respectively. Among genes encoding enzymes involved in Transcription, Similar to Agamous-like MADS Box Protein AGL12 (Potri.019G076800) and WRKY Transcription Factor 10-related (Potri.013G086000), were associated with Gal and C5-sugars, respectively. Concerning the Protein Synthesis/Modification class, examples of identified genes are those encoding a Similar to Threonyl-tRNA synthetase and Leucine Rich Repeat (LRR_1)//Leucine rich repeat (LRR_8), associated with C5-sugars and height, respectively.
Genes detected by single-SNP association and sliding windows approaches
We also verified the consistency between SNP-markers identified by single-SNP association and sliding window analyses. To this end, we considered as an example the most significant window (#154; p-value 4.70E-25) detected in chromosome 5 for leaf δ15N (Fig. 4a). The SNPs identified within the window were part of a gene (Potri.005G048900) encoding a Similar to 5'-3' Exoribonuclease (XRN4) Gene, which is involved in disease resistance, response to ethylene, RNAi, and miRNA-mediated RNA decay [53]. The associated window comprised 64 SNPs (Fig. 4b). Fourteen of these markers, were also identified by the single-marker association, indicating the consistency between both approaches. Particularly, markers such as S05_3547832, S05_3547864, S05_3547904 and S05_3548573 were in high LD (r2 > 0.75) and produced significant variation at the level of leaf δ15N means (Fig. 4c). Alternatively, alleles for those SNPs represent intronic and non-synonymous polymorphisms, involving possible effects on transcript splicing, and protein structure and function.
Significant associations for traits underlying growth, nutrient metabolism and xylem formation, among others, define SNPs and genes, which might represent logical candidates for functional studies focused on confirming their role and impact on phenotype of Populus species. Considering their high significance and the simultaneous detection by the single-SNP association and/or sliding windows approaches, we centered part of our analysis on gene Similar to 5'-3' Exoribonuclease (XRN4), which included SNPs and windows associated with leaf δ15N. The exoribonuclease function of this gene links it to transcription, RNA metabolism and RNA interference in eukaryotes [54]. In plants, it has been related to ethylene signaling [55] and response of plants to abiotic and biotic stresses [56, 57]. Mutation of members of the XNR gene family produced sensitivity to N starvation in Saccharomyces cerevisiae [58] and morphological alterations in Arabidopsis thaliana [59, 60]. Thus, association of XRN4 with leaf δ15N, an indicator of N use efficiency [61], could be related to the N metabolism and mobilization at leaves, particularly during the last third of the growing season, when sampling was done. More studies will be required to detect possible effects of SNPs at XRN4 on photosynthesis and biomass production.
Chemical composition of wood
Association analyses of the chemical composition of P. trichocarpa wood was previously performed in the same population by our group [18] using a candidate gene approach. We carried out a comparison between the results from both studies considering the 40 candidate genes utilized by Wegrzyn et al. [18], which encodes enzymes from the cellulose and lignin biosynthesis pathways and cytoskeletal proteins. Association results in the present study were significant only under a p-value < 0.0001 threshold. Results indicated both overlap and divergence between the two studies (Fig. 5 and Table S5). SNPs within genes encoding cellulose synthase (CesA1A) were significantly associated with C6-sugars in both studies. For this trait, we also detected SNPs in TUA5 gene, which were not identified by Wegrzyn et al. [18]. In the case of lignin content, members of the cellulose synthase gene family (CesA2B and CesA1A) were differentially detected (differentially) in both studies. We also identified SNPs belonging to 4- Serine Hydroxymethyl Transferase SHMT6. Finally, for the S:G ratio, our analyses detected SNPs in Laccase LAC1A, Phenylalanine Ammonia-Lyase PAL5 genes, which were not identified by Wegrzyn et al. (2010). In spite of the genotypes and wood chemical characterization methods were mostly the same in both studies, distinct trial sites (the first in Westport, Oregon, and the second in Davis, California), sampling height or differential presence of juvenile wood, among other factors, might explain the differences in the findings reported previously [18] and those described in the present work.
Forest trees are an important source for multiple wood and non-wood products. Genetic improvement aimed to develop such products, including lignocellulosic biofuels, depends on the variation underlying commercial traits. This variation is characterized by a complex genetic control and the influence of environmental factors. In this study, we identified a series of DNA polymorphisms controlling the phenotypic variation of growth, nitrogen use and wood composition of P. trichocarpa clones at different levels. Our results thus provide a starting point to define candidate genes suitable for functional characterizations addressed to confirm their biological role. At the same time, the genome-wide scale applied for the association analyses revealed a large number of SNPs which could be utilized to develop genomic selection schemes. Further efforts to define the utility of SNP polymorphisms in generating genomic breeding values will illuminate the path to breeding programs that incorporate molecular markers for bioethanol production. The upshot will be the estimation of parental hybridization values and an earlier, more precise genotypic selection from segregating F1 hybrid populations, that together will increase the overall magnitude of the realized genetic gain per unit of time.
Ade: Adenosine; C:N: Leaf C:N ratio; C5: Wood 5-carbon sugars; C6: Wood 6-carbon sugars; DBF: Days to bud flush; DBH: Diameter; GAc: Galactonic acid; Gal: Galactinol; GWAS: Genome wide association study; h: Height; H2i: Individual broad-sense heritability; HbA: 4-Hydroxybenzoic acid; LD: Linkage disequilibrium; Narea: N content:SLA ratio; S:G: Wood syringil:guayacil ratio; SLA: Specific leaf area; SNP: Single Nucleotide Polymorphism; Toc: Alpha tocopherol; Vol: Volume index; Δ: carbon isotope discrimination.
Sequences of the prepared libraries for exome capture are available at the Sequence Read Archive (http://www. ncbi.nlm.nih.gov/Traces/sra/) under the accession number SRA058855. Data used in the phenotypic characterization is available at the TreeGenes platform (http://dendrome.ucdavis.edu/treegenes/) under the accession number TGDR050.
This study was funded by the Advanced Hardwood Biofuels Northwest Project, supported by Agriculture and Food Research Initiative Competitive (Grant no. 2011-68005-30407, USDA National Institute of Food and Agriculture) and by the National Science Foundation Plant Genome Research Program (IOS Grant no. 1054444 to JAH).
B.J.S., D.B.N and F.P.G. planned and designed the research. F.P.G and H.S. analysed data. D.B.N, F.P.G, J.H. and H.S. interpreted data. F.P.G, J.H.R and R.S. conducted fieldwork and data and sample collection. J.H.R, M.D., O.F., R.F. and R.S. processed and analyzed samples. B.J.S., D.B.N, F.P.G, J.H., J.H.R, H.S. and R.F. wrote the manuscript.
We would like to thank the California Agricultural Experiment Station for the provided support and two anonymous reviewers for their helpful comments on earlier version of the manuscript.
1Department of Plant Sciences, University of California at Davis, CA 95616, USA. 2 Department of Forest Resources and Environmental Conservation, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. 3 Department of Land, Air and Water Resources, University of California, Davis, CA 95616, USA. 4 Department of Molecular and Cellular Biology & Genome Center, University of California, Davis, CA 95616, USA. 5 Biological Research Group, GreenWood Resources, Portland, OR 97201, USA. 6 National Renewable Energy Laboratory, Golden, CO 80401, USA. 7Bioenergy Research Center, University of California at Davis, Davis, CA 95616, USA. 8Instituto de Ciencias Biológicas, Universidad de Talca, P.O. Box 747, Chile.
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Table 1. Summary statistics for traits studied in the Populus trichocarpa association population. Columns "Mean", "Std. Dev.", "C.V" and "" were extracted from Guerra et al. [5].
Std. Dev.
C.V. (%)
Diameter (DBH)
Height (h)
Volume index (Vol)
Days to bud flush (DBF)
Julian days
Ecophysiology
Leaf C content
% DW
Leaf N content
Leaf C:N ratio (C:N)
kg C/kg N
Leaf Δ
Leaf δ 15N
Specific leaf area (SLA)
m2/kg DW
N content : SLA ratio (NArea)
g N/m2
Wood chem. components
Wood 5-carbon sugars (C5)
Wood lignin
Wood syringil:guayacil ratio (S:G)
Wood metabolites
Galactonic acid (GAc)
R.A.a
Galactinol (Gal)
Alpha tocopherol (Toc)
Adenosine (Ade)
4-Hydroxybenzoic acid (HbA)
Table 2. Summary of amount of analyzed SNP markers and intrachromosomal LD decay across the Populus trichocarpa genome. Linkage disequilibrium decay is referred to the physical distance (kbp) where LD = 0.2
Chr.
Size (Mbp)
Analyzed SNPs
Frequency (bp/SNP)
LD Decaya (kbp)
Additional File Legends
Additional File 1: Table S1a. Number of significant single SNP-markers.
Additional File 1: Table S1b. Proportion of significant SNPs on total analyzed SNPs.
Additional File 1: Table S2. Number of significant sliding windows .
Additional File 1: Table S3. Top three significantly associated single SNPs.
Additional File 1: Table S4. Top three significantly associated SNP-windows.
Additional File 2: Table S5. SNPs belonging to genes encoding enzymes for lignin and cellulose biosynthesis pathways.
Additional File 3: Figure S1. Linkage disequilibrium decay per chromosome.
Additional File 4: Figure S2. Manhattan plots for assessed traits.
Additional File 5: Figure S3. Manhattan plots for sliding window analysis tests.
FigS4.pdf
Equation1.png
TableS5.pdf
SupplementaryTables.xlsx
supplement2.pdf
supplement8.jpg
supplement8.xlsx
Editorial decision: Accept


Hearst House Plan - The Hearst is classic Southern charmer with its gable roof and traditional dormers. Clapboard siding and brick accents are the correct materials for this design. The courtyard entry garage allows the Hearst design to be built on many diverse sites with various conditions. The main level is anchored by the vaulted great room with a fireplace an angled wall for an interesting look. The breakfast area is located just inside from the garage making it the perfect place to talk with family after arriving home from work or school. The master suite has a vaulted ceiling, spa bath and walk-in-closet. There are two additional bedrooms and a hall bath.


The sea around Lastovo is crystal clear, and because of its beauty attractive to many tourist.
Summer events on Lastovo are being organized from June until the end of September.
Lastovo Village is an old medieval town with a long and interesting history, surrounded by mountains and forming a natural shape of amphitheatre.
There are lot's of hidden bays that one can discover by renting a small boat.


Financial services companies need to improvise their service flexibility due to constant changing technologies, customer activism, cyber attacks, terrorist attacks, regulatory changes, and natural disasters like tsunami. We are providing an end-to-end Business Resilience services to address and prioritize the services from a regulatory and customer perspective. We also focus on simplifying business processes and building controls required to mitigate risks due to critical factors.


text:
Summer Fun Along the Louisiana Culinary Trails
While the summer sun is high, carve out a little time to enjoy the best of Louisiana's homegrown vegetables, cool desserts, fresh fish, and more.
By LouisianaTravel.com Staff
With year-around growing seasons, it's easy to find fresh produce at markets like this one in Opelousas.
Just a drop of the line in one of our abundant waterways and have fresh fish on the menu that evening.
Make these summer traditions your own:
Visit the farmers market. The slow food, buy local movement that is all the rage across the country is nothing new to us. We've long known that the best-tasting fruits and veggies come from Louisiana farms and farmers. If you haven't been to a farmers' market yet this year, there's still time - many, like the Opelousas Farmers Market on the Prairie Home Cooking trail, are open year-around. Plus, you'll find homegrown goodies at roadside stands, like the ones lining both sides of Bayou Lafourche along our Bayou Bounty Trail.
Enjoy a progressive dinner. Let's be honest: Sometimes it's just too hot to be in the kitchen. Instead, invite a few friends for an evening out...with a twist. Plan a progressive dinner for the group, with drinks and appetizers at one spot, entrées and sides at another, and dessert at yet another. In Natchitoches on the Red River Riches Trail, local resident Iris Harper loves suggesting a progressive dinner to visitors. "There are nine locally owned restaurants within a three- to four-block area in the historic district," she says. In Natchitoches, why not start with appetizers at The Landing Restaurant and Bar, where Harper recommends the blackened alligator appetizer.
Fish for your supper. There's a reason we're known as a Sportman's Paradise! Redfish, catfish, bluegill, bass, and more—we've got all kinds of fish and a wealth of fishing spots. Catch your limit then cook it up yourself. You can't beat the fresh taste and the pride in serving your catch at the table. If you're in the northern part of the state, cast in Toledo Bend in Sabine Parish or hook up with Homer Humphreys, a professional fisherman and guide who sure knows his way around Lake Bistineau and the Red River. In South Louisiana, catch redfish on the Seafood Sensation trail in Lake Charles or set sail in the Gulf—which is open for recreational fishing.
Here's a cooking tip from Jean Lafitte fishing guide Theophile Bourgeois: When you're grilling redfish, leave the scales on one side of a boneless filet. Rub the meat with Worcestershire sauce and Italian dressing. Add seasonings, including Louisiana Fish Fry Cajun seasoning, and throw the filet on the grill for about 15 minutes, scale side down. When the meat cracks, squirt with a bit of margarine and let it melt over the fish.
Cool off with an icebox pie. Ice cream may be summer's star dessert, but don't overlook the icebox pie—one sweet way to cool off on a hot day. You'll find the refreshing treats on menus across the state. In New Orleans, Clancy's serves up a lemon icebox pie that is among pastry chef and author David Guas' favorite desserts (he mentions it in his book DamGoodSweet, where he includes his own recipe). In Lecompte—the Pie Capitol of Louisiana— Lea's Lunchroom is the place to go for coconut, lemon, chocolate, banana, and more. In Shreveport, locals head to Strawn's for a yummy whipped-cream-topped slice of strawberry, chocolate, or coconut. Creola Café, in Grand Coteau, serves up a chocolaty, creamy Yum Yum pie, but we'd be remiss if we didn't suggest ordering their signature Creola Italian cream cake. This chilled dessert is made with light-as-air cake, filled with mascarpone (Italian cream cheese), dusted with powdered sugar and drizzled with chocolate.
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redpajama_set_name: RedPajamaCommonCrawl


IF Charles N'Zogbia had not asked for a few dollars more he could have helped give Birmingham the penetration they lacked in this goalless affair.
Instead, the Frenchman's silky skills allowed Wigan to collect a point as Blues equalled a club-record 18 straight home games unbeaten in the top flight.
N'Zogbia agreed to join Birmingham in the summer for £9million before demanding more wages at the 11th hour.
It may well be 'Fortress St Andrews' – they were last beaten there by Bolton a year ago – but McLeish would not have been happy with the draw.
Several decent chances went begging and the late dismissal of Craig Gardner for a high tackle on Franco Di Santo summed up a frustrating day for Blues.
"Having watched the video we won't be appealing," added the Scot.
The unpopular N'Zogbia almost silenced the boo boys in the 12th minute.
His trickery set up Hugo Rodallega but his goal-bound shot was blocked by Scott Dann. Birmingham then had keeper Ben Foster to thank three minutes later when his tip-over denied N'Zogbia himself.
Blues' lone striker Cameron Jerome missed a simple header at the far post after Seb Larsson did well to cross from the right.
The match became even more scrappy and Birmingham were desperate for a spark of Alexander Hleb magic but, after his Arsenal and Barcelona days, he sulked like someone whose Porsche had been swapped for a Skoda.
"We tried to win but just lacked that killer touch," concluded McLeish.


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To prepare the manufacturing process of Power Press, Heavy Machines, Lathe Tools.
Selection of Tools for manufacturing and assembling the products.
Product & quality specifications in co-ordination with Engineering, Quality & production.
Since July 2006June 2008 worked with Dynaflow Equipment Pvt. Ltd.(Sudarshan park, New Delhi) as a CAD Operator, HRA Executive & Site Engineer (MFGAir Handling Unit, Scrubber & Duct Designing).
The Company : Dynaflow Equipment Pvt.
Ltd.(Sudarshan park, New Delhi) provides services to the world's leading AHU (AIR HANDLING UNIT) and Heat Ventilation And Air Changing in domestic areas , specializes in Product Design, Development and manufacturing.
To prepare the manufacturing process of HEAT VANTILATION & AIR CHANGING.
Interaction with customers regarding problems & the modification required in AIR Handling Unit.
Selection of Ducting designing for the products.
Vendor development for all new and existing components/materials for the Production.
Basic Knowledge of computer with Internet.
One Year computer course from O-TECH COMPUTERS, Rohtak.
Well versed to work in Windows Platform.
Three months AutoCAD diploma from WIZARD COMPUTER EDUCATION in 2007, Rohtak.
Pursuing Mechanical Diploma (Distance Learning) from CMJ University, Bangalore.


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Panini is available as an AppImage which means "one app = one file", which you can download and run on your Linux system while you don't need a package manager and nothing gets changed in your system. Awesome!
If you want to restrict what Panini can do on your system, you can run the AppImage in a sandbox like Firejail. This is entirely optional and currently needs to be configured by the user.
If you would like to update to a new version, simply download the new Panini AppImage.
The Panini AppImage also can be updated using AppImageUpdate. Using this tool, Panini can be updated by downloading only the portions of the AppImage that have actually changed since the last version.
If you would like to have the executable bit set automatically, and would like to see Panini and other AppImages integrated into the system (menus, icons, file type associations, etc.), then you may want to check the optional appimaged daemon.
Thanks for distributing Panini in the AppImage format for all common Linux distributions. Great! Here are some ideas on how to make it even better.
The screenshot for Panini has been automatically taken during a fully automated test. You can specify the URL to a nicer one by shipping an AppStream metainfo file.


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add all 94 publications to the report.
Transforming Cancer Prevention through Precision Medicine and Immune-oncology.
Preinvasive Endobronchial Lesions: Lung Cancer Precursors and Risk Markers?
AACR Cancer Progress Report 2015.
Molecular cancer prevention: Current status and future directions.
A Phase II Randomized, Controlled Trial of S-Adenosylmethionine in Reducing Serum α-Fetoprotein in Patients with Hepatitis C Cirrhosis and Elevated AFP.
Chemoprevention Clinical Trials: How Do We Move Forward? How Do We Identify Valid End Points?
Implications of the "Bad Luck" Explanation of Cancer Risk for the Field of Cancer Prevention.
Randomized Multicenter Placebo-Controlled Trial of Omega-3 Fatty Acids for the Control of Aromatase Inhibitor-Induced Musculoskeletal Pain: SWOG S0927.
Dermatomyositis paraneoplastic syndrome before symptomatic tonsillar squamous cell carcinoma: a case report.
PPARγ in head and neck cancer prevention.
Images in clinical medicine. Sunitinib-associated hair depigmentation.
The immunological and clinical effects of mutated ras peptide vaccine in combination with IL-2, GM-CSF, or both in patients with solid tumors.
Fifty years of tobacco carcinogenesis research: from mechanisms to early detection and prevention of lung cancer.
A phase I/II study of sepantronium bromide (YM155, survivin suppressor) with paclitaxel and carboplatin in patients with advanced non-small-cell lung cancer.
Effect of intermittent dosing regimens of erlotinib on methylnitrosourea-induced mammary carcinogenesis.
Characterization and management of cardiac involvement of thymic epithelial tumors.
Chemopreventive effect of a mixture of Chinese Herbs (antitumor B) on chemically induced oral carcinogenesis.
Validation of LOH profiles for assessing oral cancer risk.
A phase I combination study of olaparib with cisplatin and gemcitabine in adults with solid tumors.
Criteria of evidence to move potential chemopreventive agents into late phase clinical trials.
Lung cancer chemoprevention with celecoxib in former smokers.
Altered histology provides a positive clinical signal in the bronchial epithelium.


text:
HomeMemoirThe Cathedral of Medicine: Gareth Rees vs Serious Illness
19th February 2018 Memoir
Since December 2017, Portsmouth author and S&C regular Gareth Rees has been battling serious illness and pondering some deep questions about his own existence. Here he recounts finding solace in Charles Dickens, chirpy NHS staff and a trip to the Meon Valley.
I haven't achieved self-mastery yet. Haven't even arrived at the foothills. Haven't learned to replace cursing with blessing, to convert anger into compassion, to understand instead of to judge. Haven't found a yin and yang removed from a world riven into harsh opposites. Haven't learned to mediate between the dreadful polarity of life and death. Haven't found an inner wisdom to guide me to the truth in a world screaming out fake prescriptions.
I mean, do I eat nuts or not? And when it says on the label the cashews are honey-roasted but I find it's more sugar than honey, do I retreat to my hermitage mind in despair, unable to trust the world?
Maybe all those cashews – and the protein they contain – I ate last night caused the churning in my guts which broke my sleep. Fruit juice? Oh no, too much sugar. What about the apple and rhubarb juice from Waitrose? Something mysterious and wonderful about rhubarb. Salvation through rhubarb! Maple syrup's all right though, isn't it? But it's still sugar. What about gluten-free porridge with date syrup?
This nutrition hysteria is a substitute for church with its emphasis on sin and righteousness. The cold, marble-faced seriousness that eventually leads to the banning of fun, music, dance and even kite-flying. I heard the Puritans even banned mince pies. I'm scared to admit that all I feel like at the moment is a slice of toasted Tesco white bread with plenty of butter and marmalade slapped on it.
The Persian philosopher Rumi comes to mind. 'Beyond the fields of Right and Wrong, there is a field. I will meet you there.'
Who is Right, who is Wrong? It's an ongoing, life-long labour to disentangle the fake from the true. In the end, I have to be an empiricist and trust to my own experience. I must ask myself if the feeling I have is conducive to my own well-being and will cause no harm to others.
Cold, marble-faced seriousness. No expression. Such a face meets me when I call at the GP's to renew a prescription. The face tells me I must wait four days for my medicine. Resisting my inheritance of deference, I say, 'No, that's not acceptable.' Instead of four days, the medicine is available at the pharmacy in two hours.
I'm desperately searching for a new book. The charity shops and the local library are dried-up rivers on that score. Then I question my rule about not reading a book twice. I don't apply the same rule to music. So I grab from the library Dombey and Son by Charles Dickens.
When I read it for the first time, I felt like I'd discovered an oasis. I was, after all, sitting in a caravan in the moonlit Libyan desert in the late 1970s. As 'nodding donkeys' sucked oil outside, I disappeared into Victorian England where the people may have lacked modern 'necessities' like vacuum cleaners and electricity, but they lived much like people live today, with love existing side-by-side with cruelty. Some things about human nature never change, and I like being part of it. I'm just another frail human being, but I'm not alone.
I was distracted from my book and reminded I was in the depths of the Sahara when a huge insect flew into the caravan. I sprayed it with insecticide and watched its death agony. I felt bad. But was its suffering greater just because it was large? Would I have felt better if I'd flicked at a fly? Worse if I'd shot an elephant or harpooned a whale?
Back in a waiting room inside the cathedral of medicine. The receptionist gives me the code FFJ and instructs me to watch a screen. I'm twenty minutes early but worry I might lose myself in Dombey and Son and miss my calling.
The screen tells me to go to 'Sub-Wait Three'. Not much poetry there, not like being called to an assignation in Love Apple Lane. Feeling like a circus animal who's long forgotten its natural environment, I push through double doors and am welcomed by smiling humanity. 'Hello, I'm Ingrid. Please, this way. Let me weigh you.'
I don't ask what my weight is now. I don't want reminding of the hostile being within me which is realising its identity by stealing my substance.
My meetings with doctors or 'heads of team' are seldom useful. There's a screen, always a screen, and then a collation of data, an interpretation of that data and then an oily seepage of gloom. In contrast, the hands-on people, the nurses and the operatives who change dressings, take blood, attach tubes and so forth, are warm and upbeat. That's how it is today. I leave the hospital feeling cheerful.
A new day and I am feeling less weak and less likely to be blown over by a puff of wind upon venturing out. Instead of head bent low and looking down at the pavement, my gaze is horizontal.
I buy some more daffodils and apricot juice. Don't care for the apricot fruit, but the juice is ambrosial. I recall that, in the 1950s, a National Geographic journalist accessed a remote Shangri-La-type valley somewhere amidst the mountains of Pakistan. Apricot orchards abounded and the writer reckoned the centrality of that fruit in the diet of the inhabitants gave them health and longevity.
It's 9.30 p.m. Up until a couple of months ago, I'd be drinking beer in the tavern at this time. All I'm imbibing now is medicine from a bottle housed in the left hand pocket of my shirt. It's pumped in drips via a tube into an opening in my right arm.
I don't miss the beer nor tavern company hugely but feel a vacuum during this part of the day. It's too early to switch off the light. I fear the sleeplessness which will follow. If sleep comes, I fear that too. Both states are like a troubled dream.
But I must jump off this train of despondency. A pancake with maple syrup and a dash of double cream. And then some chords on the guitar, a bluegrass kind of thing. After that, 'Amazing Grace' on the harmonica and the foul emotion that produces a line like 'a wretch like me' is wafted away by the perfume of the music.
It seems little Dombey in Dombey and Son is dying. But I know he doesn't so I'm not sad when I put the book down and turn off the light. Compared to other recent nights, sleep comes quickly and I rest well. Brilliant sunshine – another rarity – greets me in the morning.
I stand on my head for a bit and then do some 'salute to the sun' yoga moves. Then it's a round of alternate nostril breathing. I go through the alphabet on each intake and exhalation, thinking up words as I go.
'A' is for the Aroma when I take off the cap of my wee bottle of jasmine oil.
'B' is for the Soft Footfall of Beloved.
Seeking the Divine.
Equanimity.
Faith, Gladness, Hope.
The Inner Way to the Kingdom of Light and Mercy.
Orange blossom at dawn.
The Peace of God beyond understanding.
A Quiet Mind.
Reeds on the shore of Galilee.
A man in an allotment cutting Sweet Peas for his wife.
Upswing.
The Vision of little pips.
The Well of Well-Being.
And now it's time for breakfast: apple juice and a fresh croissant soaked in coffee.
'And the child lies calm and beautiful on his little bed.' So I'm wrong, little Dombey does die. I must have left that memory behind in the Sahara. And his poor sister, who has already lost her mother as well, is in terrible grief. But is it comforting to be told at the funeral that 'all grief is unavailing and it is our duty to submit'?
When to accept and when to resist ?
To the Meon Valley. The English countryside still wintry, lifeless, bedraggled. At least snowdrops are blooming on the grass verges of the lanes. Thomas Lord, founder of Lords cricket ground, lived in the village of West Meon and there's a pub named after him. In that pub is a log fire and chips like roast potatoes that are golden and delicious, especially with a scoop of mayonnaise.
It's joy to have my appetite returned. I think of the Jesus story when a family believed its sick little girl had died. Jesus cleared the room of all the wailing hysteria. The child turned out to be asleep and, when she woke, Jesus hastened her recovery by ordering food for her.
Gareth's book Read Rees is available to purchase here.
Photography by Alexander Sebley.
gareth rees
Queen Alexandra Hospital
Star & Crescent
Event: What is Going On? The State of Local News
Election '17: Covering a Snap Election – the View from the Frontline
8th June 2017 Editorials, Politics
Editor in Chief, Sarah Cheverton, revisits the last 6 weeks of election fever in a behind-the-scenes look at how S&C worked with local residents and writers to bring alternative perspectives to local mainstream news. When [… read more ]
Dr Manfratton – an origin story
8th October 2015 Fiction
By Jon Crout. It is 1992. It is Fudger's birthday, and we are sat in his shed, hiding from his mum. She's got the hump because he re-enacted Anderton's goal at Highbury in her lounge [… read more ]
Preventing Crime, Not Picking Up The Pieces: An Interview with Simon Hayes


text:
Magic Quadrant for Cloud Core Financial Management Suites for Midsize, Large and Global Enterprises 2019
www.acumatica.com ERP Systems Comparison Comparing Acumatica vs Microsoft Dynamics GP
Comparing Acumatica vs Microsoft Dynamics GP
Microsoft Dynamics GP (formerly Great Plains) is a mid-market ERP product that integrates financial management, , manufacturing, distribution, and CRM.
Some of Dynamics GP's strengths are:
Highly customizable and flexible
Strong third-party software support
Support for multiple vertical industries
Acumatica boasts the same strengths and performs many of the same functions. But we believe Acumatica is a superior product than Dynamics GP because Acumatica was designed from the start to be accessible through the cloud using current technology. This means Acumatica can offer more features and functions than Dynamics GP and at a lower long-term price.
Take a look at how Acumatica compares with Dynamics GP:
True cloud (built for cloud)
Full function ERP
Full relational database export
Customization using industry standard tools
Upgrade on your schedule
Flexible licensing options (subscription and perpetual)
Multiple deployment options (cloud, on premise, hybrid)
Scale as you grow
Lower Total Cost of Ownership (TCO)
Significant differentiators between Acumatica and Dynamics GP
True cloud: "True cloud" means all functionality is accessible through the internet using a standard browser without the need for additional software installation on the user's device or additional software licensing. Acumatica can be deployed on premises or in the cloud without additional equipment or software. Dynamics GP is a traditional client-server product and was NOT built for the cloud. GP can be HOSTED on a remote server but is not a true cloud ERP product. Currently, the only true cloud option for GP users today is to give up GP entirely and re-implement with Dynamics 365 Business Central (formerly Dynamics NAV).
Customizations using industry standard tools: Both products are highly configurable, but Acumatica is built using industry standard C# and .NET. Dynamics GP uses a proprietary development language, making customizations more difficult and expensive.
Multiple deployment options (cloud, on premises, hybrid): Technically, both products support cloud, on-premises, and hybrid configurations. However, Acumatica supports all three deployment options natively using a web browser to access the system. Dynamics GP, on the other hand, requires SharePoint as a front-end to enable a cloud configuration. In addition, SharePoint requires user licensing, increasing TCO.
Scale as you grow: When choosing an ERP solution, you must consider your future requirements, not just your company's current needs. Both products allow you to scale resources to support a growing company, but because Acumatica only charges by the by computing power required and not by users, you can scale your equipment as you grow, allowing an unlimited number of users to access the system regardless of function. Dynamics GP, on the other hand, incurs greater costs as your company grows and more employees, customers, and suppliers need access to the system. Be aware of these costs and consider them in your final evaluation.
TCO: Acumatica charges by computing power used, not by user. Dynamics GP charges per user, and these costs increase as your company grows. Dynamics GP requires a license for SQL Server to run, while Acumatica can work not only with SQL Server but also with less expensive databases, such as MySQL. Moreover, Acumatica works natively in the cloud, but Dynamics GP requires a separate license for SharePoint for it to work in the cloud.
Calculate the cost of replacing your ERP system
Use Acumatica's pricing tool to find out the real cost of moving to a modern ERP system. Calculate ROI and total cost of ownership for any ERP. Including Microsoft Dynamics GP.
Calculate now
About Microsoft Dynamics GP
Microsoft Dynamics GP is an ERP product built using a much older system architecture, which becomes an issue in scenarios designed for the latest technologies, including cloud deployment options, customizations using current development tools, and integration with CRM.
But there are other drawbacks, for example:
Dynamics GP still uses the same proprietary programming language (Dexterity) that it used back in 1992.
Dynamics GP still uses the same client-server architecture, and requires Microsoft SharePoint to move it to the cloud.
Integration between Dynamics GP and Dynamics CRM (or any other CRM product) requires the use of separate databases.
GP has no direct migration path to Business Central, Microsoft's only true-cloud mid-market ERP product.
Acumatica offers a completely integrated ERP and CRM solution and can be deployed on premise or in the cloud. And with Acumatica, you enjoy unlimited users. If you are looking for the best price/value, performance, functionality, and ease of use of any ERP product available for your business, then make sure you consider Acumatica.
In the end, the ERP solution you choose will be an integral part of your company for several years. We encourage you to perform due diligence when considering the purchase of any ERP system, not just ours. Ask questions. Look seriously at your business requirements over the next three to five years. Sure, we want you to choose Acumatica, but, more importantly, we want to make sure you get the ERP system that is right for your company now and in the future.
Take an in-depth look at
our Cloud ERP solution
Get a free consultation to help
accelerate your business
See Acumatica's integrated
solution in action
What our customers are saying after replacing Microsoft Dynamics with Acumatica?
In my old system, it would take me about a week to do a reconciliation. With Acumatica, I did September's reconciliation in two hours. It's saving me so much time.
Sheila Lavin,
Target Enterprises
Acumatica is bringing functionality to the small/medium business sector that was out of reach budget-wise four years ago.
Scott Starkweather,
President,
We finally have a package that is all-in-one and the data is a lot more detailed. This way we don't have to switch from one program to the next to get our data. All the data is right there in one integrated program.
Curt Norton,
Office Manager,
C&O Nursery
With DMS solution developed by DMSpro on top of Acumatica, at the headquarters we now have exact knowledge of all daily sales activities on the field and real-time information including product display at hundreds of thousands of outlets… Real-time visibility into daily business activities and timely business data now empowers every decision….
Dr Tran Qui,
President and CEO,
Tan Hiep Phat Beverage Group
From being able to enter orders and production information directly in the field, to minimizing travel time, saving on vehicles, saving on fuel, there's a lot of things Acumatica will make a big difference with.
Jim Lieffort,
Director of Planning & IT,
IMG Enterprises
From Day One we saw how valuable Acumatica was–from tracking expenses to getting invoices approved to running reports that showed where people were working, who was being underutilized, and how we could reallocate people to where they were needed.
Francisco Callegari,
CIO,
Guardian SealTech
[Acumatica is] extremely reliable—a platform that is up and running 24/7 around the world. It's accessible to everyone, any time they need it; there's nothing from an Information System perspective that is stopping us.
Adam Rosenbaum,
Director of Information Systems,
Acumatica provided a solution to integrate our financial reporting across several systems, currencies, and offices. Since the solution is web-based, it was cost-effective to deploy across our international offices.
Parallels Employee,
More customer stories
Watch video testimonials from our customers
Watch video stories
Read what analysts are saying about Acumatica
Insights from Analysts
See how Acumatica supports your industry
Return to Comparison page
ERP Comparision
Use Acumatica's pricing tool to find out the real cost of moving to a modern ERP system. Calculate ROI and total cost of ownership for any ERP.
Calculate now No thanks, I love my legacy ERP


text:
Watch Alice in Chains Perform at Washington State University in 1989
Michael Haskoor
On Dec. 1, 1989 at Washington State University, grunge legends Alice in Chains performed a harrowing set for their college fans -- and thanks to the fine folks at Alternative Nation, we can now watch the full, never-before-seen footage 25 years later. Check it out above.
The performance was one of the band's first after signing with Columbia Records, the label that put out their debut LP Facelift. WSU promoter Ken Cardwell scrounged together nearly $2,000 (which included fronting his second semester tuition money) to book the band. Tickets for the show sold rapidly and Cardwell, along with WSU student Tony Harrell, were forced to find a larger venue, the CUB Ballroom, where no other band had played before.
"I didn't have much of an advertising budget," Cardwell said. "So I worked with KUGR, the campus radio station, to cut some promos and they ran ads promoting the show." You can listen to the radio show's commercial below:
The show's setlist included many of the band's greatest hits to date, including show opener "Killing Yourself," as well as "Man in the Box," "We Die Young," "Sunshine," "Suffragette City" and many more. Alice in Chains' Facelift, which was released on Aug. 21, 1990, has since been certified double-platinum by the RIAA, selling more than 2 million copies in the U.S.
Worst to First: Every Alice in Chains Album Ranked
Next: Chicago Cubs Sing 'All the Way' With Eddie Vedder at Benefit Concert
Filed Under: Alice in Chains
Categories: Music, News, Videos


Did you see Big Hero 6 when it was in the theaters? Emma and I did and we absolutely adored it. I swear there were some random ninja onions in the theater because my eyes were watering... *sniff*. If you've been around these parts once or twice, you will know I am a big nerd. With that being said, San Fransokyo is awesome and I wish it was real because I would totally live there. /end nerd rant.
Now Big Hero 6 is out on DVD/Blu-Ray (Yaaaayyyy) and you can get a copy at Walmart. The nice this is with your purchase of Big Hero 6 Blu-Ray/DVD + Gift with Purchase Set includes a digital movie on Vudu while supplies last. You can simply scan your receipt using the Savings Catcher in the Walmart app. Then look for a message on your phone or your email and now you can watch Big Hero 6 on any device! I love the free digital copy. I watch most of my movies/tv series in bed late at night and since I don't have a DVD/Blu-Ray player in my room, I stream via my Roku and I love having my movies available on Vudu.
Of course, you can't have a Big Hero 6 movie night without friends. Oh hai, Fred and Honey Lemon! Of course,when you have some friends over, you gotta have popcorn. Movies. Friends. Popcorn. Must have.
Of course, since it's San Fransokyo, a hybrid of San Francisco and Tokyo... wasabi popcorn just seems to be the perfect pairing.
The best part? It's ridiculously easy to make.
I popped a bag of microwave popcorn but you can make homemade.
Add wasabi (I recommend starting with a small amount and building up to your taste), soy sauce, ginger, and sea salt to butter and stir.
Pour the wasabi butter over popcorn and mix well. I keep the popcorn in the bag so I can shake it up but you can do this part however you please.
And... turn on Big Hero 6, grab some friends and popcorn.
What do you think the cities of the future will be like?
Would you like to see a San Fransokyo kind of city?


Robert Charles Wilson's _Vortex_ (July). I really liked _Spin_, and _Axis_ hopefully suffered from an "unsatisfying book in the middle" effect (though it was still pretty good).
Charles Stross' _Rule 34_ (July).
Steven Gould's _7th Sigma_ (July). If you haven't read _Jumper_ or _Wildside_, go do that.
Neal Stephenon's _Readme_ (September). I really liked _Zodiac_, _Snow Crash_ and _The Diamond Age_, but didn't get through his bug-crushing System of the World trilogy. I'd like to see another _Anathem_, but I'd be happy if it was as good as _The Cobweb_.
Jay Lake's _Endurance (Nov). Follow-on to the quite excellent fantasy _Green_.
If you liked Jumper and Wildside, give The Walls of the Universe (Paul Melko) a shot.
@JOsborne: _Walls of the Universe_ was a lot of fun, yes. (However, I didn't like Melko's _Singularity's Ring_. Melko's book of short stories was excellent, YMMV).
Another good "quick"read: Jeffrey Olton's _The Getaway Special_. Yet another: Duncan Long's _Anti-Grav Unlimited_. You'd think that Olton and Long were channeling each other, but they're both worth a read.
Have you read Leviathan Wakes? I loved it. Epic sci-fi.