AssemblyQC: a Nextflow pipeline for reproducible reporting of assembly quality.

SUMMARY: Genome assembly projects have grown exponentially due to breakthroughs in sequencing technologies and assembly algorithms. Evaluating the quality of genome assemblies is critical to ensure the reliability of downstream analysis and interpretation. To fulfil this task, we have developed the AssemblyQC pipeline that performs file-format validation, contaminant checking, contiguity measurement, gene- and repeat-space completeness quantification, telomere inspection, taxonomic assignment, synteny alignment, scaffold examination through Hi-C contact-map visualization, and assessments of completeness, consensus quality and phasing through k-mer analysis. It produces a comprehensive HTML report with method descriptions, tables, and visualizations. AVAILABILITY AND IMPLEMENTATION: The pipeline uses Nextflow for workflow orchestration and adheres to the best-practice established by the nf-core community. This pipeline offers a reproducible, scalable, and portable method to assess the quality of genome assemblies-the code is available online at GitHub: https://github.com/Plant-Food-Research-Open/assemblyqc.

Identification and Characterization of Innate Immunity in Actinidia melanandra in Response to Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has decimated kiwifruit orchards growing susceptible kiwifruit Actinidia chinensis varieties. Effector loss has occurred recently in Psa3 isolates from resistant kiwifruit germplasm, resulting in strains capable of partially overcoming resistance present in kiwiberry vines (Actinidia arguta, Actinidia polygama, and Actinidia melanandra). Diploid male A. melanandra recognises several effectors, sharing recognition of at least one avirulence effector (HopAW1a) with previously studied tetraploid kiwiberry vines. Sequencing and assembly of the A. melanandra genome enabled the characterisation of the transcriptomic response of this non-host to wild-type and genetic mutants of Psa3. A. melanandra appears to mount a classic effector-triggered immunity (ETI) response to wildtype Psa3 V-13, as expected. Surprisingly, the type III secretion (T3SS) system-lacking Psa3 V-13 ∆hrcC strain did not appear to trigger pattern-triggered immunity (PTI) despite lacking the ability to deliver immunity-suppressing effectors. Contrasting the A. melanandra responses to an effectorless Psa3 V-13 ∆33E strain and to Psa3 V-13 ∆hrcC suggested that PTI triggered by Psa3 V-13 was based on the recognition of the T3SS itself. The characterisation of both ETI and PTI branches of innate immunity responses within A. melanandra further enables breeding for durable resistance in future kiwifruit cultivars.

Investigating the genetic components of tuber bruising in a breeding population of tetraploid potatoes.

BACKGROUND: Tuber bruising in tetraploid potatoes (Solanum tuberosum) is a trait of economic importance, as it affects tubers' fitness for sale. Understanding the genetic components affecting tuber bruising is a key step in developing potato lines with increased resistance to bruising. As the tetraploid setting renders genetic analyses more complex, there is still much to learn about this complex phenotype. Here, we used capture sequencing data on a panel of half-sibling populations from a breeding programme to perform a genome-wide association analysis (GWAS) for tuber bruising. In addition, we collected transcriptomic data to enrich the GWAS results. However, there is currently no satisfactory method to represent both GWAS and transcriptomics analysis results in a single visualisation and to compare them with existing knowledge about the biological system under study. RESULTS: When investigating population structure, we found that the STRUCTURE algorithm yielded greater insights than discriminant analysis of principal components (DAPC). Importantly, we found that markers with the highest (though non-significant) association scores were consistent with previous findings on tuber bruising. In addition, new genomic regions were found to be associated with tuber bruising. The GWAS results were backed by the transcriptomics differential expression analysis. The differential expression notably highlighted for the first time the role of two genes involved in cellular strength and mechanical force sensing in tuber resistance to bruising. We proposed a new visualisation, the HIDECAN plot, to integrate the results from the genomics and transcriptomics analyses, along with previous knowledge about genomic regions and candidate genes associated with the trait. CONCLUSION: This study offers a unique genome-wide exploration of the genetic components of tuber bruising. The role of genetic components affecting cellular strength and resistance to physical force, as well as mechanosensing mechanisms, was highlighted for the first time in the context of tuber bruising. We showcase the usefulness of genomic data from breeding programmes in identifying genomic regions whose association with the trait of interest merit further investigation. We demonstrate how confidence in these discoveries and their biological relevance can be increased by integrating results from transcriptomics analyses. The newly proposed visualisation provides a clear framework to summarise of both genomics and transcriptomics analyses, and places them in the context of previous knowledge on the trait of interest.

Implementation of different relationship estimate methodologies in breeding value prediction in kiwiberry (Actinidia arguta).

In dioecious crops such as Actinidia arguta (kiwiberries), some of the main challenges when breeding for fruit characteristics are the selection of potential male parents and the long juvenile period. Currently, breeding values of male parents are estimated through progeny tests, which makes the breeding of new kiwiberry cultivars time-consuming and costly. The application of best linear unbiased prediction (BLUP) would allow direct estimation of sex-related traits and speed up kiwiberry breeding. In this study, we used a linear mixed model approach to estimate narrow sense heritability for one vine-related trait and five fruit-related traits for two incomplete factorial crossing designs. We obtained BLUPs for all genotypes, taking into consideration whether the relationship was pedigree-based or marker-based. Owing to the high cost of genome sequencing, it is important to understand the effects of different sources of relationship matrices on estimating breeding values across a breeding population. Because of the increasing implementation of genomic selection in crop breeding, we compared the effects of incorporating different sources of information in building relationship matrices and ploidy levels on the accuracy of BLUPs' heritability and predictive ability. As kiwiberries are autotetraploids, multivalent chromosome formation and occasionally double reduction can occur during meiosis, and this can affect the accuracy of prediction. This study innovates the breeding programme of autotetraploid kiwiberries. We demonstrate that the accuracy of BLUPs of male siblings, without phenotypic observations, strongly improved when a tetraploid marker-based relationship matrix was used rather than parental BLUPs and female siblings with phenotypic observations. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01419-8.

sismonr: simulation of in silico multi-omic networks with adjustable ploidy and post-transcriptional regulation in R.

SUMMARY: We present sismonr, an R package for an integral generation and simulation of in silico biological systems. The package generates gene regulatory networks, which include protein-coding and non-coding genes along with different transcriptional and post-transcriptional regulations. The effect of genetic mutations on the system behaviour is accounted for via the simulation of genetically different in silico individuals. The ploidy of the system is not restricted to the usual haploid or diploid situations but can be defined by the user to higher ploidies. A choice of stochastic simulation algorithms allows us to simulate the expression profiles of the genes in the in silico system. We illustrate the use of sismonr by simulating the anthocyanin biosynthesis regulation pathway for three genetically distinct in silico plants. AVAILABILITY AND IMPLEMENTATION: The sismonr package is implemented in R and Julia and is publicly available on the CRAN repository (https://CRAN.R-project.org/package=sismonr). A detailed tutorial is available from GitHub at https://oliviaab.github.io/sismonr/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Validation of a hospital clinical pharmacy workforce calculator: A methodology for pharmacy?

BACKGROUND: The benefits of clinical pharmacy services are established within hospital practice but staff numbers required for service delivery are not well described and staffing levels vary. The need for a consistent, objective method of determining staffing levels was recognised at a UK University Hospital and a Clinical Pharmacy Workforce Calculator (CPWC) was developed. OBJECTIVE: To develop the Activity Standard (AS) for pharmaceutical care and establish the reliability of the CPWC across acute hospital settings in UK. SETTING: Acute hospital in-patient clinical pharmacy services on medical and surgical wards. METHOD: Using the World Health Organisation's Workload Indicators of Staffing Need (WISN) methodology, a two-round Delphi study was undertaken. This developed the Activity Standard for pharmaceutical care and identified the staff-time unavailable for clinical work. Consenting panel members then tested the CPWC, calculating the staff required for three scenarios to determine whether it could be reliably used by different operators. RESULTS: Thirty-six participants consented to participate. Data were returned from 22 (61%) of whom 20 (56%) supplied analysable data. Consensus was achieved on the tasks required for pharmaceutical care delivery, the mean time each takes, how frequently they should be completed and the time unavailable for clinical work for each grade of staff. The CPWC calculates staffing requirements using these data. Eleven participants (55%) tested the CPWC and analysis of responses demonstrated that 30 of 33 (91%) calculations were accurately completed. DISCUSSION: This study defined the WISN Activity Standard for UK pharmaceutical care delivery to hospital inpatients and showed content validity for the CPWC in acute medical and surgical hospital settings. Different operators used the CPWC reliably and applied it to local sites. CONCLUSION: The CPWC offers hospital pharmacy managers a useful tool to negotiate adequate staffing to deliver pharmaceutical care. Its development methodology could be applied widely in pharmacy practice.

TriPoly: haplotype estimation for polyploids using sequencing data of related individuals.

Motivation: Knowledge of haplotypes, i.e. phased and ordered marker alleles on a chromosome, is essential to answer many questions in genetics and genomics. By generating short pieces of DNA sequence, high-throughput modern sequencing technologies make estimation of haplotypes possible for single individuals. In polyploids, however, haplotype estimation methods usually require deep coverage to achieve sufficient accuracy. This often renders sequencing-based approaches too costly to be applied to large populations needed in studies of Quantitative Trait Loci. Results: We propose a novel haplotype estimation method for polyploids, TriPoly, that combines sequencing data with Mendelian inheritance rules to infer haplotypes in parent-offspring trios. Using realistic simulations of both short and long-read sequencing data for banana (Musa acuminata) and potato (Solanum tuberosum) trios, we show that TriPoly yields more accurate progeny haplotypes at low coverages compared to existing methods that work on single individuals. We also apply TriPoly to phase Single Nucleotide Polymorphisms on chromosome 5 for a family of tetraploid potato with 2 parents and 37 offspring sequenced with an RNA capture approach. We show that TriPoly haplotype estimates differ from those of the other methods mainly in regions with imperfect sequencing or mapping difficulties, as it does not rely solely on sequence reads and aims to avoid phasings that are not likely to have been passed from the parents to the offspring. Availability and implementation: TriPoly has been implemented in Python 3.5.2 (also compatible with Python 2.7.3 and higher) and can be freely downloaded at https://github.com/EhsanMotazedi/TriPoly. Supplementary information: Supplementary data are available at Bioinformatics online.

Kiwifruit SVP2 controls developmental and drought-stress pathways.

KEY MESSAGE: Genome-wide targets of Actinidia chinensis SVP2 confirm roles in ABA- and dehydration-mediated growth repression and reveal a conservation in mechanism of action between SVP genes of taxonomically distant Arabidopsis and a woody perennial kiwifruit. The molecular mechanisms underlying growth and dormancy in woody perennials are largely unknown. In Arabidopsis, the MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) plays a key role in the progression from vegetative to floral development, and in woody perennials SVP-like genes are also proposed to be involved in controlling dormancy. During kiwifruit development SVP2 has a role in growth inhibition, with high-chill kiwifruit Actinidia deliciosa transgenic lines overexpressing SVP2 showing suppressed bud outgrowth. Transcriptomic analyses of these plants suggests that SVP2 mimics the well-documented abscisic acid (ABA) effect on the plant dehydration response. To corroborate the growth inhibition role of SVP2 in kiwifruit development at the molecular level, we analysed the genome-wide direct targets of SVP2 using chromatin immunoprecipitation followed by high-throughput sequencing in kiwifruit A. chinensis. SVP2 was found to bind to at least 297 target sites in the kiwifruit genome, and potentially modulates 252 genes that function in a range of biological processes, especially those involved in repressing meristem activity and ABA-mediated dehydration pathways. In addition, our ChIP-seq analysis reveals remarkable conservation in mechanism of action between SVP genes of taxonomically distant plant species.

When Men Grieve: Widowers' Stories of Coping With Their Wives' Deaths.

To increase our understanding of the role gender plays in spousal loss, this qualitative study analyzes bereavement narratives of 33 recent widowers, aged 45-89 years, all of whom accessed the National Widowers' Organization website. In particular, we look at how these widowers' lives changed, the impact of changing gender norms, and coping strategies. To illuminate the temporal process evident in these narratives, we utilize Silverman's nonlinear characteristics of bereavement and the anthropological concept of liminality. In contrast to studies of widowers in earlier decades, our analysis reveals questioning and rejection of stereotypical masculine gender norms, a wide variety of creative responses to spousal loss, and the need for individualized support.

Cerebral Small Vessel Disease Burden Is Increased in Systemic Lupus Erythematosus.

BACKGROUND AND PURPOSE: Systemic lupus erythematosus (SLE) increases stroke risk, but the mechanism is uncertain. This study aimed to determine the association between SLE and features on neuroimaging of cerebral small vessel disease (SVD), a risk factor for stroke. METHODS: Consecutive patients attending a clinic for SLE were recruited. All patients underwent brain magnetic resonance imaging; had blood samples taken for markers of inflammation, endothelial dysfunction, cholesterol, and autoantibodies; and underwent cognitive and psychiatric testing. The data were compared with sex- and age-matched healthy controls and patients with minor stroke. Features of SVD were measured, a total SVD score calculated, and associations sought with vascular risk factors, cognition, SLE activity, and disease duration. RESULTS: Fifty-one SLE patients (age: 48.8 years; SD: 14.3 years) had a greater total SVD score compared with healthy controls (1 versus 0; P<0.0001) and stroke patients (1 versus 0; P=0.02). There were higher perivascular spaces and deep white matter hyperintensity scores and more superficial brain atrophy in SLE patients versus healthy controls. Despite fewer vascular risk factors than similarly aged stroke patients, SLE patients had similar or more of some SVD features. The total SVD score was not associated with SLE activity, cognition, disease duration, or any blood measure. CONCLUSIONS: In this data set, SLE patients had a high burden of SVD features on magnetic resonance imaging, particularly perivascular spaces. A larger longitudinal study is warranted to determine the causes of SVD features in SLE and clinical implications.

Drag-Reducing Polymer Enhances Microvascular Perfusion in the Traumatized Brain with Intracranial Hypertension.

Current treatments for traumatic brain injury (TBI) have not focused on improving microvascular perfusion. Drag-reducing polymers (DRP), linear, long-chain, blood-soluble, nontoxic macromolecules, may offer a new approach to improving cerebral perfusion by primary alteration of the fluid dynamic properties of blood. Nanomolar concentrations of DRP have been shown to improve hemodynamics in animal models of ischemic myocardium and ischemic limb, but have not yet been studied in the brain. We recently demonstrated that DRP improved microvascular perfusion and tissue oxygenation in a normal rat brain. We hypothesized that DRP could restore microvascular perfusion in hypertensive brain after TBI. Using in vivo two-photon laser scanning microscopy we examined the effect of DRP on microvascular blood flow and tissue oxygenation in hypertensive rat brains with and without TBI. DRP enhanced and restored capillary flow, decreased microvascular shunt flow, and, as a result, reduced tissue hypoxia in both nontraumatized and traumatized rat brains at high intracranial pressure. Our study suggests that DRP could constitute an effective treatment for improving microvascular flow in brain ischemia caused by high intracranial pressure after TBI.

Tuber shape and eye depth variation in a diploid family of Andean potatoes.

BACKGROUND: Tuber appearance is highly variable in the Andean cultivated potato germplasm. The diploid backcross mapping population 'DMDD' derived from the recently sequenced genome 'DM' represents a sample of the allelic variation for tuber shape and eye depth present in the Andean landraces. Here we evaluate the utility of morphological descriptors for tuber shape for identification of genetic loci responsible for the shape and eye depth variation. RESULTS: Subjective morphological descriptors and objective tuber length and width measurements were used for assessment of variation in tuber shape and eye depth. Phenotypic data obtained from three trials and male-female based genetic maps were used for quantitative trait locus (QTL) identification. Seven morphological tuber shapes were identified within the population. A continuous distribution of phenotypes was found using the ratio of tuber length to tuber width and a QTL was identified in the paternal map on chromosome 10. Using toPt-437059, the marker at the peak of this QTL, the seven tuber shapes were classified into two groups: cylindrical and non-cylindrical. In the first group, shapes classified as 'compressed', 'round', 'oblong', and 'long-oblong' mainly carried a marker allele originating from the male parent. The tubers in this group had deeper eyes, for which a strong QTL was found at the same location on chromosome 10 of the paternal map. The non-cylindrical tubers classified as 'obovoid', 'elliptic', and 'elongated' were in the second group, mostly lacking the marker allele originating from the male parent. The main QTL for shape and eye depth were located in the same genomic region as the previously mapped dominant genes for round tuber shape and eye depth. A number of candidate genes underlying the significant QTL markers for tuber shape and eye depth were identified. CONCLUSIONS: Utilization of a molecular marker at the shape and eye depth QTL enabled the reclassification of the variation in general tuber shape to two main groups. Quantitative measurement of the length and width at different parts of the tuber is recommended to accompany the morphological descriptor classification to correctly capture the shape variation.

Structure and expression of GSL1 and GSL2 genes encoding gibberellin stimulated-like proteins in diploid and highly heterozygous tetraploid potato reveals their highly conserved and essential status.

BACKGROUND: GSL1 and GSL2, Gibberellin Stimulated-Like proteins (also known as Snakin-1 and Snakin-2), are cysteine-rich peptides from potato (Solanum tuberosum L.) with antimicrobial properties. Similar peptides in other species have been implicated in diverse biological processes and are hypothesised to play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. To help resolve the biological roles of GSL1 and GSL2 peptides we have undertaken an in depth analysis of the structure and expression of these genes in potato. RESULTS: We have characterised the full length genes for both GSL1 (chromosome 4) and GSL2 (chromosome 1) from diploid and tetraploid potato using the reference genome sequence of potato, coupled with further next generation sequencing of four highly heterozygous tetraploid cultivars. The frequency of SNPs in GSL1 and GSL2 were very low with only one SNP every 67 and 53 nucleotides in exon regions of GSL1 and GSL2, respectively. Analysis of comprehensive RNA-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. Expression analysis based on the frequency of next generation sequence reads established that GSL2 was expressed at a higher level than GSL1 in 30 out of 32 tissue and treatment libraries. Furthermore, both the GSL1 and GSL2 genes exhibited constitutive expression that was not up regulated in response to biotic or abiotic stresses, hormone treatments or wounding. Potato transformation with antisense knock-down expression cassettes failed to recover viable plants. CONCLUSIONS: The potato GSL1 and GSL2 genes are very highly conserved suggesting they contribute to an important biological function. The known antimicrobial activity of the GSL proteins, coupled with the FPKM analysis from RNA-seq data, implies that both genes contribute to the constitutive defence barriers in potatoes. The lethality of antisense knock-down expression of GSL1 and GSL2, coupled with the rare incidence of SNPs in these genes, suggests an essential role for this gene family. These features are consistent with the GSL protein family playing a role in several aspects of plant development in addition to plant defence against biotic stresses.

Genetic analyses of bolting in bulb onion (Allium cepa L.).

We present the first evidence for a QTL conditioning an adaptive trait in bulb onion, and the first linkage and population genetics analyses of candidate genes involved in photoperiod and vernalization physiology. Economic production of bulb onion (Allium cepa L.) requires adaptation to photoperiod and temperature such that a bulb is formed in the first year and a flowering umbel in the second. 'Bolting', or premature flowering before bulb maturation, is an undesirable trait strongly selected against by breeders during adaptation of germplasm. To identify genome regions associated with adaptive traits we conducted linkage mapping and population genetic analyses of candidate genes, and QTL analysis of bolting using a low-density linkage map. We performed tagged amplicon sequencing of ten candidate genes, including the FT-like gene family, in eight diverse populations to identify polymorphisms and seek evidence of differentiation. Low nucleotide diversity and negative estimates of Tajima's D were observed for most genes, consistent with purifying selection. Significant population differentiation was observed only in AcFT2 and AcSOC1. Selective genotyping in a large 'Nasik Red × CUDH2150' F2 family revealed genome regions on chromosomes 1, 3 and 6 associated (LOD > 3) with bolting. Validation genotyping of two F2 families grown in two environments confirmed that a QTL on chromosome 1, which we designate AcBlt1, consistently conditions bolting susceptibility in this cross. The chromosome 3 region, which coincides with a functionally characterised acid invertase, was not associated with bolting in other environments, but showed significant association with bulb sucrose content in this and other mapping pedigrees. These putative QTL and candidate genes were placed on the onion map, enabling future comparative studies of adaptive traits.

Visual Integration of Genome-Wide Association Studies and Differential Expression Results with the Hidecan R Package.

BACKGROUND/OBJECTIVES: We present hidecan, an R package for generating visualisations that summarise the results of one or more genome-wide association studies (GWAS) and differential expression analyses, as well as manually curated candidate genes, e.g., extracted from the literature. This tool is applicable to all ploidy levels; we notably provide functionalities to facilitate the visualisation of GWAS results obtained for autotetraploid organisms with the GWASpoly package. RESULTS: We illustrate the capabilities of hidecan with examples from two autotetraploid potato datasets. CONCLUSIONS: The hidecan package is implemented in R and is publicly available on the CRAN repository and on GitHub. A description of the package, as well as a detailed tutorial, is made available alongside the package. It is also part of the VIEWpoly tool for the visualisation and exploration of results from polyploids computational tools.

Effect of a dietitian-managed bone algorithm on serum phosphorus level in maintenance hemodialysis patients.

OBJECTIVE: This study examined the effectiveness of a registered dietitian (RD)-managed bone metabolism algorithm compared with a non-RD (registered nurse and the nephrologist)-managed one on serum phosphorus (PO4) and related clinical outcomes (corrected serum calcium [cCa] level, intact parathyroid hormone [iPTH] level, incidence of parathyroidectomy) among in-center maintenance hemodialysis (MHD) patients. DESIGN AND SETTING: The study was an 18-month retrospective review of adult MHD patients (n = 252) at 5 outpatient dialysis centers in western Massachusetts and Connecticut before and after change in the management of a comprehensive bone metabolism treatment algorithm (intravenous vitamin D, phosphate-binding medication, calcimimetic) from non-RD to RD. Calendar-matched timepoints representing 3-month averages during the non-RD- and RD-managed periods of the same algorithm were used for analyses. Comparisons of outcomes at non-RD-managed timepoint 2 (February 2009-April 2009) and RD-managed timepoint 6 (February 2010-April 2010) were performed considering potential demographic and clinical confounders. RESULTS: On average, serum PO4 level was lower during the RD-managed timepoint 6 (5.17 ± 1.23 mg/dL; mean ± standard deviation) compared with non-RD-managed timepoint 2 (5.23 ± 1.24 mg/dL), although the difference between these calendar-matched timepoints was not statistically significant (F = .108, P = .74) after controlling for age, dietary intake (equilibrated normalized protein catabolic rate), and dialysis adequacy (equilibrated Kdrt/V). Mean cCa at RD-managed timepoint 6 (8.76 ± 0.65 mg/dL) was not significantly different from non-RD-managed timepoint 2 (8.79 ± 0.74), and the difference between serum iPTH level at timepoint 6 (363.0 ± 296.8 pg/mL) compared with timepoint 2 (319.8 ± 251.5 pg/mL) was nonsignificant (F = .650, P = .42) after controlling for age. There were fewer parathyroidectomies during the RD-managed period (0.8%) compared with the non-RD-managed period (1.6%). CONCLUSIONS: RDs may be equally effective as non-RDs in bone metabolism algorithm management with respect to serum PO4, cCa, and iPTH control in MHD patients. Further research is needed to prospectively evaluate the effect of RD management on these bone mineral outcomes.

A toolkit for bulk PCR-based marker design from next-generation sequence data: application for development of a framework linkage map in bulb onion (Allium cepa L.).

BACKGROUND: Although modern sequencing technologies permit the ready detection of numerous DNA sequence variants in any organisms, converting such information to PCR-based genetic markers is hampered by a lack of simple, scalable tools. Onion is an example of an under-researched crop with a complex, heterozygous genome where genome-based research has previously been hindered by limited sequence resources and genetic markers. RESULTS: We report the development of generic tools for large-scale web-based PCR-based marker design in the Galaxy bioinformatics framework, and their application for development of next-generation genetics resources in a wide cross of bulb onion (Allium cepa L.). Transcriptome sequence resources were developed for the homozygous doubled-haploid bulb onion line 'CUDH2150' and the genetically distant Indian landrace 'Nasik Red', using 454™ sequencing of normalised cDNA libraries of leaf and shoot. Read mapping of 'Nasik Red' reads onto 'CUDH2150' assemblies revealed 16836 indel and SNP polymorphisms that were mined for portable PCR-based marker development. Tools for detection of restriction polymorphisms and primer set design were developed in BioPython and adapted for use in the Galaxy workflow environment, enabling large-scale and targeted assay design. Using PCR-based markers designed with these tools, a framework genetic linkage map of over 800cM spanning all chromosomes was developed in a subset of 93 F(2) progeny from a very large F(2) family developed from the 'Nasik Red' x 'CUDH2150' inter-cross. The utility of tools and genetic resources developed was tested by designing markers to transcription factor-like polymorphic sequences. Bin mapping these markers using a subset of 10 progeny confirmed the ability to place markers within 10 cM bins, enabling increased efficiency in marker assignment and targeted map refinement. The major genetic loci conditioning red bulb colour (R) and fructan content (Frc) were located on this map by QTL analysis. CONCLUSIONS: The generic tools developed for the Galaxy environment enable rapid development of sets of PCR assays targeting sequence variants identified from Illumina and 454 sequence data. They enable non-specialist users to validate and exploit large volumes of next-generation sequence data using basic equipment.

Allele diversity for the apoplastic invertase inhibitor gene from potato.

In planta the enzymatic activity of apoplastic and vacuolar invertases is controlled by inhibitory proteins. Although these invertase inhibitors (apoplastic and vacuolar forms) have been implicated as contributing to resistance to cold-induced sweetening (CIS) in tubers of potato (Solanum tuberosum L.), there is a lack of information on the structure and allelic diversity of the apoplastic invertase inhibitor genes. We have PCR-isolated and sequenced the alleles of the apoplastic invertase inhibitor gene (Stinh1) from three tetraploid potato genotypes: 1021/1 (a genotype with very high tolerance to CIS), 'Karaka' and 'Summer Delight' (two cultivars that are highly susceptible to CIS). In total, five alleles were identified in these genotypes, of which four (Stinh1-c, Stinh1-d, Stinh1-e, Stinh1-f) were novel. An analysis of allele diversity was conducted by incorporating previously published sequences of apoplastic invertase inhibitors from potato. Eight alleles were assessed for sequence polymorphism in the two exons and the single hypervariable intron. Contrary to the hypervariable intron, only 65 single nucleotide polymorphisms were observed in the exons, of which 42 confer amino acid substitutions. Phylogenetic analysis of amino acid sequences indicates that the alleles of the invertase inhibitor are highly conserved amongst members of the Solanaceae family.

Understanding suicide clusters through exploring self-harm: Semi-structured interviews with individuals presenting with near-fatal self-harm during a suicide cluster.

There was a highly publicised cluster of at least ten suicides in South Wales, United Kingdom, in 2007-2008. We carried out a qualitative descriptive study using cross-case thematic analysis to investigate the experiences and narratives of eight individuals who lived in the area where the cluster occurred and who survived an episode of near-fatal self-harm at the time of the cluster. Interviews were conducted from 01.01.2015 to 31.12.2015. All interviewees denied that the other deaths in the area had affected their own suicidal behaviour. However, in other sections of the interviews they spoke about the cluster contributing to difficulties they were experiencing at the time, including damage to social relationships, feelings of loss and being out of control. When asked about support, the interviewees emphasized the importance of counselling, which they would have found helpful but in most cases did not receive, even in the case of close contacts of individuals who had died. The findings suggest that effective prevention messaging must be subtle, since those affected may not be explicitly aware of or acknowledge the imitative aspects of their behaviour. This could be related to stigma attached to suicidal behaviour in a cluster context. Lessons for prevention include changing the message from asking if people 'have been affected by' the suicide deaths to emphasising the preventability of suicide, and directly reaching out to individuals rather than relying on people to come forward.

High-density linkage map construction in an autotetraploid blueberry population and detection of quantitative trait loci for anthocyanin content.

Highbush blueberry (Vaccinium corymbosum, 2n = 4x = 48) is the most cultivated type of blueberry, both in New Zealand and overseas. Its perceived nutritional value is conferred by phytonutrients, particularly anthocyanins. Identifying the genetic mechanisms that control the biosynthesis of these metabolites would enable faster development of cultivars with improved fruit qualities. Here, we used recently released tools for genetic mapping in autotetraploids to build a high-density linkage map in highbush blueberry and to detect quantitative trait loci (QTLs) for fruit anthocyanin content. Genotyping was performed by target sequencing, with ∼18,000 single nucleotide polymorphism (SNP) markers being mapped into 12 phased linkage groups (LGs). Fruits were harvested when ripe for two seasons and analyzed with high-performance liquid chromatography-mass spectrometry (HPLC-MS): 25 different anthocyanin compounds were identified and quantified. Two major QTLs that were stable across years were discovered, one on LG2 and one on LG4, and the underlying candidate genes were identified. Interestingly, the presence of anthocyanins containing acylated sugars appeared to be under strong genetic control. Information gained in this study will enable the design of molecular markers for marker-assisted selection and will help build a better understanding of the genetic control of anthocyanin biosynthesis in this crop.