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Aim: Our goal was to describe a precision medicine program in a regional academic hospital, characterize features of included patients and present early data on clinical impact. Materials and methods: We prospectively included 163 eligible patients with late-stage cancer of any diagnosis from June 2020 to May 2022 in the Proseq Cancer trial. Molecular profiling of new or fresh frozen tumor biopsies was done by WES and RNAseq with parallel sequencing of non-tumoral DNA as individual reference. Cases were presented at a National Molecular Tumor Board (NMTB) for discussion of targeted treatment. Subsequently, patients were followed for at least 7 months. Results: 80% (N = 131) of patients had a successful analysis done, disclosing at least one pathogenic or likely pathogenic variant in 96%. A strongly or potentially druggable variant was found in 19% and 73% of patients, respectively. A germline variant was identified in 2.5%. Median time from trial inclusion to NMTB decision was one month. One third (N = 44) of patients who underwent molecularly profiling were matched with a targeted treatment, however, only 16% were either treated (N = 16) or are waiting for treatment (N = 5), deteriorating performance status being the primary cause of failure. A history of cancer among 1st degree relatives, and a diagnosis of lung or prostate cancer correlated with greater chance of targeted treatment being available. The response rate of targeted treatments was 40%, the clinical benefit rate 53%, and the median time on treatment was 3.8 months. 23% of patients presented at NMTB were recommended clinical trial participation, not dependent on biomarkers. Conclusions: Precision medicine in end-stage cancer patients is feasible in a regional academic hospital but should continue within the frame of clinical protocols as few patients benefit. Close collaboration with comprehensive cancer centers ensures expert evaluations and equality in access to early clinical trials and modern treatment.

ObjectivesTo facilitate clinical implementation and research in precision oncology, notably the pairing of patients, variants and treatments to identify candidates for clinical trials, we have built a data infrastructure to 1) capture and store data, 2) reduce manual tasks for clinical and genomic data collection and management, 3) combine data for quality controls, reporting and findability.InfrastructureThe infrastructure uses REDCap repositories to capture and store data. The structure of these repositories is customized for each project. Additionally, a cross-project web platform was developed using software development best practices and state-of-the-art web technologies to circumvent REDCap’s limitations and integrate other third-party resources. Using REDCap’s application programming interfaces, this platform allowed validation of data across multiple repositories, easy import of data from external sources, generation of overviews of included patients and available data, combination of genomic and clinical data to generate tumour board reports and the findability of data. Its design was driven by data stewardship best practices.UsageAcross four precision medicine projects, the infrastructure has been used to collect data for 1921 patients, including 453 genomic data files. The custom-built web platform made it possible to import, validate, and present data in a comprehensive manner. This included building tumour board reports for clinicians, combining clinical and genomic data, and search functionalities for researchers.DiscussionREDCap allowed us to capitalize on the numerous data capture and management features developed in this solution. Designing a cross-project platform guarantees long-term relevance where developments can be mutualised across projects and allowed us to make the overall solution more compliant with the FAIR (Findable, Accessible, Interoperable, Reusable) data principles. Further developments should be considered, notably automatic retrieval of data from electronic health records to limit the number of manual tasks.ConclusionThe proposed infrastructure allowed our precision oncology projects to gain efficiency in data collection and increase data quality by reducing manual work, and it gave a straightforward and customized access to data for researchers and clinicians.

Tuber dormancy and sprouting are commercially important potato traits as long-term tuber storage is necessary to ensure year-round availability. Premature dormancy release and sprout growth in tubers during storage can result in a significant deterioration in product quality. In addition, the main chemical sprout suppressant chlorpropham has been withdrawn in Europe, necessitating alternative approaches for controlling sprouting. Breeding potato cultivars with longer dormancy and slower sprout growth is a desirable goal, although this must be tempered by the needs of the seed potato industry, where dormancy break and sprout vigour are required for rapid emergence. We have performed a detailed genetic analysis of tuber sprout growth using a diploid potato population derived from two highly heterozygous parents. A dual approach employing conventional QTL analysis allied to a combined bulk-segregant analysis (BSA) using a novel potato whole-exome capture (WEC) platform was evaluated. Tubers were assessed for sprout growth in storage at six time-points over two consecutive growing seasons. Genetic analysis revealed the presence of main QTL on five chromosomes, several of which were consistent across two growing seasons. In addition, phenotypic bulks displaying extreme sprout growth phenotypes were subjected to WEC sequencing for performing BSA. The combined BSA and WEC approach corroborated QTL locations and served to narrow the associated genomic regions, while also identifying new QTL for further investigation. Overall, our findings reveal a very complex genetic architecture for tuber sprouting and sprout growth, which has implications both for potato and other root, bulb and tuber crops where long-term storage is essential.

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid neoplasm among adults,and approximately 30–40% of patients will experience relapse while 5–10% will suffer from primary refractory disease caused by different mechanisms, including treatment-induced resistance. For refractory and relapsed DLBCL (rrDLBCL) patients, early detection and understanding of the mechanisms controlling treatment resistance are of great importance to guide therapy decisions. Here, we have focused on genetic variations in immune surveillance genes in diagnostic DLBCL (dDLBCL) and rrDLBCL patients to elaborate on the suitability of new promising immunotherapies.MethodsBiopsies from 30 dDLBCL patients who did not progress or relapse during follow up and 17 rrDLBCL patients with refractory disease or who relapsed during follow up were analyzed by whole-exome sequencing, including matched individual germline samples to include only somatic genetic variants in downstream analysis of a curated list of 58 genes involved in major immune surveillance pathways.ResultsMore than 70% of both dDLBCLs and rrDLBCLs harbored alterations in immune surveillance genes, but rrDLBCL tumor samples have a lower number of genes affected compared to dDLBCL tumor samples. Increased gene mutation frequencies in rrDLBCLs were observed in more than half of the affected immune surveillance genes than dDLBCLs.ConclusionGenetic variants in the antigen-presenting genes affect a higher number of rrDLBCL patients supporting an important role for these genes in tumor progression and development of refractory disease and relapse.

Scotch broom (Cytisus scoparius) is a global invasive species disrupting native species and habitats in several parts of the world. Its natural distribution spans across Europe, but seeds have also been transferred between countries, mixing plants of different genetic origins. In Denmark, Scotch broom consists of two gene pools: one is found in relatively small populations and is considered native; the other one is presumably introduced and is spreading as a noxious weed. To assess climate thresholds controlling the geographic ranges of the two gene pools, we evaluated seasonal changes in freezing tolerance (electrolyte leakage) and underlying variations in carbohydrate metabolism (HPLC) and water status of genetically different source populations representing the two gene pools. The data reveal that the introduced type is less freezing tolerant than the native type with differences being greatest in early autumn and when plants are most cold hardy, lending support to the hypothesis that insufficient freezing tolerance currently limits range expansion of the introduced Danish Scotch broom. In both gene pools, parallel increases in freezing tolerance and concentrations of sucrose and raffinose and decreasing levels of starch indicate the mobilization of soluble sugars from storage carbohydrates to achieve maximum freezing tolerance. However, genotypic differences in freezing tolerance are not explained by differences in carbohydrate metabolism, but may be related to different water contents. The observed low temperature limit for the introduced C. scoparius could provide valuable information for building process-based species distribution models predicting its geographic distribution and range shift following climate change.

Within recent years, many precision cancer medicine initiatives have been developed. Most of these have focused on solid cancers, while the potential of precision medicine for patients with hematological malignancies, especially in the relapse situation, are less elucidated. Here, we present a demographic unbiased and observational prospective study at Aalborg University Hospital Denmark, referral site for 10% of the Danish population. We developed a hematological precision medicine workflow based on sequencing analysis of whole exome tumor DNA and RNA. All steps involved are outlined in detail, illustrating how the developed workflow can provide relevant molecular information to multidisciplinary teams. A group of 174 hematological patients with progressive disease or relapse was included in a non-interventional and population-based study, of which 92 patient samples were sequenced. Based on analysis of small nucleotide variants, copy number variants, and fusion transcripts, we found variants with potential and strong clinical relevance in 62% and 9.5% of the patients, respectively. The most frequently mutated genes in individual disease entities were in concordance with previous studies. We did not find tumor mutational burden or micro satellite instability to be informative in our hematologic patient cohort.

Compelling research has recently shown that cancer is so heterogeneous that single research centres cannot produce enough data to fit prognostic and predictive models of sufficient accuracy. Data sharing in precision oncology is therefore of utmost importance. The Findable, Accessible, Interoperable and Reusable (FAIR) Data Principles have been developed to define good practices in data sharing. Motivated by the ambition of applying the FAIR Data Principles to our own clinical precision oncology implementations and research, we have performed a systematic literature review of potentially relevant initiatives. For clinical data, we suggest using the Genomic Data Commons model as a reference as it provides a field-tested and well-documented solution. Regarding classification of diagnosis, morphology and topography and drugs, we chose to follow the World Health Organization standards, i.e. ICD10, ICD-O-3 and Anatomical Therapeutic Chemical classifications, respectively. For the bioinformatics pipeline, the Genome Analysis ToolKit Best Practices using Docker containers offer a coherent solution and have therefore been selected. Regarding the naming of variants, we follow the Human Genome Variation Society's standard. For the IT infrastructure, we have built a centralized solution to participate in data sharing through federated solutions such as the Beacon Networks.

Colonization of new habitats is expected to require genetic adaptations to overcome environmental challenges. Here we use full genome re-sequencing and extensive common garden experiments to investigate demographic and selective processes associated with the recent colonization of Japan byLotus japonicus. We carefully track the colonization process whereL. japonicusgradually spread from subtropical conditions to much colder climates in northern Japan. We characterize the loss of diversity during this process and identify genomic regions with extreme genetic differentiation. Next, we perform population structure-corrected association mapping of phenotypic traits measured in a common garden and discover a number of genome-wide significant associations. Contrasting these analyses, we find that there is a strong concordance between phenotypic variation and extreme differentiation for overwintering and flowering time traits. Our results provide evidence that these traits were direct targets of selection by local adaptation during the colonization process and point to associated candidate genes.

Potatoes and other Solanaceae species produce biologically active secondary metabolites called steroidal glycoalkaloids (GAs) which have antimicrobial, fungicidal, antiviral and insecticidal properties. GAs are, however, also toxic to animals and humans. Compared to wild species of potato, the elite cultivars primarily used for everyday consumption have very low contents of GAs. Breeding for important agronomical traits, like e.g. pathogen resistance, often requires the use of wild species and a situation where offspring have unacceptable high contents of GAs quite frequently arises. Knowledge of metabolic pathways leading to the synthesis of GAs, as well as of the genes that are responsible for the observed differences in plant and tuber GA content is only partial. The primary purpose of this study was to identify genomic regions and candidate genes responsible for differential GA content within a diploid potato mapping population (n = 90) that shows a high variation in GA accumulation. The analysis was performed using a novel method based on next generation genome sequencing. A region on chromosome 1 was found to be associated with differential GA content. Within that region, sterol 24-C-methyltransferase (SMT1), sterol desaturase (SD) and C-4 sterol methyl oxidase (SMO) genes were found, all encoding critical enzymes in the synthesis of the GAs precursor cholesterol. Potatoes and other Solanaceae species produce biologically active secondary metabolites called steroidal glycoalkaloids (GAs) which have antimicrobial, fungicidal, antiviral and insecticidal properties. GAs are, however, also toxic to animals and humans. Compared to wild species of potato, the elite cultivars primarily used for everyday consumption have very low contents of GAs. Breeding for important agronomical traits, like e.g. pathogen resistance, often requires the use of wild species and a situation where offspring have unacceptable high contents of GAs quite frequently arises. Knowledge of metabolic pathways leading to the synthesis of GAs, as well as of the genes that are responsible for the observed differences in plant and tuber GA content is only partial. The primary purpose of this study was to identify genomic regions and candidate genes responsible for differential GA content within a diploid potato mapping population (n = 90) that shows a high variation in GA accumulation. The analysis was performed using a novel method based on next generation genome sequencing. A region on chromosome 1 was found to be associated with differential GA content. Within that region, sterol 24-C-methyltransferase (SMT1), sterol desaturase (SD) and C-4 sterol methyl oxidase (SMO) genes were found, all encoding critical enzymes in the synthesis of the GAs precursor cholesterol.

Colonization of new habitats is expected to require genetic adaptations to overcome environmental challenges. Here, we use full genome re-sequencing and extensive common garden experiments to investigate demographic and selective processes associated with colonization of Japan by Lotus japonicus over the past ~20,000 years. Based on patterns of genomic variation, we infer the details of the colonization process where L. japonicus gradually spread from subtropical conditions to much colder climates in northern Japan. We identify genomic regions with extreme genetic differentiation between northern and southern subpopulations and perform population structure-corrected association mapping of phenotypic traits measured in a common garden. Comparing the results of these analyses, we find that signatures of extreme subpopulation differentiation overlap strongly with phenotype association signals for overwintering and flowering time traits. Our results provide evidence that these traits were direct targets of selection during colonization and point to associated candidate genes., Local adaptation contributes to plant colonization across extreme environmental gradients. Here, the authors reconstruct the colonization history of Lotus japonicus in Japan and identify extreme genetic signatures of local adaptation to a cold climate using genome resequencing and common garden experiments.

Despite the recent progress in treatment of multiple myeloma (MM), it is still an incurable malignant disease, and we are therefore in need of new risk stratification tools that can help us to understand the disease and optimize therapy. Here we propose a new subtyping of myeloma plasma cells (PCs) from diagnostic samples, assigned by normal B-cell subset associated gene signatures (BAGS). For this purpose, we combined fluorescence-activated cell sorting and gene expression profiles from normal bone marrow (BM) Pre-BI, Pre-BII, immature, naïve, memory, and PC subsets to generate BAGS for assignment of normal BM subtypes in diagnostic samples. The impact of the subtypes was analyzed in 8 available data sets from 1772 patients’ myeloma PC samples. The resulting tumor assignments in available clinical data sets exhibited similar BAGS subtype frequencies in 4 cohorts from de novo MM patients across 1296 individual cases. The BAGS subtypes were significantly associated with progression-free and overall survival in a meta-analysis of 916 patients from 3 prospective clinical trials. The major impact was observed within the Pre-BII and memory subtypes, which had a significantly inferior prognosis compared with other subtypes. A multiple Cox proportional hazard analysis documented that BAGS subtypes added significant, independent prognostic information to the translocations and cyclin D classification. BAGS subtype analysis of patient cases identified transcriptional differences, including a number of differentially spliced genes. We identified subtype differences in myeloma at diagnosis, with prognostic impact and predictive potential, supporting an acquired B-cell trait and phenotypic plasticity as a pathogenetic hallmark of MM.

BACKGROUND: Radiation-therapy (RT) induces mucositis, a clinically challenging condition with limited prophylactic interventions and no predictive tests. In this pilot study, we applied global gene-expression analysis on serial human oral mucosa tissue and blood cells from patients with tonsil squamous cell cancer (TSCC) to identify genes involved in mucositis pathogenesis.METHODS AND FINDINGS: Eight patients with TSCC each provided consecutive buccal biopsies and blood cells before, after 7 days of RT treatment, and 20 days following RT. We monitored clinical mucositis and performed gene-expression analysis on tissue samples. We obtained control tissue from nine healthy individuals. After RT, expression was upregulated in apoptosis inducer and inhibitor genes, EDA2R and MDM2, and in POLH, a DNA-repair polymerase. Expression was downregulated in six members of the histone cluster family, e.g., HIST1H3B. Gene expression related to proliferation and differentiation was altered, including MKI67 (downregulated), which encodes the Ki-67-proliferation marker, and KRT16 (upregulated), which encodes keratin16. These alterations were not associated with the clinical mucositis grade. However, the expression of LY6G6C, which encodes a surface immunoregulatory protein, was upregulated before treatment in three cases of clinical none/mild mucositis, but not in four cases of ulcerative mucositis.CONCLUSION: RT caused molecular changes related to apoptosis, DNA-damage, DNA-repair, and proliferation without a correlation to the severity of clinical mucositis. LY6G6C may be a potential protective biomarker for ulcerative mucositis. Based on these results, our study model of consecutive human biopsies will be useful in designing a prospective clinical validation trial to characterize molecular mucositis and identify predictive biomarkers.

Verticillium dahliae Kleb. is a pathogenic fungus causing wilting, chlorosis, and early dying in potato (Solanum tuberosum L.). Genetic mapping of resistance to V. dahliae was done using a diploid population of potato. The major quantitative trait locus (QTL) for Verticillium resistance was found on chromosome 5. The StCDF1 gene, controlling earliness of maturity and tuberization, was mapped within the interval. Another QTL on chromosome 9 co-localized with the Ve2 Verticillium wilt resistance gene marker. Epistasis analysis indicated that the loci on chromosomes 5 and 9 had a highly significant interaction, and that StCDF1 functioned downstream of Ve2. The StCDF1 alleles were sequenced and found to encode StCDF1.1 and StCDF1.3. Interaction between the Ve2 resistance allele and the StCDF1.3 was demonstrated, but not for StCDF1.1. Genome-wide expression QTL (eQTL) analysis was performed and genes with eQTL at the StCDF1 and Ve2 loci were both found to have similar functions involving the chloroplast, including photosynthesis, which declines in both maturity and Verticillium wilt. Among the gene ontology (GO) terms that were specific to genes with eQTL at the Ve2, but not the StCDF1 locus, were those associated with fungal defense. These results suggest that Ve2 controls fungal defense and reduces early dying in Verticillium wilt through affecting genetic pathway controlling tuberization timing.

Brown bears (Ursus arctos) hibernate for 5-7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 degrees C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general. Funding Agencies|Lundbech Foundation [R126-2012-12408]; Aalborg University

TFIIS is a transcript elongation factor that facilitates transcription by RNA polymerase II through blocks to elongation. Arabidopsis plants lacking TFIIS are affected in seed dormancy, which represents a block to complete germination under favourable conditions. We have comparatively profiled the transcript levels of seeds of tfIIs mutants and control plants. Among the differentially expressed genes, the DOG1 gene was identified that is a QTL for seed dormancy. The reduced expression of DOG1 in tfIIs seeds was confirmed by quantitative RT-PCR and Northern analyses, suggesting that down-regulation of DOG1 expression is involved in the seed dormancy phenotype of tfIIs mutants.

Potato (Solanum tuberosum L.) is the world's most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop., La lista completa de autores que integran el documento puede consultarse en el archivo., Facultad de Ciencias Exactas

Key message WUE phenotyping and subsequent QTL analysis revealed cytosolic GS genes importance for limiting N loss due to photorespiration under well-watered and well-fertilized conditions. Abstract Potato (Solanum tuberosum L.) closes its stomata at relatively low soil water deficits frequently encountered in normal field conditions resulting in unnecessary annual yield losses and extensive use of artificial irrigation. Therefore, unraveling the genetics underpinning variation in water use efficiency (WUE) of potato is important, but has been limited by technical difficulties in assessing the trait on individual plants and thus is poorly understood. In this study, a mapping population of potatoes has been robustly phenotyped, and considerable variation in WUE under well-watered conditions was observed. Two extreme WUE bulks of clones were identified and pools of genomic DNA from them as well as the parents were sequenced and mapped to reference potato genome. Following a novel data analysis approach, two highly resolved QTLs were found on chromosome 1 and 9. Interestingly, three genes encoding isoforms of cytosolic glutamine synthase were located in the QTL at chromosome 1 suggesting a major contribution of this enzyme to photosynthetic efficiency and thus WUE in potato. Indeed, Glutamine synthetase enzyme activity of leaf extracts was measured and found to be correlated with contrasting WUE phenotypes. Electronic supplementary material The online version of this article (doi:10.1007/s00122-015-2573-2) contains supplementary material, which is available to authorized users.

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker-based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new similar to 936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (similar to 93%) of the 723 Mb genome assembly and 37,482 (similar to 96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal pseudomolecules. Potato Genome Sequencing grant, UK; Scottish Government Rural and Environmental Science and Analytical Services Division (RESAS); Department for Environment, Food and Rural Affairs (DEFRA)Department for Environment, Food & Rural Affairs (DEFRA); Agriculture and Horticulture Development Board (AHDB)-Potato Council; Biotechnology and Biological Sciences Research Council (BBSRC)Biotechnology and Biological Sciences Research Council (BBSRC) [BB/F012640]; New Zealand Institute for Crop & Food Research Ltd Strategic Science Initiative; New Zealand Institute for Plant & Food Research Ltd Capability Fund, New Zealand; NMEA (Netherlands Ministry of Economic Affairs); CBSG (Centre for BioSystems Genomics); STW (Netherlands Technology Foundation), The Netherlands [07796]; Teagasc Core Funding; DAFF-Research Stimulus Fund, Ireland; International Potato Center (CIP-CGIAR)/CRP RTB, Peru; CONICYTComision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) [Fondap 1509007, PBCT-PSD-03]; CONICYT (Basal CMM); CIRIC INRIA; INIA-Ministry of Agriculture of Chile, Chile; FEMCIDI OEA [PE/09/02 MINCyT-CONCyTEC]; Instituto Nacional de Tecnologia Agropecuaria (INTA-Core Funds); Ministerio de Ciencia y Tecnologia (MINCyT), Argentina; Proyecto FEMCIDI-OEA [SEDI/AE-305 /09]; Proyecto Bilateral Argentina, Per; FINCyT [099-FINCyT-EQUIP-2009) / (076-FINCyT-PIN-2008)]; Prestamo BID [1663/OC-PE]; Instituto Nacional de Innovacion Agraria, Ministry of Agriculture of Peru; Peruvian Ministry of Agriculture, Technical Secretariat of coordination; CGIAR; Consejo Nacional de Ciencia, Tecnologia e Innovacion Tecnologica, Peru (CONCYTEC); Special Multilateral Fund of the Inter-American Council for Integral Development (FEMCIDI-Peru); Biotechnology and Biological Sciences Research CouncilBiotechnology and Biological Sciences Research Council (BBSRC) [BB/F012640/1] We thank Andrzej Kilian (Diversity Arrays Technology, Australia) for DArT genotyping of the DMDD mapping population. We acknowledge Peter E. Hedley and Clare Booth (The James Hutton Institute, UK) for help with SNP genotyping. We thank S. B. Divito (Instituto Nacional de Tecnologia Agropecuaria, Balcarce, Argentina) for technical assistance. We are also grateful to Luke Ramsay and Peter E. Hedley (The James Hutton Institute, UK) for comments on the manuscript. AFLP and WGP are (registered) trademarks owned by KeyGene N.V. We acknowledge the funding made available by the Potato Genome Sequencing grant, UK [Scottish Government Rural and Environmental Science and Analytical Services Division (RESAS), Department for Environment, Food and Rural Affairs (DEFRA), Agriculture and Horticulture Development Board (AHDB)-Potato Council, Biotechnology and Biological Sciences Research Council (BBSRC, Grant BB/F012640)]; New Zealand Institute for Crop & Food Research Ltd Strategic Science Initiative and the New Zealand Institute for Plant & Food Research Ltd Capability Fund, New Zealand; NMEA (Netherlands Ministry of Economic Affairs), CBSG (Centre for BioSystems Genomics), STW (Netherlands Technology Foundation grant 07796), The Netherlands; Teagasc Core Funding, DAFF-Research Stimulus Fund, Ireland; International Potato Center (CIP-CGIAR)/CRP RTB, Peru; CONICYT (Fondap 1509007, Basal CMM, PBCT-PSD-03), CIRIC INRIA, INIA-Ministry of Agriculture of Chile, Chile; FEMCIDI OEA, PE/09/02 MINCyT-CONCyTEC, 2010-2011, Instituto Nacional de Tecnologia Agropecuaria (INTA-Core Funds) and Ministerio de Ciencia y Tecnologia (MINCyT), Argentina; Proyecto FEMCIDI-OEA SEDI/AE-305 /09 (2008-2012), Proyecto Bilateral Argentina, Per; FINCyT (099-FINCyT-EQUIP-2009) / (076-FINCyT-PIN-2008), Prestamo BID no. 1663/OC-PE, Instituto Nacional de Innovacion Agraria, Ministry of Agriculture of Peru, Peruvian Ministry of Agriculture, Technical Secretariat of coordination with the CGIAR, Consejo Nacional de Ciencia, Tecnologia e Innovacion Tecnologica, Peru (CONCYTEC), Special Multilateral Fund of the Inter-American Council for Integral Development (FEMCIDI-Peru).

The traditional approach to potato breeding, the classical “mate and phenotype” approach is relatively costly and because phenotyping and growth capacity is limited, this are being slowly replaced by Marker Assisted Selection (MAS) breeding schemes. MAS is based on the presence of DNA polymorphic markers closely associated with trait of interest and as such allows screening of much larger effective populations in a cost-effective manner already at the seed or seedling stages. However, it requires discovery and development of relevant molecular markers, which up to recently has been both costly and time consuming process.Here, a novel method for Quantitative Trait Locus (QTL) analysis has been developed, that allows for development of specific markers by use of genomic sequence reads and the recently published reference genome sequence for potato. Prior to sequencing the mapping population is phenotyped for the trait of interest and contrasting bulks of individuals are formed. Following validation on a simulated dataset, the new method was applied to a mapping population of 186 dihaploid potato clones originating from a cross between 90-HAF-01 (S. tuberosum1) and 90-HAG-15 (S.tuberosum2 x S.sparsipilum), phenotyped for water use efficiency. This population has also previously been phenotyped for the total glycoalkaloid (TGA) content.

Background Malignant cells in tumours of B-cell origin account for 0.1% to 98% of the total cell content, depending on disease entity. Recently, gene expression profiles (GEPs) of B-cell lymphomas based on microarray technologies have contributed significantly to improved sub-classification and diagnostics. However, the varying degrees of malignant B-cell frequencies in analysed samples influence the interpretation of the GEPs. Based on emerging next-generation sequencing technologies (NGS) like tag sequencing (tag-seq) for GEP, it is expected that the detection of mRNA transcripts from malignant B-cells can be supplemented. This study provides a quantitative assessment and comparison of the ability of microarrays and tag-seq to detect mRNA transcripts from malignant B-cells. A model system was established by eight serial dilutions of the malignant B-cell lymphoma cell line, OCI-Ly8, into the embryonic kidney cell line, HEK293, prior to parallel analysis by exon microarrays and tag-seq. Results We identified 123 and 117 differentially expressed genes between pure OCI-Ly8 and HEK293 cells by exon microarray and tag-seq, respectively. There were thirty genes in common, and of those, most were B-cell specific. Hierarchical clustering from all dilutions based on the differentially expressed genes showed that neither technology could distinguish between samples with less than 1% malignant B-cells from non-B-cells. A novel statistical concept was developed to assess the ability to detect single genes for both technologies, and used to demonstrate an inverse proportional relationship with the sample purity. Of the 30 common genes, the detection capability of a representative set of three B-cell specific genes - CD74, HLA-DRA, and BCL6 - was analysed. It was noticed that at least 5%, 13% and 22% sample purity respectively was required for detection of the three genes by exon microarray whereas at least 2%, 4% and 51% percent sample purity of malignant B-cells were required for tag-seq detection. Conclusion A sample purity-dependent loss of the ability to detect genes for both technologies was demonstrated. Taq-seq, in comparison to exon microarray, required slightly less malignant B-cells in the samples analysed in order to detect the two most abundantly expressed of the selected genes. The results show that malignant cell frequency is an important variable, with fundamental impact when interpreting GEPs from both technologies.

We determined the genome-wide digital gene expression (DGE) profiles of primary acute lymphoblastic leukemia (ALL) cells from 21 patients taking advantage of `second-generation sequencing technology. Patients included in this study represent four cytogenetically distinct subtypes of B-cell precursor (BCP) ALL and T-cell lineage ALL (T-ALL). The robustness of DGE combined with supervised classification by nearest shrunken centroids (NSC) was validated experimentally and by comparison with published expression data for large sets of ALL samples. Genes that were differentially expressed between BCP ALL subtypes were enriched to distinct signaling pathways with dic(9;20) enriched to TP53 signaling, t(9;22) to interferon signaling, as well as high hyperdiploidy and t(12;21) to apoptosis signaling. We also observed antisense tags expressed from the non-coding strand of similar to 50% of annotated genes, many of which were expressed in a subtype-specific pattern. Antisense tags from 17 gene regions unambiguously discriminated between the BCP ALL and T-ALL subtypes, and antisense tags from 76 gene regions discriminated between the 4 BCP subtypes. We observed a significant overlap of gene regions with alternative polyadenylation and antisense transcription (Pless than1 x 10(-15)). Our study using DGE profiling provided new insights into the RNA expression patterns in ALL cells.

Potato breeding is a slow and costly affair, primarily done as a classical "mate and phenotype" approach using relatively small populations. In contrast, Marker Assisted Selection (MAS) allows cost-effective screening of much larger effective populations sizes because most of the offspring is discarded based on the absence of desired molecular marker already at the seed or seedling stage. However, the number of molecular markers known in potato with appropriate linkage to agronomical traits is presently insufficient to establish a comprehensive MAS breeding platform for potato.

Summary Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid-like structure verified. Partial sequencing by MS/MS combined with full genomic sequencing of the Pseudomonas strain identified the gene coding for the major subunit of the amyloid fibril, termed fapC. FapC contains a thrice repeated motif that differs from those previously found in curli fimbrins and prion proteins. The lack of aromatic residues in the repeat shows that aromatic side chains are not needed for efficient amyloid formation. In contrast, glutamine and asparagine residues seem to play a major role in amyloid formation as these are highly conserved in curli, prion proteins and FapC. fapC is conserved in many Pseudomonas strains including the opportunistic pathogen P. aeruginosa and is situated in a conserved operon containing six genes, of which one encodes a fapC homologue. Heterologous expression of the fapA–F operon in Escherichia coli BL21(DE3) resulted in a highly aggregative phenotype, showing that the operon is involved in biofilm formation.

Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid-like structure verified. Partial sequencing by MS/MS combined with full genomic sequencing of the Pseudomonas strain identified the gene coding for the major subunit of the amyloid fibril, termed fapC. FapC contains a thrice repeated motif that differs from those previously found in curli fimbrins and prion proteins. The lack of aromatic residues in the repeat shows that aromatic side chains are not needed for efficient amyloid formation. In contrast, glutamine and asparagine residues seem to play a major role in amyloid formation as these are highly conserved in curli, prion proteins and FapC. fapC is conserved in many Pseudomonas strains including the opportunistic pathogen P. aeruginosa and is situated in a conserved operon containing six genes, of which one encodes a fapC homologue. Heterologous expression of the fapA-F operon in Escherichia coli BL21(DE3) resulted in a highly aggregative phenotype, showing that the operon is involved in biofilm formation.

Population analyzed during the experiments originated from a cross between 90-HAF-01 (S.tuberosum1) and 90-HAG-15 (S.tuberosum2xS.sparsipilum), which was previously evaluated for total glycoalkaloid content (TGA)1. Responses of stomatal conductance to different combinations of light, humidity, CO2 concentration on leaf surface as well as temperature were analyzed and their consequences on water use efficiency were evaluated during the climate chamber experiments in 2010 and 2011.We have previously participated in development of a de novo sequence-based alternative to positional cloning (SHOREMap)2. Here we employ a new method that allows QTL mapping exclusively from genomic sequence reads and a single reference genome sequence3. The procedure was applied to the population for which the low and high WUE groups in respect to dry matter / water use and photosynthesis / transpiration were identified. Four bulks (two of high and two of low WUE) and the parents were subsequently sequenced to ~30x coverage. Non-random distribution of sequence read-based polymorphisms between the WUE pools and the parental pool delimited highly resolved regions on several chromosomes. Within these regions candidate genes were found, namely glutamate ammonia ligase and proline synthase, that are presently being analyzed.1. Sorensen, K.K. et al. Theor Appl Genet (2008).2. Schneeberger, K. et al. Nature Methods 6: 550-551 (2009).3. The potato genome sequencing consortium, Nature 475: 189-195 (2011).