Isolation of Cells from Glioblastoma Multiforme Grade 4 Tumors for Infection with Zika Virus prME and ME Pseudotyped HIV-1.

This study aimed to isolate cells from grade 4 glioblastoma multiforme tumors for infection experiments with Zika virus (ZIKV) prME or ME enveloped HIV-1 pseudotypes. The cells obtained from tumor tissue were successfully cultured in human cerebrospinal fluid (hCSF) or a mixture of hCSF/DMEM in cell culture flasks with polar and hydrophilic surfaces. The isolated tumor cells as well as the U87, U138, and U343 cells tested positive for ZIKV receptors Axl and Integrin αvß5. Pseudotype entry was detected by the expression of firefly luciferase or green fluorescent protein (gfp). In prME and ME pseudotype infections, luciferase expression in U-cell lines was 2.5 to 3.5 logarithms above the background, but still two logarithms lower than in the VSV-G pseudotype control. Infection of single cells was successfully detected in U-cell lines and isolated tumor cells by gfp detection. Even though prME and ME pseudotypes had low infection rates, pseudotypes with ZIKV envelopes are promising candidates for the treatment of glioblastoma.

Insights into breeding history, hotspot regions of selection, and untapped allelic diversity for bread wheat breeding.

Breeding has increasingly altered the genetics of crop plants since the domestication of their wild progenitors. It is postulated that the genetic diversity of elite wheat breeding pools is too narrow to cope with future challenges. In contrast, plant genetic resources (PGRs) of wheat stored in genebanks are valuable sources of unexploited genetic diversity. Therefore, to ensure breeding progress in the future, it is of prime importance to identify the useful allelic diversity available in PGRs and to transfer it into elite breeding pools. Here, a diverse collection consisting of modern winter wheat cultivars and genebank accessions was investigated based on reduced-representation genomic sequencing and an iSelect single nucleotide polymorphism (SNP) chip array. Analyses of these datasets provided detailed insights into population structure, levels of genetic diversity, sources of new allelic diversity, and genomic regions affected by breeding activities. We identified 57 regions representing genomic signatures of selection and 827 regions representing private alleles associated exclusively with genebank accessions. The presence of known functional wheat genes, quantitative trait loci, and large chromosomal modifications, i.e., introgressions from wheat wild relatives, provided initial evidence for putative traits associated within these identified regions. These findings were supported by the results of ontology enrichment analyses. The results reported here will stimulate further research and promote breeding in the future by allowing for the targeted introduction of novel allelic diversity into elite wheat breeding pools.

Recommendations for the formatting of Variant Call Format (VCF) files to make plant genotyping data FAIR.

In this opinion article, we discuss the formatting of files from (plant) genotyping studies, in particular the formatting of (meta-) data in Variant Call Format (VCF) files. The flexibility of the VCF format specification facilitates its use as a generic interchange format across domains but can lead to inconsistency between files in the presentation of metadata. To enable fully autonomous machine actionable data flow, generic elements need to be further specified. We strongly support the merits of the FAIR principles and see the need to facilitate them also through technical implementation specifications. VCF files are an established standard for the exchange and publication of genotyping data. Other data formats are also used to capture variant call data (for example, the HapMap format and the gVCF format), but none currently have the reach of VCF. In VCF, only the sites of variation are described, whereas in gVCF, all positions are listed, and confidence values are also provided. For the sake of simplicity, we will only discuss VCF and our recommendations for its use. However, the part of the VCF standard relating to metadata (as opposed to the actual variant calls) defines a syntactic format but no vocabulary, unique identifier or recommended content. In practice, often only sparse (if any) descriptive metadata is included. When descriptive metadata is provided, proprietary metadata fields are frequently added that have not been agreed upon within the community which may limit long-term and comprehensive interoperability. To address this, we propose recommendations for supplying and encoding metadata, focusing on use cases from the plant sciences. We expect there to be overlap, but also divergence, with the needs of other domains.

DivBrowse-interactive visualization and exploratory data analysis of variant call matrices.

BACKGROUND: The sequencing of whole genomes is becoming increasingly affordable. In this context, large-scale sequencing projects are generating ever larger datasets of species-specific genomic diversity. As a consequence, more and more genomic data need to be made easily accessible and analyzable to the scientific community. FINDINGS: We present DivBrowse, a web application for interactive visualization and exploratory analysis of genomic diversity data stored in Variant Call Format (VCF) files of any size. By seamlessly combining BLAST as an entry point together with interactive data analysis features such as principal component analysis in one graphical user interface, DivBrowse provides a novel and unique set of exploratory data analysis capabilities for genomic biodiversity datasets. The capability to integrate DivBrowse into existing web applications supports interoperability between different web applications. Built-in interactive computation of principal component analysis allows users to perform ad hoc analysis of the population structure based on specific genetic elements such as genes and exons. Data interoperability is supported by the ability to export genomic diversity data in VCF and General Feature Format 3 files. CONCLUSION: DivBrowse offers a novel approach for interactive visualization and analysis of genomic diversity data and optionally also gene annotation data by including features like interactive calculation of variant frequencies and principal component analysis. The use of established standard file formats for data input supports interoperability and seamless deployment of application instances based on the data output of established bioinformatics pipelines.

Unlocking big data doubled the accuracy in predicting the grain yield in hybrid wheat.

The potential of big data to support businesses has been demonstrated in financial services, manufacturing, and telecommunications. Here, we report on efforts to enter a new data era in plant breeding by collecting genomic and phenotypic information from 12,858 wheat genotypes representing 6575 single-cross hybrids and 6283 inbred lines that were evaluated in six experimental series for yield in field trials encompassing ~125,000 plots. Integrating data resulted in twofold higher prediction ability compared with cases in which hybrid performance was predicted across individual experimental series. Our results suggest that combining data across breeding programs is a particularly appropriate strategy to exploit the potential of big data for predictive plant breeding. This paradigm shift can contribute to increasing yield and resilience, which is needed to feed the growing world population.

Implementing FAIR data management within the German Network for Bioinformatics Infrastructure (de.NBI) exemplified by selected use cases.

This article describes some use case studies and self-assessments of FAIR status of de.NBI services to illustrate the challenges and requirements for the definition of the needs of adhering to the FAIR (findable, accessible, interoperable and reusable) data principles in a large distributed bioinformatics infrastructure. We address the challenge of heterogeneity of wet lab technologies, data, metadata, software, computational workflows and the levels of implementation and monitoring of FAIR principles within the different bioinformatics sub-disciplines joint in de.NBI. On the one hand, this broad service landscape and the excellent network of experts are a strong basis for the development of useful research data management plans. On the other hand, the large number of tools and techniques maintained by distributed teams renders FAIR compliance challenging.

Identification of rye B chromosome-associated peptides by mass spectrometry.

B chromosomes (Bs) are supernumerary dispensable components of the standard genome (A chromosomes, As) that have been found in many eukaryotes. So far, it is unkown whether the B-derived transcripts translate to proteins or if the host proteome is changed due to the presence of Bs. Comparative mass spectrometry was performed using the protein samples isolated from shoots of rye plants with and without Bs. We aimed to identify B-associated peptides and analyzed the effects of Bs on the total proteome. Our comparative proteome analysis demonstrates that the presence of rye Bs affects the total proteome including different biological function processes. We found 319 of 16 776 quantified features in at least three out of five +B plants but not in 0B plants; 31 of 319 features were identified as B-associated peptide features. According to our data mining, one B-specific protein fragment showed similarity to a glycine-rich RNA binding protein which differed from its A-paralogue by two amino acid insertions. Our result represents a milestone in B chromosome research, because this is the first report to demonstrate the existence of Bs changing the proteome of the host.

Comparison Between Core Set Selection Methods Using Different Illumina Marker Platforms: A Case Study of Assessment of Diversity in Wheat.

Collections of plant genetic resources stored in genebanks are an important source of genetic diversity for improvement in plant breeding programs and for conservation of natural variation. The establishment of reduced representative collections from a large set of genotypes is a valuable tool that provides cost-effective access to the diversity present in the whole set. Software like Core Hunter 3 is available to generate high quality core sets. In addition, general clustering approaches, e.g., k-medoids, are available to subdivide a large data set into small groups with maximum genetic diversity between groups. Illumina genotyping platforms are a very efficient tool for the assessment of genetic diversity of plant genetic resources. The accumulation of genotyping data over time using commercial genotyping platforms raises the question of how such huge amount of information can be efficiently used for creating core collections. In the present study, after developing a 15K wheat Infinium array with 12,908 SNPs and genotyping a set of 479 hexaploid winter wheat lines (Triticum aestivum), a larger data set was created by merging 411 lines previously genotyped with the 90K iSelect array. Overlaying the markers from the 15K and 90K arrays enabled the identification of a common set of 12,806 markers, suggesting that the 15K array is a valuable and cost-effective resource for plant breeding programs. Finally, we selected genetically diverse core sets out of these 890 wheat genotypes derived from five collections based on the common markers from the 15K and 90K SNP arrays. Two different approaches, k-medoids and Core Hunter 3 were compared,and k-medoids was identified as an efficient method for selecting small core sets out of a large collection of genotypes while retaining the genetic diversity of the original population.

BRIDGE - A Visual Analytics Web Tool for Barley Genebank Genomics.

Genebanks harbor a large treasure trove of untapped plant genetic diversity. A growing world population and a changing climate require an increase in the production and development of stress resistant plant cultivars while decreasing the acreage. These requirements for improved plant cultivars can be supported by the broader exploitation of plant genetic resources (PGR) as inputs for genomics-assisted breeding. To support this process we have developed BRIDGE, a data warehouse and exploratory data analysis tool for genebank genomics of barley (Hordeum vulgare L.). Using efficient technologies for data storage, data transfer and web development, we facilitate access to digital genebank resources of barley by prioritizing the interactive and visual analysis of integrated genotypic and phenotypic data. The underlying data resulted from a barley genebank genomics study cataloging sequence and morphological data of 22,626 barley accessions, mainly from the German Federal ex situ genebank. BRIDGE consists of interactively coupled modules to visualize integrated, curated and quality checked data, such as variation data, results of dimensionality reduction and genome wide association studies (GWAS), phenotyping results, passport data as well as the geographic distribution of germplasm samples. The core component is a manager for custom collections of germplasm. A search module to find and select germplasm by passport and phenotypic attributes is included as well as modules to export genotypic data in gzip-compressed variant call format (VCF) files and phenotypic data in MIAPPE-compliant ISA-Tab files. BRIDGE is accessible at the following URL: https://bridge.ipk-gatersleben.de.

Negative dominance and dominance-by-dominance epistatic effects reduce grain-yield heterosis in wide crosses in wheat.

The genetics underlying heterosis, the difference in performance of crosses compared with midparents, is hypothesized to vary with relatedness between parents. We established a unique germplasm comprising three hybrid wheat sets differing in the degree of divergence between parents and devised a genetic distance measure giving weight to heterotic loci. Heterosis increased steadily with heterotic genetic distance for all 1903 hybrids. Midparent heterosis, however, was significantly lower in the hybrids including crosses between elite and exotic lines than in crosses among elite lines. The analysis of the genetic architecture of heterosis revealed this to be caused by a higher portion of negative dominance and dominance-by-dominance epistatic effects. Collectively, these results expand our understanding of heterosis in crops, an important pillar toward global food security.

Suitability of Single-Nucleotide Polymorphism Arrays Versus Genotyping-By-Sequencing for Genebank Genomics in Wheat.

Genebank genomics promises to unlock valuable diversity for plant breeding but first, one key question is which marker system is most suitable to fingerprint entire genebank collections. Using wheat as model species, we tested for the presence of an ascertainment bias and investigated its impact on estimates of genetic diversity and prediction ability obtained using three marker platforms: simple sequence repeat (SSR), genotyping-by-sequencing (GBS), and array-based SNP markers. We used a panel of 378 winter wheat genotypes including 190 elite lines and 188 plant genetic resources (PGR), which were phenotyped in multi-environmental trials for grain yield and plant height. We observed an ascertainment bias for the array-based SNP markers, which led to an underestimation of the molecular diversity within the population of PGR. In contrast, the marker system played only a minor role for the overall picture of the population structure and precision of genome-wide predictions. Interestingly, we found that rare markers contributed substantially to the prediction ability. This combined with the expectation that valuable novel diversity is most likely rare suggests that markers with minor allele frequency deserve careful consideration in the design of a pre-breeding program.

Kmasker plants - a tool for assessing complex sequence space in plant species.

Many plant genomes display high levels of repetitive sequences. The assembly of these complex genomes using short high-throughput sequence reads is still a challenging task. Underestimation or disregard of repeat complexity in these datasets can easily misguide downstream analysis. Detection of repetitive regions by k-mer counting methods has proved to be reliable. Easy-to-use applications utilizing k-mer counting are in high demand, especially in the domain of plants. We present Kmasker plants, a tool that uses k-mer count information as an assistant throughout the analytical workflow of genome data that is provided as a command-line and web-based solution. Beside its core competence to screen and mask repetitive sequences, we have integrated features that enable comparative studies between different cultivars or closely related species and methods that estimate target specificity of guide RNAs for application of site-directed mutagenesis using Cas9 endonuclease. In addition, we have set up a web service for Kmasker plants that maintains pre-computed indices for 10 of the economically most important cultivated plants. Source code for Kmasker plants has been made publically available at https://github.com/tschmutzer/kmasker. The web service is accessible at https://kmasker.ipk-gatersleben.de.

Exome association analysis sheds light onto leaf rust (Puccinia triticina) resistance genes currently used in wheat breeding (Triticum aestivum L.).

Resistance breeding is crucial for a sustainable control of leaf rust (Puccinia triticina) in wheat (Triticum aestivum L.) while directly targeting functional variants is the Holy Grail for efficient marker-assisted selection and map-based cloning. We assessed the limits and prospects of exome association analysis for severity of leaf rust in a large hybrid wheat population of 1574 single-crosses plus their 133 parents. After imputation and quality control, exome sequencing revealed 202 875 single-nucleotide polymorphisms (SNPs) covering 19.7% of the high-confidence annotated gene space. We performed intensive data mining and found significant associations for 2171 SNPs corresponding to 50 different loci. Some of these associations mapped in the proximity of the already known resistance genes Lr21, Lr34-B, Lr1 and Lr10, while other associated genomic regions, such as those on chromosomes 1A and 3D, harboured several annotated genes putatively involved in resistance. Validation with an independent population helped to narrow down the list of putative resistance genes that should be targeted by fine-mapping. We expect that the proposed strategy of intensive data mining coupled with validation will significantly influence research in plant genetics and breeding.

Detecting Large Chromosomal Modifications Using Short Read Data From Genotyping-by-Sequencing.

Markers linked to agronomic traits are of the prerequisite for molecular breeding. Genotyping-by-sequencing (GBS) data enables to detect small polymorphisms including single nucleotide polymorphisms (SNPs) and short insertions or deletions (InDels) that can be used, for instance, for marker-assisted selection, population genetics, and genome-wide association studies (GWAS). Here, we aim at detecting large chromosomal modifications in barley and wheat based on GBS data. These modifications could be duplications, deletions, substitutions including introgressions as well as alterations of DNA methylation. We demonstrate that GBS coverage analysis is capable to detect Hordeum vulgare/Hordeum bulbosum introgression lines. Furthermore, we identify large chromosomal modifications in barley and wheat collections. Hence, large chromosomal modifications, including introgressions and copy number variations (CNV), can be detected easily and can be used as markers in research and breeding without additional wet-lab experiments.

Leaf Variegation and Impaired Chloroplast Development Caused by a Truncated CCT Domain Gene in albostrians Barley.

Chloroplasts fuel plant development and growth by converting solar energy into chemical energy. They mature from proplastids through the concerted action of genes in both the organellar and the nuclear genome. Defects in such genes impair chloroplast development and may lead to pigment-deficient seedlings or seedlings with variegated leaves. Such mutants are instrumental as tools for dissecting genetic factors underlying the mechanisms involved in chloroplast biogenesis. Characterization of the green-white variegated albostrians mutant of barley (Hordeum vulgare) has greatly broadened the field of chloroplast biology, including the discovery of retrograde signaling. Here, we report identification of the ALBOSTRIANS gene HvAST (also known as Hordeum vulgare CCT Motif Family gene 7, HvCMF7) by positional cloning as well as its functional validation based on independently induced mutants by Targeting Induced Local Lesions in Genomes (TILLING) and RNA-guided clustered regularly interspaced short palindromic repeats-associated protein 9 endonuclease-mediated gene editing. The phenotypes of the independent HvAST mutants imply residual activity of HvCMF7 in the original albostrians allele conferring an imperfect penetrance of the variegated phenotype even at homozygous state of the mutation. HvCMF7 is a homolog of the Arabidopsis (Arabidopsis thaliana) CONSTANS, CO-like, and TOC1 (CCT) Motif transcription factor gene CHLOROPLAST IMPORT APPARATUS2, which was reported to be involved in the expression of nuclear genes essential for chloroplast biogenesis. Notably, in barley we localized HvCMF7 to the chloroplast, without any clear evidence for nuclear localization.

Mutations in the gene of the Gα subunit of the heterotrimeric G protein are the cause for the brachytic1 semi-dwarf phenotype in barley and applicable for practical breeding.

BACKGROUND: Short-culm mutants have been widely used in breeding programs to increase lodging resistance. In barley (Hordeum vulgare L.), several hundreds of short-culm mutants have been isolated over the years. The objective of the present study was to identify the Brachytic1 (Brh1) semi-dwarfing gene and to test its effect on yield and malting quality. RESULTS: Double-haploid lines generated through a cross between a brh1.a mutant and the European elite malting cultivar Quench, showed good malting quality but a decrease in yield. Especially the activities of the starch degrading enzymes ß-amylase and free limit dextrinase were high. A syntenic approach comparing markers in barley to those in rice (Oryza sativa L.), sorghum (Sorghum bicolor Moench) and brachypodium (Brachypodium distachyon P. Beauv) helped us to identify Brh1 as an orthologue of rice D1 encoding the Gα subunit of a heterotrimeric G protein. We demonstrated that Brh1 is allelic to Ari-m. Sixteen different mutant alleles were described at the DNA level. CONCLUSIONS: Mutants in the Brh1 locus are deficient in the Gα subunit of a heterotrimeric G protein, which shows that heterotrimeric G proteins are important regulators of culm length in barley. Mutant alleles do not have any major negative effects on malting quality.

Correlating the Genetic and Physical Map of Barley Chromosome 3H Revealed Limitations of the FISH-Based Mapping of Nearby Single-Copy Probes Caused by the Dynamic Structure of Metaphase Chromosomes.

Genetic maps are based on the recombination frequency of molecular markers which often show different positions in comparison to the corresponding physical maps. To decipher the position and order of DNA sequences genetically mapped to terminal and interstitial regions of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes was performed with 16 genomic single-copy probes derived from fingerprinted BAC contigs. Long genetic distances at subterminal regions translated into short physical distances, confirming that recombination events occur more often at distal regions of chromosome 3H. Nonoverlapping FISH signals were frequently obtained for probes with a physical distance of at least 30-60 kb. Only 8% of the analyzed chromosomes showed a symmetric order of FISH signals on both sister chromatids. Due to the dynamic packing of metaphase chromatin, the order of 2 adjacent single-copy signals along the chromosome arms outside the (peri)centromeric region can only reliably be determined if the cytological distance is approximately 3%, corresponding to 21.6 Mb.

Construction of a map-based reference genome sequence for barley, Hordeum vulgare L.

Barley (Hordeum vulgare L.) is a cereal grass mainly used as animal fodder and raw material for the malting industry. The map-based reference genome sequence of barley cv. 'Morex' was constructed by the International Barley Genome Sequencing Consortium (IBSC) using hierarchical shotgun sequencing. Here, we report the experimental and computational procedures to (i) sequence and assemble more than 80,000 bacterial artificial chromosome (BAC) clones along the minimum tiling path of a genome-wide physical map, (ii) find and validate overlaps between adjacent BACs, (iii) construct 4,265 non-redundant sequence scaffolds representing clusters of overlapping BACs, and (iv) order and orient these BAC clusters along the seven barley chromosomes using positional information provided by dense genetic maps, an optical map and chromosome conformation capture sequencing (Hi-C). Integrative access to these sequence and mapping resources is provided by the barley genome explorer (BARLEX).

A chromosome conformation capture ordered sequence of the barley genome.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.