Nanopore Sequencing with GraphMap for Comprehensive Pathogen Detection in Potato Field Soil.

Early detection of causal pathogens is important to prevent crop loss from diseases. However, some diseases, such as soilborne diseases, are difficult to diagnose due to the absence of visible or characteristic symptoms. In the present study, the use of the Oxford Nanopore MinION sequencer as a molecular diagnostic tool was assessed due to its long-read sequencing capabilities and portability. Nucleotide samples (DNA or RNA) from potato field soils were sequenced and analyzed using a locally curated pathogen database, followed by identification via sequence mapping. We performed computational speed tests of three commonly used mapping/annotation tools (BLAST, BWA-BLAST, and BWA-GraphMap) and found BWA-GraphMap to be the fastest tool for local searching against our curated pathogen database. The data collected demonstrate the high potential of Nanopore sequencing as a minimally biased diagnostic tool for comprehensive pathogen detection in soil from potato fields. Our GraphMap-based MinION sequencing method could be useful as a predictive approach for disease management by identifying pathogens present in field soil prior to planting. Although this method still needs further experimentation with a larger sample size for practical use, the data analysis pipeline presented can be applied to other cropping systems and diagnostics for detecting multiple pathogens.

15 years of GDR: New data and functionality in the Genome Database for Rosaceae.

The Genome Database for Rosaceae (GDR, https://www.rosaceae.org) is an integrated web-based community database resource providing access to publicly available genomics, genetics and breeding data and data-mining tools to facilitate basic, translational and applied research in Rosaceae. The volume of data in GDR has increased greatly over the last 5 years. The GDR now houses multiple versions of whole genome assembly and annotation data from 14 species, made available by recent advances in sequencing technology. Annotated and searchable reference transcriptomes, RefTrans, combining peer-reviewed published RNA-Seq as well as EST datasets, are newly available for major crop species. Significantly more quantitative trait loci, genetic maps and markers are available in MapViewer, a new visualization tool that better integrates with other pages in GDR. Pathways can be accessed through the new GDR Cyc Pathways databases, and synteny among the newest genome assemblies from eight species can be viewed through the new synteny browser, SynView. Collated single-nucleotide polymorphism diversity data and phenotypic data from publicly available breeding datasets are integrated with other relevant data. Also, the new Breeding Information Management System allows breeders to upload, manage and analyze their private breeding data within the secure GDR server with an option to release data publicly.

Directed construction and analysis of a Sinorhizobium meliloti pSymA deletion mutant library.

Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid libraries were used in a medium-throughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.

Blueberry and cranberry pangenomes as a resource for future genetic studies and breeding efforts.

Domestication of cranberry and blueberry began in the United States in the early 1800s and 1900s, respectively, and in part owing to their flavors and health-promoting benefits are now cultivated and consumed worldwide. The industry continues to face a wide variety of production challenges (e.g. disease pressures) as well as a demand for higher-yielding cultivars with improved fruit quality characteristics. Unfortunately, molecular tools to help guide breeding efforts for these species have been relatively limited compared with those for other high-value crops. Here, we describe the construction and analysis of the first pangenome for both blueberry and cranberry. Our analysis of these pangenomes revealed both crops exhibit great genetic diversity, including the presence-absence variation of 48.4% genes in highbush blueberry and 47.0% genes in cranberry. Auxiliary genes, those not shared by all cultivars, are significantly enriched with molecular functions associated with disease resistance and the biosynthesis of specialized metabolites, including compounds previously associated with improving fruit quality traits. The discovery of thousands of genes, not present in the previous reference genomes for blueberry and cranberry, will serve as the basis of future research and as potential targets for future breeding efforts. The pangenome, as a multiple-sequence alignment, as well as individual annotated genomes, are publicly available for analysis on the Genome Database for Vaccinium - a curated and integrated web-based relational database. Lastly, the core-gene predictions from the pangenomes will serve useful to develop a community genotyping platform to guide future molecular breeding efforts across the family.

Blueberry and cranberry pangenomes as a resource for future genetic studies and breeding efforts.

Domestication of cranberry and blueberry began in the United States in the early 1800s and 1900s, respectively, and in part owing to their flavors and health-promoting benefits are now cultivated and consumed worldwide. The industry continues to face a wide variety of production challenges (e.g. disease pressures), as well as a demand for higher-yielding cultivars with improved fruit quality characteristics. Unfortunately, molecular tools to help guide breeding efforts for these species have been relatively limited compared with those for other high-value crops. Here, we describe the construction and analysis of the first pangenome for both blueberry and cranberry. Our analysis of these pangenomes revealed both crops exhibit great genetic diversity, including the presence-absence variation of 48.4% genes in highbush blueberry and 47.0% genes in cranberry. Auxiliary genes, those not shared by all cultivars, are significantly enriched with molecular functions associated with disease resistance and the biosynthesis of specialized metabolites, including compounds previously associated with improving fruit quality traits. The discovery of thousands of genes, not present in the previous reference genomes for blueberry and cranberry, will serve as the basis of future research and as potential targets for future breeding efforts. The pangenome, as a multiple-sequence alignment, as well as individual annotated genomes, are publicly available for analysis on the Genome Database for Vaccinium-a curated and integrated web-based relational database. Lastly, the core-gene predictions from the pangenomes will serve useful to develop a community genotyping platform to guide future molecular breeding efforts across the family.

The Breeding Information Management System (BIMS): an online resource for crop breeding.

In this era of big data, breeding programs are producing ever larger amounts of data. This necessitates access to efficient management systems to keep track of cross, performance, pedigree, geographical and image-based data, as well as genotyping data. In this article, we report the progress on the Breeding Information Management System (BIMS), a free, secure and online breeding management system that allows breeders to store, manage, archive and analyze their private breeding data. BIMS is the first publicly available database system that enables individual breeders to integrate their private phenotypic and genotypic data with public data and, at the same time, have complete control of their own breeding data along with access to tools such as data import/export, data analysis and data archiving. The integration of breeding data with publicly available genomic and genetic data enhances genetic understanding of important traits and maximizes the marker-assisted breeding utility for breeders and allied scientists. BIMS incorporates the use of the Android App Field Book, open-source phenotype data collection software for phones and tablets that allows breeders to replace hard copy field books, thus alleviating the possibility of transcription errors while providing faster access to the collected data. BIMS comes with training materials and support for individual or small group training and is currently implemented in the Genome Database for Rosaceae, CottonGEN, the Citrus Genome Database, the Pulse Crop Database, and the Genome Database for Vaccinium. Database URLs: (https://www.rosaceae.org/), (https://www.cottongen.org/), (https://www.citrusgenomedb.org/), (https://www.pulsedb.org/) and (https://www.vaccinium.org/).

Tripal MegaSearch: a tool for interactive and customizable query and download of big data.

Tripal MegaSearch is a Tripal module for querying and downloading biological data stored in Chado. This module allows site users to select data types, restrict the dataset by applying various filters and then customizing fields to view and download through a single interface. Set by site administrators, example data types include gene, germplasm, marker, map, QTL, genotype, phenotype and expression data. When querying for genes, users can restrict the gene dataset using various filters such as name, chromosome position and functional annotation. They can then customize fields to download, such as name, organism, type, chromosome position, various functional annotations such as BLAST, KEGG, InterPro and GO term. FASTA files can also be downloaded for the sequence data. Site administrators can choose from two different data sources to serve data: Tripal MegaSearch materialized views or Chado tables. If neither data source is desired, administrators may also create their own materialized views and serve them through the flexible dynamic Tripal MegaSearch query form. Tripal MegaSearch is currently implemented in several databases including the Genome Database for Rosaceae www.rosaceae.org and TreeGenes www.https://treegenesdb.org/.

Defining the 'HoneySweet' insertion event utilizing NextGen sequencing and a de novo genome assembly of plum (Prunus domestica).

'HoneySweet' plum (Prunus domestica) is resistant to Plum pox potyvirus, through an RNAi-triggered mechanism. Determining the precise nature of the transgene insertion event has been complicated due to the hexaploid genome of plum. DNA blots previously indicated an unintended hairpin arrangement of the Plum pox potyvirus coat protein gene as well as a multicopy insertion event. To confirm the transgene arrangement of the insertion event, 'HoneySweet' DNA was subjected to whole genome sequencing using Illumina short-read technology. Results indicated two different insertion events, one containing seven partial copies flanked by putative plum DNA sequence and a second with the predicted inverted repeat of the coat protein gene driven by a double 35S promoter on each side, flanked by plum DNA. To determine the locations of the two transgene insertions, a phased plum genome assembly was developed from the commercial plum 'Improved French'. A subset of the scaffolds (2447) that were >10 kb in length and representing, >95% of the genome were annotated and used for alignment against the 'HoneySweet' transgene reads. Four of eight matching scaffolds spanned both insertion sites ranging from 157,704 to 654,883 bp apart, however we were unable to identify which scaffold(s) represented the actual location of the insertion sites due to potential sequence differences between the two plum cultivars. Regardless, there was no evidence of any gene(s) being interrupted as a result of the insertions. Furthermore, RNA-seq data verified that the insertions created no new transcriptional units and no dramatic expression changes of neighboring genes.

CottonGen: The Community Database for Cotton Genomics, Genetics, and Breeding Research.

Over the last eight years, the volume of whole genome, gene expression, SNP genotyping, and phenotype data generated by the cotton research community has exponentially increased. The efficient utilization/re-utilization of these complex and large datasets for knowledge discovery, translation, and application in crop improvement requires them to be curated, integrated with other types of data, and made available for access and analysis through efficient online search tools. Initiated in 2012, CottonGen is an online community database providing access to integrated peer-reviewed cotton genomic, genetic, and breeding data, and analysis tools. Used by cotton researchers worldwide, and managed by experts with crop-specific knowledge, it continuous to be the logical choice to integrate new data and provide necessary interfaces for information retrieval. The repository in CottonGen contains colleague, gene, genome, genotype, germplasm, map, marker, metabolite, phenotype, publication, QTL, species, transcriptome, and trait data curated by the CottonGen team. The number of data entries housed in CottonGen has increased dramatically, for example, since 2014 there has been an 18-fold increase in genes/mRNAs, a 23-fold increase in whole genomes, and a 372-fold increase in genotype data. New tools include a genetic map viewer, a genome browser, a synteny viewer, a metabolite pathways browser, sequence retrieval, BLAST, and a breeding information management system (BIMS), as well as various search pages for new data types. CottonGen serves as the home to the International Cotton Genome Initiative, managing its elections and serving as a communication and coordination hub for the community. With its extensive curation and integration of data and online tools, CottonGen will continue to facilitate utilization of its critical resources to empower research for cotton crop improvement.

Regulatory and DNA repair genes contribute to the desiccation resistance of Sinorhizobium meliloti Rm1021.

Sinorhizobium meliloti can form a nitrogen-fixing symbiotic relationship with alfalfa after bacteria in the soil infect emerging root hairs of the growing plant. To be successful at this, the bacteria must be able to survive in the soil between periods of active plant growth, including when conditions are dry. The ability of S. meliloti to withstand desiccation has been known for years, but genes that contribute to this phenotype have not been identified. Transposon mutagenesis was used in combination with novel screening techniques to identify four desiccation-sensitive mutants of S. meliloti Rm1021. DNA sequencing of the transposon insertion sites identified three genes with regulatory functions (relA, rpoE2, and hpr) and a DNA repair gene (uvrC). Various phenotypes of the mutants were determined, including their behavior on several indicator media and in symbiosis. All of the mutants formed an effective symbiosis with alfalfa. To test the hypothesis that UvrC-related excision repair was important in desiccation resistance, uvrA, uvrB, and uvrC deletion mutants were also constructed. These strains were sensitive to DNA damage induced by UV light and 4-NQO and were also desiccation sensitive. These data indicate that uvr gene-mediated DNA repair and the regulation of stress-induced pathways are important for desiccation resistance.

Structural and Functional Annotation of Eukaryotic Genomes with GenSAS.

The Genome Sequence Annotation Server (GenSAS, https://www.gensas.org ) is a secure, web-based genome annotation platform for structural and functional annotation, as well as manual curation. Requiring no installation by users, GenSAS integrates popular command line-based, annotation tools under a single, easy-to-use, online interface. GenSAS integrates JBrowse and Apollo, so users can view annotation data and manually curate gene models. Users are guided step by step through the annotation process by embedded instructions and a more in-depth GenSAS User's Guide. In addition to a genome assembly file, users can also upload organism-specific transcript, protein, and RNA-seq read evidence for use in the annotation process. The latest versions of the NCBI RefSeq transcript and protein databases and the SwissProt and TrEMBL protein databases are provided for all users. GenSAS projects can be shared with other GenSAS users enabling collaborative annotation. Once annotation is complete, GenSAS generates the final files of the annotated gene models in common file formats for use with other annotation tools, submission to a repository, and use in publications.

Tripal MapViewer: A tool for interactive visualization and comparison of genetic maps.

Tripal is an open-source, resource-efficient toolkit for construction of genomic, genetic and breeding databases. It facilitates development of biological websites by providing tools to integrate and display biological data using the generic database schema, Chado, together with Drupal, a popular website creation and content management system. Tripal MapViewer is a new interactive tool for visualizing genetic map data. Developed as a Tripal replacement for Comparative Map Viewer (CMap), it enables visualization of entire maps or linkage groups and features such as molecular markers, quantitative trait loci (QTLs) and heritable phenotypic markers. It also provides graphical comparison of maps sharing the same markers as well as dot plot and correspondence matrices. MapViewer integrates directly with the Tripal application programming interface framework, improving data searching capability and providing a more seamless experience for site visitors. The Tripal MapViewer interface can be integrated in any Tripal map page and linked from any Tripal page for markers, QTLs, heritable morphological markers or genes. Configuration of the display is available through a control panel and the administration interface. The administration interface also allows configuration of the custom database query for building materialized views, providing better performance and flexibility in the way data is stored in the Chado database schema. MapViewer is implemented with the D3.js technology and is currently being used at the Genome Database for Rosaceae (https://www.rosaceae.org), CottonGen (https://www.cottongen.org), Citrus Genome Database (https://citrusgenomedb.org), Vaccinium Genome Database (https://www.vaccinium.org) and Cool Season Food Legume Database (https://www.coolseasonfoodlegume.org). It is also currently in development on the Hardwood Genomics Web (https://hardwoodgenomics.org) and TreeGenes (https://treegenesdb.org). Database URL: https://gitlab.com/mainlabwsu/tripal_map.

Construction and expression of sugar kinase transcriptional gene fusions by using the Sinorhizobium meliloti ORFeome.

The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to beta-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput beta-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.

AgBioData consortium recommendations for sustainable genomics and genetics databases for agriculture.

The future of agricultural research depends on data. The sheer volume of agricultural biological data being produced today makes excellent data management essential. Governmental agencies, publishers and science funders require data management plans for publicly funded research. Furthermore, the value of data increases exponentially when they are properly stored, described, integrated and shared, so that they can be easily utilized in future analyses. AgBioData (https://www.agbiodata.org) is a consortium of people working at agricultural biological databases, data archives and knowledgbases who strive to identify common issues in database development, curation and management, with the goal of creating database products that are more Findable, Accessible, Interoperable and Reusable. We strive to promote authentic, detailed, accurate and explicit communication between all parties involved in scientific data. As a step toward this goal, we present the current state of biocuration, ontologies, metadata and persistence, database platforms, programmatic (machine) access to data, communication and sustainability with regard to data curation. Each section describes challenges and opportunities for these topics, along with recommendations and best practices.

Extension modules for storage, visualization and querying of genomic, genetic and breeding data in Tripal databases.

Tripal is an open-source database platform primarily used for development of genomic, genetic and breeding databases. We report here on the release of the Chado Loader, Chado Data Display and Chado Search modules to extend the functionality of the core Tripal modules. These new extension modules provide additional tools for (1) data loading, (2) customized visualization and (3) advanced search functions for supported data types such as organism, marker, QTL/Mendelian Trait Loci, germplasm, map, project, phenotype, genotype and their respective metadata. The Chado Loader module provides data collection templates in Excel with defined metadata and data loaders with front end forms. The Chado Data Display module contains tools to visualize each data type and the metadata which can be used as is or customized as desired. The Chado Search module provides search and download functionality for the supported data types. Also included are the tools to visualize map and species summary. The use of materialized views in the Chado Search module enables better performance as well as flexibility of data modeling in Chado, allowing existing Tripal databases with different metadata types to utilize the module. These Tripal Extension modules are implemented in the Genome Database for Rosaceae (rosaceae.org), CottonGen (cottongen.org), Citrus Genome Database (citrusgenomedb.org), Genome Database for Vaccinium (vaccinium.org) and the Cool Season Food Legume Database (coolseasonfoodlegume.org). Database URL: https://www.citrusgenomedb.org/, https://www.coolseasonfoodlegume.org/, https://www.cottongen.org/, https://www.rosaceae.org/, https://www.vaccinium.org/.

VirE1-Mediated Resistance to Crown Gall in Transgenic Arabidopsis thaliana.

ABSTRACT Crown gall disease, caused by Agrobacterium tumefaciens, remains a serious agricultural problem despite current biocontrol methods. Agrobacterium tumefaciens transfers single-stranded DNA (T-strands) into plant cells along with several virulence proteins, including a single-stranded DNA-binding protein (VirE2). In plant cells, T-strands are protected from nucleases and targeted to the nucleus by VirE2, which is essential for efficient transmission (transfer and integration) of T-strands. VirE1 is the secretory chaperone for VirE2; it prevents VirE2 from forming aggregates and from binding the T-strands in bacterial cells. Therefore, we hypothesized that sufficient quantities of VirE1 expressed in plant cells might block T-DNA transmission by preventing VirE2 from binding T-strands. Here we show that root explants from Arabidopsis thaliana plants that expressed virE1 formed 3.5-fold fewer tumors than roots from plants without virE1. Also, this resistance was specific for VirE2-mediated Agrobacterium transformation. Plants that have been genetically altered to resist crown gall may prove more effective than biological control.

Genome Sequence of Bibersteinia trehalosi Strain Y31 Isolated from the Pneumonic Lung of a Bighorn Sheep.

Here, we report the genome sequence for Bibersteinia trehalosi strain Y31, isolated from the lungs of a bighorn sheep (Ovis canadensis) that had succumbed to pneumonia, which exhibits proximity-dependent inhibition (PDI) of Mannheimia haemolytica The sequence will be used to understand the mechanism of PDI for these organisms.

Complete genome of the switchgrass endophyte Enterobacter clocace P101.

The Enterobacter cloacae complex is genetically very diverse. The increasing number of complete genomic sequences of E. cloacae is helping to determine the exact relationship among members of the complex. E. cloacae P101 is an endophyte of switchgrass (Panicum virgatum) and is closely related to other E. cloacae strains isolated from plants. The P101 genome consists of a 5,369,929 bp chromosome. The chromosome has 5,164 protein-coding regions, 100 tRNA sequences, and 8 rRNA operons.

Nuclease released by Verticillium dahliae is a signal for non-host resistance.

A DNase released from the fungal pathogen of bean, Fusarium solani f. sp. phaseoli (Fsph), was previously shown to signal the activation of total disease resistance and activate pathogenesis-related (PR) genes in pea. Data in the current study which used the pea-endocarp model to research non-host resistance, indicated that DNase released by Verticillium dahliae (Vd), pathogenic on potato also has non-host resistance-inducing capabilities in peas. Other strains of Vd that release DNase are pathogenic on other plant species. DNase catalytic activity was also released from representative genera of other pathogenic fungi. Purified VdDNase induced pisatin and pea gene DRR49 (PR-10 gene) in pea endocarp tissue. VdDNase reduced the in vitro growth of Vd but completely inhibited that of F. solani f. sp. pisi (Fspi) and a Colletotrichum pathogen of potato. VdDNase (2 units) applied to pea endocarp tissue both broke resistance to Fsph and increased resistance to Fspi. Pea DNA damage generated both by the VdDNase enzyme and the intact Vd spores indicated that the host DNA alteration is a component of the non-host resistance response (innate immunity). These data support the previously reported inductive potential of fungal DNase and further implicate fungal DNases as signals in activating non-host resistance responses.