Andrew J. Robinson

New computational tools for Brassica genome research

With the increasing quantities of Brassica genomic data being entered into the public domain and in preparation for the complete Brassica genome sequencing effort, there is a growing requirement for the structuring and detailed bioinformatic analysis of Brassica genomic information within a user-friendly database. At the Plant Biotechnology Centre, Melbourne, Australia, we have developed a series of tools and computational pipelines to assist in the processing and structuring of genomic data, to aid its application to agricultural biotechnology research. These tools include a sequence database, ASTRA, a sequence processing pipeline incorporating annotation against GenBank, SwissProt and Arabidopsis Gene Ontology (GO) data and tools for molecular marker discovery and comparative genome analysis. All sequences are mined for simple sequence repeat (SSR) molecular markers using ‘SSR primer’ and mapped onto the complete Arabidopsis thaliana genome by sequence comparison. The database may be queried using a text-based search of sequence annotation or GO terms, BLAST comparison against resident sequences, or by the position of candidate orthologues within the Arabidopsis genome. Tools have also been developed and applied to the discovery of single nucleotide polymorphism (SNP) molecular markers and the in silico mapping of Brassica BAC end sequences onto the Arabidopsis genome. Planned extensions to this resource include the integration of gene expression data and the development of an EnsEMBL-based genome viewer.

GlobalAnts: a new database on the geography of ant traits (Hymenoptera: Formicidae)

In recent years the focus in ecology has shifted from species to a greater emphasis on functional traits. In tandem with this shift, a number of trait databases have been developed covering a range of taxa. Here, we introduce the GlobalAnts database. Globally, ants are dominant, diverse and provide a range of ecosystem functions. The database represents a significant tool for ecology in that it (i) contributes to a global archive of ant traits (morphology, ecology and life history) which complements existing ant databases and (ii) promotes a trait‐based approach in ant and other insect ecology through a broad set of standardised traits. The GlobalAnts database is unique in that it represents the largest online database of functional traits with associated georeferenced assemblage‐level data (abundance and/or occupancy) for any animal group with 9056 ant species and morphospecies records for entire local assemblages across 4416 sites. We describe the structure of the database, types of traits included and present a summary of data coverage. The value of the database is demonstrated through an initial examination of trait distributions across subfamilies, continents and biomes. Striking biogeographic differences in ant traits are highlighted which raise intriguing questions as to the mechanisms generating them.

The complete mitochondrial genome of Haliotis laevigata (Gastropoda: Haliotidae) using MiSeq and HiSeq sequencing

Abstract The mitochondrial genome of greenlip abalone, Haliotis laevigata, is reported. MiSeq and HiSeq sequencing of one individual was assembled to yield a single 16,545 bp contig. The sequence shares 92% identity to the H. rubra mitochondrial genome (a closely related species that hybridize with H. laevigata in the wild). The sequence will be useful for determining the maternal contribution to hybrid populations, for investigating population structure and stock-enhancement effectiveness.

Comparative analysis of the predicted secretomes of Rosaceae scab pathogens Venturia inaequalis and V. pirina reveals expanded effector families and putative determinants of host range

BackgroundFungal plant pathogens belonging to the genus Venturia cause damaging scab diseases of members of the Rosaceae. In terms of economic impact, the most important of these are V. inaequalis, which infects apple, and V. pirina, which is a pathogen of European pear. Given that Venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. Thus the predicted secretomes of a range of isolates of Venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity.ResultsGenomes of Venturia pirina (one European pear scab isolate) and Venturia inaequalis (three apple scab, and one loquat scab, isolates) were sequenced and the predicted secretomes of each isolate identified. RNA-Seq was conducted on the apple-specific V. inaequalis isolate Vi1 (in vitro and infected apple leaves) to highlight virulence and pathogenicity components of the secretome. Genes encoding over 600 small secreted proteins (candidate effectors) were identified, most of which are novel to Venturia, with expansion of putative effector families a feature of the genus. Numerous genes with similarity to Leptosphaeria maculans AvrLm6 and the Verticillium spp. Ave1 were identified. Candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. Candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats (e.g. transposable elements), compared with core eukaryotic genes.ConclusionsComparative secretomics has revealed candidate effectors from Venturia fungal plant pathogens that attack pome fruit. Effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host-species specificity. Since many of the effector candidates are in close proximity to repetitive sequences this may point to a possible mechanism for the effector gene family expansion observed and a route to diversification via transposition and repeat-induced point mutation.

Structural homology guided alignment of cysteine rich proteins

BackgroundCysteine rich protein families are notoriously difficult to align due to low sequence identity and frequent insertions and deletions.ResultsHere we present an alignment method that ensures homologous cysteines align by assigning a unique 10 amino acid barcode to those identified as structurally homologous by the DALI webserver. The free inter-cysteine regions of the barcoded sequences can then be aligned using any standard algorithm. Finally the barcodes are replaced with the original columns to yield an alignment which requires the minimum of manual refinement.ConclusionsUsing structural homology information to constrain sequence alignments allows the alignment of highly divergent, repetitive sequences that are poorly dealt with by existing algorithms. Tools are provided to perform this method online using the CysBar web-tool (http://CysBar.science.latrobe.edu.au) and offline (python script available from http://github.com/ts404/CysBar).

A Large Family of AvrLm6-like Genes in the Apple and Pear Scab Pathogens, Venturia inaequalis and Venturia pirina

Venturia inaequalis and V. pirina are Dothideomycete fungi that cause apple scab and pear scab disease, respectively. Whole genome sequencing of V. inaequalis and V. pirina isolates has revealed predicted proteins with sequence similarity to AvrLm6, a Leptosphaeria maculans effector that triggers a resistance response in Brassica napus and B. juncea carrying the resistance gene, Rlm6. AvrLm6-like genes are present as large families (>15 members) in all sequenced strains of V. inaequalis and V. pirina, while in L. maculans, only AvrLm6 and a single paralog have been identified. The Venturia AvrLm6-like genes are located in gene-poor regions of the genomes, and mostly in close proximity to transposable elements, which may explain the expansion of these gene families. An AvrLm6-like gene from V. inaequalis with the highest sequence identity to AvrLm6 was unable to trigger a resistance response in Rlm6-carrying B. juncea. RNA-seq and qRT-PCR gene expression analyses, of in planta- and in vitro-grown V. inaequalis, has revealed that many of the AvrLm6-like genes are expressed during infection. An AvrLm6 homolog from V. inaequalis that is up-regulated during infection was shown (using an eYFP-fusion protein construct) to be localized to the sub-cuticular stroma during biotrophic infection of apple hypocotyls.

Genome Database Integration

This paper presents a solution to many of the problems in Genome Database Integration including an integrated interface for accessing all genome databases simultaneously and the problem of a common interchange data format. The solution is the addition of a middle or mediation layer of a three layer approach. The solution provides a simple step by step approach to connect other existing genome databases quickly and efficiently. The internal data format used is a commonly used bioinformatics format called BSML, a subset of the XML standard. The architecture also allows easy addition and deletion of functionality. Finally an implementation of this solution is presented with the required support functionality to validate the proposed integration method.

AgriSeqDB: an online RNA-Seq database for functional studies of agriculturally relevant plant species

BackgroundThe genome-wide expression profile of genes in different tissues/cell types and developmental stages is a vital component of many functional genomic studies. Transcriptome data obtained by RNA-sequencing (RNA-Seq) is often deposited in public databases that are made available via data portals. Data visualization is one of the first steps in assessment and hypothesis generation. However, these databases do not typically include visualization tools and establishing one is not trivial for users who are not computational experts. This, as well as the various formats in which data is commonly deposited, makes the processes of data access, sharing and utility more difficult. Our goal was to provide a simple and user-friendly repository that meets these needs for data-sets from major agricultural crops.DescriptionAgriSeqDB (https://expression.latrobe.edu.au/agriseqdb) is a database for viewing, analysing and interpreting developmental and tissue/cell-specific transcriptome data from several species, including major agricultural crops such as wheat, rice, maize, barley and tomato. The disparate manner in which public transcriptome data is often warehoused and the challenge of visualizing raw data are both major hurdles to data reuse. The popular eFP browser does an excellent job of presenting transcriptome data in an easily interpretable view, but previous implementation has been mostly on a case-by-case basis. Here we present an integrated visualisation database of transcriptome data-sets from six species that did not previously have public-facing visualisations. We combine the eFP browser, for gene-by-gene investigation, with the Degust browser, which enables visualisation of all transcripts across multiple samples. The two visualisation interfaces launch from the same point, enabling users to easily switch between analysis modes. The tools allow users, even those without bioinformatics expertise, to mine into data-sets and understand the behaviour of transcripts of interest across samples and time. We have also incorporated an additional graphic download option to simplify incorporation into presentations or publications.ConclusionPowered by eFP and Degust browsers, AgriSeqDB is a quick and easy-to-use platform for data analysis and visualization in five crops and Arabidopsis. Furthermore, it provides a tool that makes it easy for researchers to share their data-sets, promoting research collaborations and data-set reuse.

WAD Workflow System: Data-Centric Workflow System

To date, most scientific workflows center on the processcentric variety. This paper presents a novel approach to the workflow field by taking the view-point of a datacentric workflow. The key features of the Workflow Activity Diagram (WAD) system are its record and field level views of workflow connections. It makes use of existing standards for visualization and provides automation features using existing domain ontologies. To execute the workflow, the WAD system makes use of the existing Business Process Execution Language (BPEL). The visualization system can be used in both process and data-centric workflows, thus lending itself towards a standard for workflow visualization. WAD handles multiple levels of user experience by using a multi-level drill-down approach. The user can drill-down as far as they are comfortable and allow the system to automate the remaining levels. The WAD system comes with two methods to automate the record and field level connections. The first is based on domain knowledge from the domain ontologies and the second is based of previous user experiences collected by the system. By connecting processes on a record and field level the system is able to connect processes that do not use the same data format. That is, it provides ad-hoc data format conversions. This removes the need for static data format convertors and thus increases system flexibility.