ARCHIVED - National Research Council Canada

Notes:

1. Commercially relevant advances in areas of genomics R&D related to human health (e.g. genetic testing, diagnostics, microbial genomic applications, treating and preventing human diseases such as cancer and cardiovascular disease, and pathogen detection).

1.1 Integrated devices that provide rapid, point of care diagnosis of pathogens based on their genomic signature. Central to the success of these devices are on-chip assays that can achieve detection with the sensitivity and specificity necessary to inform sound clinical decisions. In 2008-2009, the Biochips team developed conditions for simultaneous PCR amplification and hybridization of target molecules on the chips, allowing detection of less than 5 molecules of pathogen DNA sequence with a specificity distinguishing a single nucleotide mismatch. This advance brings the detection sensitivity of the assay within the useful clinical range of less than 10 molecules of pathogen per assay, and represents a breakthrough in actualizing on-chip real-time PCR, hybridization and detection of biological samples.

1.2 Building a sustainable pipeline of protein-based tumour-targeting agents that will act as prospective therapeutics is the goal of the GHI Cancer project. On July 7, 2008 Alethia Biotherapeutics, a Canadian biotechnology company engaged in the discovery and development of therapeutics in areas of unmet medical needs, signed an option to license anti-Clusterin monoclonal antibodies (mAbs) developed by the project team. In 2008-2009, in vivo animal studies showed that three of the anti-Clusterin mAbs caused a significant reduction in metastatic nodule production. In addition, an isolated clusterin-binding peptide has been shown effective as an optical imaging agent for tumor cells in animal models and a provisional patent has been filed.

2. Improvements in crop value in cereals, soybean and canola through quality improvements in areas related to plant adaptation to biotic and abiotic stresses (e.g. resistance to disease, tolerance to drought and cold), as well as seed development and metabolism (e.g. related to oil content for biofuels and nutraceutical applications).

2.1 AAFC genomics scientists leveraged GRDI funds to participate in an international collaboration between Canada and Germany. An AAFC scientist and a German colleague co-lead a dedicated group of Canadian and German scientists from academia, government and industry, developing solutions for Fusarium infection in wheat. Infection by Fusarium is a global problem in cereal crops which can result in severe yield loss as well as an accumulation of toxin in the grain. The team is using a combination of breeding and genomics methods to understand the disease and develop new varieties of wheat for producers in both countries. In 2008 progress in identifying wheat types with enhanced resistance to Fusarium resulted in the presentation of an award to the research team from the City of Freising, Germany.

2.2 As part of a team of 23 researchers from 6 countries AAFC scientists contributed to constructing a physical map of wheat chromosome 3B which is the first step toward sequencing the first chromosome of wheat. Each wheat chromosome is about the size of the entire rice genome so sequencing of wheat is a massive project. AAFC expertise in genetic mapping was used to assist colleagues at INRA (France) with ordering more than 1,400 genetic markers on chromosome 3B, which was critical for ordering the bacterial artificial chromosome (BAC) clones and contigs in preparation for future sequencing of the chromosome. The work was reported in the prestigious journal Science.

2.3 In 1975 a major virulence factor in Phytophthora root rot of soybean was described. Despite many efforts over the last 35 years it was not possible to conclusively identify this factor until AAFC scientists reported the genetic sequence and description of the Avr1a factor in 2009. The identification of Avr1a adds to a growing list of P. sojae Avr factors that have recently been identified. These successes in finding Avr factors have been driven by technological changes such as genome sequencing and advances in knowledge. The isolation of P. sojae Avr1a is notable, given its long history in molecular plant pathology, but more importantly the identification of the Avr genes in Phytophthora sojae will aid pathogen diagnostics and cultivar development for one of the world's largest crops. A more rational approach to soybean breeding, diagnostics, and cultivar deployment will arise from such solid genetic information. It will also lead to a better mechanistic understanding of disease and how it may be managed.

2.4 A serious fungal pathogen of cereal crops such as wheat, barley, and corn, Fusarium graminearum produces a number of toxic metabolites or mycotoxins that accumulate in infected grain. Using a proteomics approach, AAFC researchers were able to identify hundreds of Fusarium proteins that were present when the fungus is induced to synthesize these mycotoxins. The relative quantities of seventy-two proteins increased significantly with the induction of mycotoxin production and provide leads in the search for mechanisms and markers of plant disease and novel antifungal targets.

2.5 Sclerotinia sclerotiorum is a necrotrophic plant pathogen that infects important agricultural crops including canola (Brassica napus), soybean (Glycine max) and sunflower (Helianthus annuus) (Boland and Hall, 1994). It secretes a battery of enzymes that break down plant tissues including polygalacturonases (PG), pectin lyases, pectin methyl and acetyl esterases that degrade plant pectin. Most plants genomes encode a limited set of polygalacturonase inhibitor proteins PGIP) that may inactivate the PG secreted by pathogens. AAFC scientists characterized a diverse set of 16 PGIP Brassica napus genes. Several BnPgip genes responded to infection by S. sclerotiorum or were responsive to other stresses and signalling molecules. The large number of PGIP genes and the different manners in which they are regulated likely ensures that B. napus can respond to attack from a broad spectrum of pathogens and pests. These are currently being examined for their ability to provide resistance to diseases caused by fungal pathogens.

2.6 Baculoviruses are insect specific and have proved to be effective insect control agents that are non-harmful to both the environment and humans. As baculoviruses have been identified for many insects of economic importance they represent a significant resource for the future development of organic insecticides. The baculovirus Autographa californica nucleopolyhedrovirus has the broadest host range of any known baculovirus. These studies have been investigating key genes that baculovirus genomics have identified to be present in all baculovirus species completely sequenced to date. Conservation of these genes suggest they play critical roles in virus replication By generating viruses that have these genes (ac142 and ac143) deleted from the genome we have shown they are critical for virus replication. One of them ac142 appears to be essential for recruiting membranes to virus particles a unique property. These studies will help to understand the molecular basis of baculovirus disease and lead to improved use of these natural agents as insect control agents.

2.7 The NRC Biorenewable Oil for Food and Fuel team is characterizing 29 genes that may play an important role in canola crop improvement. Elite candidate genes will be incorporated into the pre-commercialization. Five prototypes were tested in the field during the first year in collaboration with AAFC. Compared to the controls, two prototypes confirmed improved oil content and one prototype confirmed early flowering and increased seed size. The summary outputs of the program include the publication of 11 papers in refereed journals, 18 other publications / presentations / book chapters and one patent application. PIs from this program have established formal international collaborations with scientists from Germany, the Netherlands, China and India in areas directly related to the objectives of the program.

3. Sustainable management of aquatic resources through the use of genomics tools to: manage fishery openings; generate increased understanding of population genetics and structure; further understanding of behavioural, physiological and immunological responses to the environment; and through the management of aquatic animal diseases.

3.1 Manage fishery openings - DFO continues to develop information geared at understanding the changing behaviour of fish stocks due to climate change and pollution. Genomics tools including gene expression profiling are being used by DFO to determine physiological changes along the migration route to natal streams that is associated with early entry, a behavioural shift in late run Fraser River sockeye salmon. This research directly feeds into the management of fisheries openings through the 1) use of functional genomics technology in fisheries management, 2) use of wild caught organisms in large-scale ecological genomics research.

3.2 Increased understanding of population genetics and structure - Adding to previous work, several new projects were undertaken in order to understand the population structure of various species of socio-economic importance, including wild salmon, capelin, redfish, arctic char and beluga whales.

3.3 Further understanding of behavioural, physiological and immunological responses to the environment - Predictive biomarkers are being developed for monitoring relevant genes to enable pre-season prediction of migration behaviour and fish survival of pacific salmon for fishery managers in order to monitor the impacts of climate change and pollution.

3.4 Management of aquatic animal diseases - DFO continues to study aquatic animal diseases of import, such as the immune function of salmon and disease resistance against infectious salmon anemia virus (ISAV), a major pathogen of Atlantic salmon The viral mechanisms leading either to fish death or survival and resistance is not well understood at the immune or molecular levels. Another project includes using a functional genomics approach to characterising salmonid responses to the salmon louse, a distinct cause for concern for Pacific salmon growers

3.5 Monitor and mitigation of environmental contaminants including invasive species - New projects were undertaken to monitor and mitigate the effects of environmental contaminants. One project seeks to characterize microbial populations from produced water and its influence on the microbial community in the marine environment around the Hibernia Production Platform. A further project looks at the application of genetic markers to resolve species identification and population structure of invasive tunicate species.

4. Positioning the Canadian regulatory system for health to enable innovation while minimizing the risks to Canadians through a focused genomics R&D program aimed at strengthening capacity in priority areas such as genetic information, biotechnology products, human genomics and microbial genomics, on human, animal and environmental health.

4.1 Molecular Genomics Applied to Radiation Risk Assessment: Specialized custom-exposure systems have been constructed and characterized for radiofrequency (RF) fields and alpha-radiation for use in this project. Differential gene expression has been assessed in three human derived cell-lines after exposure to alpha-radiation. A list of alpha-radiation responsive genes has been generated and validated by RT-PCR.

4.2 Environmental Health Applications of toxico-genomics and proteomics. Identification of exposure and effect biomarkers for mutagenic carcinogens in complex environmental matrices: Gene expression responsive to low concentrations of diesel exhaust (SRM 1650b) using high density microarrays is completed and bioinformatic analysis is underway. In addition, genomics funding is being used to train additional HC staff. Thirteen proteins were identified as being potential candidate biomarkers of exposure for mutagenic carcinogens in complex environmental matrices. Confirmation and validation of the method are currently under investigation.

4.3 Development and validation of toxicogenomic tools, and integrated systems biology approaches in regulatory toxicology: Confirmation and validation of gene expression responsive and measured following low (environmentally-relevant) doses and further development of the NOTEL (no observable transcript effect level) below the doses inducing histological or biochemical changes. The following research methodologies were developed for identification and investigation of biomarkers. Technical validation of microRNA arrays: cross-platform correlation, sample storage, quality control/assurance development and statistical analysis. Biological validation of an in vitro mutation screening cell line (FE1) using DNA microarrays - comparison of response in vitro versus in vivo. Establishing the biological activity and the similarities/differences to the cells in vivo to improve interpretation of data acquired using in vitro systems. Use of ChIP-chip to identify regions of DNA that bind the thyroid receptor in the brains of developing mice. New reporter assays are being developed to identify chemicals which operate through disruption of TH pathways. Explorations of the cardio-pulmonary health effects of inhaled nanoparticles in utero (ongoing) and in the adult and induction of acute phase response.

4.4 Investigations on Colon Epithelial Cell Genetic Responses to Dietary Fibre were been established. The changes manifested in the intestinal bacteria will be used to determine what influence these changes may have on colonocyte responses. This work also represents methodological development for later work in this rat system.

4.5 Cell lines expressing a number of viral immunomodulatory proteins have been constructed. These include the influenza virus NS1 protein and the HCV NS3/NS4a proteins. MiRNA gene expression has been determined in these cell lines and a number of deregulated MiRNAs have been identified. MiRNAs expressed by microglia in the brains of prion infected mice have also been identified. Potential targets of one of these MiRNAs have been identified using a combination of functional genomic analyses.

4.6 Prototype gene and microarray assays to genotype Campilobacter jejuni samples per day were developed and tested, and are currently being deployed to perform molecular epidemiological analysis of various datasets, and used to investigate genome variability among virulent C. jejuni isolates that are highly prevalent among human clinical cases.

4.7 Work is undertaken to generate, synthesize, and translate new knowledge in pathogen and human genomics and related sciences (proteomics, transcriptomics, bioinformatics, etc.) to: strengthen infectious disease surveillance, prevention and control through applications such as diagnostics, molecular risk assessment, vaccines, intervention strategies, mitigation of antimicrobial resistance; and, develop applications to diagnose and prevent diseases and promote health through predictive genetic screening and/or modulation of gene-environment interactions.

5. Increased knowledge for forest generation and protection methods, and for addressing environmental impact considerations, through a focused genomics R&D effort on species and traits that are of economic importance to Canada.

5.1 The production of effective biological control methods based on genomics requires the understanding of the interactions between host and pathogen. Novel genomic resources were created by CFS researchers for two indigenous pests and one alien invasive pest. Eight cDNA libraries were created: one for the white tussock moth, two for the hemlock looper and five for the Emerald Ash Borer (EAB). Other investigations of pest species found the existence of microsporidia, a group of unicellular insect pathogens, in Mountain Pine Beetles (MPB) from 4 of the 10 collection sites and in one population of EAB. Several isolates of fungal pathogens were found in field collections of MPB from old infestations sites. Furthermore, primers were developed for various viruses of coleopteran species.

5.2 Early evidence has shown that the co-evolution of insects and their viruses also saw a co-dependency of the interacting proteins to initiate and establish infection in the host. Research on the occlusion derived virus of the balsam fir sawfly led to the identification of 18 proteins, three of which are novel.

5.3 Building on the genome scan performed by NRCan scientists in 2007-08, QTL analysis was completed and it was determined that bud flush, bud set and growth characters are mainly controlled by 7, 14 and 9 genomic regions respectively. Refinement of the QTL results is underway by association analysis. A high density genetic map of white spruce is in development to position 1800 of the 28000 genes (identified by the Arborea effort). This is the starting point of a physical map for spruce.

5.4 In white spruce, 214 candidate genes were rated as high priority and are being used to search for variants in association studies. Exploratory association studies identified 6 to 20 polymorphic SNPs to be significantly associated with one of the 25 wood traits investigated. White spruce primers were tested on black spruce DNA, identifying over 1800 SNPs. Primers were designed for candidate genes relating to wood formation.

5.5 2008-09 saw novel discoveries in the white pine-blister rust interaction, the identification of proteins and genes that function in disease resistance. In collaboration with the USDA-FS and BC Ministry of Forest and Range, several white pine candidate genes were investigated for single nucleotide polymorphisms (SNPs) and SNP genetic association with resistance phenotypes in multiple white pine families with quantitative resistance phenotypes. Research on Douglas Fir (DF) identified defence related proteins in DF roots infected with laminated root rot. Further testing is underway on 45 genes with potential pathogenicity function.

6. Development of genome-science applications to support regulatory and enforcement activities in key areas such as environmental risk assessment and management; enforcement and compliance; pollution detection, monitoring and prevention; conservation biology and wildlife genetics; technology forecasting and assessment; and the responsible and sustainable development and use of bio-based products and industrial processes.

6.1 In 2008-09 EC made significant advances in the application of the genomic tools and data that have been generated over the life span of the GRDI. Using GRDI funding, EC developed an in-situ technique to conduct pathogenicity and toxicity testing of microbial substances in soil which has helped to validate a new national guidance document under the Canadian Environmental Protection Act. EC has also developed and applied genomics methods to investigate the toxic effects of environmental contaminants on aquatic microbial communities. This research has lead to the application of tools and approaches for assessing priority substances and emerging contaminants such as personal care products and pharmaceuticals. EC's wildlife research has provided the scientific (genomic) data in which conservation and wildlife management decisions (i.e for bird species Yellow-breasted chats, Ontario's goose populations, Loggerhead Shrikes etc.) have been based (both at the federal and provincial level).

6.2 Numerous environmental genomics-based approaches were used in 2008-09 by EC:

• Application of genomics to microbial source tracking: Identification of genetic markers specific for animal fecal pollution in aquatic environments.
• Application of genomics in environmental risk assessment.
• Validation of genomics tools for the prediction of environmental effects: response of fish to contaminated sediments.
• Development and validation of a lobster eco-toxicogenomic array - Correlation of gene expression profiles with traditional toxicological end-points for contaminant exposure, histological effects, and behavioural changes.
• Environmental safety of biotechnology to aquatic ecosystems - Impacts of transgenic crops and commercial bacterial consortia to aquatic invertebrates.
• Application of genomics to monitor the health and activities of microbial communities in contaminated soils and stressed riverine environments.
• Gene expression technologies - Application of genomics to wildlife toxicology.
• Application of genomics methods and end-point measurements for aquatic toxicological testing and environmental effects monitoring.
• Development and application of novel genomics-based techniques for the detection of environmental microorganisms and the assessment of chronic toxicity in wastewater.
• Development and standardization of test methods using environmental genomics techniques.
• Breeding behavior and population history of the red-listed Yellow-breasted chat in British Columbia.
• Establishing molecular assessment techniques for emerging infectious diseases in native amphibians; laboratory and sampling technique comparisons.
• Comparative Genetic Diversity of Captive and Wild of Loggerhead Shrikes.
• Conservation Genetics of Leach's Storm-petrels.
• Genetic characterization of strains of Pasteurella multocida isolated from large-scale avian cholera outbreaks across Canada.
• Ontario Canada goose population harvest discrimination.
• Characterization and sequencing of avian influenza viruses from birds in eastern Newfoundland.
• Hybridization between Eastern wolves (Canis lycaon) and other canids: An example of contemporary evolution in human-modified landscapes.