Gene Expression Profiling
Gene expression profiling experiments in the Arborea project aim to delineate groups of co-ordinately regulated genes and regulons, and investigate the function of putative regulatory genes in tree growth and development. Large-scale profiling using custom microarrays as well as high resolution QPCR approaches are used side-by-side to meet our objectives.
Phase I – Expression profiling of wood formation and defense response with custom cDNA arrays (2002-2006)
Custom cDNA arrays were developed for spruce (11,000 low redundancy elements) and poplar (3,400 low redundancy elements). Microarray and QPCR studies were carried out to investigate wood formation and defense response in trees, using physiological and transgenic approaches. An integrated transcript profiling laboratory was established and a bioinformatics infrastructure implemented to analyze, archive and mining microarray data. Microarrays were produced in partnership with the BC Array Facility (Jack Bell Center, Vancouver) and the Biotechnology Research Institute (Montreal).
Phase II - Large-scale expression profiling and Expression polymorphism mapping in spruce (2006-2009)
The overarching objective is to identify bud formation and wood formation regulons. Genes whose patterns of expression are correlated with these developmental events will be identified as candidate genes for genotyping and mapping in within our studies of genetic diversity and for our experiments aimed at developing molecular breeding methods.
In this phase of the Arborea project we will develop a long-oligonucleotide array. The design will encompass a set of 25-30K long nucleotide probes (50-70 mers) from white and other spruce sequences obtained from collaborators, namely UBC. We will also augment our capabilities for Quantitative RT-PCR analysis and develop assays for approximately 200 transcription factors for high resolution profiling.
First, we will develop a budset roadmap based on regulons identified by microarray profiling, quantitative RT-PCR and metabolite analyses of developmental time course studies of budset. Our second goal is to conduct expression profiling for budset, in order to identify loci responsible for expression polymorphism using the approaches of QTL mapping (eQTLs) and association studies. We will carry out microarray and Q-RTPCR analyses on individuals representing the phenotypic extremes and on larger random samples from QTL populations. We will also target phenotypic extremes for expression profiling from the population used for association studies of budset.
First, we will identify wood formation regulons by microarray and Q-RTPCR analyses of transgenics developed in the Arborea I project, which mis-express diverse transcription factors. Secondly, we will carry out expression profiling related to wood properties, in order to identify loci responsible for expression polymorphism, using a transmission disequilibrium testing approach. Microarray and Q-RTPCR analyses will target the phenotypic extremes for selected wood properties (two or more) and the phenotypic extremes for stored N content from the TDT population. We will also use population-wide approaches for microarray and Q-RTPCR analyses, of randomly selected individuals from the TDT population.