Project 2 - Transcript analysis for specific cells and tissues

1. Planned Outcome / Aims

• A series of transcript databases to support gene discovery projects
  
  
• Micro-array based screens of target cells and tissues
  
  
• Staff trained in the development and utilisation of transcript databases for gene discovery

2. Contracted Output(s)

• Transcript databases covering 5,000 to 10,000 sequences and micro-array-based expression information for developing meiocytes, embryo, endosperm and other grain cells
  
  
• Micro-array and transcript screen data on the target tissues from normal and mutant barley
  
  
• Accurate evaluation of transcript databases versus micro-array information for candidate gene identification in wheat and barley
  
  
• Staff trained in the development and use of transcript and microarray databases for gene discovery

3. Research Methodology

• The tissues for analysis will be prepared by micro-dissection as outlined for Project 2.  Preliminary experiments have demonstrated that we can isolate cells from pre-meiotic interphase to pollen maturity and embryos endosperm from about 5 hours after fertilisation.  The earliest stages for aleurone, and testa isolation are yet to be determined.  RNA from these tissues will be used for solid phase cDNA for analysis.  The cDNA is covalently bound to beads and is stable and suitable for analysis.  The AFLP technique using 2 base selective extension will be used to screen for polymorphisms.  Between 5,000 and 10,000 diverse mRNAs can be detected and quantified by this technique.  The bound cDNAs can also be used to produce labelled (with fluorescent tags) second strand DNA for microarray analysis.  At least 40,000 Triticeae ESTs will be available by mid 2000.  These will be used to select a unique set for arraying.  The arrays will be probed with the labelled second strand cDNA to determine probable mRNA concentrations.  Sequences showing variation in expression across the time series of tissues being analysed will be taken for further analysis.  Data management will be in collaboration with the GrainGenes database in USA. A bioinformatics position will be provided by the USDA to support this project and a computer officer has also been provided through an existing GRDC project for barley ESTs.
  
  An important part of the project will be the application of these techniques to screening mRNA populations from barley mutants and wheat barley translocation lines.  Over 2,000 mutants of barley are available.  These include over 50 male sterile mutants and a wide range of lines with abnormal grain development.  The screening of expression from these lines will also help identify candidates for genes involved in the development of pollen, endosperm and embryo.

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