project 3.4.2
Grapevine gene discovery
The science of genomics and gene mapping is a major new approach for gene discovery and isolation not only in plants, but also in animals, humans and other organisms. With the development of transgenic technology which allows us to produce both genetically modified wine grape cultivars and wine yeasts it has become necessary to identify additional genes which can be used for genetic improvement.
This project will construct cDNA libraries from genes expressed during different stages of inflorescence and berry development, sequence cDNA clones from the cDNA libraries and establish an expressed sequence tag (EST) database.
It will also identify new genes by data analysis of ESTs, begin isolation of full length clones for functional analysis, extend the EST database to include more genes including those from other tissues and look at the functional analysis of identified key genes by introduction into transgenic grapevines.
project 3.4.2 - update
Pat Iocco, who has moved from CRCV project 3.4.3, has replaced Ms Chi Hua who has resigned. Normalised libraries have been constructed from RNA previously isolated in the 01/02 season from flower and berry samples.
EST DNA sequencing from these normalised libraries has increased the number of unique genes sequences obtained to more than 4000.
Microarray technology for analysing global gene expression has been given priority over functional analysis of full length clones as this method has the potential to investigate viticulture problems in the field at the molecular level. The technology has been established with an initial 1200 grapevine gene array produced for testing.
Initial trials with this microarray have shown that there are large global gene expression differences during berry development with the expression of many stress-related genes at post-veraison. During the 01/02 season berry samples were collected, with the help of a number of wine companies, from different regions and vineyards known to produce wine of different quality. Preliminary analysis of these samples indicates that there are gene expression differences between the grapes sourced from the different sites.
Microarray experimental plans are being prepared for the 02/03 season to help determine the reproducibility of the technology. Following assessment of the 2001/02 microarray data and further discussions with other researchers and with the technical and viticultural staff of Orlando, BRL Hardy and Southcorp a much expanded sampling regime was deemed necessary. This sampling just been completed. If suitable data (wine making sensory evaluation etc.) is available from the participating wine companies then an exploratory attempt will be made to try and link environmental and berry quality data to the gene expression data.
New software for the analysis of microarray data has been purchased and used to do in-depth comparisons. Considerable effort has been exerted to verify the reliability and reproducibility of the data. Other analysis freeware is also being assessed for usefulness. Considerable effort has been put into re-examining data in the light of new developments and a paper is in preparation.
A collaboration is continuing with Dr. Roberto Botta, Italy to sequence additional ESTs. Discussions are taking place within the International Grape Genome Program to expand cooperation between international groups with the goals of sharing EST sequences and developing a common grapevine microarray.
