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Title: IDENTIFICATION OF COLD-RESPONSIVE GENES IN BLUEBERRY (VACCINIUM CORYMBOSUM L.) USING A SUBTRACTIVE HYBRIDIZATION APPROACH

Author
item DHANANJAY, NAIK - U. IOWA STATE UNIV
item DHANARAJ, ANIK - U. IOWA STATE UNIV
item Rowland, Lisa

Submitted to: Mid Atlantic Plant Molecular Biology Society Conference
Publication Type: Abstract Only
Publication Acceptance Date: 7/21/2005
Publication Date: 8/11/2005
Citation: Dhananjay, N., Dhanaraj, A., Rowland, L.J. 2005. Identification of cold-responsive genes in blueberry (vaccinium corymbosum l.) using a subtractive hybridization approach. Mid Atlantic Plant Molecular Biology Society Conference. Page 36.

Interpretive Summary:

Technical Abstract: The United States is the world’s leading blueberry producer. The blueberry industry in the U.S., however, suffers from a lack of cold hardy cultivars. Consequently, there is a need to identify genes associated with cold hardiness and use this information to develop cold hardy blueberry cultivars. Toward this direction, our laboratory previously has been using the Expressed Sequence Tag (EST) approach to identify cold-responsive genes from standard cDNA libraries prepared from flower bud RNA from cold acclimated and non-acclimated plants. However, random picking and sequencing of even several thousand clones from standard cDNA libraries will result in selection of clones representing more highly abundant transcripts because these clones will be present in the libraries at a higher frequency than those representing less abundant transcripts. Important regulatory genes, such as transcription factors, are often expressed at rather low levels and over a shorter timeframe. Thus, they can be missed using this approach. Therefore, here we report on the preparation of subtracted and reverse subtracted libraries using procedures that help to increase the chances of finding rarer classes of transcripts by helping to normalize the distribution of clones. The subtracted library was prepared in such a way to enrich for transcripts that are expressed at higher levels in blueberry flower buds at 400 hours of cold exposure than at 0 hours of cold exposure and vice versa for the reverse subtracted library. Clones from each library have been picked and sequenced and confirm that the libraries are of good quality and enriched for cold-responsive genes. Contig analyses and BLAST searches have been performed to categorize the genes. Quantification of expression of specific cold up-regulated genes using qRT-PCR is in progress.