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Title: ANALYSIS OF GENE EXPRESSION ASSOCIATED WITH COLD ACLIMATION IN BLUBERRY FLORAL BUDS USING EXPRESSED SEQUENCE TAGS

Author
item DHANARAJ, ANIK - IOWA STATE, HORT. DEPT.
item Slovin, Janet
item Rowland, Lisa

Submitted to: Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/17/2003
Publication Date: 1/20/2004
Citation: Dhanaraj, A.L., Slovin, J.P., Rowland, L.J. 2003. Analysis of gene expression associated with cold aclimation in bluberry floral buds using expressed sequence tags. Plant Science. 166:863-872.

Interpretive Summary: The United States is the world's largest producer of blueberries, but there is a need to develop more winter hardy and spring frost-tolerant cultivars for the industry. Previously, we identified proteins known as dehydrins that accumulate in cold hardy flower buds of blueberry and found a good correlation between levels of dehydrins and cold hardiness levels. However, based on work done on herbaceous plants, it is known that many genes are involved in development of cold hardiness (cold acclimation), in addition to dehydrins. Here, we have used a genomics approach to identify other genes potentially involved in cold acclimation in blueberry. We prepared two collections of genes, called libraries, that represent genes that are expressed midwinter (when plants are their cold hardiest) and genes that are expressed in the early fall (prior to the development of cold hardiness). About 600 genes were picked from each library, sequenced, and studied to compare the types of genes expressed under the two conditions. In this way, several new genes were identified that may play a role in development of cold hardiness. These genes are now available to scientists to test their involvement in cold tolerance directly. Additionally, the ~1200 gene sequences represent the first database of its kind (called an expressed sequence tag or EST database) for blueberry available to scientists for use in gene isolation, gene expression studies, and marker development.

Technical Abstract: To gain a better understanding of changes in gene expression associated with cold acclimation in the woody perennial blueberry (Vaccinium spp.), a genomics approach based on the analysis of expressed sequence tags (ESTs) was undertaken. Two cDNA libraries were constructed using RNA from cold acclimated and non-acclimated floral buds of the blueberry cultivar Bluecrop and about 600 5'-end ESTs were generated from each of the libraries. About 100 3'-end ESTs were generated from the cold acclimated library as well. Putative functions were assigned to 57% of the cDNAs that yielded high quality sequences based on homology to other genes/ESTs from Genbank, and these were classified into 14 functional categories. From a contig analysis, which clustered sequences derived from the same or very similar genes, 430 and 483 unique transcripts were identified from the cold acclimated and non-acclimated libraries, respectively. Of the total unique transcripts, only 4.3% were shared between the libraries, suggesting marked differences in the genes expressed under the two conditions. The most highly abundant cDNAs that were picked many more times from one library than from the other were identified as representing potentially differentially expressed transcripts. Northern analyses were performed to examine expression of eight selected transcripts and seven of these were confirmed to be preferentially expressed under either cold acclimating or non-acclimating conditions. Only one of the seven transcripts, encoding a dehydrin, had been found previously to be up-regulated during cold stress of blueberry. This study demonstrates that analysis of ESTs is an effective strategy to identify candidate cold-responsive transcripts in blueberry.