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Title: Understanding Cold Tolerance in Blueberry Using EST Libraries, cDNA Microarrays, and Subtractive Hybridization

item Rowland, Lisa
item Matthews, Benjamin - Ben

Submitted to: HortScience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2008
Publication Date: 12/1/2008
Citation: Rowland, L.J., Dhanaraj, A.L., Naik, D., Alkharouf, N., Matthews, B.F., Arora, R. 2008. Understanding Cold Tolerance in Blueberry Using EST Libraries, cDNA Microarrays, and Subtractive Hybridization. HortScience. 43:1975-1981.

Interpretive Summary: Blueberry harvests are often reduced by cold damage; plant breeders can develop blueberry plants that are more resistant to cold damage but need to screen literally thousands of genes to find the best ones. In this study, we used new methods to screen over 2500 genes and found genes that may be involved in helping blueberry plants resist cold. To be sure this information is readily available to those who need it, we developed a public, web-based database called the Blueberry Genomics Database (BBGD). Scientists will use these data to develop new varieties for blueberry growers. BBGD also has basic information on blueberries useful to the general public.

Technical Abstract: To gain a better understanding of changes in gene expression associated with cold acclimation in the woody perennial blueberry (Vaccinium corymbosum L.) and ultimately use this information to develop more freeze tolerant cultivars, a genomics approach based on the analysis of expressed sequence tags (ESTs) and microarrays was undertaken. Initially, two standard cDNA libraries, constructed using RNA from cold acclimated (CA) and non-acclimated (NA) floral buds of the blueberry cultivar Bluecrop, were used for the generation of about 2400 ESTs, half from each library. Putative functions were assigned to cDNAs based on homology to other genes/ESTs from GenBank. From contig analyses, 796 and 865 unique transcripts were identified from the CA and NA libraries, respectively. 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. A cDNA microarray was constructed and used to study gene expression under cold acclimating conditions in the field and cold room. Results indicated that the abundance of transcripts of numerous blueberry genes change during cold acclimation including genes not found previously to be cold-responsive in Arabidopsis, and, interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Finally, forward and reverse subtracted cDNA libraries were prepared from ‘Bluecrop’ RNA, in such a way to enrich for transcripts that are expressed at higher levels in floral buds at 400 hours and at 0 hours of low temperature exposure, respectively. Many genes encoding putative transcription factors and other proteins related to signal transduction were identified from both libraries.