Identification of genes associated with cold acclimation in blueberry (Vaccinium corymbosum L.) using a subtractive hybridization approach

Enhanced cold tolerance, including tolerance to winter freezing and spring frosts, is needed for genetic improvement of current highbush blueberry (Vaccinium corymbosum L.) cultivars. To gain a better understanding of changes in gene expression associated with development of cold tolerance in blueberry and other woody perennials, forward and reverse subtracted cDNA libraries were prepared in such a way to enrich for transcripts that are expressed at higher levels in dormant blueberry flower buds at 400 h and at 0 h of low temperature exposure, respectively. Of the clones picked and single-pass sequenced, 503 clones from the forward subtracted library and 167 clones from the reverse subtracted library had inserts and yielded high quality sequences; and of these, 291 (57.9%) and 51 (31.0%), respectively, were assigned putative identities from BLAST searches of GenBank. From contig analyses to cluster genes of like or identical sequences, 275 unigenes (unique clones) from the forward subtracted library and 99 unigenes from the reverse subtracted library were obtained. Many interesting cold and light-stress related genes were identified from the forward subtracted library and several drought-stress related genes were identified from the reverse subtracted library. Many genes, not previously characterized from blueberry or other woody perennials, encoding putative transcription factors and proteins related to signal transduction were identified from both the forward and reverse subtracted libraries. Eleven genes of interest (six from the forward subtracted library and five from the reverse subtracted library) were selected and their expression was analyzed in floral buds by quantitative real-time RT-PCR over a time course from ∼0 to 1200 h of low temperature exposure. Expression profiles validated the quality of the libraries.