The 2011 ASHS Annual Conference

Blueberry is a rapidly emerging fruit crop. Progress in our understanding of blueberry physiology can be greatly enhanced through the application of transcriptomics tools. RNA of high yield and quality is an indispensable prerequisite for transcriptome analysis. However, isolation of RNA of high quality and yield in blueberry is difficult due to the abundance of polyphenolics and polysaccharides, especially in the fruit. Quantitative real-time polymerase chain reaction (qRT-PCR) is a robust method to quantify transcript abundance. Accuracy of qRT-PCR data greatly relies on the reference genes used for normalization, as it can influence interpretation of the gene expression data. Therefore, prior to qRT-PCR analysis, analysis of the stability of the reference genes is essential. Currently, no studies on identification of reference genes in blueberry are available. The objectives of this study were to develop an effective method for extracting RNA from different tissues and to identify potential reference genes for qRT-PCR analyses in blueberry. Total RNA of high quality and yield was successfully extracted from eight different tissues (stem, leaf, flower bud, flower, fruit, immature fruit, branch and fruit abscission zone) using a modified CTAB extraction method. The average yield of RNA ranged from 18.43 to 303 µg g^-1 fresh wt. The expression stability of 12 potential reference genes was evaluated using three different statistical methods (geNorm, NormFinder and Coefficient of variance analysis). Ubiquitin conjugating enzyme (UCE), Ubiquitin 2 (UBQ2), Clathrin adapter subunit complex 1 (CACS1), Phosphoprotein phosphatase 2A (PP2A), and eukaryotic initiation factor 4 a1 (eIF4a1) were found to be the most stably expressed genes in blueberry. Further, the expression stability of the above genes was tested in the branch abscission zone following treatment with abscission inducing compounds. CACS1, eIF4a1, and UCE were found to be the most stably expressed genes and were sufficient for accurate normalization under these experimental conditions. We recommend a preliminary evaluation with the above five genes to identify reference genes suitable for the experimental conditions under consideration in blueberry.