2018 ASHS Annual Conference

Cold hardiness and chill hour requirement in blueberries are functions of germplasm composition. Southern highbush blueberries (SHB) are less cold tolerant than northern highbush blueberries and have a lower chill hour requirement, causing buds to deacclimate and break earlier. Deacclimation and bud swell heightens susceptibility to spring freezes, a recurring event in southeastern U.S. The objective of this study was to determine the differentially expressed genes in floral buds prior to bud swell and post-bud break of SHB cv. ‘O’Neal’, selected for its unique flowering behavior. Flower buds at two stages, bud swell and tight cluster, were exposed to either non-freezing (4 °C) or freezing conditions (-12 °C) achieved through an environmental control chamber decreasing 4 °C h^-1. Following temperature treatment, the buds recovered at 4 °C for periods of either 1 day or 1 week and were subsequently flash frozen in liquid nitrogen. A total of 24 stranded mRNA-Seq libraries (8 treatments x 3 biological replications) were paired-end sequenced to generate 185 Gbp of raw reads. After trimming, 150 Gbp of data was retained. A 156 Mbp transcriptome assembly with 180,487 contigs (N50=1,100) was constructed and functionally annotated with BLAST2GO (V5) software package; 2,482 (43%) differentially expressed unigenes had BLAST hits out of which 1,900 (77%) were annotated. Bioconductor package DeSeq2 implemented in Trinity (V2.5.1) was used to map the clean reads to the assembly and identify differentially expressed genes (DEGs) between tissue, temperature, and recovery periods. RNA-Seq analysis (log₂ fold change >|2|; p ≤ 0.05) revealed that there were 1,913 DEGs related to tissue, 3,810 DEGs related to temperature, and 4,440 DEGs related to recovery that were upregulated. With 4 °C as a reference, DEGs associated with -12 °C temperature treatment had more upregulated unigenes associated with tight cluster tissue type at either recovery treatment. Between tissue types, tight cluster universally had more upregulated unigenes considering both freezing treatments in conjunction with either recovery period. Unigenes corresponding to treatments of -12 °C, prior to bud swell, at one-day recovery had more uniquely expressed unigenes across the three treatments. Future work involves mapping and analyzing the assembly against the ‘O’Neal’ genome and its PacBio Iso-Seq data as well as gene network analysis. This research will provide the first data of its kind in genetic regulation of bud cold tolerance and recovery from spring freeze events, which establishes a molecular foundation for future molecular breeding projects.