Blueberry breeding faces substantial challenges in efficiently producing plants with desired traits through traditional methods, limiting the industry's ability to meet increasing demands. Tissue culture (TC) and regeneration methods, while promising, are hindered by high costs, cultivar-specific outcomes, and unpredictability. Recent research has identified the significant roles of morphogenic genes such as WUSCHEL (WUS) and BABY BOOM (BBM) in shoot regeneration, early shoot formation, and transformation processes. This study investigates the gene morphology and function of native BBM and WUS genes in various Vaccinium corymbosum (highbush blueberry) varieties exhibiting different levels of tissue culture recalcitrance. Previously, we identified the locations of WUS and two BBM gene variants (BBM1 and BBM2) in blueberry transcriptome data, followed by primer design, PCR, and Sanger sequencing. BBM variants and WUS were analyzed in blueberry varieties ‘ONeal’ and ‘Legacy,’ which are capable of propagation via TC, and ‘Pinnacle,’ ‘NC 4499,’ and ‘NC5271,’ which lack transcriptome data and are known to be recalcitrant to TC. Initial WUS sequencing revealed a three-nucleotide deletion in the hard-to-culture varieties ‘NC4499,’ ‘NC5271,’ and ‘Pinnacle,’ resulting in the loss of a single amino acid. Additionally, two non-synonymous SNPs with high mutation rates were identified in ‘Pinnacle’ and ‘NC5271.’ To further investigate the impact of these genetic differences, we conducted protein sequence analysis to determine how the codon deletion in WUS affects protein structure. This could uncover potential alterations in protein folding or function, which may contribute to reduced regenerative capacity in recalcitrant varieties. We are currently working to extract RNA from the five blueberry varieties at different growth phases for cDNA synthesis and qPCR to investigate gene expression levels and copy number. This project aims to address a critical gap in blueberry cultivation by uncovering the genetic basis for propagation challenges in high-value varieties. By focusing on the WUS and BBM genes, the study seeks to identify genetic variations contributing to TC resistance, informing future genetic and breeding strategies.
