RNA-seq combined with a bulked-segregant analysis identifies candidate genes for the waxy coating on blueberry fruit

The most significant difference between blueberries with a light blue fruit color and black fruit color is the visible layer of an epicuticular waxy coating. This layer functions in disease defense and prevention of water loss. In this study, we constructed a northern-adapted rabbiteye hybrid breeding population, ‘Nocturne’ x T 300, which segregated for fruit color (light blue versus black). We screened this population and selected plants of each extreme phenotype, waxy- coated plants with light blue colored fruit versus non-waxy plants with black colored fruit, then isolated RNA from fruit tissue of each bulk, respectively. We sequenced the transcriptome of each bulk using RNA-seq, which resulted in a total of 167,093,354 reads for both libraries combined. We de novo assembled this data set into 171,678 contigs and used the assembled transcriptome as a reference for differential expression analysis using EdgeR. A total of 515 differentially expressed genes were identified with at least a four-fold difference in expression, and efforts were made to functionally annotate them using publicly available databases. From these, one excellent candidate ‘waxy’ gene has emerged, which we are investigating further.