Background: Blueberry is of high economic value. Most blueberry varieties selected for the fresh market have an appealing light blue coating or "bloom" on the fruit due to the presence of a visible heavy epicuticular wax layer. This waxy layer also serves as natural defense against fruit desiccation and deterioration.
Results: In this study, we attempted to identify gene(s) whose expression is related to the protective waxy coating on blueberry fruit utilizing two unique germplasm populations that segregate for the waxy layer. We bulked RNA from waxy and non-waxy blueberry progenies from the two northern-adapted rabbiteye hybrid breeding populations ('Nocturne' x T 300 and 'Nocturne' x US 1212), and generated 316.85 million RNA-seq reads. We de novo assembled this data set integrated with other publicly available RNA-seq data and trimmed the assembly into a 91,861 blueberry unigene collection. All unigenes were functionally annotated, resulting in 79 genes potentially related to wax accumulation. We compared the expression pattern of waxy and non-waxy progenies using edgeR and identified overall 1125 genes in the T 300 population and 2864 genes in the US 1212 population with at least a two-fold expression difference. After validating differential expression of several genes by RT-qPCR experiments, a candidate gene, FatB, which encodes acyl-[acyl-carrier-protein] hydrolase, emerged whose expression was closely linked to the segregation of the waxy coating in our populations. This gene was expressed at more than a five-fold higher level in waxy than non-waxy plants of both populations. We amplified and sequenced the cDNA for this gene from three waxy plants of each population, but were unable to amplify the cDNA from three non-waxy plants that were tested from each population. We aligned the Vaccinium deduced FATB protein sequence to FATB protein sequences from other plant species. Within the PF01643 domain, which gives FATB its catalytic function, 80.08% of the amino acids were identical or had conservative replacements between the blueberry and the Cucumis melo sequence (XP_008467164). We then amplified and sequenced a large portion of the FatB gene itself from waxy and non-waxy individuals of both populations. Alignment of the cDNA and gDNA sequences revealed that the blueberry FatB gene consists of six exons and five introns. Although we did not sequence through two very large introns, a comparison of the exon sequences found no significant sequence differences between the waxy and non-waxy plants. This suggests that another gene, which regulates or somehow affects FatB expression, must be segregating in the populations.
Conclusions: This study is helping to achieve a greater understanding of epicuticular wax biosynthesis in blueberry. In addition, the blueberry unigene collection should facilitate functional annotation of the coming chromosomal level blueberry genome.
Keywords: Bulked segregant analysis; Differential expression; Fruit wax; RNA-seq; Vaccinium spp..