Studying the Expression of Bilberry Fruit Softening-related Cellulase (GH9) and Hemicellulase (XTH) Gene Families by Developing a Primer Selection Tool

Abstract

Fruit softening is linked to degradation of cell walls by hydrolytic enzymes, such as members of GH9 and XTH family. In this study, we aim to identify the members of the large gene families that cause the cell wall modifying effects, as they can become a target of future research related to food waste prevention and biofuel production. A tool is developed to help in the process of finding qPCR primers that are unique to a single member of large gene families. GH9 and XTH genes were identified from the bilberry genome (GDV, Vaccinium.org). Targets were identified by connecting the genes to the ripening transcriptome. A phylogenetic analysis of the bilberry GH9 genes with known members of the Arabidopsis thaliana and Oryza sativa GH9 genes was performed, thus allowing for functional estimates of bilberry genes. Similarly, bilberry XTH genes were phylogenetically compared to known XTH genes of several species, providing functional estimates based on the subclassification. The expression of 8 GH9 and 12 XTH genes is measured by qPCR at five different ripening stages of the bilberry fruit. Out of the 18 GH9 genes assessed in this study, 8 were found to be associated with fruit ripening. For XTH genes, 12 out of 39 were associated with fruit ripening. Of the fruit ripening-related GH9 genes, 4 genes showed significant upregulation during the stages of fruit ripening most associated with softening. Two group IIIb genes and six genes from groups I and II, are suggested targets for future research. Despite the bilberry's large GH9 and XTH families, we managed to study the expression of specific members of the family. Based on the phylogenetic analyses, it was found that strong upregulation of GH9 genes was associated with GH9b, although genes two GH9c genes also showed some upregulation during ripening. As for the XTH genes, upregulated genes from the III-a subclassification are most interesting, as those are most likely to show XEH activity. The self-developed primer selection tool was not found to outperform the freely available alternative PrimerBLAST in the case of finding unique primers for large gene families. We identified GH9-4, 6, 22, and 23 and XTH-5, 8, 15, 17, 28, 38, 39 and 42 as potential future targets based on their role during fruit ripening.