Expression of the olfactory receptor genes
Transcriptome sequencing using RNA-seq was performed on the nasal mucosa of the putative olfactory organ (R-001, R-006, R-046), the olfactory bulb (R-008), and the external skin (R-214 and R-215) of common minke whales. The FPKM values of β-actin were as follows: R-001, 983; R-006, 285; R-046, 549; R-008, 371; R-214, 161; R-215, 143 (Table 2), indicating successful RNA extraction from all the samples. In this study, 81 intact, 12 pseudo, and 266 truncated genes were annotated. The maximum expression level (as a percentage of FPKM for β-actin) of intact ORs in negative controls (the olfactory bulb, R-008, and external skin, R-214 and R-215) was 0.75 in R-008 (Fig. 3). The average expression of intact ORs with non-zero expression across all samples was 1.014. This expression level was used as the criterion for significant expression in this study.
Among the nasal mucosa samples, R-006 exhibit the presence of 22 significantly expressed intact ORs, while five of them were also expressed in R-001. OMP, on the other hand, was exclusively expressed in R-006, with an expression level greater than 1.014. In contrast, R-046, obtained from the anterior portion of the nasal chamber, did not demonstrate significant expression of ORs or OMP. Out of the 81 annotated intact ORs, all the genes expressed in the nasal mucosa samples belonged to Class-2 OR. Although four copies of intact Class-1 OR were annotated, none of them were expressed in the six samples investigated. The significantly expressed intact ORs did not form a cluster in the phylogenetic tree (Fig. 4). It is worth noting that some pseudogenes were expressed in the nasal mucosa, and one pseudogene was exclusively expressed in the external skin, as per our investigation.