RNA expression
The RNA expression analysis in the present study employed the same methods as described in Kishida et al. (2019). The OR genes were queried against the common minke whale genome assembly (GenBank accession GCA_000493695.1) (Yim et al., 2014) using the TBLASTN program in the BLAST+ v. 2.6.0 package (Camacho et al., 2009) with a cut-off E-value of 1 × 10−5. Deduced amino acid sequences of all intact ORs of green anole (Anolis carolinensis ) and western clawed frog (Xenopus tropicalis ), cow (Bos tauros ), and mouse identified by Niimura (2009b) and Niimura et al. (2014) were used as queries. Each obtained sequence was searched against the GenBank protein database using the BLASTX program and if its best hit did not correspond to an OR, it was discarded. A sequence was deemed a non-functional pseudogene if it contained premature stop codons and/or frame shifts, or it lacked five or more consecutive amino acids, including a transmembrane domain. Sequences interrupted by contig-gaps, though not classified as pseudogenes, were labelled as ‘truncated’.
To search for the OMP gene, FATE (https://github.com/Hikoyu/FATE/blob/master/fate.pl) was employed to search the common minke whale genome assembly (GenBank accession GCA_000493695.1) using the annotated query sequence NW_006728793.1, identified as OMP, from GenBank Refseq GCF_000493695.1. The resulting single sequence found in GCA_000493695.1 was used for OMP gene mapping.
Total RNA was extracted from the nasal chamber mucosa (R-001, R-006, R-046), olfactory bulb (R-008) and external skin (R-214, R-215) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s guidelines. The olfactory bulb and external skin samples served as negative controls. The extracted RNA was used to construct paired-end sequencing libraries using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina Inc., San Diego, CA, USA). Subsequently, Illumina NovaSeq platform (2×101 bp) was employed for sequencing, generating RNA-seq reads with the following sizes: R-001, 5.89 G bp; R-006, 4.57 G bp; R-046, 5.54 G bp; R-008, 5.37 G bp; R-214, 5.35 G bp; R-215, 6.03 G bp. Low-quality sequences and adapters were removed using Trimmomatic v. 0.38 (Bolger et al., 2014) with the following parameters: ILLUMINACLIP:TruSeq3-PE-2.fa: 2:30:10, LEADING:20, TRAILING: 20, SLIDINGWINDOW: 4:20, and MINLEN: 36. HISAT2 (Kim et al., 2015) v. 2.1.0 with default parameters was used to map trimmed RNA-seq reads to the conspecific genome assembly. The expression levels of genes were quantified using fragments per kilobase of exon per million mapped fragments (FPKM) values with Cufflinks (Trapnell et al., 2010; Roberts et al., 2011) v. 2.2.1 after removing duplicated reads. The expression level of ORs was calculated by dividing it by that of β-actin and multiplying it by 100, giving expression-percentage.
The annotated intact ORs of common minke whales were incorporated into a phylogenetic tree. The nucleotide sequences were aligned using MAFFT (Kuraku et al., 2013; Katoh et al., 2019) v. 7, and a suitable model was determined by Modeltest, IQtree (Trifinopoulos et al., 2016) v. 1.6.12. Subsequently, the TIM3+F+I+G4 model was selected, and the phylogenetic tree was constructed using RAxML-ng (Kozlov et al., 2019) v. 0.9.0 with the root set as Class-1 ORs.