2.5 | Primer design
Beal et al . (2019) reported a correlation between methylation rate and age in the common bottlenose dolphin (a closely related species to the Indo-Pacific bottlenose dolphin) using three gene regions:TET2 , GRIA2 , and CDKN2A from DNA extracted from skin samples. Similar correlations using the same genes were seen in humpback whales (Polanowski et al ., 2014) and Antarctic minke whales (Tanabe et al., 2020). These findings suggest that these genes can be commonly used for epigenetic clock analyses in cetaceans. However, we were unable to find the genomic information on Indo-Pacific bottlenose dolphins. Therefore, primer designs were referred to the genomic information on common bottlenose dolphins, available at the National Centerfor Biotechnology Information (NCBI,https://www.ncbi.nlm.nih.gov/) database using the Standard Nucleotide Basic Local Alignment Search Tool (BLAST) (Altschul et al ., 1990). We attempted to design PCR primers to amplify three target genes (reference genomes: TET2 &GRIA2 : NC_047038; CDKN2A : NC_04739). Primers were designed using Methyl Primer Express v1.0 (Thermo Fisher Scientific, San Jose, CA, USA, Table 1). However, we were unable to design a primer for the TET2 gene that would successfully amplify the target region. As a result, we proceeded with the analysis using only GRIA2 andCDKN2A genes.
There was a need for additional verification to ensure that the designed primers specifically amplified the DNA of the target species and not the prey species. To confirm this, we used the NCBI database using the BLASTN tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and homogenous gene regions were searched against reference genomes to ensure that the same sequences were not found in the prey species for this population, as reported by Takahashi et al . (2020) and Kitaet al. (2018).