Gene Expression Analysis of erbb3b in Anemonefish

In total, ten juveniles (five orange and five black) were assayed to measure gene expression of the three erbb3b genes identified in A. clarkii. Specific primers for each A. clarkii erbb3b gene (two short genes containing 1,911 bp and one long gene of 4,275 bp) were designed manually based on their genomic sequence (Supplementary Table 3). Primers previously used by Roux et al. (2022) (link) with A. ocellaris were used to target the housekeeping genes ribosomal protein L7 (rpl7) and ribosomal protein L32 (rpl32). Extracted RNA from each juvenile (as described in Supplementary Methods 1) was converted to cDNA using PrimeScript RT-PCR Kit (Takara Bio, Shiga, Japan). The efficiency and specificity of the designed primers was tested through PCR using the GoTaq Green Master kit (Promega, Madison, USA) with thermal cycling conditions of 2 min at 95°C, followed by 30 cycles of 45 s at 95°C, 45 s at 60/63/65°C, and 30 s 72°C, a final extension step of 5 min at 72°C, preservation at 4°C, and subsequent agarose gel electrophoresis (Supplementary Fig. 3). The specificity was also tested through direct forward and reverse Sanger sequencing by aligning the forward and reverse outputs and blasting the obtained amplicons against the reference genomic sequences (Supplementary Fig. 4).
The expression of each erbb3b gene and the two housekeeping genes (rpl7 and rpl32) was obtained by RT-qPCR at 65°C (PrimeScript transcriptase, Takara, SYBRgreen) and normalized with the Pfaffle equation (Ståhlberg et al. 2004 (link)):
where RE is the relative expression, E(x) is the efficiency of the amplification for isoform x, and Ct(x) is the quantification cycle of gene x.