Identification of SNPs related to hypermelanosis of the blind side by transcriptome profiling in the Japanese flounder (Paralichthys olivaceus)

Abstract

In flatfish, body coloration asymmetry and malpigmentation are major issues faced by many aquaculture practitioners. However, the molecular mechanism underlying malpigmentation in flatfish is still unclear. In this study, we used de novo transcriptome sequencing and assembly to construct the skin transcriptome of the Japanese flounder, Paralichthys olivaceus; we collected skin samples from the ocular side of normal fish and pigmented skin and non-pigmented skin from the blind side of hypermelanotic fish. Twenty-two SNP loci at the coding regions of 12 genes were identified as candidate SNPs in six pigment synthesis-related KEGG pathways, and two SNPs located on the cyclic AMP-dependent transcription factor (ATF)-4-like gene, named ATF-3004-G/A and ATF-3155-C/T, may be associated with hypermelanosis in the blind side. To the best of our knowledge, this is the first time that SNPs associated with malpigmentation has been identified in flatfish. The predominant genotypes of normal flounder at the ATF-3004-G/A locus were GG homozygous (accounting for 64.71%), whereas the predominant genotypes of hypermelanotic flounder were GA heterozygous (accounting for 46%), followed by GG (38%) and AA (16%). At the ATF-3155-C/T locus, CC homozygotes accounted for 92.16% in the normal flounders, whereas the predominant genotypes of hypermelanotic flounder were GA heterozygous (accounting for 52.94%), followed by GG (45.10%) and AA (1.96%). The two SNPs may be used to screen and eliminate the abnormal parents for breeding fine varieties of P. olivaceus. The two SNPS are associated with hypermelanosis and located in ATF4, which is involved in both thyroid hormone synthesis and pigment synthesis-related pathways. The expression of ATF4 was higher in the skin on the blind side of hypermelanotic P. olivaceus, whatever the skin was pigmented or non-pigmented. The relative expression of ATF4 mRNA on the ocular side skin was significantly different from that on the blind side of normal P. olivaceus as well as that of hypermelanotic P. olivaceus, whereas no significant differences were detected between the blind sides pigment skin and non-pigment skin of hypermelanotic P. olivaceus.