Integrated analysis of RNA-seq and microRNA-seq depicts miRNA–mRNA networks involved in stripe patterns of Botia superciliaris skin

Botia superciliaris, an endemic cobitid fish in China, is widely accepted by Chinese consumers because its edibility. Recently, the black and yellow stripes of B. superciliaris skin have made this species increasingly popular as a novel ornamental fish. However, the genetic basis of the stripe patterns in B. superciliaris skin has not been extensively studied. In this study, Illumina sequencing was employed to identify the mRNAs and miRNAs involved in stripe pattern formation in B. superciliaris skin. A total of 147.25 and 155.15 million (M) high-quality transcriptome reads were generated from three black and yellow skin libraries respectively, which resulted in 159,327 unigenes that were used as reference sequences. A total of 3169 genes exhibited significantly differential expression patterns (fold-change ≥ 2 or ≤ 0.5 and q ≤ 0.05), including 1891 upregulated genes (59.67%) and 1278 downregulated genes (40.33%) in black vs yellow skin. These genes were enriched in 50 GO terms and 10 KEGG pathways (q ≤ 0.05), including melanogenesis, with 21 upregulated genes and 5 downregulated genes in black vs yellow skin. Based on the zebrafish genome, miRNA-seq identified a total of 355 miRNAs, which included 38 novel miRNAs. Furthermore, 87 differentially expressed miRNAs including 50 upregulated and 37 downregulated miRNAs were identified in different color skin (fold-change ≥ 2 or ≤ 0.5 and q ≤ 0.05). Then, target prediction revealed a variety of putative target genes; differentially expressed mRNAs and miRNAs patterns of high-throughput sequencing were validated in 5 mRNAs and miR-217-5p by qRT-PCR. In vivo tests and dual-luciferase reporter assay revealed that overexpression of miR-217-5p can inhibit pheomelanin formation by targeting Zgc. In this study, a comparative analysis was conducted to profile the transcriptome of black and yellow skin for B. superciliaris, and we detected key genes and important miRNAs involved in the B. superciliaris skin pigmentation process. These results will enhance understanding of molecular mechanisms underlying skin pigmentation and facilitate molecular-assisted selection of highly valued skin colors.