Detection of genes with gender-specific CNV and miRNA affecting sex determination in Nile tilapia (Oreochromis niloticus)

The main objective was to detect genes and microRNAs that were differentially expressed by gender in early embryonic development. Artificial fertilization of Oreochromis niloticus XX females with either sex-reversed ΔXX males or genetically-modified YY ‘supermales’ resulted in all-female and all-male embryos, respectively. Total RNA pools of all-female and all-male embryos at 2, 5 and 9 dpf were used as template for a custom Agilent eArray hybridization and Small-RNA sequencing. Fifty-nine genes differentially expressed between genders were identified. The most overexpressed and validated genes at the early embryonic development, with gender-specific CNV and gonad-specific expression were cr/20β-hsd in females, and amh in males. cr/20β-hsd has four distinct copies in the genome. PCR amplification of transcript 3 at the 5' UTR region resulted in two products only in females, thus displaying female-specific CNV. Sequencing of amh identified a male-specific duplication of this gene, denoted amhy, differing from the sequence of amh by a 233 bp deletion on exon VII, hence lacking the capability to encode the protein motif that binds to the transforming growth factor beta receptor (TGF-β domain). Amh and amhy segregated in the mapping family in full concordance with SD-linked marker on LG23 signifying the QTL for SD. We discovered 831 microRNAs in tilapia embryos of which nine had sexually dimorphic expression patterns. An up-regulated microRNA in males, pma-mir-4585, was characterized with all six predicted target genes including cr/20β-hsd, down-regulated in males. This study reports on two major genes with sexually differential expression patterns and gender-specific CNV.