BackgroundThe probable influence of genes and the environment on sex determination in Nile tilapia suggests that it should be regarded as a complex trait. Detection of sex determination genes in tilapia has both scientific and commercial importance. The main objective was to detect genes and microRNAs that were differentially expressed by gender in early embryonic development.ResultsArtificial 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. RNA of 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 next generation sequencing. Fifty-nine genes differentially expressed between genders were identified by a false discovery rate of p < 0.05. The most overexpressed genes were amh and tspan8 in males, and cr/20β-hsd, gpa33, rtn4ipl and zp3 in females (p < 1 × 10−9). Validation of gene expression using qPCR in embryos and gonads indicated copy number variation in tspan8, gpa33, cr/20β-hsd and amh. 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 exonVII, 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 by a false discovery rate of p < 0.05. 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.ConclusionsThis study reports the first discovery of sexually differentially expressed genes and microRNAs at a very early stage of tilapia embryonic development, i.e. from 2 dpf. Genes with sexually differential expression patterns are enriched for copy number variation. A novel male-specific duplication of amh, denoted amhy, lacking the TGF-β domain was identified and mapped to the QTL region on LG23 for SD, thus indicating its potential role in SD.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-774) contains supplementary material, which is available to authorized users.

Poecilia reticulata is one of the most popular ornamental fish species with a higher demand for males due to their large colorful fins. The objectives of this study were mapping of the sex-determining (SD) region on linkage group 12 of guppy, and identification of a sex specific marker. We generated eight polymorphic microsatellite markers distributed along the distal 5.4 Mbp sequence of the previously identified SD region on linkage group (LG) 12. The markers were tested for association with sex in 156 individuals of the Red Blonde and Flame strains, and 126 progeny of four full-sibs Red Blonde families. A male-specific allele was found for microsatellite gu1066 at position of 25.3 Mbp on LG12 for both strains, and gu832 at position 24.4 Mbp for the Flame strain. Thus, a region of 0.9 Mbp between these markers, harboring 27 annotated genes, was selected for analysis. Based on association of copy number variation and sex determination we mapped a duplicated region between LGs 9 and 12, of 1.26 Mbp, containing 59 genes on LG12. The common interval between the segment bounded by gu1066 and gu832, and the duplicated region of 0.43 Mbp on LG12 harbors 11 genes of which 6 have multiple copies (54%). Growth arrest and DNA damage inducible gamma-like (GADD45G-like) is a plausible positional and functional candidate gene for its role in male fertility. We characterized the genomic structure of the gene in guppy, and found two isoforms; but no sex-biased differences were evident in genomic sequence and gene expression.

Sex determination (SD) mechanisms are ancient and conserved, yet much diversity is exhibited in primary sex-determining signals that trigger male or female development. In O. niloticus, SD is associated with a male-specific locus on linkage group (LG) 23 which harbors the Y-linked Anti-Müllerian hormone (amh) gene, and a truncated duplication, denoted amhΔy. We have evaluated the possible role of identified indels and SNPs in the amh gene on SD, based on conservation in different O. niloticus strains. A fluorescent assay for the detection of a 5 bp insertion in amhΔy exon VI, efficiently discriminated between XX, XY, and YY genotypes. Concordance rate between amhΔy and sex varied in six Oreochromis strains, from 100% (Ghana) through 90% (Swansea) to 85% (Thai-Chitralada). The association of amhΔy with sex was found to be conserved in all tested O. niloticus strains, and thus supports its key role in SD. However, the previously identified missense SNP (C/T) in amh exon II was found only in the Swansea strain, thus excluding its candidacy for the causal variation of SD across all strains. Effects of markers on LGs 1, 3, and 23 (amhΔy) fully explained sex distribution in one Thai-Chitralada family (R 2 = 1.0), whereas in another family only the major effect of LG23 (amhΔy) was significant (R 2 = 0.37). Thus, amhΔy on LG23 is associated with genetic SD, either as a single causal gene in different O. niloticus strains, or in combination with segregating genes on LGs 1 and 3 in the Thai-Chitralada hybrid strain.