Owing to the demand for sustainable sex-control protocols in aquaculture, research in tilapia sex determination is gaining momentum. The mutual influence of environmental and genetic factors hampers disentangling the complex sex determination mechanism in Nile tilapia (Oreochromis niloticus). Previous linkage analyses have demonstrated quantitative trait loci for the phenotypic sex on linkage groups 1, 3, and 23. Quantitative trait loci for temperature-dependent sex reversal similarly reside on linkage group 23. The anti-Müllerian hormone gene (amh), located in this genomic region, is important for sexual fate in higher vertebrates, and shows sexually dimorphic expression in Nile tilapia. Therefore this study aimed at detecting allelic variants and marker-sex associations in the amh gene. Sequencing identified six allelic variants. A significant effect on the phenotypic sex for SNP ss831884014 (p<0.0017) was found by stepwise logistic regression. The remaining variants were not significantly associated. Functional annotation of SNP ss831884014 revealed a non-synonymous amino acid substitution in the amh protein. Consequently, a fluorescence resonance energy transfer (FRET) based genotyping assay was developed and validated with a representative sample of fish. A logistic linear model confirmed a highly significant effect of the treatment and genotype on the phenotypic sex, but not for the interaction term (treatment: p<0.0001; genotype: p<0.0025). An additive genetic model proved a linear allele substitution effect of 12% in individuals from controls and groups treated at high temperature, respectively. Moreover, the effect of the genotype on the male proportion was significantly higher in groups treated at high temperature, giving 31% more males on average of the three genotypes. In addition, the groups treated at high temperature showed a positive dominance deviation (+11.4% males). In summary, marker-assisted selection for amh variant ss831884014 seems to be highly beneficial to increase the male proportion in Nile tilapia, especially when applying temperature-induced sex reversal.
BackgroundIn Nile tilapia sex determination is governed by a male heterogametic system XX/XY either on LG1 or LG23. The latter carries a Y-specific duplicate of the amh gene, which is a testis-determining factor. Allelic variants in the amh gene demonstrated to be major triggers for autosomal and temperature-dependent sex reversal. Further, QTL on LG23 and LG20 show a temperature-responsiveness with influence on the phenotypic sex relative to the sex chromosomes. Here we present a ddRADseq based approach to identify genomic regions that show unusual large differentiation in terms of fixation index (FST) between temperature-treated pseudomales and non-masculinized females using a comparative genome-scan. Genome-wide associations were identified for the temperature-dependent sex using a genetically all-female population devoid of amh-ΔY.ResultsTwenty-two thousand three hundred ninety-two SNPs were interrogated for the comparison of temperature-treated pseudomales and females, which revealed the largest differentiation on LG23. Outlier FST-values (0.35–0.44) were determined for six SNPs in the genomic interval (9,190,077–11,065,693) harbouring the amh gene (9,602,693–9,605,808), exceeding the genome-wide low FST of 0.013. Association analysis with a set of 9104 selected SNPs confirmed that the same genomic region on LG23 exerts a significant effect on the temperature-dependent sex.ConclusionsThis study highlights the role of LG23 in sex determination, harbouring major determinants for temperature-dependent sex reversal in Nile tilapia. Furthermore FST outlier detection proves a powerful tool for detection of sex-determining regions in fish genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3930-0) contains supplementary material, which is available to authorized users.
Objective: A genome-wide high-throughput single nucleotide polymorphism (SNP) typing method was tested with respect of the applicability to ancient and degraded DNA. The results were compared to mini-sequencing data achieved through single base extension (SBE) typing. The SNPs chosen for the study allow to determine the hair colors and eye colors of humans. Material and methods: The DNA samples were extracted from the skeletal remains of 59 human individuals dating back to the Late Bronze Age. The 3,000 years old bones had been discovered in the Lichtenstein Cave in Lower Saxony, Germany. The simultaneous typing of 24 SNPs for each of the ancient DNA samples was carried out using the 192.24 Dynamic Array™ by Fluidigm ® . Results: Thirty-eight of the ancient samples (=64%) revealed full and reproducible SNP genotypes allowing hair and eye color phenotyping. In 10 samples (=17%) at least half of the SNPs were unambiguously determined, in 11 samples (=19%) the SNP typing failed. For 23 of the 59 individuals, a comparison of the SNP typing results with genotypes from an earlier performed SBE typing approach was possible.The comparison confirmed the full concordance of the results for 90% of the SNP typings. In the remaining 10% allelic dropouts were identified.Discussion: The high genotyping success rate could be achieved by introducing modifications to the preamplification protocol mainly by increasing the DNA input and the amplification cycle number. The occurrence of allelic dropouts indicates that a further increase of DNA input to the preamplification step is desirable. K E Y W O R D S ancient and degraded DNA, eye color, genome wide, hair color, high-throughput SNP typing, phenotype
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