Abstract
Sex in Oreochromis niloticus (Nile tilapia) is principally determined by an XX/XY locus but other genetic and environmental factors also influence sex ratio. Restriction Associated DNA (RAD) sequencing was used in two families derived from crossing XY males with females from an isogenic clonal line, in order to identify Single Nucleotide Polymorphisms (SNPs) and map the sex-determining region(s). We constructed a linkage map with 3,802 SNPs, which corresponded to 3,280 informative markers, and identified a major sex-determining region on linkage group 1, explaining nearly 96% of the phenotypic variance. This sex-determining region was mapped in a 2 cM interval, corresponding to approximately 1.2 Mb in the O. niloticus draft genome. In order to validate this, a diverse family (4 families; 96 individuals in total) and population (40 broodstock individuals) test panel were genotyped for five of the SNPs showing the highest association with phenotypic sex. From the expanded data set, SNPs Oni23063 and Oni28137 showed the highest association, which persisted both in the case of family and population data. Across the entire dataset all females were found to be homozygous for these two SNPs. Males were heterozygous, with the exception of five individuals in the population and two in the family dataset. These fish possessed the homozygous genotype expected of females. Progeny sex ratios (over 95% females) from two of the males with the “female” genotype indicated that they were neomales (XX males). Sex reversal induced by elevated temperature during sexual differentiation also resulted in phenotypic males with the “female” genotype. This study narrows down the region containing the main sex-determining locus, and provides genetic markers tightly linked to this locus, with an association that persisted across the population. These markers will be of use in refining the production of genetically male O. niloticus for aquaculture.
Highlights
Patterns of sex determination and differentiation in fish are very varied, with a wide range of gonochoristic and hermaphroditic species
The Stacks package [26] was used to make the assembly of the sampled loci from each individual: 95,791 and 127,014 Restriction Associated DNA (RAD)-tags were retrieved for Families 1 and 2 respectively, covering 152,916 RAD-tags in total including 69,889 of these shared between the two families (Figure 1)
Current evidence suggests that O. niloticus possess an XY/XX male heterogametic system complicated by genetic variance at this and other loci, environmental factors and probably by their interaction [36]
Summary
Patterns of sex determination and differentiation in fish are very varied, with a wide range of gonochoristic and hermaphroditic species. Several other fish sexdetermining genes have been isolated: Gsdf(Y) in Oryzias luzonensis (Luzon ricefish) [6,7]; amhy in Odontesthes hatcheri (Patagonian pejerrey) [2,8]; Amhr in Takifugu rubripes (tiger pufferfish) [1]; and sdY in Oncorhynchus mykiss (rainbow trout) [4]. Four of these genes come from those involved in sexual differentiation, while one derives from an immune-related gene (the sdY protein in O. mykiss is similar to part of the interferon regulatory factor 9). While we might search for genes from the sex differentiation pathway in a region where a sex determination gene has been mapped, it is by no means certain that we can identify a master switch gene
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