Figure 1.
Wide-ranging sex ratios were observed among 62 zebrafish families.
We have crossed randomly picked zebrafish individuals, grown their offspring to sexual maturity and determined their sex ratio based on presence/absence of sexual dimorphic phenotypic markers.
Figure 2.
Sex ratios of offspring groups generated by repeated single pair crossings show close correlation.
Nineteen randomly selected breeding pairs were crossed twice; eighteen of them are shown here. The high R2 value indicates that sex ratios between 1st and 2nd crosses from the same breeding pair are very similar. Red circles indicate pairs producing offspring with female-biased sex ratio, orange diamond labels the pairs with unbiased sex ratio, whereas blue squares indicate pairs producing offspring with male-biased sex ratio.
Table 1.
The percentage of males from repeated single pair mating of 19 randomly selected zebrafish pairs.
Figure 3.
Coefficients of variation for each generation family sex ratios show selection effect on sex ratio.
Panels A & B: For both families, CV for the F0 generation (unselected) was more than two-folds higher than those for the subsequent generations, which underwent selection. Panel C: In the control experiment, after selecting for pairs that produced high proportion of males at F0 generation, CV for F1 generation family sex ratio decreased by about three-folds. However, when selection pressure was removed at F2 generation by performing a random mass cross, CV for F3 generation family sex ratio returned to a level similar to that of unselected F0 generation.
Figure 4.
Comparative analysis of CNVRs in four zebrafish families.
Out of 255 CNVRs detected, only five were present in all four families tested, however, those common CNVRs have not shown any association with sex. The number of CNVRs detected for each family is indicated in the bracket.
Figure 5.
PCR-based validation of aCGH results that showed apparent family specific sex-linked inheritance pattern confirms that none of the three CNVRs analyzed are sex-linked.
A) The lack of sex-linkage for CNV regions 2 and 5 as confirmed by PCR. Size of the amplified fragments for CNVR2 and CNVR5 are 157 bp and 183 bp, respectively. CNVR2 was present only in males from the Toh1 family (Father and Son 1 and 2), while CNVR5 was only seen in female samples from the AB2 family (Mother and Daughter 1 and 2). As they showed a family-specific, sex-linked pattern, additional offspring (one son and one daughter; red boxes) were used for the validation. Upon further validation, CNVR2 and CNVR5 were found not to be sex-linked. B) CNV region 3 could only be validated by real time qPCR. As the three female samples from Toh2 family used for aCGH showed a loss with reference to the father's genome, additional offspring (one son and one daughter; red bar) were used for validation. Further validation also showed that this is not a sex-linked CNVR.
Table 2.
CNV regions selected for further validation due to their apparent association to sex based on preliminary aCGH.
Figure 6.
High rearing densities yield higher male to female sex ratios compared to lower ones.
Two individual experiments were performed consisting of forty-four populations for experiment 1 and thirty populations for experiment 2. The final percentage of males was assayed for each population and the averages for each population group, denoted by the starting density, were calculated. For each experiment the overall sex ratios varied, but both showed about a twenty percent increase in male percentage in populations with starting densities of 100 fish per 1.5 liters compared to populations with starting densities of 50 or 25 fish per 1.5 liters. Each datapoint represents the percentage of males for a given parental pair, whereas the horizontal line indicates the mean male ratio.