The Journal of Heredity 2002:93(4)
© 2002 The American Genetic Association 93:270-276
Association Between Loci With Deleterious Alleles and Distorted Sex Ratios in an Inbred Line of Tilapia (Oreochromis aureus)
From the Laboratory of Fish Immunology and Genetics, Faculty of Life Sciences, Bar Ilan University, Ramat Gan 52900, Israel (Shirak and Avtalion); Institute of Animal Science, Agricultural Research Organization, P.O. Box 6, Bet Dagan 50250, Israel (Shirak, Palti, Cnaani, Hulata, and Ron); and Institute of Evolution, University of Haifa 31905, Israel (Korol). Y. Palti is currently at NCCCWA-USDA/ARS 11876 Leetown Rd., Kearneysville, WV 25430.
Address correspondence to Ramy R. Avtalion, at the address above, or e-mail: avtalr{at}mail.biu.ac.il.
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Abstract |
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Three microsatellite markers (UNH159, UNH231, and UNH216) were examined for association with both deleterious genes and sex-ratio distortions in a full-sib family of 222 progeny from the fourth generation of a meiogynogenetic tilapia line (Oreochromis aureus). The three markers were mapped previously to different linkage groups and were shown to be associated with genes with deleterious alleles in this line. A restricted maximum likelihood model was used for analysis of major effects and their interactions on sex ratio and viability. This model was based on selective mortality of genders, ignoring effects of possible sex-determining genes. The results showed that deleterious genes linked to UNH216 and UNH231 exert higher lethality in females than in males (P < .0005 and P < .05, respectively). UNH159 was not associated directly with sex ratio distortion, but acts strongly as a modifier of sex ratio in combination with UNH216 and UNH231. Each of the three loci was found to have a significant effect on viability (P < .05) in the maximum likelihood analysis. The deleterious single-locus effects act strongly against females, while most of the epistatic interactions exert higher lethality in males. This contradiction results in a close to 1:1 sex ratio at maturity. The genetic mechanism and significance of such a balance between genders are still unknown. A detailed analysis of sex-specific lethality may be applied by screening in appropriate series of matings and fine mapping with additional markers. Our data suggest that UNH216 and UNH231 are linked to sex ratio distortion genes and that UNH159 may be linked to a modifier of these genes.
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Introduction |
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Tilapias are fishes of great importance to aquaculture in tropical and subtropical countries and provide an important source of protein for developing countries in these regions (Food and Drugs Organization 1992). One of the major goals of tilapia breeding programs is to prevent overpopulation of grow-out ponds by producing monosex populations. Males grow much more rapidly than females, hence most breeding programs are designed to produce all-male populations (Hulata 1995; Pruginin et al. 1975; Tave 1995).
Hickling (1960) and Fishelson (1966) found that crosses between certain related tilapia species (e.g., Oreochromis mossambicus females with O. hornorum males, or O. niloticus females with O. aureus males) generated all-male hybrid broods. This finding stimulated further study of sex determination in tilapia (McAndrew 1993; reviewed by Trombka and Avtalion 1993). Gynogenesis (all-maternal inheritance) was used to demonstrate different gonosomal models of sex determination among tilapia (heterogametic females [WZ] with homogametic males [ZZ] in O. aureus or homogametic females [XX] and heterogametic males [XY] in O. niloticus). However, this four-gonosomal model could not explain the experimental sex ratio results obtained in hybrids of the second generation (Chen 1969; Jalabert et al. 1971). The autosomal theory (Avtalion and Hammerman 1978; Hammerman and Avtalion 1979) gave a better explanation for the second-generation sex ratios. This theory was able to explain most of the experimental results assuming that sex is determined by the sum of effects of three alleles (W, X, and Z, where Y = Z) of a major sex-determining locus and two alleles (A, a) of an autosomal locus. Thus O. mossambicus and O. niloticus are classified as AAXX females and AAXY males, while O. hornorum and O. aureus are classified as aaWY females and aaYY males. According to this theory, 10 female and 8 male genotypes are expected to give rise to 8 different sex ratios (1:0, 3:1, 5:3, 9:7, 1:1, 3:5, 1:3, and 0:1). Still, this theory cannot explain distortions from the 1:1 sex ratio in purebred species. Additional studies that employed sex reversal and gynogenesis led other authors to suggest multigenic involvement in sex determination of pure tilapia species (Calhoun and Shelton 1983; Hopkins et al. 1979; Lester et al. 1989; Mair et al. 1991; Wohlfarth and Wedekind 1991). Several studies have also shown an influence of ambient temperature on sex ratios in O. niloticus (Argue and Phelps 1995; Baroiller et al. 1995).
To date, no sex-linked markers have been detected in tilapias by biochemical, electrophoretic, or other genetic methods (Chen and Tsusuki 1970; Cruz et al. 1982; Trombka and Avtalion 1993). Nijjhar et al. (1983) suggested the two long chromosomes as the candidate gonosomes in tilapia. Carrasco et al. (1999) reached the same conclusion after visualizing the synaptonemal complex in O. niloticus, but recent fluorescent in situ hybridization (FISH) results suggested these irregularities are due to interstitial telomeric repeats left over from the fusion of smaller chromosomes (Wright J, personal communication). Recently Lee and Kocher (1996) developed more than 150 microsatellite DNA markers, which led to construction of several linkage maps for different tilapia species (Agresti et al. 2000; Kocher et al. 1998; McConnell et al. 2000). The suggested multigenic influence on sex determination and the recent availability of genetic maps for tilapia enabled application of genomic screening (Soller et al. 1976) to map sex-determining genes. We previously used the map of Kocher et al. (1998) for large-scale genomic screening and detected an association of three unlinked microsatellite loci (UNH159, UNH216, and UNH231) to genes with deleterious alleles in a well-characterized gynogenetic line of O. aureus (Avtalion and Don 1990; Don and Avtalion 1988; Palti et al. 2002; Shirak et al. 1998).
In Drosophila melanogaster, sex determination was found to be controlled by the Sex-lethal (Sxl) gene (reviewed by Cline and Meyer 1996). Misregulation of this gene results in sex-specific lethality. Failure to express Sxl is lethal only to females, while constitutive expression is lethal to males (Stitzinger et al. 1999). Skewed sex ratios (e.g., 2 female:1 male ratio) previously were observed in many of the crosses between O. aureus meiogynogens in Bar Ilan University (Shirak A and Avtalion RR, unpublished data). These observations led us to hypothesize that, similar to the Drosophila model, sex determination in this line is associated with genes with deleterious alleles. In the present study we tested this hypothesis by examining associations between the three microsatellite markers and distortions from the 1:1 sex ratio.
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Materials and Methods |
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Fish
The meiogynogenetic O. aureus line (BIU-1) was produced in the Laboratory of Fish Immunology and Genetics, Bar Ilan University, by four successive generations of meiogynogenesis followed by full-sib mating (Avtalion and Don 1990; Don and Avtalion 1988; Shirak et al. 1998). A stock of 36 progeny was produced in a cross of two females and one male from the F4 generation (F4 x F4). Selected progeny that were heterozygous for UNH159, UNH216, and UNH231 were mated and produced 222 grandchildren to test for association between the microsatellites and sex determination.
Fish Reproduction and Rearing
One-year-old fish of the F4 x F4 generation were sexed, tagged, and divided into families of one male and five to six females, which were kept in 200 L aquaria. Fertilized eggs were collected from the mouth cavity of the selected F4 x F4 females 1–2 h after fertilization. The resulting embryos were incubated in 750 ml Zuger bottles at a constant temperature of 28.2 ± 0.2°C until yolk sac resorption. Full sibs from two different spawns were stocked and reared for 4–5 months in thermoregulated (25 ± 2°C) 750 L plastic tanks connected to a recirculating aquaculture system. At this stage, sexing was carried out by microscopic observation of the gonads (Guerrero and Shelton 1974).
Genotyping With Microsatellite Markers
Three microsatellite DNA markers (UNH159, UNH216, and UNH231) with the forward primer dye labeled were purchased from Research Genetics, Inc. (Huntsville, AL). Genotypes were obtained by automated sizing of fluorescently tagged polymerase chain reaction (PCR) amplification products. Amplification reactions were carried out in a 10 µl reaction volume containing 1x PCR buffer with 2 mM MgCl2, 1 U Taq DNA polymerase (Quantum Biotechnologies Inc., Montreal, Canada), 187.5 µM each dNTP, 0.8 µM each primer, and 30 ng genomic DNA. The amplification conditions were as follows: 92°C for 40 s, 50–58°C for 40 s, and 72°C for 1 min, for 30 cycles. Electrophoretic analysis was conducted with an ABI-377 DNA sequencer (4% acrylamide gel) as previously described (Palti et al. 2002). The DNA fragments were automatically sized by comparison with an internal standard using Genescan (version 2.1). Genotypes of individuals were determined by Genotyper (version 2.0) and automatically exported to a database. Fast (f) (117 bp, 170 bp, and 226 bp) and slow (s) (121 bp, 174 bp, and 232 bp) migrating amplification products for UNH216, UNH231, and UNH159, respectively, could be distinguished. These amplification products were found in three different combinations, interpreted as homozygous F (ff), S (ss), and heterozygous H (fs) genotypes.
Statistical Analysis
Goodness-of-fit to the expected 1:2:1 Mendelian segregation (see 
2F, 2M, and 2F+M in Table 1) and to a 1:1 sex ratio (see Table 2) were assessed using Pearson's chi-square test. Contingency tables (2 x 3) were used to test distortions in genotypic segregation between genders (see 2F:M in Table 1).
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Statistical Model of Gender Viability Selection
A two-locus model of viability selection for gender was applied for analysis of segregation results using the marker pairs UNH159-UNH216, UNH159-UNH231, and UNH216-UNH231. This model is based on the hypothesis that viability of the two sexes depends on allele combinations at two autosomal loci (A/a and B/b). In order to test this hypothesis we need to compare the observed segregation with the expected one. The expected segregation can be obtained by multiplying the two-locus genotypic frequencies expected in the absence of selection by corresponding viability coefficients. Viability for each genotype-sex combination is presented in Matrix 1.
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An additional parameter (
) was included as a coefficient for female viability to test the hypothesis that females are less viable than males (when < 1 it means more males survived to sexual maturity than females). Four possible types of epistatic interactions between two loci were considered as: homo(aa) - homo(bb) = e1, homo(aa) - hetero(Bb) = e2, hetero(Aa) - homo(bb) = e3, and hetero(Aa) - hetero(Bb) = e4. These additional parameters are included in Matrix 2.
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The viability coefficients for each locus represent selection against one of the homozygotes relative to the other, which is presumed to be subject to zero selection. The range of possible values of l coefficients is 0
lam, lbm, laf, lbf 1, where l = 0 indicates equal viability of the two homozygote classes and l = 1 indicates elimination of one class. Likewise, ham, hbm, haf, hbf are the coefficients of dominance, where h = 1 means complete dominance of the A or B allele, h = 0 means recessiveness of the A or B allele, and 0 < h < 1 means codominance of the two. If values of the coefficients e1, e2, e3, e4 equal 1 it means completely independent effects of the two loci, if e < 1 it means lower viability than expected due to "negative" epistatic selection, and if e > 1 it means higher viability than expected due to "positive" epistatic effects.
The expected frequencies of the genotypes of two loci with no selection are defined in Matrix 3. Expected frequencies of females (pijf) and males (pijm) in each genotypic class ij can be calculated using Matrices 2 and 3 (as a product of corresponding elements of these two matrices). Altogether there were 9 x 2 = 18 functions pij that depend on a set 
of 18 parameters: = {r, , lam, laf, lbm, lbf, ham, haf, hbm, hbf, e1m, e1f, e2m, e2f, e3m, e3f, e4m, and e4m}. Based on the observed numbers Nijm and Nijf (i = 1,2,3; j = 1,2,3) of the respective genotypic classes, one can evaluate the unknown set of parameters in such a way that it will provide the maximum of the likelihood function L() which is equivalent to its logarithm:
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) are the expected numbers of the classes in the foregoing scheme. The maximum likelihood values of the parameters for vector can be found by numerical optimization of the function lnL() using the procedure described by Korol et al. (2000). Let = 1 be a vector with restrictions corresponding to assumptions of "no linkage" and "no epistasis" (all em and ef equal to 1). Let = 2 be another vector with one or more relaxed restrictions [e.g., e1f is not fixed, so that the model allows estimation of female epistatic selection homo(aa) - homo(bb) = e1, provided that all other epistatic components are not important]. To compare two such hypotheses represented by vectors 2 and 1, one can use the following statistic
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2 with df = k2 - k1 degrees of freedom, where k2 and k1 are the number of parameters in the compared hypotheses as described by Kendall and Stuart (1967).
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Results |
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The sex ratio among the 222 full sibs was 114 males:108 females. Significant (P < .005) distortions from the expected 1:2:1 genotypic segregation ratios were observed for UNH216 and for UNH231 (Table 1). These distortions were the result of (1) reduction in the homozygous class of the fast-migrating allele of the UNH216 (F216), (2) reduction in the homozygous class of the slow-migrating allele of UNH231 (S231), (3) reduction in the heterozygous class H216, and (4) reduction in the heterozygous class H231. The genotypic segregation of UNH159 was not significantly different from the expected 1:2:1 ratio and the differences were not detected between genders. Significant differences between the genders' genotypic distributions were detected for UNH216 (P < .005) and for UNH231 (P < .05). These differences are the result of a stronger selection against the F216, S231, H216, and H231 genotypes in females (Table 1).
Two of the possible 27 two-locus combinations (F216S231 and F216S159) produced only males (0 females:7 males). The main effect of UNH216 and UNH231 genotypic combinations on sex ratio is illustrated in Figure 1. The s allele of UNH216 and f allele of UNH231 simultaneously increased the proportion of females. The main effect of the UNH216 s allele was modified by interactions with UNH159 alleles (Figure 2). An interaction between UNH231 and UNH159 genotypes is shown in Figure 3. Analysis of sex ratios in the three-locus combinations showed that 2 of the possible 27 combinations (S216F231H159 and H216H231H159) produced all females (21:0) and all males (0:10), respectively (Table 2).
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The maximum likelihood approach was applied to all three pairs of the marker loci as described in Materials and Methods. The estimation of coefficients with the highest LOD score for each marker pair are shown in Table 3. The values for the r coefficient derived from the model were not significantly different from 0.5, indicating an absence of linkage between the markers. The
coefficient represents the overall gender effect on viability due to the two-locus interactions. In all three equations, is significantly less than one, reflecting the lower viability of females. The effect is more pronounced for UNH216-UNH231 (Table 3). The l values compare the viability of two homozygous classes: a significant difference between viability of homozygotes was evident at UNH159 irrespective of sex (laf and lam = 0.30–0.41). In contrast, the effect for the other two markers was evident in females only (lbf = 0.58, 0.59). The h value, characterizing the viability of the three genotypic groups for each marker, was in the range of 0.45–0.78, showing that a deleterious allele reduced viability in the heterozygote state. However, an haf = 0 for marker pair UNH216-UNH231 indicated that only one class of homozygote female for UNH216 had low viability. On the other hand, hb values for marker pair UNH159-UNH216 approached zero, indicating that one class of homozygotes for both males and females at UNH216 had low viability. In addition, a negative value for ham represented low viability for heterozygote males at UNH159. Only values of e1 and e4 were different from unity, indicating epistatic interactions between homozygotes for two markers and between heterozygotes for two markers. For all double heterozygotes, epistatic interactions decreased male viability (e4m ranged from 0.20 to 0.45). For marker pair UNH216-UNH231, the viability of double-homozygous females was greatly enhanced (e1f = 6.40). However, for marker pair UNH159-UNH216, the viability of double-homozygous individuals was high irrespective of sex (e1f = e1m = 2.42) (Table 3).
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Discussion |
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Skewed sex ratios were previously shown in O. aureus and O. niloticus intraspecific spawns. Distortions from 1:1 that were repeatable in same-pair spawnings of brood fish were suggested to be the result of differential mortality of sex during incubation or early hatching (Shelton et al. 1983). Three microsatellite loci (UNH216, UNH231, and UNH159) from the fourth generation of the BIU-1 meiogynogenetic line of O. aureus were previously found to be linked to genes with deleterious alleles at hatching (Palti et al. 2002). The results of the present work show that distortions from Mendelian segregation ratios are associated with the loci UNH216 and UNH231. These distortions stem from strong selection against homozygous genotypes at each of these two loci (Table 1). Further, genotypic frequencies at these markers indicated significant differences between genders. Such differences were expressed as lower frequencies of females with genotypes F216, S231, H216, and H231 than of males. Nine possible genotypic combinations between the markers UNH216 and UNH231 showed a diagonal elevation of the sex ratio (females:males) from 0 in genotype F216S231 to 2.3 in genotype S216F231 (Figure 1). This elevation indicated a female-specific mortality that was exerted independently by each of the two loci.
No significant distortion from expected Mendelian segregation ratio and no differences between the genders' genotypic distributions were associated with UNH159 (Table 1). However, this marker acted strongly as a modifier of sex ratio in combination with UNH216 and UNH231. In combination with UNH216, sex ratios ranged from 0 in genotype F216S159 to 2.7 in genotype S216H159 (Figure 2), and with UNH231 the observed sex ratios ranged from 0.3 in genotype S231F159 to 1.5 in genotype F231H159 (Figure 3). Analysis of the three-loci combinations showed that carriers of genotype S216F231H159 were all females (21:0 ratio), as opposed to genotype H216H231H159 with all males (0:10 ratio). It is noteworthy that in both monosexual genotypes, the UNH159 marker was in the heterozygous form (Table 2). Of interest is that in progeny of heterozygous males and females for all three markers (H216, H159, H231), only males inherited the parental genotype. This unexpected result could be explained by crossing over between the microsatellite markers and the genes involved in sex ratio distortion, thereby altering the linkage phase. Alternatively it may be due to the involvement of additional yet unknown genes. The strong modifier effect of UNH159 on sex ratio may be supported by the finding that UNH159 is involved in an epistatic interaction with UNH216 that affects viability, as shown in a different sibling cross of the BIU-1 gynogenetic line (Palti et al. 2002). For a thorough investigation of major effects and interactions on sex ratio and viability, we used a restricted maximum likelihood model.
This model was based on the hypothesis of selective mortality of genders ignoring the effects of possible sex-determining genes. This model confirmed that the three microsatellite loci (UNH216, UNH231, and UNH159) are unlinked as previously reported (Agresti et al. 2000; Kocher et al. 1998). In contrast to the results at the separate chi-square analysis for each of the three loci, the deleterious single-locus effects in the model were all significant. Locus UNH159, in contrast to the two other loci, showed a fixed deleterious effect on survival in both sexes.
Epistatic interactions between loci were revealed by the maximum likelihood model (Figure 4). There were three interactions that decreased viability and two that increased viability. All unfavorable interactions acted through heterozygous males for any two of the three loci. The two favorable interactions involved UNH216 with either one of the two loci. Females with the S216F231 genotype and both genders with F216S159 were more viable than expected. The deleterious single-locus effects acted strongly against females, whereas most epistatic interactions exert higher lethality in males. Thus this contradiction between two types of effects equally stabilized the viability of genders toward maturity. The significance of such balance between genders is still unknown.
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The role of sex-determining genes may be inferred based on the observation of monosex genotypes in the three-locus combination analysis. A cross between two individuals heterozygous for three loci produces a Punnett square of 64 different zygotes, which can be used for calculating the expected frequency of each of the 27 possible genotypes (Table 2). The homozygous genotype S216F231F159 had the highest observed frequency (15/222) compared to that expected (1/64). Thus it was used as an estimated 100% viability reference. The all-female genotype (S216F231H159), with 70% relative viability, exceeds 50%, and thus cannot be explained by male-specific mortality alone (assuming an expected sex ratio of 1:1). A comprehensive model with both sex-specific lethality and sex-determining factors should be employed for analysis of genotype segregation for three loci. However, many parameters are needed for such a model, which could not be used in the current study because of the restricted dataset. Alternatively, parents may be selected with one locus in a homozygous state, with the other two loci as heterozygotes. By an appropriate series of matings, a detailed analysis of gene involvement in sex-specific lethality and sex determination may be applied. The three loci we studied do not appear to be associated with the mechanism underlying the increase of male proportion (from 33% at 27°C to 81% at 36°C) due to high larvae incubation temperatures at 13–23 days after fertilization (Argue and Phelps 1995; Baroiller et al. 1995). These authors suggested that the underlying mechanism that caused skewed sex ratio in their study was sex reversal, since the overall survivorship was not affected by temperature. In contrast, the action of deleterious alleles was clearly evident in the sex ratio distortions observed in this study.
In D. melanogaster, sex determination is controlled by the Sex-lethal (Sxl) gene (reviewed by Cline and Meyer 1996). Successful activation of Sxl requires both maternally and zygotically provided gene products, which also are essential for viability and have other non-sex-specific functions. Misregulation of Sxl results in sex-specific lethality (Stitzinger et al. 1999). An Sxl analogue (dsx) was identified in the fly Megaselia scalaris, and was found to have similar influence on sex determination (Sievert et al. 1997). Our data suggest that sex determination in tilapia may be similar to the Drosophila model.
In conclusion, we demonstrated an association between distorted sex ratios and genes with deleterious alleles in an inbred line of O. aureus. Application of a maximum likelihood model provided estimates for genetic parameters underlying gender viability. Further molecular dissection is needed to develop a complete model.
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Acknowledgments |
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The contribution of A. Shirak to this research was part of his doctoral dissertation at Bar-Ilan University, Israel. This study also was supported by the U.S.-Israel Binational Agricultural Research and Development Program (BARD), postdoctoral grant number FU-268–97 (awarded to Y.P.).
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Footnotes |
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Corresponding Editor: Lisa Seeb
Received May 14, 2001
Accepted May 17, 2002
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References |
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