Mol Biol Rep (2014) 41:1501–1509 DOI 10.1007/s11033-013-2995-3
A sex-associated sequence identified by RAPD screening in gynogenetic individuals of turbot (Scophthalmus maximus) Luis Vale • Rebeca Dieguez • Laura Sa´nchez Paulino Martı´nez • Ana Vin˜as
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Received: 10 September 2012 / Accepted: 28 December 2013 / Published online: 11 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Understanding the genetic basis of sex determination mechanisms is essential for improving the productivity of farmed aquaculture fish species like turbot (Scophthalmus maximus). In culture conditions turbot males grow slower than females starting from eight months post-hatch, and this differential growth rate is maintained until sexual maturation is reached, being mature females almost twice as big as males of the same age. The goal of this study was to identify sex-specific DNA markers in turbot using comparative random amplified polymorphism DNA (RAPD) profiles in males and females to get new insights of the genetic architecture related to sex determination. In order to do this, we analyzed 540 commercial 10-mer RAPD primers in male and female pools of a gynogenetic family because of its higher inbreeding, which facilitates the detection of associations across the genome. Two sex-linked RAPD markers were identified in the female pool and one in the male pool. After the analysis of the three markers on individual samples of each pool and also in unrelated individuals, only one RAPD showed significant association with females. This marker was isolated, cloned and sequenced, containing two sequences, a microsatellite (SEX01) and a minisatellite (SEX02), which were mapped in the turbot reference map. From this map position, through a comparative mapping approach, we
L. Vale R. Dieguez A. Vin˜as (&) Departamento de Gene´tica, Facultad de Biologı´a (CIBUS), Universidad de Santiago de Compostela Avda. Lope Go´mez de Marzoa, 15782 Santiago de Compostela, Spain e-mail: [email protected] L. Sa´nchez P. Martı´nez Departamento de Gene´tica, Facultad de Veterinaria, Universidad de Santiago de Compostela, Avda das Ciencias, s/n, 27002 Lugo, Spain
identified Foxl2, a relevant gene related to initial steps of sex differentiation, and Wnt4, a gene related with ovarian development, close to the microsatellite and minisatellite markers, respectively. The position of Foxl2 and Wnt4 was confirmed by linkage mapping in the reference turbot map. Keywords RAPD Sex Gynogenetic Turbot Map Foxl2 Wnt4
Introduction Fish display an amazing diversity of sex determining mechanisms relaying on genetic, environmental cues or a combination of both. This diversity is related to the large amount of fish species and the great diversity of aquatic environments where they inhabit [32]. Important progresses have been made in this field in the last years [28, 33, 44, 71], but for many farmed species the mechanism responsible of sex determination is still a to be solved key issue. Since several traits of high economical value are associated to sex in aquaculture: body shape, time and age of maturation and growth rate [16]; monosex populations are often a desirable goal for farm production [22, 23]. Thus, all-male populations are produced in tilapias, the first group of fish in which male monosex culture became a common practice, because male grow faster than females [29]; in catfish, where males grow faster than females [26], although the industrial production of all-male stock was held back due to severe reproduction problems [21]; or in guppies, since male body shape and color pattern have higher commercial value [43]. Conversely, all-female populations are produced in carps because of their faster growth and the value of their eggs [16, 25] also females are preferentially harvested in eels: both, in the wild because of
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their higher size, and in farms, because females grow faster than males, thus showing higher market value [19]; in salmonid fish species females are preferred because males mature earlier and have a lower flesh quality than females; also in sturgeons females have been cultured for meat and caviar since the 1990s [9]. Since size differences between males and females in turbot are among the largest ones in marine aquaculture [50] understanding the genetic basis of sex should be very useful to improve growth rate by obtaining all-female populations. This would also aid to avoid size dispersion which makes feeding difficult at initial stages of growth in fish farming. Additionally, it is necessary to get balanced sex ratios in breeding programs, because selection for growth is performed at 1 year old, before growth sexual dimorphism and maturation take place [30]. Different approaches have been carried out to elucidate the mechanism of sex determination in turbot. Cytogenetic analysis on mitotic and meiotic chromosomes did not detect any heteromorphism that could be related to a sex chromosome pair [6, 18]. Analyses of gynogenetic and triploid families resulted controversial, some studies pointing toward a XX/XY sex determination system [10, 11], while others invoked a ZZ/ZW one [4]. Analysis based on the sex ratios of offspring from sex-reversed parents suggested for the first time a ZZ/ZW sex determination system in turbot [27]. In the same way, the application of a microsatellite genetic map for genomic screening demonstrated the existence of a major sex-related genomic region located on linkage group (LG) 5 and confirmed a ZZ/ZW system [39]. To achieve all female populations when a ZZ/ ZW sex determination system occurs, it is necessary to produce WW neomales or WW females [49]. The existence of sex-associated markers would facilitate the identification of the genotypic sex in these progenies [27]. However, both these studies suggested that other genetic/environmental factors are present in order to explain the observed sex ratio differences between families [27, 39]. Recently, a cDNAAFLP analysis detected sex-biased expression for several genes in male adult gonads [59] and a sex-associated random amplified polymorphism DNA (RAPD) marker permitted a sexing efficiency of 90 % in males and 83, 3 % in females in a wild population [12]. These RAPD markers along with ten genes involved in the sex development pathway in vertebrates were mapped [63], and some of them were positioned on linkage groups where sex-related QTLs had been previously located [39]. Other genes and one RAPD mapped close to a growth-related QTL [57], which suggested a possible association between sex and growth in this species. In this study we employed the RAPD methodology [65, 66] to identify sex-associated markers in turbot using a meiogynogenetic full-sib family. Although the use of gynogenetics to look for sex markers
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would be more successful win a XY sex determination system, their employment can still be justified for a ZW sex determination system depending on the distance between the SD gene and the centromere. The closer the SD gene to the centromere, the lower the proportion of ZW individuals, thus facilitates the detection of sex associations. In the case of turbot, the SD gene is close to the centromere [39], rendering only 17.8 % ZW males in gynogenetic progenies. Further, the high inbreeding of gynogenetics would enhance the genetic differences at alternative phenotypes making easier the detection of marker associations. On the other hand, the RAPD approach has the advantage that no prior knowledge of the genome is necessary and the use of universal sets of primers commercially available. Furthermore, it is a fast, cheap and reliable tool [66] which has been successfully used to detect sex-associated markers in several fish species, even in turbot [12]. However, dominance (polymorphism is reflected as presence or absence of bands) and concerns about reproducibility limit the application of these molecular markers [1]. This approach allowed us to detect of two significant sex-associated genetic markers closely linked to relevant genes related to gonad differentiation, confirming the existence of minor genetic factors underlying sex determination in the turbot.
Materials and methods Samples A meiogynogenetic family was obtained at the Instituto Oceanogra´fico de Vigo facilities (NW Spain) following the protocol by Piferrer et al. [51]. Fishes were cultured according to the usual procedures for turbot in tanks at 18 °C [56]. Its maternal constitution was confirmed by using microsatellite markers according to Castro et al. [13]. All the full-sibs of this family were successfully sexed at maturation age. RAPD analysis Turbot DNA samples were obtained from fin clips following the protocol of Sambrook et al. [55] , but using the SSTNE extraction buffer according to Blanquer [5]. In order to average the effects of inter-individual variation and to improve the efficiency of the screening process, a bulk segregant analysis was performed on two separate pools of 11 males and 11 females. Five hundred and forty 10-mer primers of series A (1–20) to Z (1–20) from Sigma Genosys (Woodlands, TX, USA) were tested in both DNA pools. We used two different pools for each sex; one of them constituted by 20 ng/ll of each individual and the other one with 5 ng/ll.
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Total reaction volumes of 25 ll were used with final concentrations of 2 mM Cl2Mg, 200 lM dNTPs, BIOTAQ Taq Polymerase (Bioline, London, UK) (0.5 U), RAPD primer (2 lM) and 20 or 5 ng of genomic DNA depending on the pool. Amplifications were performed in a thermocycler ‘‘My cycler’’ (BioRad) following the next steps: a initial denaturation step of 2 min at 94 °C following by 35 cycles: 15 s at 94 °C, 45 s at 34 °C and 2 min at 72 °C and a last step of 5 min at 72 °C. Amplification products were analyzed in 1.5 % agarose (Bio-Rad, CA, USA) in TBE buffer gel electrophoresis, stained with SYBRÒ Gold (Invitrogen) and visualized under blue light. Electrophoresis was run at 80 V during 6 h. The molecular weight of bands in the gel was estimated using Bench Top 100 bp DNA ladder and Lambda DNA/HindIII (Promega, USA). The same RAPD protocol was applied to individual DNA analyses. In addition, to confirm the consistency of a possible association of specific RAPDs to sex, we repeated the analysis on unrelated individuals from a wild population (18 males and 18 females). Gels were digitalized using the Gel Doc 2000 (Biorad) software. Images were edited with Adobe Photoshop 7.0. RAPD sex association was checked by applying the Pearson Chi square test. DNA cloning and sequencing Sex-associated RAPD bands obtained from amplification of ten individuals (five males and five females) were excised from the gels and sequenced. DNA was purified using the extraction kit ‘‘QIAEX II Gel extraction kit’’ (Qiagen) following the protocol provided by the manufacturer. Ligation and transformation were carried out with the ‘‘pGEMÒ-T Easy Vector System’’ (Promega) using high efficiency competent cells JM19. Cells were grown in plates with solid LB-Agar/X-Gal/IPTG (LENNOX) medium during 24 h to ensure the maximum number of clones as possible. Positive white colonies were selected and incubated overnight at 37 °C in liquid LB (LENNOX) medium supplemented with ampicillin (Roche; 100 lg/ml). The last step was to purify the plasmid using the ‘‘QiAprepÒ Spin Miniprep’’ kit (Qiagen) following the protocol provided by the manufacturer. Sequencing was performed using the ‘‘Big Dye Terminator v3.1 Cycle sequencing’’ kit (Applied Biosystems). Both strands of each insert were sequenced for accuracy. Four clones containing an insert of correct size of the C20 RAPD marker were sequenced. Sequencing reactions were carried out in a total volume of 20 ll containing 2 ll 5X BigDye Sequencing buffer, 4 ll 2.5X Terminator Ready Reaction mix, 3.2 pmol universal primers T7 or SP6 and 1 ll of DNA from purified Minipreps. Amplifications were performed following the next program: 1 cycle at 95 °C, 3 min;
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25 cycles at 95 °C during 10 s, 5 s at 50 °C and at 60 °C 4 min and finally 1 cycle at 60 °C for 4 s. The sequences obtained were edited with VecScreen (http://www.ncbi.nlm. nih.gov/VecScreen/VecScreen.html) software to delete the vector parts of the sequence; aligned for sequence comparison and to obtain a consensus sequence by Clustal v2 (http:// www.ebi.ac.uk/Tools/clustalw2/index.html); annotated by searching for homologies in public databases using Blastn and Blastx (http://blast.ncbi.nlm.nih.gov/Blast.cgi); and finally, used as template for primer design for further analysis using Primer 3 (http://frodo.wi.mit.edu/cgi-bin/pri mer3/primer3_www.cgi). Comparative mapping and gene mining Sex-associated turbot sequences were used as queries against the model fish genomes of Gasterosteus aculeatus and Takifugu rubripes which were dowloaded from the Ensembl public database (http://www.ensembl.org). The same markers were employed to perform gene mining in approximately 2 Mb of stickleback genome using BioMart tool of Ensembl (http://www.ensembl.org/biomart/). SNP genotyping for association and mapping Foxl2 and Wnt4, two relevant sex-related genes, were mapped using SNPs following Vera et al. [62] because their genomic location was predicted to be close to the RAPD sex-associated sequences. Primers for amplification of one exon of Foxl2 were designed in the conserved regions of homologous sequences from Gasterosteus aculeatus, Oryzias latipes, Danio rerio and Takifugu rubripes obtained from Ensembl genome browser (http://www.ensembl.org). In a similar way, primers for amplification of turbot Wnt4 gene were designed from exonic sequences of Gasterosteus aculeatus, Oryzias latipes and Oreochromis niloticus obtained from Ensembl genome browser. In this case, we detected a SNP variation in an intronic sequence. Linkage analysis was performed according to the methodology described by Bouza et al. [7]. JOINMAP 3.0 was used for linkage analysis using a LOD threshold [3 and a recombination frequency threshold \0.4 for a consistent mapping. The offspring of the reference family employed for mapping were genotyped in an ABI 3730xl DNA sequencer using the GENEMAPPER 4.0 software (Applied Biosystem). Chi square tests using Bonferroni correction (a = 0.05) were used to evaluate Mendelian segregation at these loci. Population analysis A sample of 34 turbot individuals from a wild population was employed for analyzing population parameters of the
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microsatellite and the minisatellite described in our study and also for the SNP markers developed for Foxl2 and Wnt4 mapping. Expected heterozygosity (He) and mean number of alleles per locus (A) were computed to estimate genetic diversity. Hardy–Weinberg proportion was checked by using exact tests. Genetic differentiation between males and females was estimated using the relative coefficient of genetic differentiation (FST) and checked by using exact probablility homogeneity test. All these analysis were carried out using the GENEPOP 3.1 sofware [53].
Results and discussion Gynogenesis is a useful tool for both basic and applied investigation in fish [15, 52, 60]. Several biological phenomena, like the study of sex determination, can be approached or enhanced by using mito- or meio-gynogenetic progenies. One of the first evidences of the genetic basis of sex determination in fish was drawn from biased sex ratios observed in gynogenetic progenies [48]. In particular in turbot, several studies using gynogenetics suggested different mechanisms of sex determination [4, 10]. All data obtained until now in turbot sex determination suggest a genetic-based sex determining mechanism with a major sex determining region on LG5, but other minor genetic or environmental factors cannot be discarded [27, 39]. This scenario postulates turbot as a candidate species to identify the major sex-determining switching gene, but also to identify putative interactions with other minor genes out of LG5, and with environmental factors such as temperature. The high genomic resources and tools recently developed in turbot should facilitate this task [8]. To address the identification of genomic associations for dychotomic traits with a few major genes underlying, RAPD markers constitute an interesting choice because of their low cost and the high amount of markers genotyped per run. In fact, this technique has been used to search for sex-specific genome markers in several fish species like Atlantic salmon [41], pufferfish [37], beluga [34], and several species of the genus Acipenser [40, 69]. Positive associations were found in catfish after screening with 300 RAPD primers [36], in the common carp with 220 [14] and in rainbow trout applying 900 primers [31]. In the present work, we combined a genomic RAPD screening with the advantage of using inbred gynogenetic progenies to enhance molecular divergence at the sex determining region/s between sexes. A recent RAPD screening in this species identified four sex-associated DNA markers [12], however this study did not count with the advantages of gynogenetic progenies. Five hundred and forty 10-mer RAPD primers were tested on pooled DNA of female and male gynogenetic
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siblings. For each sex, amplifications were carried out with 5 and 20 ng DNA pools, which in some cases can facilitate band identification. We achieved amplification products with the 96 % of the primers tested, comprising band sizes ranging from 300 to 3 kb. From 540 primers analyzed, only three yielded putative sex-associated bands: (i) C20 produced a 840 bp band in female pools; (ii) F8 gave a band around 1,300 in female pools; and (iii) G9 rendered a band of similar size in male pools (Fig. 1). These potential sex-associated markers were then tested in individual samples within each pool (four pools, two per sex) to confirm if the sex-related bands detected were due to individual polymorphisms or represented true sexmarkers. None of the three primers were able to fully classify individuals by sex. However, we observed remarkable sex differences in the presence/absence of polymorphic bands, especially for the C20 primer. For this marker, we observed the 840 bp band in 7 out of 11 females and in 2 out of 11 males. Aiming to confirm this sex-association polymorphism at the species level, we analyzed diploid unrelated wild individuals, 18 males and 18 females, with these markers. As shown in Fig. 2, the polymorphic C20 band was observed in 11 out of 18 females, while no band was apparent in the males analyzed (Fig. 3). Pearson’s Chi square (13.75, P = 0.000) confirmed RAPD association with sex. Sex differences observed in the pools with F8 and G9 primers were not confirmed at individual level. The polymorphic C20 band was excised from the gels, and then purified, cloned and sequenced. Sequence analysis revealed two different clones, one containing a microsatellite motif and the other one a minisatellite one (GenBank number SEX01: JX270672; SEX02:JX270673). None of these bands revealed significant homology with public databases. After the design of appropriate primers, both markercontaining sequences demonstrated to be polymorphic when amplified in 34 turbot individuals (SEX01: Expected Heterozygosity (He) = 0.848; Number of Alleles (A) = 12; SEX02: He = 0.859; A = 12). Also, no significant genetic differences were detected between males and females using a Mann–Whitney non-parametric test on allelic frequencies both at SEX01 (P = 0.543) and SEX02 (P = 0.705), thus, being suitable for mapping. The reference DM family was used for mapping [7] and their position was compared with that of the previously detected sex-related QTLs [39]. The SEX01 marker was located at linkage group 15 (LG15), between the microsatellite markers SmaUSC32 and SmaUSC211 (Fig. 4), within a genomic region significativelly associated with growth [57]. Comparative mapping analysis had revealed that LG15 is synthenic with the homologous linkage groups of stickleback (LG1), medaka (LG13) and fugu (LG11). The
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Fig. 1 RAPD banding profile with C20, F8 and G9 primers in pooled samples of males and females. The amplification obtained with 20 or 5 ng of each pool is shown at left and right of each marker gel.
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Arrows indicate the putative sex associated markers: an 840 bp band in female pools in C20 profile; a 1,300 bp band in female pools in F8 pattern; and a band of similar size in male pools using G9 primer
Fig. 2 C20 RAPD in unrelated females. Arrows indicate the band of *840 bp present in the female gynogenetic pool present in 11 out of 18 individual females
conservation was higher between turbot and stickleback [8]. In the stickleback genome,very close to the homologus sequences of SmaUSC32 and SmaUSC211 is located Foxl2, a key gene regulating oestrogen balance at the first stages of gonad differentiation [17, 64]. A SNP marker located in an exonic region of Foxl2 was put forward for genotyping and characterized in the same turbot sample as SEX01 and SEX02 (He = 0.395; A = 2), no genetic differences between males and females were detected (P = 1.000). The predicted position of Foxl2 at LG15 was confirmed by genotyping the DM turbot reference family, being located at 1 cM of SEX01 marker (Fig. 4) with a
recombination frequency of 0.0 and a LOD value of 19.8. This gene is a member of the winged helix/forkhead proteins and is one of the earliest markers of ovarian differentiation in vertebrates. Foxl2 was suggested to be a repressor of the male pathway during female gonad development in mouse. In this species, complete loss of ovarian expression of this gene was found to be associated with sex reversal of XX female to male in mouse [47, 61]. As in mammals, aromatase and Foxl2 were found to be colocalized in the adult ovaries of medaka, tilapia and flounder [45, 64, 70] playing a decisive role in early ovarian sex differentiation by activating the Cyp19a
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Fig. 3 C20 RAPD profile in unrelated males. Arrow indicates the position of the C20 band, lacking in the 18 tested males
Fig. 4 Turbot genetic map of linkage groups(LG) 11 and 15 showing the position of the microsatellite marker SEX01 placed on LG 15 and the minisatellite marker SEX02 located on LG11. Also, it is higligthed the position of Foxl2 (LG 15) and Wnt4 (LG 11) genes in this species
transcription directly or indirectly by enhancing estrogen production. Foxl2 has been identified and characterized in several fish such as tilapia [64], rainbow trout [2], medaka [45], Japanese flounder [70], zebrafish [58], dogfish [67] and half-smooth tongue sole [20], proving to be a key
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factor for female development in most of the species analyzed. Our results also seem to support the relationship established between sex and growth in different fish species like Salmo salar, where a QTL for body weight was identified close to a QTL for maturation [42]; gilthead sea bream, where weight and sex-related QTL co-mapped in the same region [3, 38] and turbot, where some genes related to sex differentiation co-mapped with growth-related QTL [63]. The minisatellite sequence (SEX02) was mapped using the DM reference family in LG11 at 51.5 cM, close to the Sma-USC235 (3,6 cM) and USC275 (4 cM) markers (Fig. 4). In this region, a growth-related QTL was detected significantly associated with the SmaUSC-22 marker [54]. The sequence of the SmaUSC22 was used to query the stickleback genome, being located in a highly conserved genomic region which contains several genes related to sex differentiation such as Wnt4, Sox13, Wnt7 and FoxP1 [24]. Given the importance of Wnt4 in the Wnt/b catenin pathway which is crucial for female differentiation in mammals, we decided to map this gene in turbot, despite its role is not well known in fish. In the black porgy, Wu and Chang [68] showed that this gene is related to sex change and ovarian growth, but in rainbow trout its expression was slightly higher in males at the initial steps of gonadal differentiation [46] suggesting no relationship with ovarian differentiation. A Wnt4 SNP was identified at an intron region showing a low genetic diversity in the 34 turbot sample analyzed (He = 0.053; A = 2) and no genetic differences were detected between males and females (P = 1.000). As shown in Fig. 4, this gene was mapped in
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LG11 at 38.3 cM using the DM family, quite distant from SEX02. However, the recombination frequency and LOD value of several markers close to SEX02 (Sma-USC275, Sma-USC62 and SmaUSC-E24) ranged between 0.0 and 3.9, with LOD scores above ten leaving open the question of whether not only Wnt4, but also other genes like Wnt7 and FoxP1, may actually be located close to SEX02. Further turbot map refinement is necessary to check their actual positions. In this sense, it is interesting to highlight that comparative genome analysis showed significant homology between the Sma-USC62 and a region of fugu LG 19, where the sex determining region of this species is located [35]. In our study, a RAPD analysis in turbot gynogenetic individuals resulted in a successful identification of sexassociated markers. Sequence analysis of the most consistent associated RAPD marker revealed two different clones, one containing a microsatellite (SEX01) and the other a minisatellite (SEX02) sequence. These two sequences mapped on two different regions where no sexrelated QTLs had been previously identified in turbot [39]. With a ZZ/ZW sex determination system, the production of all-female populations requires obtaining WW neomales or WW females [49]. Individuals with a WW genotype can be produced by gynogenesis and neomales WW by gynogenesis followed by masculinization. Nevertheless, this is a long process because the identification of WW individuals following progeny testing needs several years, which prevents its application in the aquaculture industry. The availability of a reliable sex-associated marker could shorten this time and would allow the transfer of these individuals obtained by gynogenesis to the industry [27]. In order to distinguish one sex of the other, in species with a ZZ/ZW system, it would be desirable to get specific markers for both chromosomes: a marker of the Z chromosome would enable the identification of WW females and a marker of W chromosome the identification of ZZ individuals. Unfortunately, the markers described in this work do not permit differentiating ZW from WW females which could be really helpful to the turbot farming. However both markers were located close to genomic regions where growth-related QTL were detected. Furthermore, SEX01 mapped at 1 cM of Foxl2, a gene related to ovarian differentiation, and comparative mapping analysis of SEX02 showed that this marker is in the vicinity of Wnt4, a gene related to ovarian development. Mapping of both genes confirmed their predicted localization by comparative genome which could suggests a possible relationship between sex and growth in this species. Acknowledgments This study was supported by the Spanish Ministerio de Ciencia y Tecnologı´a (AGL2003-05539) and by the Xunta the Galicia Government (07MMA004200PR) projects. We thank
1507 Cristina Gianzo for technical support, Miguel Hermida for markers mapping and Carmen Bouza for population analysis.
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A sex-associated sequence identified by RAPD screening in gynogenetic individuals of turbot (Scophthalmus maximus) Luis Vale • Rebeca Dieguez • Laura Sa´nchez Paulino Martı´nez • Ana Vin˜as
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Received: 10 September 2012 / Accepted: 28 December 2013 / Published online: 11 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Understanding the genetic basis of sex determination mechanisms is essential for improving the productivity of farmed aquaculture fish species like turbot (Scophthalmus maximus). In culture conditions turbot males grow slower than females starting from eight months post-hatch, and this differential growth rate is maintained until sexual maturation is reached, being mature females almost twice as big as males of the same age. The goal of this study was to identify sex-specific DNA markers in turbot using comparative random amplified polymorphism DNA (RAPD) profiles in males and females to get new insights of the genetic architecture related to sex determination. In order to do this, we analyzed 540 commercial 10-mer RAPD primers in male and female pools of a gynogenetic family because of its higher inbreeding, which facilitates the detection of associations across the genome. Two sex-linked RAPD markers were identified in the female pool and one in the male pool. After the analysis of the three markers on individual samples of each pool and also in unrelated individuals, only one RAPD showed significant association with females. This marker was isolated, cloned and sequenced, containing two sequences, a microsatellite (SEX01) and a minisatellite (SEX02), which were mapped in the turbot reference map. From this map position, through a comparative mapping approach, we
L. Vale R. Dieguez A. Vin˜as (&) Departamento de Gene´tica, Facultad de Biologı´a (CIBUS), Universidad de Santiago de Compostela Avda. Lope Go´mez de Marzoa, 15782 Santiago de Compostela, Spain e-mail: [email protected] L. Sa´nchez P. Martı´nez Departamento de Gene´tica, Facultad de Veterinaria, Universidad de Santiago de Compostela, Avda das Ciencias, s/n, 27002 Lugo, Spain
identified Foxl2, a relevant gene related to initial steps of sex differentiation, and Wnt4, a gene related with ovarian development, close to the microsatellite and minisatellite markers, respectively. The position of Foxl2 and Wnt4 was confirmed by linkage mapping in the reference turbot map. Keywords RAPD Sex Gynogenetic Turbot Map Foxl2 Wnt4
Introduction Fish display an amazing diversity of sex determining mechanisms relaying on genetic, environmental cues or a combination of both. This diversity is related to the large amount of fish species and the great diversity of aquatic environments where they inhabit [32]. Important progresses have been made in this field in the last years [28, 33, 44, 71], but for many farmed species the mechanism responsible of sex determination is still a to be solved key issue. Since several traits of high economical value are associated to sex in aquaculture: body shape, time and age of maturation and growth rate [16]; monosex populations are often a desirable goal for farm production [22, 23]. Thus, all-male populations are produced in tilapias, the first group of fish in which male monosex culture became a common practice, because male grow faster than females [29]; in catfish, where males grow faster than females [26], although the industrial production of all-male stock was held back due to severe reproduction problems [21]; or in guppies, since male body shape and color pattern have higher commercial value [43]. Conversely, all-female populations are produced in carps because of their faster growth and the value of their eggs [16, 25] also females are preferentially harvested in eels: both, in the wild because of
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their higher size, and in farms, because females grow faster than males, thus showing higher market value [19]; in salmonid fish species females are preferred because males mature earlier and have a lower flesh quality than females; also in sturgeons females have been cultured for meat and caviar since the 1990s [9]. Since size differences between males and females in turbot are among the largest ones in marine aquaculture [50] understanding the genetic basis of sex should be very useful to improve growth rate by obtaining all-female populations. This would also aid to avoid size dispersion which makes feeding difficult at initial stages of growth in fish farming. Additionally, it is necessary to get balanced sex ratios in breeding programs, because selection for growth is performed at 1 year old, before growth sexual dimorphism and maturation take place [30]. Different approaches have been carried out to elucidate the mechanism of sex determination in turbot. Cytogenetic analysis on mitotic and meiotic chromosomes did not detect any heteromorphism that could be related to a sex chromosome pair [6, 18]. Analyses of gynogenetic and triploid families resulted controversial, some studies pointing toward a XX/XY sex determination system [10, 11], while others invoked a ZZ/ZW one [4]. Analysis based on the sex ratios of offspring from sex-reversed parents suggested for the first time a ZZ/ZW sex determination system in turbot [27]. In the same way, the application of a microsatellite genetic map for genomic screening demonstrated the existence of a major sex-related genomic region located on linkage group (LG) 5 and confirmed a ZZ/ZW system [39]. To achieve all female populations when a ZZ/ ZW sex determination system occurs, it is necessary to produce WW neomales or WW females [49]. The existence of sex-associated markers would facilitate the identification of the genotypic sex in these progenies [27]. However, both these studies suggested that other genetic/environmental factors are present in order to explain the observed sex ratio differences between families [27, 39]. Recently, a cDNAAFLP analysis detected sex-biased expression for several genes in male adult gonads [59] and a sex-associated random amplified polymorphism DNA (RAPD) marker permitted a sexing efficiency of 90 % in males and 83, 3 % in females in a wild population [12]. These RAPD markers along with ten genes involved in the sex development pathway in vertebrates were mapped [63], and some of them were positioned on linkage groups where sex-related QTLs had been previously located [39]. Other genes and one RAPD mapped close to a growth-related QTL [57], which suggested a possible association between sex and growth in this species. In this study we employed the RAPD methodology [65, 66] to identify sex-associated markers in turbot using a meiogynogenetic full-sib family. Although the use of gynogenetics to look for sex markers
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would be more successful win a XY sex determination system, their employment can still be justified for a ZW sex determination system depending on the distance between the SD gene and the centromere. The closer the SD gene to the centromere, the lower the proportion of ZW individuals, thus facilitates the detection of sex associations. In the case of turbot, the SD gene is close to the centromere [39], rendering only 17.8 % ZW males in gynogenetic progenies. Further, the high inbreeding of gynogenetics would enhance the genetic differences at alternative phenotypes making easier the detection of marker associations. On the other hand, the RAPD approach has the advantage that no prior knowledge of the genome is necessary and the use of universal sets of primers commercially available. Furthermore, it is a fast, cheap and reliable tool [66] which has been successfully used to detect sex-associated markers in several fish species, even in turbot [12]. However, dominance (polymorphism is reflected as presence or absence of bands) and concerns about reproducibility limit the application of these molecular markers [1]. This approach allowed us to detect of two significant sex-associated genetic markers closely linked to relevant genes related to gonad differentiation, confirming the existence of minor genetic factors underlying sex determination in the turbot.
Materials and methods Samples A meiogynogenetic family was obtained at the Instituto Oceanogra´fico de Vigo facilities (NW Spain) following the protocol by Piferrer et al. [51]. Fishes were cultured according to the usual procedures for turbot in tanks at 18 °C [56]. Its maternal constitution was confirmed by using microsatellite markers according to Castro et al. [13]. All the full-sibs of this family were successfully sexed at maturation age. RAPD analysis Turbot DNA samples were obtained from fin clips following the protocol of Sambrook et al. [55] , but using the SSTNE extraction buffer according to Blanquer [5]. In order to average the effects of inter-individual variation and to improve the efficiency of the screening process, a bulk segregant analysis was performed on two separate pools of 11 males and 11 females. Five hundred and forty 10-mer primers of series A (1–20) to Z (1–20) from Sigma Genosys (Woodlands, TX, USA) were tested in both DNA pools. We used two different pools for each sex; one of them constituted by 20 ng/ll of each individual and the other one with 5 ng/ll.
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Total reaction volumes of 25 ll were used with final concentrations of 2 mM Cl2Mg, 200 lM dNTPs, BIOTAQ Taq Polymerase (Bioline, London, UK) (0.5 U), RAPD primer (2 lM) and 20 or 5 ng of genomic DNA depending on the pool. Amplifications were performed in a thermocycler ‘‘My cycler’’ (BioRad) following the next steps: a initial denaturation step of 2 min at 94 °C following by 35 cycles: 15 s at 94 °C, 45 s at 34 °C and 2 min at 72 °C and a last step of 5 min at 72 °C. Amplification products were analyzed in 1.5 % agarose (Bio-Rad, CA, USA) in TBE buffer gel electrophoresis, stained with SYBRÒ Gold (Invitrogen) and visualized under blue light. Electrophoresis was run at 80 V during 6 h. The molecular weight of bands in the gel was estimated using Bench Top 100 bp DNA ladder and Lambda DNA/HindIII (Promega, USA). The same RAPD protocol was applied to individual DNA analyses. In addition, to confirm the consistency of a possible association of specific RAPDs to sex, we repeated the analysis on unrelated individuals from a wild population (18 males and 18 females). Gels were digitalized using the Gel Doc 2000 (Biorad) software. Images were edited with Adobe Photoshop 7.0. RAPD sex association was checked by applying the Pearson Chi square test. DNA cloning and sequencing Sex-associated RAPD bands obtained from amplification of ten individuals (five males and five females) were excised from the gels and sequenced. DNA was purified using the extraction kit ‘‘QIAEX II Gel extraction kit’’ (Qiagen) following the protocol provided by the manufacturer. Ligation and transformation were carried out with the ‘‘pGEMÒ-T Easy Vector System’’ (Promega) using high efficiency competent cells JM19. Cells were grown in plates with solid LB-Agar/X-Gal/IPTG (LENNOX) medium during 24 h to ensure the maximum number of clones as possible. Positive white colonies were selected and incubated overnight at 37 °C in liquid LB (LENNOX) medium supplemented with ampicillin (Roche; 100 lg/ml). The last step was to purify the plasmid using the ‘‘QiAprepÒ Spin Miniprep’’ kit (Qiagen) following the protocol provided by the manufacturer. Sequencing was performed using the ‘‘Big Dye Terminator v3.1 Cycle sequencing’’ kit (Applied Biosystems). Both strands of each insert were sequenced for accuracy. Four clones containing an insert of correct size of the C20 RAPD marker were sequenced. Sequencing reactions were carried out in a total volume of 20 ll containing 2 ll 5X BigDye Sequencing buffer, 4 ll 2.5X Terminator Ready Reaction mix, 3.2 pmol universal primers T7 or SP6 and 1 ll of DNA from purified Minipreps. Amplifications were performed following the next program: 1 cycle at 95 °C, 3 min;
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25 cycles at 95 °C during 10 s, 5 s at 50 °C and at 60 °C 4 min and finally 1 cycle at 60 °C for 4 s. The sequences obtained were edited with VecScreen (http://www.ncbi.nlm. nih.gov/VecScreen/VecScreen.html) software to delete the vector parts of the sequence; aligned for sequence comparison and to obtain a consensus sequence by Clustal v2 (http:// www.ebi.ac.uk/Tools/clustalw2/index.html); annotated by searching for homologies in public databases using Blastn and Blastx (http://blast.ncbi.nlm.nih.gov/Blast.cgi); and finally, used as template for primer design for further analysis using Primer 3 (http://frodo.wi.mit.edu/cgi-bin/pri mer3/primer3_www.cgi). Comparative mapping and gene mining Sex-associated turbot sequences were used as queries against the model fish genomes of Gasterosteus aculeatus and Takifugu rubripes which were dowloaded from the Ensembl public database (http://www.ensembl.org). The same markers were employed to perform gene mining in approximately 2 Mb of stickleback genome using BioMart tool of Ensembl (http://www.ensembl.org/biomart/). SNP genotyping for association and mapping Foxl2 and Wnt4, two relevant sex-related genes, were mapped using SNPs following Vera et al. [62] because their genomic location was predicted to be close to the RAPD sex-associated sequences. Primers for amplification of one exon of Foxl2 were designed in the conserved regions of homologous sequences from Gasterosteus aculeatus, Oryzias latipes, Danio rerio and Takifugu rubripes obtained from Ensembl genome browser (http://www.ensembl.org). In a similar way, primers for amplification of turbot Wnt4 gene were designed from exonic sequences of Gasterosteus aculeatus, Oryzias latipes and Oreochromis niloticus obtained from Ensembl genome browser. In this case, we detected a SNP variation in an intronic sequence. Linkage analysis was performed according to the methodology described by Bouza et al. [7]. JOINMAP 3.0 was used for linkage analysis using a LOD threshold [3 and a recombination frequency threshold \0.4 for a consistent mapping. The offspring of the reference family employed for mapping were genotyped in an ABI 3730xl DNA sequencer using the GENEMAPPER 4.0 software (Applied Biosystem). Chi square tests using Bonferroni correction (a = 0.05) were used to evaluate Mendelian segregation at these loci. Population analysis A sample of 34 turbot individuals from a wild population was employed for analyzing population parameters of the
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microsatellite and the minisatellite described in our study and also for the SNP markers developed for Foxl2 and Wnt4 mapping. Expected heterozygosity (He) and mean number of alleles per locus (A) were computed to estimate genetic diversity. Hardy–Weinberg proportion was checked by using exact tests. Genetic differentiation between males and females was estimated using the relative coefficient of genetic differentiation (FST) and checked by using exact probablility homogeneity test. All these analysis were carried out using the GENEPOP 3.1 sofware [53].
Results and discussion Gynogenesis is a useful tool for both basic and applied investigation in fish [15, 52, 60]. Several biological phenomena, like the study of sex determination, can be approached or enhanced by using mito- or meio-gynogenetic progenies. One of the first evidences of the genetic basis of sex determination in fish was drawn from biased sex ratios observed in gynogenetic progenies [48]. In particular in turbot, several studies using gynogenetics suggested different mechanisms of sex determination [4, 10]. All data obtained until now in turbot sex determination suggest a genetic-based sex determining mechanism with a major sex determining region on LG5, but other minor genetic or environmental factors cannot be discarded [27, 39]. This scenario postulates turbot as a candidate species to identify the major sex-determining switching gene, but also to identify putative interactions with other minor genes out of LG5, and with environmental factors such as temperature. The high genomic resources and tools recently developed in turbot should facilitate this task [8]. To address the identification of genomic associations for dychotomic traits with a few major genes underlying, RAPD markers constitute an interesting choice because of their low cost and the high amount of markers genotyped per run. In fact, this technique has been used to search for sex-specific genome markers in several fish species like Atlantic salmon [41], pufferfish [37], beluga [34], and several species of the genus Acipenser [40, 69]. Positive associations were found in catfish after screening with 300 RAPD primers [36], in the common carp with 220 [14] and in rainbow trout applying 900 primers [31]. In the present work, we combined a genomic RAPD screening with the advantage of using inbred gynogenetic progenies to enhance molecular divergence at the sex determining region/s between sexes. A recent RAPD screening in this species identified four sex-associated DNA markers [12], however this study did not count with the advantages of gynogenetic progenies. Five hundred and forty 10-mer RAPD primers were tested on pooled DNA of female and male gynogenetic
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siblings. For each sex, amplifications were carried out with 5 and 20 ng DNA pools, which in some cases can facilitate band identification. We achieved amplification products with the 96 % of the primers tested, comprising band sizes ranging from 300 to 3 kb. From 540 primers analyzed, only three yielded putative sex-associated bands: (i) C20 produced a 840 bp band in female pools; (ii) F8 gave a band around 1,300 in female pools; and (iii) G9 rendered a band of similar size in male pools (Fig. 1). These potential sex-associated markers were then tested in individual samples within each pool (four pools, two per sex) to confirm if the sex-related bands detected were due to individual polymorphisms or represented true sexmarkers. None of the three primers were able to fully classify individuals by sex. However, we observed remarkable sex differences in the presence/absence of polymorphic bands, especially for the C20 primer. For this marker, we observed the 840 bp band in 7 out of 11 females and in 2 out of 11 males. Aiming to confirm this sex-association polymorphism at the species level, we analyzed diploid unrelated wild individuals, 18 males and 18 females, with these markers. As shown in Fig. 2, the polymorphic C20 band was observed in 11 out of 18 females, while no band was apparent in the males analyzed (Fig. 3). Pearson’s Chi square (13.75, P = 0.000) confirmed RAPD association with sex. Sex differences observed in the pools with F8 and G9 primers were not confirmed at individual level. The polymorphic C20 band was excised from the gels, and then purified, cloned and sequenced. Sequence analysis revealed two different clones, one containing a microsatellite motif and the other one a minisatellite one (GenBank number SEX01: JX270672; SEX02:JX270673). None of these bands revealed significant homology with public databases. After the design of appropriate primers, both markercontaining sequences demonstrated to be polymorphic when amplified in 34 turbot individuals (SEX01: Expected Heterozygosity (He) = 0.848; Number of Alleles (A) = 12; SEX02: He = 0.859; A = 12). Also, no significant genetic differences were detected between males and females using a Mann–Whitney non-parametric test on allelic frequencies both at SEX01 (P = 0.543) and SEX02 (P = 0.705), thus, being suitable for mapping. The reference DM family was used for mapping [7] and their position was compared with that of the previously detected sex-related QTLs [39]. The SEX01 marker was located at linkage group 15 (LG15), between the microsatellite markers SmaUSC32 and SmaUSC211 (Fig. 4), within a genomic region significativelly associated with growth [57]. Comparative mapping analysis had revealed that LG15 is synthenic with the homologous linkage groups of stickleback (LG1), medaka (LG13) and fugu (LG11). The
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Fig. 1 RAPD banding profile with C20, F8 and G9 primers in pooled samples of males and females. The amplification obtained with 20 or 5 ng of each pool is shown at left and right of each marker gel.
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Arrows indicate the putative sex associated markers: an 840 bp band in female pools in C20 profile; a 1,300 bp band in female pools in F8 pattern; and a band of similar size in male pools using G9 primer
Fig. 2 C20 RAPD in unrelated females. Arrows indicate the band of *840 bp present in the female gynogenetic pool present in 11 out of 18 individual females
conservation was higher between turbot and stickleback [8]. In the stickleback genome,very close to the homologus sequences of SmaUSC32 and SmaUSC211 is located Foxl2, a key gene regulating oestrogen balance at the first stages of gonad differentiation [17, 64]. A SNP marker located in an exonic region of Foxl2 was put forward for genotyping and characterized in the same turbot sample as SEX01 and SEX02 (He = 0.395; A = 2), no genetic differences between males and females were detected (P = 1.000). The predicted position of Foxl2 at LG15 was confirmed by genotyping the DM turbot reference family, being located at 1 cM of SEX01 marker (Fig. 4) with a
recombination frequency of 0.0 and a LOD value of 19.8. This gene is a member of the winged helix/forkhead proteins and is one of the earliest markers of ovarian differentiation in vertebrates. Foxl2 was suggested to be a repressor of the male pathway during female gonad development in mouse. In this species, complete loss of ovarian expression of this gene was found to be associated with sex reversal of XX female to male in mouse [47, 61]. As in mammals, aromatase and Foxl2 were found to be colocalized in the adult ovaries of medaka, tilapia and flounder [45, 64, 70] playing a decisive role in early ovarian sex differentiation by activating the Cyp19a
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Fig. 3 C20 RAPD profile in unrelated males. Arrow indicates the position of the C20 band, lacking in the 18 tested males
Fig. 4 Turbot genetic map of linkage groups(LG) 11 and 15 showing the position of the microsatellite marker SEX01 placed on LG 15 and the minisatellite marker SEX02 located on LG11. Also, it is higligthed the position of Foxl2 (LG 15) and Wnt4 (LG 11) genes in this species
transcription directly or indirectly by enhancing estrogen production. Foxl2 has been identified and characterized in several fish such as tilapia [64], rainbow trout [2], medaka [45], Japanese flounder [70], zebrafish [58], dogfish [67] and half-smooth tongue sole [20], proving to be a key
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factor for female development in most of the species analyzed. Our results also seem to support the relationship established between sex and growth in different fish species like Salmo salar, where a QTL for body weight was identified close to a QTL for maturation [42]; gilthead sea bream, where weight and sex-related QTL co-mapped in the same region [3, 38] and turbot, where some genes related to sex differentiation co-mapped with growth-related QTL [63]. The minisatellite sequence (SEX02) was mapped using the DM reference family in LG11 at 51.5 cM, close to the Sma-USC235 (3,6 cM) and USC275 (4 cM) markers (Fig. 4). In this region, a growth-related QTL was detected significantly associated with the SmaUSC-22 marker [54]. The sequence of the SmaUSC22 was used to query the stickleback genome, being located in a highly conserved genomic region which contains several genes related to sex differentiation such as Wnt4, Sox13, Wnt7 and FoxP1 [24]. Given the importance of Wnt4 in the Wnt/b catenin pathway which is crucial for female differentiation in mammals, we decided to map this gene in turbot, despite its role is not well known in fish. In the black porgy, Wu and Chang [68] showed that this gene is related to sex change and ovarian growth, but in rainbow trout its expression was slightly higher in males at the initial steps of gonadal differentiation [46] suggesting no relationship with ovarian differentiation. A Wnt4 SNP was identified at an intron region showing a low genetic diversity in the 34 turbot sample analyzed (He = 0.053; A = 2) and no genetic differences were detected between males and females (P = 1.000). As shown in Fig. 4, this gene was mapped in
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LG11 at 38.3 cM using the DM family, quite distant from SEX02. However, the recombination frequency and LOD value of several markers close to SEX02 (Sma-USC275, Sma-USC62 and SmaUSC-E24) ranged between 0.0 and 3.9, with LOD scores above ten leaving open the question of whether not only Wnt4, but also other genes like Wnt7 and FoxP1, may actually be located close to SEX02. Further turbot map refinement is necessary to check their actual positions. In this sense, it is interesting to highlight that comparative genome analysis showed significant homology between the Sma-USC62 and a region of fugu LG 19, where the sex determining region of this species is located [35]. In our study, a RAPD analysis in turbot gynogenetic individuals resulted in a successful identification of sexassociated markers. Sequence analysis of the most consistent associated RAPD marker revealed two different clones, one containing a microsatellite (SEX01) and the other a minisatellite (SEX02) sequence. These two sequences mapped on two different regions where no sexrelated QTLs had been previously identified in turbot [39]. With a ZZ/ZW sex determination system, the production of all-female populations requires obtaining WW neomales or WW females [49]. Individuals with a WW genotype can be produced by gynogenesis and neomales WW by gynogenesis followed by masculinization. Nevertheless, this is a long process because the identification of WW individuals following progeny testing needs several years, which prevents its application in the aquaculture industry. The availability of a reliable sex-associated marker could shorten this time and would allow the transfer of these individuals obtained by gynogenesis to the industry [27]. In order to distinguish one sex of the other, in species with a ZZ/ZW system, it would be desirable to get specific markers for both chromosomes: a marker of the Z chromosome would enable the identification of WW females and a marker of W chromosome the identification of ZZ individuals. Unfortunately, the markers described in this work do not permit differentiating ZW from WW females which could be really helpful to the turbot farming. However both markers were located close to genomic regions where growth-related QTL were detected. Furthermore, SEX01 mapped at 1 cM of Foxl2, a gene related to ovarian differentiation, and comparative mapping analysis of SEX02 showed that this marker is in the vicinity of Wnt4, a gene related to ovarian development. Mapping of both genes confirmed their predicted localization by comparative genome which could suggests a possible relationship between sex and growth in this species. Acknowledgments This study was supported by the Spanish Ministerio de Ciencia y Tecnologı´a (AGL2003-05539) and by the Xunta the Galicia Government (07MMA004200PR) projects. We thank
1507 Cristina Gianzo for technical support, Miguel Hermida for markers mapping and Carmen Bouza for population analysis.
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