Mar Biotechnol (2012) 14:655–663
DOI 10.1007/s10126-012-9451-6
ORIGINAL ARTICLE
Mapping of DNA Sex-Specific Markers and Genes Related
to Sex Differentiation in Turbot (Scophthalmus maximus)
Ana Viñas & Xoana Taboada & Luis Vale &
Diego Robledo & Miguel Hermida & Manel Vera &
Paulino Martínez
Received: 15 December 2011 / Accepted: 3 April 2012 / Published online: 3 May 2012
# Springer Science+Business Media, LLC 2012
Abstract Production of all-female populations in turbot can
increase farmer’s benefits since sexual dimorphism in
growth in this species is among the highest within marine
fish, turbot females reaching commercial size 3–6 months
earlier than males. Puberty in males occurs earlier than in
females, which additionally slows their growth. Thus, elucidating the mechanisms of sex determination and gonad
differentiation is a relevant goal for turbot production. A ZZ/
ZW sex determination mechanism has been suggested for
this species, and four sex-related quantitative trait loci
(QTL) were detected, the major one located in linkage group
(LG) 5 and the three minor ones in LG6, LG8, and LG21. In
the present work, we carried out a linkage analysis for
several sex-related markers: (1) three anonymous sexassociated RAPD and (2) several candidate genes related
to sex determination and gonad differentiation in other species (Sox3, Sox6, Sox8, Sox9, Sox17, Sox19, Amh, Dmrta2,
Cyp19a, Cyp19b). We focused our attention on their colocalization with the major and minor sex-related QTL
trying to approach to the master sex-determining gene of
this species. Previously described growth-related QTL were
also considered since the association observed between
growth and sex determination in fish. Amh, Dmrta2, and
one RAPD were located in LG5, while Sox9 and Sox17
A. Viñas (*) : X. Taboada : L. Vale : D. Robledo
Departamento de Genética, Facultad de Biología (CIBUS),
Universidad de Santiago de Compostela,
Rúa Lope Gómez de Marzoa, s/n 15782,
Santiago de Compostela, Spain
e-mail: [email protected]
M. Hermida : M. Vera : P. Martínez
Departamento de Genética, Facultad de Veterinaria,
Universidad de Santiago de Compostela,
Campus de Lugo, Avda das Ciencias, s/n 27002,
Lugo, Spain
(LG21), Cyp19b (LG6), and a second RAPD (LG8) comapped with suggestive sex-related QTL, thus supporting
further analyses on these genes to elucidate the genetic basis
of this relevant trait for turbot farming.
Keywords Turbot . Linkage map . Sex . Growth . Candidate
genes . QTL
Introduction
Among vertebrates, fish display an enormous variation of
sex determining (SD) mechanisms. Pure genetic (GSD) and
environmental (ESD) SD mechanisms as well as a continuous range between both extremes have been reported in this
group (Devlin and Nagahama 2002; Mank and Avise 2009).
Different GSD systems have been reported within the same
fish genus and even in different populations of the same
species (Volff 2005), which indicates the rapid evolutionary
turnover of the SD system in fish (Böhne et al. 2009). Only
one master SD gene has been identified to date in fish, the
dmy/dmrt1bY in Oryzias latipes (Matsuda et al. 2002).
Several traits of high economical value are associated to
sex in aquaculture species. Sexual dimorphism has been
observed in growth rate, time and age of maturation, color
pattern or fin shape (Cnaani and Levavi-Sivan 2009). This is
the case of turbot, one of the most appreciated aquaculture
species in Europe, which shows extreme differential growth
rates between sexes (Piferrer et al. 1995). This dimorphism
is among the largest ones in farmed marine fishes and
continues after sexual maturation (Piferrer et al. 2004).
Females grow faster than males and reach puberty later,
which makes all-female population production desirable
for turbot industry.
656
Several studies have been carried out in turbot aiming to
elucidate the SD mechanism. The balanced sex ratio in
families suggests the existence of a major genetic sexrelated factor (Imsland et al. 1997) and a limited influence
of environmental factors such as temperature (Haffray et al.
2009). Mitotic and meiotic chromosome analysis have not
revealed any heteromorphic sex-associated chromosome
pair in this species (Bouza et al. 1994; Cuñado et al.
2001). Sex ratio of gynogenetic progenies rendered different
results, a ZZ/ZW system being reported by Baynes et al.
(2006), while other authors suggested a XX/XY mechanism
(Cal et al. 2006). Recently, a major SD region on LG5 and
three additional suggestive sex-related QTL were identified
by a medium scale genome scan for QTL detection (Martínez et al. 2009). Segregation analysis demonstrated a ZZ/
ZW mechanism in the major SD region. These results agree
with those obtained by Haffray et al. (2009) based on the
analysis of progenies obtained from steroid sex-reversal
treated parents. However, the studies by Martínez et al.
(2009) and Haffray et al. (2009) also suggested the influence
of other genetic or environmental factors underlying the SD
mechanism in this species. It should be noted that hybridization between turbot (Scophthalmus maximus) and brill
(Scophthalmus rhombus) produces monosex progenies
depending on the sex of the parents, which could suggest
different SD mechanisms for these congeneric species
(Purdom and Thacker 1980). A recent screening with 2030
RAPD DNA markers by Casas et al. (2011) on pooled
genomic DNA from both sexes detected three sexassociated RAPDs, which combined reached a molecular
sexing accuracy of 90 % in males and 83.3 % in females.
Finally, a transcriptome analysis based on cDNA-AFLP
allowed the identification of a set of differentially expressed
genes in male and female adult gonads among which Mns1
and Nek10, genes both related to spermatogenesis in mammals, showed significant male-biased expression by Q-PCR
(Taboada et al. 2012).
Recently, the relationship between sex and growth in fish
is gaining importance. Thus, in Salmo salar a QTL for
maturation close to a QTL for body weight was identified
(Moghadam et al. 2007) and a genetic correlation between
body weight and sex reversal in gilthead sea bream was
established (Batargias et al. 1998), being recently confirmed
by Loukovitis et al. (2011). These authors found two significant QTL one for weight and one for sex co-mapping in the
same position in Sparus aurata.
The objective of this study was to map several markers
and candidate genes associated with sex determination in
turbot, trying to ascertain their association with previously
reported sex- (Martínez et al. 2009) and growth-related QTL
(Sánchez-Molano et al. 2011) using the turbot map (Bouza
et al. 2007, 2008). These included three sex-associated
RAPD markers in turbot (Casas et al. 2011) and some
Mar Biotechnol (2012) 14:655–663
orthologous genes of the vertebrate sex determination pathway (Sox3, Sox6, Sox8, Sox9, Sox17, Sox19, Amh, Dmrta2,
Cyp19a, Cyp19b). Results showed relationships of some of
these genes/markers with sex- or growth-related QTL, thus
suggesting the convenience of carrying additional analysis
on them for elucidating the genetic basis of the SD mechanism in turbot.
Materials and Methods
PCR Conditions
Genomic DNA was extracted from muscle tissue in all
analyzed individuals using standard phenol–chloroform procedures (Sambrook et al. 1989). Amplicons of the selected
genes and sex-associated markers (see below) were obtained
in a volume of 50 μl, 75 ng of genomic DNA, 20 pmol of
each primer, 0.2 mM of each dNTP, 1× PCR reaction buffer,
and 2.5 U of GreenTaq DNA polymerase (GenScript). PCR
was performed in a MyCycler Thermal cycler (Bio-Rad) as
follows: initial denaturation at 94 °C for 3 min; 30 cycles
including 94 °C during 30 s, 50 s at an annealing temperature between 58 and 60 °C, and an extension time dependent
on amplicon size (about 1 kb/min) at 72 °C; and a final
extension step at 72 °C for 7 min. The PCR products were
separated on agarose gels and stained with SYBR gold
(Invitrogen). Amplification products were excised from the
gel and purified with Qiaquick gel extraction kit (Qiagen) or
obtained directly from PCR amplification using SpinCLean
(Mbiotech) kit.
Searching for Polymorphism in Sex-Related Genes
Orthologous sequences of several genes involved in the sex
development pathway of vertebrates (Sox3, Sox6, Sox8,
Sox9, Amh, Dmrta2, Cyp19a, and Cyp19b) were obtained
from stickleback, fugu, zebrafish, tetraodon, and medaka
using ENSEMBL database (http://www.ensembl.org/
index.html) and were aligned by CLUSTALW (http://
www.ebi.ac.uk/Tools/clustalw2/index.html). Primers to amplify putative polymorphic fragments within introns were
designed in conserved adjacent exon regions using the
Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/
primer3_www.cgi) for most genes. In Sox3, the amplicon
was obtained from an exon region since this gene lacks
introns. In Sox17 and Sox19, the polymorphism was identified in the 3′ UTR (untranslated region) using the turbot
EST database (Pardo et al. 2008; Vera et al. 2011). When
primers did not work, as in Amh and Cyp19a, degenerated
primers were designed from sequences of close related fish
species deposited in GenBank: Amh from Acanthopagrus
schlegelii (GU256046), Dicentrachus labrax (AM232701),
Mar Biotechnol (2012) 14:655–663
Odontesthes hatchery (DQ441594), and Paralichthys olivaceus (AB166791); and Cyp19a from D. labrax (AJ318516),
Monopterus albus (AY583785), Oncorhynchus mykiss
(AJ311937), and P. olivaceus (AB017182). After amplification with these primers, bands were cloned with “pGEM®-T
Easy Vector System” (Promega) using high efficiency competent cells JM19 and sequenced following the ABI Prism
BigDye™ Terminator v3.1 Cycle Sequencing Kit protocol
on an ABI 3730xl Genetic Analyzer (Applied Biosystems).
Turbot sequences were aligned using the software SeqScape
v2.5 (Applied Biosystems) and new primers were designed
to look for polymorphism using 10 individuals (five males
and five females) (Table 1). These sequences were aligned
and analyzed with SeqScape v2.5 (Applied Biosystems) to
identify polymorphisms.
Searching for Polymorphism in Sex-Associated DNA
Markers
Three sex RAPD markers, ScmM1, ScmF1, and ScmF3,
obtained in a previous work on pooled genomic DNA
(Casas et al. 2011), were amplified from sequences deposited in GenBank (Table 1).
Genotyping and Linkage Maps
Two different sources of variation (length polymorphism
and SNP) were used for mapping the sex-related genes
and markers following Vera et al. (2011). Primers flanking
length polymorphic regions were designed for ScmM1 and
ScmFe2 sex-associated markers, and for Sox8, Sox9,
Dmrta2 genes. SNP variation was detected at ScmFe1 sexassociated marker, and for Sox3, Sox6, Sox17, Sox19, Amh,
Cyp19a, and Cyp19b genes.
Informativeness of the markers for mapping was checked
in the parents of eight reference families used for mapping
(Bouza et al. 2007; Sánchez-Molano et al. 2011). This sample
was constituted by a total of 35 individuals corresponding to
the parents (eight males and eight females) and grandparents
(eleven males and eight females) available from the mapping
families. Progenies of families ranged between 85 and 96
offspring. All SNPs were successfully adjusted for Snapshot
reactions. Offspring genotyping of the most informative family was carried out in an ABI 3730xl DNA sequencer using
the GENEMAPPER 4.0 software (Applied Biosystems). Segregation at each locus was evaluated for conformance to Mendelian segregation using chi-square tests using Bonferroni
correction (α00.05). Linkage analysis was performed according to the methodology described by Bouza et al. (2007) on
the consensus map previously reported (Bouza et al. 2007,
2008). JoinMap 3.0 was used for map construction, with a
LOD threshold >3 and a recombination frequency threshold
<0.4 for a consistent mapping.
657
Results and Discussion
Sex determination depends on an initial signal transmitted
through a regulatory cascade which leads to the development of a bipotential gonad as a testis or as an ovary
(Graham et al. 2002; Penman and Piferrer 2008). Wilkins
(1995) proposed the “bottom-up hypothesis”, according to
which this hierarchy would evolve from downstream components that would acquire new upstream regulatory functions. This is not the case of medaka’s (Oryzias latipes)
dmy/Dmrt1bY, the only known sex-determination gene
described to date in fish (Matsuda et al. 2002), since it arose
as a Dmrt1 duplicate, first located in the autosomal LG9 and
then inserted into another chromosome (proto-Y chromosome), finally becoming the sex-determination master gene
(Schartl 2004).
Linkage mapping provides a powerful tool to detect
genomic regions associated with relevant traits. In our study,
we mapped several genes reported to be associated to sexual
differentiation in other vertebrate species and some anonymous sex-associated markers previously reported in turbot
(Casas et al. 2011). The existence of a major sex-associated
QTL in LG5 of this species and other three minor QTL in
LG6, LG8, and LG21 (Martínez et al. 2009) provided a
reference framework for this task. Also, the genetic association observed between growth and sex in several fish
species (Badyaev 2002; McCormick et al. 2010; Loukovitis
et al. 2011) suggested to take the growth-related QTL previously identified in turbot (Sánchez-Molano et al. 2011) as
an additional reference.
Figure 1 shows the eight linkage groups where the 10
sex-related candidate genes and the three sex-associated
RAPD markers were positioned on turbot map. It is noteworthy that five out of 10 genes and two out of three RAPD
analyzed markers were located in the four linkage groups
(LG5, LG6, LG8, and LG21) where sex-associated QTLs
had been previously reported (Martínez et al. 2009). In
Table 2, the mapping positions, within each linkage group,
of sex-related genes and markers, the nearest marker with
the low recombination frequency, and the LOD score for
consistent mapping are detailed. With the exception of Sox3,
the low recombination frequency to the closest marker and
the LOD score values indicate the reliability of mapping
and, consequently, the conclusions derived from it. It is
important to note that comparative mapping using syntenies
of turbot map against the stickleback (Gasterosteus aculeatus)
genome (Bouza et al. unpublished) also confirmed the positions obtained in our mapping analysis. Furthermore, in this
analysis a 1.6:1 female/male ratio of recombination frequency
(RF) was observed (Bouza et al. unpublished). This value
corroborated the female/male RF result obtained in a previous
work (Bouza et al. 2007) and differs slightly from that found
by Ruan et al. (2010) which reported a 1.3:1 ratio of RF.
JQ403637
JQ300536
JQ300535
JQ403638
JQ403639
JQ403642
JQ403644
JQ403643
JQ403645
Sox6
Sox8
Sox9
Sox17
Sox19
Amh
Dmrta2
Cyp19a
Cyp19b
F: CCTGATTGTAATGCCGTCGAT
F: GTACCTGTCGATGGTGAGGG
R: CCTGCCGCTGGTTCCTTCTT
R: AGACCCTTCTCATGGTCTGG
65
691
545
250
600
SNP
SNP
197
378
Length polymorphism 596
SNP
SNP
539
Length polymorphism 311
Length polymorphism 224
SNP
SNP
Length polymorphism 183
SNP
Ninth exon
Fourth intron
First intron
Sixth exon
3′ UTR
Second exon + 3′ UTR
First intron
Second intron
19th intron
Exon
Amplicon size (bp) Marker position
Length polymorphism 419
Polymorphism
F: CCTCAAATCTCTGTAGCAAATTTTTT SNP
F: CGCTATCTTGTTGTTTCTTACCATTT
F: GGCCATCACGTCCCACTC
SNP internal primers
F and R indicate forward and reverse primers, respectively, and DegF and DegR degenerate forward and reverse primers, respectively
JQ300537
Sox3
ScmFe2 EF612194
ScmFe1 EF612192
EF612196
ScmM1
F: CAGATCACTGGGAGGAGAGC
R: TGAGGAGTGTGCAAATCTGTCT
F: ACTCCAGAATTTGCATCAG
R: TCTCTTTTCCCACAGCATCA
F: TGTTCATGTTGAATGGGACA
R: AAAGAGCCAACTCATCTCCTCT
F: AAGACCAAGACCCTGCTCAA
R: GTCCCTGCGCTCTGATAGTG
F: CCACTGAGGCGAGAGTCTTC
R: CTCTTGATAGCCCTGCCAAT
F: CCCTGAGGAGAAAGCATCAG
R:GAACCAGGGATTGGAGGATT
F: GCGCGTCCAAATTTGGCGGA
R: GAACGGCCGCTTCTCGCCTT
F: GAGCAGATGCACCACTCTGA
R: GCAAAAAGTATAAAAACACCGTCA
F: TGTGCCTTGCCTTTTGATTT
R: GGTTTGCTGGAGATTTAGTCTGA
DegF: GASAGYTGRCYCTGTCTCC
DegR: GAGCCDYBGCAGTTGTTRAT
F: AGGAGGAGGTGGGTCACAG
R: CACGCAGGAGAGAAAACAGA
F: GAGGTGTTTGGTTCCGTCAG
R: CTCCGTCTTTCAAAGGCTTG
DegF: GGVACNGCCWGYAACTACTAC
DegR: TTBAVBAGCABCAGCATRAA
F: GGAGGTTTGTGTCTCCTCCA
R: AGATGTCGGGTTTGATCAGC
F: CTTTCTTTGTCCGTTCTTCCA
R: GTTGACTTCACCATGCGAAA
Accession number Primers
Gene
Table 1 Technical conditions for amplification of polymorphic regions associated to sex-related genes (Sox3, Sox6, Sox8, Sox9, Sox17, Sox19, Amh, Dmrta2, Cyp19a, Cyp19b) and RAPD markers
(SmM1, ScmFe1, ScmFe1) used for their mapping in the turbot (Scophthalmus maximus) genetic map
658
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Mar Biotechnol (2012) 14:655–663
LG02
LG04
0.0
Sma-USC90
0.0
8.9
Sma-USC46
13.1
16.1
18.4
19.2
19.5
31.0
33.8
35.6
37.0
43.8
44.8
45.5
45.7
47.7
53.0
54.2
57.5
57.8
59.1
66.0
71.8
Sma-USC242
Sma-USC64
Sma-USC171
Sma-USC168
Sma-USC36
Sma-USC84
SmaUSC-E6
Sma-USC245
Sma-USC44
Sma-USC249
Sma-USC185
Sma-USC187
Sma-USC109
Sma-USC43
Sma-USC161
Sma-USC219
86.9
Sma-USC112(a)
100.0
103.1
Sma-USC47
CYP19a
Sma-USC177
SOX6
28.0
36.6
Sma-USC102
Sma-USC167
Sma-USC100
Sma-USC277
B12-IGT14
42.6
SmaUSC-E3
49.5
Sma-USC7
66.3
67.9
Sma-USC205
Sma-USC230
19.3
659
LG06
LG05
0,0
Sma-USC270
6,3
SmaUSC-E30
18,0
22,3
24,7
35,9
42,4
43,0
44,3
48,2
51,2
54,1
58,3
58,5
62,0
64,7
66,5
Sma-USC65
Sma-USC254
0,0
4,7
8,1
8,3
9,6
14,8
19,5
20,8
24,2
29,1
30,7
ScmM1
Sma-USC247
Dmrta2
Sma-USC10
Sma-USC225
LG7
3/3GT
Sma-USC147
Sma-USC28
Sma-USC107
Sma3-12INRA
Sma-USC188
SOX19
0,0
2,1
5,7
SOX3
Sma-USC194
Sma-USC59
ScmFe1
cyp19b
SmaUSC-E29
Sma-USC110
Sma-USC132
Sma-USC227
Amh
Sma-USC88
Sma-USC278
Sma-USC198
Sma-USC12
Sma-USC202
Sma-USC265
Sma1-152INRA
0.0
LG13
LG8
64,1
Sma-USC159
76,7
78,0
Sma-USC264
7/1TC18
Sma-USC186
Sma-USC166
36.4
Sma4-14INRA
43.1
Sma-USC37(a)
60.0
ScmFe2
72.8
76.9
78.5
Sma-USC178
Sma-USC238
Sma-USC206
84.3
Sma-USC154
92.0
94.4
B11-I12/6/3
Sma-USC135
101.9
102.0
107.1
Sma-USC272
Sma-USC204
Sma-USC174
35,2
Sma-USC170
56,8
59,9
Sma-USC18
Sma-USC48
66,4
Sma-USC208
0.0
3.1
6.3
7.1
11.9
14.1
15.4
15.6
15.7
15.8
16.6
18.4
23.0
35.3
44.3
51.4
Sma-USC280
Sma1-125INRA
Sma-USC9
Sma-USC267
Sma-USC125
Sma-USC76
Sma-USC215
Sma-USC94
Sma-USC34
Sma-USC203
LG21
0,0
0,4
5,5
7,4
8,6
9,1
17,4
18,0
Sma-USC87
Sma-USC41
Sma-USC75
Sma-USC148
Sma-USC117
SOX17
SOX9
Sma-USC231
Sma-USC234
SOX8
Sma-USC115
Sma-USC16
SmaUSC-E10
SmaUSC-E38
Sma-USC155(a)
Fig. 1 Map positions (highlighted in red) of sex-related genes (Amh,
Dmrta2, Cyp19a, Cyp19b, Sox3, Sox8, Sox6, Sox9, Sox17, Sox19) and
sex-associated RAPD markers (SmM1, ScmFe1, ScmFe1) on turbot
(Scophthalmus maximus) genetic map. The number of each linkage
group (LG) is indicated above, genetic distances in centimorgans on
the left, and marker codes on the right. Framework markers (LOD > 3)
are presented in bold face. Gray shading indicates the position of the
SD QTLs in LG5, LG6, LG8, and LG21
However, it is important to note that it is a global data and
differences were found in different linkage groups as we will
discuss below for LG5 and LG21.
Shirak et al. (2006) considered Amh and Dmrta2 SD
candidate genes in tilapia because they co-localized with
QTL related to sex-specific mortality and sex determination,
respectively. In this species, a detailed analysis of the sexassociated QTL region in LG23 based on linkage and physical map of 33 genetic markers reduced the sex-related
interval to 1.5 Mb, where 51 genes were identified. Among
these genes, Amh is particularly relevant since it is located in
the center of the sex-related region showing the highest
over-expression in male embryos between 3 and 7 days
post-fertilization (Eshel et al. 2012). Furthermore, Poonlaphdecha et al. (2011) reported a dimorphic expression of
Amh both in the testis and brain of tilapia males during the
critical period of sex differentiation. In a previous work,
Martínez et al. (2009) demonstrated a macrosyntenic relationship between turbot LG5 and tilapia LG23 using model
fish genomes as a bridge, indicating the relevance of mapping Amh and Dmrta2. These genes were located at 10–
13 Mb from a sequence homologous to Sma-USC30, the
closest SD turbot marker, using comparative mapping. Amh
codifies the anti-Müllerian hormone, which determines the
regression of Müllerian ducts in mammals, birds, and reptiles during early testis differentiation (Rey et al. 2003)
likely acting as an antiaromatase factor, but its role is not
clear in fish. Besides tilapia, Amh expression was demonstrated to be higher in males than in females of rainbow trout
(Vizziano et al. 2008), flounder (Yoshinaga et al. 2004),
zebrafish (Wang and Orban 2007), and Squalius alburnoides
(Pala et al. 2008). On the other hand, Dmrta2, a gene
Table 2 Location of the sexrelated genes (Sox3, Sox6, Sox8,
Sox9, Sox17, Sox19, Dmrta2,
Amh, Cyp19a, Cyp19b) and
RAPD markers (ScmFe2,
ScmFe1, ScmM1) on turbot
linkage map
The last three columns show the
nearest marker with low RF on
the map to each gene or RAPD
tested, the recombination frequency (RF), and the LOD score
value
LG linkage group
Gene
LG
Nearest marker with low RF
Sox3
Sox6
Sox8
Sox9
Sox17
Sox19
Dmrta2
Amh
Cyp19a
Cyp19b
ScmFe2
ScmFe1
ScmM1
7
4
13
21
21
2
5
5
4
6
7
8
5
Sma4-14INRA
Sma-USC102
Sma-USC16
Sma-USC231
Sma-USC117
Sma-USC219
Sma-USC247
Sma-USC88
Sma-USC177
Sma-USC110
Sma-USC238
Sma-USC194
Sma-USC10
RF to nearest marker
LOD score
0.3111
0.0000
0.0106
0.0000
0.0000
0.0405
0.0638
0.0312
0.0460
0.1364
0.0635
0.0500
0.1538
2.08
11.15
24.21
23.38
26.94
16.42
17.63
23.17
18.98
9.40
12.36
21.33
9.24
660
belonging to the DM gene family, encodes a protein containing a zinc finger motif called the DM domain. Dmy (dmrt1bY),
an ortholog of the mammalian DMRT1, is implicated in testis
development and is the responsible for sex determination in O.
latipes (Matsuda et al. 2002), the only sex determination gene
detected in fish to date.
Amh and Dmrta2 genes mapped at LG5 in the turbot
consensus map (Fig. 1), as comparative mapping suggested
(Martínez et al. 2009), but they were far apart from the
closest major SD marker (SmaUSC-30), at 31.9 and
36.1 cM, respectively. SmaUSC-30 correctly sexed 98.4 %
of the offspring in the four families analyzed and showed
significant sex association in a panmictic natural sample
(Martínez et al. 2009). In this work, it was estimated that
the SD gene would be at 2.6 cM of SmaUSC-30. Although
the localization of markers in linkage maps depends on the
family or population analyzed, and on the density of
markers, recent data in a new turbot consensus map (Bouza
et al. unpublished) confirm the position of Sma-USC30
previously reported. It is remarkable that no relevant recombination differences were detected between sexes in LG5
(Martínez et al. 2009). Thus, our results very likely discard
Amh and Dmrta2 as primary sex determination genes in
turbot or, in other words, indicate that both genes are not
responsible for the association values observed between the
QTL and sex in LG5. However, this does not mean that both
genes are unimportant in the sex differentiation of turbot.
Other studies such as their expression analysis in different
stages of gonad development are needed to unravel the role
of Amh and Dmrta2 in the sexual development of turbot.
Cytochrome P450 aromatase is a crucial enzyme for
gonad differentiation because it drives the conversion of
androgens into estrogens. In lower vertebrates, inactivation
of this enzyme both by environmental factors, such as
temperature, or by genes of the differentiation cascade determines testis differentiation, its activity being on the basis of
ovary differentiation (Guiguen et al. 2010). In teleost, two
genes of this family, Cyp19a and Cyp19b, encode gonad and
brain aromatases, respectively, which have been described
in most species with some exceptions such as European and
Japanese eel (Ijiri et al. 2003; Tzchori et al. 2004). As shown
in Fig. 1, Cyp19a was located in turbot map at 13.1 cM in
LG4 (Table 2) a linkage group where neither sex- nor
growth-related QTL have been identified to date. However,
Cyp19b mapped at 19. 5 cM in LG6, only at 4. 7 cM from
the closest marker (SmaUSC-110) to a minor sex-related
turbot QTL (Martínez et al. 2009). There are evidences of
Cyp19b aromatase brain sexual dimorphism (Diotel et al.
2010; Munakata and Kobayashi 2010; Vizziano-Cantonet et
al. 2011) and some studies indicate its putative role in sexual
differentiation (Diotel et al. 2010).
SRY-related high-mobility-group box (Sox) genes constitute a family that encodes transcription factors including a
Mar Biotechnol (2012) 14:655–663
DNA-binding HMG box domain of conserved 79 amino
acid protein motif (Gubbay et al. 1990) and additional
domains implicated in transcriptional regulation (Wegner
1999). Most mammalian Sox genes have two orthologs in
teleost fish as a result of the teleost-specific genome duplication (Koopman et al. 2004). In fish, several Sox genes
have demonstrated gonad expression, but their exact role in
sex determination and gonad differentiation is mostly
unknown. Sex differentiation and Sox genes have been
related since the identification of Sry (Lefebvre et al.
2007), a gene expressed in the XY gonad which induces
the differentiation of Sertoli cells and the subsequent testis
development in mammals (Sinclair et al. 1990).
In this group, Sox9 is expressed after Sry and is required
for testis differentiation, but unlike Sry, this gene is conserved in all vertebrates and its expression has been detected
in birds, reptiles, amphibians, and fish. In mammals, Sox9
activates the Amh gene (Bagheri-Fam et al. 2009). Q-PCR
and in situ hybridization analysis performed on gonad tissues have suggested a role for Sox17 in the spermatogenesis
and during gonad differentiation from ovary to testis in the
rice eel, a freshwater species that undergoes natural sex
reversal from male to female during its life (Wang et al.
2003). In sea bass, this gene was discarded as sex determinant because its expression was detected after gonad differentiation started. However, a sexual dimorphic expression of
Sox17 was described, showing higher expression in females
as ovarian differentiation progresses (Navarro-Martín et al.
2009). Sox17 and Sox9 mapped in turbot LG21 at 0.5 cM
between them and at 8 cM from the SmaUSC231 microsatellite (Fig. 1, Table 2), the highest associated marker to a
suggestive sex-related QTL in LG21 (Martínez et al. 2009).
Remarkably, this linkage group showed sharp recombination frequency differences between sexes, no recombination
being detected in males (Bouza et al. 2007). Diminished
recombination is a property associated with the sexdetermining region in the heterogametic sex which avoids
breakage of beneficial sex-associated allele combinations
(Charlesworth and Charlesworth 1997). Our data strongly
suggest further analysis on these genes to understand their
putative role in sex determination.
Sox8, together with Sox9 and Sox10, is expressed in testis
development in mouse. Several experiments revealed that
Sox8 and Sox9 show overlapping functions during testis
differentiation (Chaboissier et al. 2004). Sox 8 mapped at
16.6 cM in turbot LG13 (Fig. 1); in this linkage group, a
suggestive weight QTL was detected at 10 cM (SánchezMolano et al. 2011). Sox3 has been located in the pseudoautosomal X–Y homologous region of the X chromosome in
humans and marsupials (Graves 1998) and is the putative
ancestor of SRY (Foster and Graves 1994). Despite not being
essential for sex determination, this gene has been related to
normal male testis differentiation and gametogenesis in
Mar Biotechnol (2012) 14:655–663
black porgy (Shin et al. 2009), while other studies suggested
its relationship to ovary rather to testis development as in
Rana rugosa (Oshima et al. 2009). Also, Yao et al. (2007)
pointed out that Sox3 is more important for oogenesis than
for spermatogenesis in protogynous hermaphroditic fish.
Sox3 gene was located in turbot LG7 in our study, but its
position was only suggestive (LOD <3) (Fig. 1, Table 2).
Anyway, no sex-associated or growth-related QTL has been
identified to date in turbot close to this position. No sexassociated or growth-related QTL was previously identified
in the region of LG2 where Sox19 was located (Fig. 1).
Koopman et al. (2004) proposed that this gene could have
evolved from Sox3. Sox19 seems to be fish specific and has
been described in zebrafish (Okuda et al. 2006), sturgeon
(Hett and Ludwig 2005), fugu (Koopman et al. 2004), rice
field eel (Liu and Zou 2001), and now in turbot.
Expression of Sox6, together with Sox5 and Sox13, has
been detected in a variety of vertebrate tissues (Lefebvre
2010), and these genes may be involved in sperm maturation in vertebrates (Hagiwara 2011). In fish, there are few
works focused on the analysis of Sox6 function. In rainbow
trout, this gene was excluded as the primary sexdetermining gene (Alfaqih et al. 2009), but interestingly it
is only expressed in testis (Yamashita et al. 1998). Similarly,
Sox6 expression has been detected in mouse in the last
stages of spermatogenesis (Takamatsu et al. 1995). In turbot,
this gene was located at 18.4 cM in LG4 and at 5 cM of
Cyp19a.
Several sex-associated markers had been previously identified in turbot (Casas et al. 2011). ScmM1 RAPD marker
mapped at 24.7 cM in the major sex-determinant LG5 of
turbot, but at 18.4 cM from the closest marker to the major
sex QTL previously reported (SmaUSC-30; Martínez et al.
2009). This distance could explain the limited sexing of
turbot individuals when using this marker (81.7 %; Casas
et al. 2011). Interestingly a weight QTL is located in LG5 in
a confidence interval between 10 and 30 cM, suggesting the
association of sex- and growth-related QTL in turbot. Mining of genes around this marker by using comparative
mapping could provide information to identify relevant candidates. ScmFe1 RAPD marker was located at 5.7 cM in
LG8 only at 3.6 cM of the closest marker (USC59) to a
suggestive sex-associated QTL (Martínez et al. 2009). This
marker yielded a 76.7 % of sexing efficiency (Casas et al.
2011), thus, suggesting the involvement of this LG8 region
in sex determination of turbot. Finally, the ScmFe2 RAPD
marker mapped at 60 cM in LG7 associated to Sma-USC
135 maker (Fig. 1) where a Fulton factor QTL was
described (confidence interval 10–30 cM; Sánchez-Molano
et al. 2011). As outlined before, a linkage between sex and
growth appears to be reflected.
This is the first report where sex-related candidate genes
have been mapped to look for their relationship with sex
661
determination in turbot. Some genes like Dmrta2 and Amh,
and a sex-associated RAPD marker were mapped on LG5,
where the main sex-related QTL had been previously identified. However, most evidences suggest that they are far
apart from the main SD region and further work should be
performed. Other genes like Cyp19b, Sox9, and Sox17, and
several sex-related RAPD markers co-mapped with sexrelated suggestive QTL in different linkage groups, indicating their possible relationship with sex determination and
suggesting a complex regulation of sex determination in
turbot. Finally, some candidate genes co-mapped with
growth-related QTL showing a possible association between
sex determination and growth in this species. This work
provides baseline information for further studies on sex
determination/differentiation in turbot focusing on their
expression along gonad development to shed some light
on the sex determination mechanism in this species.
Acknowledgments We thank María López, Lucía Insua, and Sonia
Gómez for technical assistance. This research work was supported by the
Consellería de Educación e Ordenación Universitaria and the Dirección
Xeral de I + D Xunta de Galicia (project 10MMA200027PR) and by the
Spanish Government (Consolider Ingenio Aquagenomics—CSD200700002 project).
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