BACKGROUND
the mechanisms of sex determination in animals are remarkably diverse.
gonochoristic animals show genetic and/or environmental sex-determining mechanisms.
genetic sex-determining systems can be either chromosomal, and involve a master sex-determining gene/region on a sex chromosome, or can be polygenic and involve several genes/regions on multiple chromosomes.
in most fish species with xx/xy or zz/zw mechanism, the sex chromosomes do not show clear differences in length or gene content  <cit> .
several fish sex determining genes have been isolated from species with xx/xy mechanisms: dmy/dmrt1by in oryzias latipes   <cit> ; gsdf in oryzias luzonensis   <cit> ; amhy in odontesthes hatcheri   <cit> ; amhr <dig> in takifugu rubripes   <cit> ; and sdy in oncorhynchus mykiss   <cit> .
in environmental sex-determining systems, the environment plays a decisive role, such as temperature in turtles, alligators and fish  <cit> .
both systems can interact in some species such as in o. latipes, which has an xx/xy genetic system, where high temperatures can cause female-to-male sex reversal  <cit> .
additionally, autosomal loci can also contribute to sex determination in many species  <cit> .
overall, the understanding of sex determination systems in fish has direct commercial applications, given the strong sexual dimorphism exhibited in a wide variety of aquaculture fish species for a range of commercially important traits like growth or age at maturity.
hippoglossus hippoglossus  has been a high-value species for cold-water marine aquaculture for several decades in northern europe and america, although production has been limited by a series of bottlenecks.
among these, sexual dimorphism in growth, with males maturing earlier and growing significantly slower than females, reduces productivity and profitability of the sector.
females can reach market size  at around 36 months while males require at least an extra year, making the production of all-female stocks particularly appealing for the aquaculture industry  <cit> .
flatfish  show a range of sex-determining mechanisms, including xx/xy and zz/zw, with significant effects of environmental factors, principally temperature, in some species  <cit> .
meiotic gynogenetic h. hippoglossus were all-female, suggesting an xx/xy sex-determining system  <cit> .
temperature has not been shown to have an effect on h. hippoglossus sex ratio  <cit> .
gonadal sex differentiation can be manipulated through in-feed synthetic steroid treatments  or aromatase inhibitor treatments .
however, direct sex reversal is not a commercially acceptable means to alter sex ratios in food fish within the eu  <cit> .
thus indirect sex reversal is required, whereby masculinised genotypic females  are crossed to normal females  to produce genetically all-female progeny, a process which has yet to be proven in h. hippoglossus.
the crux of successful indirect sex reversal is the non-lethal identification of the neomales.
currently the main technique for such verification is progeny testing of treated animals which is time consuming and costly, taking at least four or five years due to the timing of puberty in halibut .
direct genetic sexing, instead of progeny testing, would be preferable using non-lethal and cheap genotyping techniques.
this is only likely to be possible in simple cases of male or female heterogamety.
sex-specific genomic sequences are only available in a limited number of aquaculture species  <cit> .
although a genetic linkage map based on microsatellites and amplified fragment length polymorphism  is available for h. hippoglossus <cit> , this does not contain any information about sex-determination.
restriction associated dna  sequencing is a powerful technique for generation of high-density linkage maps and conducting quantitative trait locus  analysis  <cit>  including the mapping of sex-determining loci in fish  <cit> .
the aim of the current research was to demonstrate that indirect sex reversal was possible and thereafter to develop sex-associated markers through rad-sequencing.
an in-feed mdht treatment was given to weaned halibut juveniles during the labile period, which resulted in 97% phenotypic males.
a sub-population of these treated fish was then reared to maturity and from this stock, two neomales and two normal males were verified by progeny testing, a process that took four years to complete.
the sex-determining locus was mapped to the end of the linkage group  <dig>  in the two mixed sex families from the sex reversal study, using polymorphic single nucleotide polymorphisms .
a combination of four markers predicted sex with 97% accuracy in any individual fish, from a panel of progeny and broodstock.
synteny analysis showed that dna sequences containing atlantic halibut sex-associated snps were consistently clustered in several other fish genomes.
these results suggested that sex determination in h. hippoglossus is likely to be monogenic  and localised within a  <dig>  cm window on linkage group  <dig>
RESULTS
hormonal sex reversal and neomale verification
the control group exhibited a sex ratio not significantly different from 1: <dig> , whereas 97% of the group treated for six weeks  and 70% of the group treated for three weeks  were confirmed as phenotypic males .
both in-feed treatments significantly altered the natural sex ratio in favour of males.
of the seven putative neomales that were progeny tested, only four crosses produced enough survivors at the age of sexing, at approximately one year of age.
from these four crosses, two gave 100% female progeny  while the other two gave balanced sex ratios .
obs.
; observed; exp.
: expected under h <dig> hypothesis .
rad sequencing
two crosses with  <dig>  and  <dig> offspring  and their parents  were analysed .
the dna samples were barcoded, pooled and sequenced in two lanes of an illuminia hiseq  <dig> sequencer .
in total,  <dig> , <dig> raw reads  were produced .
after removing low quality sequences , ambiguous barcodes and orphaned paired-end reads,  <dig> % of the raw reads were retained .
the stacks package  <cit>  was then used to make a de-novo assembly of the sampled loci from each individual:  <dig>  and  <dig>  rad-tags were retrieved for families b and c respectively, covering  <dig>  rad-tags in total including  <dig>  of these shared between the two families .
the number of reads and rad-tags for each sample are reported in additional file  <dig>
genetic map
in order to maximise the number of informative markers and minimise the amount of missing or erroneous data, we used only paired-end rad-tags retrieved in at least 75% of the samples in each family, and carrying one or two snps.
<dig> and  <dig> rad-tags were retained for the families b, and c respectively .
since family b had only  <dig> offspring, the genetic map was constructed with the family c data only .
the map consists of  <dig> snps and  <dig> microsatellites  in  <dig> linkage groups  and spanning 1514 cm .
<dig> of the above snps were common in the two families and were used to incorporate the data from family b into a joint linkage map.
sex-specific genetic maps were constructed, with the female-specific map spanning 1496 cm and the male-specific map spanning 1378 cm .
three linkage maps were constructed using all  <dig> segregating polymorphic markers from males, females or both.
qtl-association mapping
the results from the single-qtl model for binary traits provided evidence for the existence of a major qtl in lg  <dig> for both families in the male informative dataset .
the highest logarithm of odds  scores for families b and c were  <dig>  and  <dig>  respectively .
the genome-wide thresholds  were calculated from permutation tests  to be  <dig>  and  <dig>  respectively.
the highest lod scores were observed in the region between 30 cm and 48 cm in lg  <dig> for both families.
even though models that take into account the existence of a major qtl  or ones that test for existence of multiple qtls simultaneously reduce the residual variation , no additional qtls were detected.
the calculated 95% bayesian density intervals for the qtl location spanned a region of 22 cm , while the  <dig> -lod support interval spanned a region of 28 cm .
the variance explained by the qtl was around 82%.
there were no significant qtl in the female informative dataset.
the polymorphism information content  of the markers in lg  <dig> ranged from  <dig>  to  <dig> .
the joint qtl analysis of the two families showed a maximum at 46 cm with a likelihood ratio  of  <dig> .
the chromosome-wide threshold calculated from permutation tests  was lr =  <dig>  the association analysis identified  <dig> snps being strongly associated  with sex in the two families.
models scanning simultaneously for the existence of two qtl or multiple qtl did not reveal any extra qtl.
verification of snp sex association and sex prediction
sex association of  <dig> snp markers  selected from lg  <dig> was investigated using allele specific endpoint-genotyping assays .
the p-value thresholds  after taking into account multiple testing  according to the permutations and the bonferroni correction tests were found to be  <dig>  and  <dig>  respectively.
hhi <dig> , hhi <dig> , hhi <dig>  and hhi <dig>  showed the highest association with sex, over 97% in the three test families .
in each case the parents were a heterozygous ♂ and a homozygous ♀ for each marker.
furthermore when tested in  <dig> wild sourced broodstock from locations across the species native range , the same four markers individually showed between 78% and 89% association with sex , with snp hhi <dig> showing the highest association .
these four markers were located within a  <dig>  cm region of lg  <dig>
the combined prediction power of these four markers was tested on the  <dig> broodstock and  <dig> progeny using the jrip classifier, as the derived rules have straightforward interpretation.
the combined prediction is based on two rules using three markers  and produced 97% corrected classification with the 3% of errors being phenotypic males predicted to be females.
the male prediction precision is  <dig>  and the female prediction precision is  <dig>  .
within the  <dig> tested male broodstock, two had “female” genotypes for all four of these markers .
one of these two broodstock had previously been crossed with four females and had produced only female offspring .
the phenotypic sex of these  <dig> offspring was verified by post-mortem examination three years post-fertilisation.
synteny searches
we selected the  <dig> markers within the 95% confidence interval around the lod score peak and mapped them onto the genomes of related species to identify syntenic regions.
we performed this search against danio rerio , gadus morhua , gasterosteus aculeatus , latimeria chalumnae , oreochromis niloticus , oryzias latipes , takifugu rubripes  and tetraodon nigroviridis  genomes.
<dig> markers had unique hits across at least five out of eight species .
g. aculeatus and o. niloticus show the highest level of synteny with h. hippoglossus and each other .
the order of the markers selected for snp genotyping in the regions point toward one  <dig>  mb region embedding more than  <dig> annotated genes .
no genes associated with sexual differentiation or determination were identified in this region.
discussion
h. hippoglossus is a species of increasing commercial interest for cold-water marine aquaculture.
however one of the main limitations to profitable culture of the species is the sexual dimorphism in age at maturation related to gender specific growth performance  <cit> .
to address this bottleneck, the current study demonstrates, for the first time in this species, that indirect monosex female production is possible for commercial h. hippoglossus aquaculture.
while having strong commercial application, this research also had the fundamental aim to investigate the genomic regulation of sex determination in the species through state-of-the-art high-throughput sequencing methodologies.
rad-tag sequencing has recently been used with a number of different fish species since the technology became available in  <dig>  one of the aims of the baird et al.
<cit>  study, which first validated the technique in fish, was to fine map qtls in g. aculeatus.
a number of different restriction-digest methodologies already existed, using high-throughput sequencers.
however, what sets the rad-tag methodology apart is the fact that it combines control over the fragments that result from the digestion with deep sequencing across individuals, making the identified snp reproducible  <cit> .
this makes the rad platform very efficient for constructing genetic maps and qtl studies.
in the present study, a genetic map of  <dig> snps and  <dig> microsatellites spanning 1514 cm was constructed.
to our knowledge this is the first dense genetic map incorporating snps in any flatfish species.
the map has  <dig> linkage groups, corresponding to the number of chromosome pairs in h. hippoglossus <cit> .
in a similar study, amores et al.
<cit>  constructed a genetic map for lepisosteus oculatus  consisting of  <dig> snps.
the above map was used to prove that l. oculatus diverged from teleosts before the teleost genome duplication.
genetic maps of more than  <dig> snps using rad-seq were also constructed in o. mykiss and in d. rerio <cit> .
the high lod , which was used to assign the genetic markers in linkage groups in our study, ensures that the map is of high quality.
however, it must be acknowledged that even though the assignment of markers in linkage groups is robust, none of the available algorithms used for ordering markers provides an accurate positioning of closely spaced markers due to the relatively low number of meioses represented in our sample size.
in a species like h. hippoglossus with no sequenced genome available, a genetic map is an invaluable tool for mapping any trait of interest in a qtl study.
apart from mapping qtl, the identified snp of the genetic map can be used to construct a genomic relationship matrix, which can replace the relationship matrix inferred by pedigree for calculating breeding values.
this would improve accuracy of estimated breeding values  under best linear unbiased prediction  methodology in a breeding program  <cit> .
the improvement in accuracy is due to the fact that the genomic relationship matrix accounts for the random segregation of chromosome segments at meiosis between siblings.
in this study we associated mapped rad-tag markers to sex determination.
a major qtl involved in sex determination was identified in lg  <dig> in both families .
the location of the above qtl spans a region of around 22 cm.
this region should contain one or more genes responsible for sex determination in h. hippoglossus.
the reduced recombination in this region resulted in an almost flat likelihood surface for this region.
genome regions with reduced recombination are a common characteristic of sex chromosomes.
in a similar study by anderson et al.
<cit>  where the objective was to identify qtls involved in sex determination in zebrafish using rad-tag, a region in chromosome  <dig> spanning more than 20 cm showed reduced recombination.
in general suppression of recombination keeps together genes  with functions that are advantageous for one sex and avoids their transfer to the other sex chromosome, where they might have negative effects on the opposite sex  <cit> .
our data support the hypothesis that the h. hippoglossus has an xx/xy sex determination system.
among flatfish species, paralichthys olivaceus  has also been shown to possess an xx/xy system  <cit> , although temperature also influences sex ratio.
on the other hand other closely related species, in which sex associated genetic markers have been identified, such as hippoglossus stenolepis   <cit> , verasper variegates   <cit> , scophthalmus maximus   <cit>  and cynoglossus semilaevis   <cit>  were all shown to have a zz/zw sex determination system.
unusually in this group, c. semilaevis has differentiated w and z chromosomes  <cit> .
validating the results of the qtl-association analysis is of the utmost importance.
the fact that the sex-associated snps showed strong association when tested in a wider panel of three families and  <dig> wild broodstock provides clear evidence that those markers are in strong linkage disequilibrium with the sex-determining gene.
marker-assisted selection  could be conducted using these snps, providing a valuable tool towards more efficient production of all-female stocks for the aquaculture industry.
in the current study it took four years from initiation of sex reversal treatment to completion of progeny testing for neomale identification with guaranteed all-female production from the following year.
by employing mas however it would be possible to confirm sex associated genotype from a non-destructive biopsy sample in hormonally-treated fish within 6-12 months of treatment, allowing neomales to be isolated and used from first maturation at three-four years post-treatment.
snps hhi <dig>  hhi <dig>  hhi <dig> and hhi <dig> are the strongest candidates for mas since they correctly assign sex in more than 97% of the screened individuals.
they span a narrow region of  <dig>  cm.
genotyping a larger population for the snps in this region would allow fine mapping of the sex-determining locus.
other genetic factors involved in sex determination might also be involved.
the application of this technology will enable the industry to include a greater number of neomales from a wider genetic base to be included in future breeding programmes without the reduction in effective population size  associated with the use of a small number of neomales from these initial sex-reversed families.
limited examples exist of practical application of mas in breeding programmes in aquaculture.
a y-specific dna marker was used to assist in the development of monosex female culture in oncorhynchus tshawytscha   <cit> .
more recently, mas has been apply to a qtl for infectious pancreatic necrosis virus resistance in salmo salar : initially microsatellite markers were used, and more recently snps derived from rad sequencing have been added  <cit> .
CONCLUSIONS
overall this work has demonstrated that all-female halibut production is commercially possible using indirect monosex production techniques.
this in itself confirms that h. hippoglossus has an xx/xy sex determination system.
rad-tag sequencing produced  <dig>  unique loci, and a single sex determination locus was mapped to lg  <dig>  a further set of  <dig> markers that were present only or predominantly in dna from male fish was isolated from two families and validated in a wider population screening, opening the possibility of mas for sex in the species.
synteny analysis showed that dna sequences containing h. hippoglossus sex-associated snps were consistently clustered in several other genomes, which provides a new focus for research into the sex determination mechanism in this species.

 the mechanisms of sex determination in animals are remarkably diverse. gonochoristic animals show genetic and/or environmental sex-determining mechanisms. genetic sex-determining systems can be either chromosomal, and involve a master sex-determining gene/region on a sex chromosome, or can be polygenic and involve several genes/regions on multiple chromosomes. in most fish species with xx/xy or zz/zw mechanism, the sex chromosomes do not show clear differences in length or gene content. several fish sex determining genes have been isolated from species with xx/xy mechanisms: dmy/dmrt1by in oryzias latipes; gsdf in oryzias luzonensis; amhy in odontesthes hatcheri; amhr in takifugu rubripes; and sdy in oncorhynchus mykiss. in environmental sex-determining systems, the environment plays a decisive role, such as temperature in turtles, alligators and fish. both systems can interact in some species such as in o. latipes, which has an xx/xy genetic system, where high temperatures can cause female-to-male sex reversal. additionally, autosomal loci can also contribute to sex determination in many species. overall, the understanding of sex determination systems in fish has direct commercial applications, given the strong sexual dimorphism exhibited in a wide variety of aquaculture fish species for a range of commercially important traits like growth or age at maturity. hippoglossus hippoglossus has been a high-value species for cold-water marine aquaculture for several decades in northern europe and america, although production has been limited by a series of bottlenecks. among these, sexual dimorphism in growth, with males maturing earlier and growing significantly slower than females, reduces productivity and profitability of the sector. females can reach market size at around 36 months while males require at least an extra year, making the production of all-female stocks particularly appealing for the aquaculture industry. flatfish show a range of sex-determining mechanisms, including xx/xy and zz/zw, with significant effects of environmental factors, principally temperature, in some species. meiotic gynogenetic h. hippoglossus were all-female, suggesting an xx/xy sex-determining system. temperature has not been shown to have an effect on h. hippoglossus sex ratio. gonadal sex differentiation can be manipulated through in-feed synthetic steroid treatments or aromatase inhibitor treatments. however, direct sex reversal is not a commercially acceptable means to alter sex ratios in food fish within the eu. thus indirect sex reversal is required, whereby masculinised genotypic females are crossed to normal females to produce genetically all-female progeny, a process which has yet to be proven in h. hippoglossus. the crux of successful indirect sex reversal is the non-lethal identification of the neomales. currently the main technique for such verification is progeny testing of treated animals which is time consuming and costly, taking at least four or five years due to the timing of puberty in halibut. direct genetic sexing, instead of progeny testing, would be preferable using non-lethal and cheap genotyping techniques. this is only likely to be possible in simple cases of male or female heterogamety. sex-specific genomic sequences are only available in a limited number of aquaculture species. although a genetic linkage map based on microsatellites and amplified fragment length polymorphism is available for h. hippoglossus, this does not contain any information about sex-determination. restriction associated dna sequencing is a powerful technique for generation of high-density linkage maps and conducting quantitative trait locus analysis including the mapping of sex-determining loci in fish. the aim of the current research was to demonstrate that indirect sex reversal was possible and thereafter to develop sex-associated markers through rad-sequencing. an in-feed mdht treatment was given to weaned halibut juveniles during the labile period, which resulted in 97% phenotypic males. a sub-population of these treated fish was then reared to maturity and from this stock, two neomales and two normal males were verified by progeny testing, a process that took four years to complete. the sex-determining locus was mapped to the end of the linkage group in the two mixed sex families from the sex reversal study, using polymorphic single nucleotide polymorphisms. a combination of four markers predicted sex with 97% accuracy in any individual fish, from a panel of progeny and broodstock. synteny analysis showed that dna sequences containing atlantic halibut sex-associated snps were consistently clustered in several other fish genomes. these results suggested that sex determination in h. hippoglossus is likely to be monogenic and localised within a  cm window on linkage group  hormonal sex reversal and neomale verification the control group exhibited a sex ratio not significantly different from 1:, whereas 97% of the group treated for six weeks and 70% of the group treated for three weeks were confirmed as phenotypic males. both in-feed treatments significantly altered the natural sex ratio in favour of males. of the seven putative neomales that were progeny tested, only four crosses produced enough survivors at the age of sexing, at approximately one year of age. from these four crosses, two gave 100% female progeny while the other two gave balanced sex ratios. obs.; observed; exp.: expected under h hypothesis. rad sequencing two crosses with and offspring and their parents were analysed. the dna samples were barcoded, pooled and sequenced in two lanes of an illuminia hiseq sequencer. in total,, raw reads were produced. after removing low quality sequences, ambiguous barcodes and orphaned paired-end reads, % of the raw reads were retained. the stacks package was then used to make a de-novo assembly of the sampled loci from each individual: and rad-tags were retrieved for families b and c respectively, covering rad-tags in total including of these shared between the two families. the number of reads and rad-tags for each sample are reported in additional file genetic map in order to maximise the number of informative markers and minimise the amount of missing or erroneous data, we used only paired-end rad-tags retrieved in at least 75% of the samples in each family, and carrying one or two snps. and rad-tags were retained for the families b, and c respectively. since family b had only offspring, the genetic map was constructed with the family c data only. the map consists of snps and microsatellites in linkage groups and spanning 1514 cm. of the above snps were common in the two families and were used to incorporate the data from family b into a joint linkage map. sex-specific genetic maps were constructed, with the female-specific map spanning 1496 cm and the male-specific map spanning 1378 cm. three linkage maps were constructed using all segregating polymorphic markers from males, females or both. qtl-association mapping the results from the single-qtl model for binary traits provided evidence for the existence of a major qtl in lg for both families in the male informative dataset. the highest logarithm of odds scores for families b and c were and respectively. the genome-wide thresholds were calculated from permutation tests to be and respectively. the highest lod scores were observed in the region between 30 cm and 48 cm in lg for both families. even though models that take into account the existence of a major qtl or ones that test for existence of multiple qtls simultaneously reduce the residual variation, no additional qtls were detected. the calculated 95% bayesian density intervals for the qtl location spanned a region of 22 cm, while the -lod support interval spanned a region of 28 cm. the variance explained by the qtl was around 82%. there were no significant qtl in the female informative dataset. the polymorphism information content of the markers in lg ranged from to. the joint qtl analysis of the two families showed a maximum at 46 cm with a likelihood ratio of. the chromosome-wide threshold calculated from permutation tests was lr =  the association analysis identified snps being strongly associated with sex in the two families. models scanning simultaneously for the existence of two qtl or multiple qtl did not reveal any extra qtl. verification of snp sex association and sex prediction sex association of snp markers selected from lg was investigated using allele specific endpoint-genotyping assays. the p-value thresholds after taking into account multiple testing according to the permutations and the bonferroni correction tests were found to be and respectively. hhi, hhi, hhi and hhi showed the highest association with sex, over 97% in the three test families. in each case the parents were a heterozygous ♂ and a homozygous ♀ for each marker. furthermore when tested in wild sourced broodstock from locations across the species native range, the same four markers individually showed between 78% and 89% association with sex, with snp hhi showing the highest association. these four markers were located within a  cm region of lg the combined prediction power of these four markers was tested on the broodstock and progeny using the jrip classifier, as the derived rules have straightforward interpretation. the combined prediction is based on two rules using three markers and produced 97% corrected classification with the 3% of errors being phenotypic males predicted to be females. the male prediction precision is and the female prediction precision is. within the tested male broodstock, two had “female” genotypes for all four of these markers. one of these two broodstock had previously been crossed with four females and had produced only female offspring. the phenotypic sex of these offspring was verified by post-mortem examination three years post-fertilisation. synteny searches we selected the markers within the 95% confidence interval around the lod score peak and mapped them onto the genomes of related species to identify syntenic regions. we performed this search against danio rerio, gadus morhua, gasterosteus aculeatus, latimeria chalumnae, oreochromis niloticus, oryzias latipes, takifugu rubripes and tetraodon nigroviridis genomes. markers had unique hits across at least five out of eight species. g. aculeatus and o. niloticus show the highest level of synteny with h. hippoglossus and each other. the order of the markers selected for snp genotyping in the regions point toward one  mb region embedding more than annotated genes. no genes associated with sexual differentiation or determination were identified in this region. discussion h. hippoglossus is a species of increasing commercial interest for cold-water marine aquaculture. however one of the main limitations to profitable culture of the species is the sexual dimorphism in age at maturation related to gender specific growth performance. to address this bottleneck, the current study demonstrates, for the first time in this species, that indirect monosex female production is possible for commercial h. hippoglossus aquaculture. while having strong commercial application, this research also had the fundamental aim to investigate the genomic regulation of sex determination in the species through state-of-the-art high-throughput sequencing methodologies. rad-tag sequencing has recently been used with a number of different fish species since the technology became available in one of the aims of the baird et al. study, which first validated the technique in fish, was to fine map qtls in g. aculeatus. a number of different restriction-digest methodologies already existed, using high-throughput sequencers. however, what sets the rad-tag methodology apart is the fact that it combines control over the fragments that result from the digestion with deep sequencing across individuals, making the identified snp reproducible. this makes the rad platform very efficient for constructing genetic maps and qtl studies. in the present study, a genetic map of snps and microsatellites spanning 1514 cm was constructed. to our knowledge this is the first dense genetic map incorporating snps in any flatfish species. the map has linkage groups, corresponding to the number of chromosome pairs in h. hippoglossus. in a similar study, amores et al. constructed a genetic map for lepisosteus oculatus consisting of snps. the above map was used to prove that l. oculatus diverged from teleosts before the teleost genome duplication. genetic maps of more than snps using rad-seq were also constructed in o. mykiss and in d. rerio. the high lod, which was used to assign the genetic markers in linkage groups in our study, ensures that the map is of high quality. however, it must be acknowledged that even though the assignment of markers in linkage groups is robust, none of the available algorithms used for ordering markers provides an accurate positioning of closely spaced markers due to the relatively low number of meioses represented in our sample size. in a species like h. hippoglossus with no sequenced genome available, a genetic map is an invaluable tool for mapping any trait of interest in a qtl study. apart from mapping qtl, the identified snp of the genetic map can be used to construct a genomic relationship matrix, which can replace the relationship matrix inferred by pedigree for calculating breeding values. this would improve accuracy of estimated breeding values under best linear unbiased prediction methodology in a breeding program. the improvement in accuracy is due to the fact that the genomic relationship matrix accounts for the random segregation of chromosome segments at meiosis between siblings. in this study we associated mapped rad-tag markers to sex determination. a major qtl involved in sex determination was identified in lg in both families. the location of the above qtl spans a region of around 22 cm. this region should contain one or more genes responsible for sex determination in h. hippoglossus. the reduced recombination in this region resulted in an almost flat likelihood surface for this region. genome regions with reduced recombination are a common characteristic of sex chromosomes. in a similar study by anderson et al. where the objective was to identify qtls involved in sex determination in zebrafish using rad-tag, a region in chromosome spanning more than 20 cm showed reduced recombination. in general suppression of recombination keeps together genes with functions that are advantageous for one sex and avoids their transfer to the other sex chromosome, where they might have negative effects on the opposite sex. our data support the hypothesis that the h. hippoglossus has an xx/xy sex determination system. among flatfish species, paralichthys olivaceus has also been shown to possess an xx/xy system, although temperature also influences sex ratio. on the other hand other closely related species, in which sex associated genetic markers have been identified, such as hippoglossus stenolepis, verasper variegates, scophthalmus maximus and cynoglossus semilaevis were all shown to have a zz/zw sex determination system. unusually in this group, c. semilaevis has differentiated w and z chromosomes. validating the results of the qtl-association analysis is of the utmost importance. the fact that the sex-associated snps showed strong association when tested in a wider panel of three families and wild broodstock provides clear evidence that those markers are in strong linkage disequilibrium with the sex-determining gene. marker-assisted selection could be conducted using these snps, providing a valuable tool towards more efficient production of all-female stocks for the aquaculture industry. in the current study it took four years from initiation of sex reversal treatment to completion of progeny testing for neomale identification with guaranteed all-female production from the following year. by employing mas however it would be possible to confirm sex associated genotype from a non-destructive biopsy sample in hormonally-treated fish within 6-12 months of treatment, allowing neomales to be isolated and used from first maturation at three-four years post-treatment. snps hhi hhi hhi and hhi are the strongest candidates for mas since they correctly assign sex in more than 97% of the screened individuals. they span a narrow region of  cm. genotyping a larger population for the snps in this region would allow fine mapping of the sex-determining locus. other genetic factors involved in sex determination might also be involved. the application of this technology will enable the industry to include a greater number of neomales from a wider genetic base to be included in future breeding programmes without the reduction in effective population size associated with the use of a small number of neomales from these initial sex-reversed families. limited examples exist of practical application of mas in breeding programmes in aquaculture. a y-specific dna marker was used to assist in the development of monosex female culture in oncorhynchus tshawytscha. more recently, mas has been apply to a qtl for infectious pancreatic necrosis virus resistance in salmo salar: initially microsatellite markers were used, and more recently snps derived from rad sequencing have been added.  overall this work has demonstrated that all-female halibut production is commercially possible using indirect monosex production techniques. this in itself confirms that h. hippoglossus has an xx/xy sex determination system. rad-tag sequencing produced unique loci, and a single sex determination locus was mapped to lg a further set of markers that were present only or predominantly in dna from male fish was isolated from two families and validated in a wider population screening, opening the possibility of mas for sex in the species. synteny analysis showed that dna sequences containing h. hippoglossus sex-associated snps were consistently clustered in several other genomes, which provides a new focus for research into the sex determination mechanism in this species. 
atlantic halibut is a high-value, niche market species for cold-water marine aquaculture. altogether our findings support the hypothesis that the atlantic halibut has an xx/xy sex determination system. understanding the sex determination mechanism and developing sex-associated markers will shorten the time for the development of monosex female production, thus decreasing the costs of farming. these should also help to speed up the inclusion of neomales derived from many families to maintain a larger effective population size and ensure long-term improvement through selective breeding. a linkage map was constructed based on single nucleotide polymorphism markers and microsatellites consisting of linkage groups, which corresponds to the number of chromosome pairs in this species. two of these groups consisted of only females, while the other two contained balanced sex ratios. using a variety of machine-learning algorithms 97% correct classification could be obtained with the 3% of errors being phenotypic males predicted to be females. assays for snps with significant association with phenotypic sex were tested in both population data and in additional families. halibut juveniles were masculinised with α-methyldihydrotestosterone and grown to maturity.
 atlantic halibut is a high-value, niche market species for cold-water marine aquaculture. production of monosex female stocks is desirable in commercial production since females grow faster and mature later than males. understanding the sex determination mechanism and developing sex-associated markers will shorten the time for the development of monosex female production, thus decreasing the costs of farming.  halibut juveniles were masculinised with α-methyldihydrotestosterone and grown to maturity. progeny groups from four treated males were reared and sexed. two of these groups consisted of only females, while the other two contained balanced sex ratios. dna from parents and offspring from the two mixed-sex families were used as a template for restriction-site associated dna sequencing. the million raw reads produced unique rad-tags. a linkage map was constructed based on single nucleotide polymorphism markers and microsatellites consisting of linkage groups, which corresponds to the number of chromosome pairs in this species. a major sex determining locus was mapped to linkage group in both families. assays for snps with significant association with phenotypic sex were tested in both population data and in additional families. using a variety of machine-learning algorithms 97% correct classification could be obtained with the 3% of errors being phenotypic males predicted to be females.  altogether our findings support the hypothesis that the atlantic halibut has an xx/xy sex determination system. assays are described for sex-associated dna markers developed from the rad sequencing analysis to fast track progeny testing and implement monosex female halibut production for an immediate improvement in productivity. these should also help to speed up the inclusion of neomales derived from many families to maintain a larger effective population size and ensure long-term improvement through selective breeding. hippoglossus hippoglossussex determinationmonosexqtl mappingrad-seqaquaculture 