A gene-based radiation hybrid map of the gilthead sea bream Sparus aurata refines and exploits conserved synteny with Tetraodon nigroviridis
- Elena Sarropoulou1, 2Email author,
- Rafaella Franch3,
- Bruno Louro4,
- Deborah M Power4,
- Luca Bargelloni3,
- Antonios Magoulas1,
- Fabrice Senger5, 7,
- Matina Tsalavouta1, 8,
- Tomaso Patarnello3, 5,
- Francis Galibert6,
- Georgios Kotoulas†1 and
- Robert Geisler†2
© Sarropoulou et al; licensee BioMed Central Ltd. 2007
Received: 30 September 2006
Accepted: 07 February 2007
Published: 07 February 2007
Comparative teleost studies are of great interest since they are important in aquaculture and in evolutionary issues. Comparing genomes of fully sequenced model fish species with those of farmed fish species through comparative mapping offers shortcuts for quantitative trait loci (QTL) detections and for studying genome evolution through the identification of regions of conserved synteny in teleosts. Here a comparative mapping study is presented by radiation hybrid (RH) mapping genes of the gilthead sea bream Sparus aurata, a non-model teleost fish of commercial and evolutionary interest, as it represents the worldwide distributed species-rich family of Sparidae.
An additional 74 microsatellite markers and 428 gene-based markers appropriate for comparative mapping studies were mapped on the existing RH map of Sparus aurata. The anchoring of the RH map to the genetic linkage map resulted in 24 groups matching the karyotype of Sparus aurata. Homologous sequences to Tetraodon were identified for 301 of the gene-based markers positioned on the RH map of Sparus aurata. Comparison between Sparus aurata RH groups and Tetraodon chromosomes (karyotype of Tetraodon consists of 21 chromosomes) in this study reveals an unambiguous one-to-one relationship suggesting that three Tetraodon chromosomes correspond to six Sparus aurata radiation hybrid groups. The exploitation of this conserved synteny relationship is furthermore demonstrated by in silico mapping of gilthead sea bream expressed sequence tags (EST) that give a significant similarity hit to Tetraodon.
The addition of primarily gene-based markers increased substantially the density of the existing RH map and facilitated comparative analysis. The anchoring of this gene-based radiation hybrid map to the genome maps of model species broadened the pool of candidate genes that mainly control growth, disease resistance, sex determination and reversal, reproduction as well as environmental tolerance in this species, all traits of great importance for QTL mapping and marker assisted selection. Furthermore this comparative mapping approach will facilitate to give insights into chromosome evolution and into the genetic make up of the gilthead sea bream.
Fish species constitute an exceedingly diverse group representing roughly half of the extant vertebrate species. More than 95 % of all living fish species are represented by the ray-finned fishes (actinopterygians) of which more than 99.8 % are teleosts. Their high level of morphological, behavioral, and ecological diversity makes the study of teleosts of real importance in attempts to address and resolve evolutionary questions. Furthermore teleost studies are of great intrinsic interest since they are economically important in both fisheries and aquaculture. In recent years due to the efforts made in genome studies of many fish species, especially of model fish species like zebrafish and Tetraodon, genomic information of vertebrates has shown a substantial increase and comparative genomics studies have become a very important method for studying genome evolution in teleosts and vertebrates in general  as well as for the identification of regions of conserved synteny (e.g. for review ).
The opportunity of comparing genomes of model fish species with those of farmed fish species can facilitate functional studies, such as the detection of candidate genes and regions for the identification of qualitative and quantitative trait loci (QTLs). Furthermore comparative genomics can improve on the time-consuming work of identifying genes affecting trait variability through QTL mapping by offering shortcuts and hypothesis-based approaches rather than random scan approaches. Nevertheless, this promising approach has until now been hampered by the limited number of genome projects because of the expensive technology involved. A powerful method that allows comparative genome analysis to be conducted by simple means constitutes comparative mapping, enabling comparison of syntenies and gene orders to be carried out [3–7]. Whereas for model fish species such as the zebrafish, Tetraodon, fugu and medaka, comparative mapping is a common practice, in non-model fish species of commercial as well as of evolutionary and ecological interest only a few studies have so far been published e.g. .
In contrast to studies concerning agricultural animals, maps of DNA markers and genes allowing QTL analysis are relatively rare for cultured fish species. However, linkage maps among aquaculture fish species are available for salmonid species [9, 10], tilapia , channel catfish [12, 13], Japanese flounder  and the common carp . Among Mediterranean species linkage maps for Sparus aurata  and for another important marine aquaculture species, Dicentrarchus labrax  have recently been published. In addition to the genetic linkage map of the gilthead sea bream, a first generation of RH map has also been constructed . Radiation hybrid mapping results in dense and reliable genome maps for comparative use, since, unlike linkage mapping, it is not dependent on polymorphism and permits easy mapping of genes and of neutral polymorphic markers.
In the present study comparative mapping is taken with the gilthead sea bream (Sparus aurata), a key species for large-scale Mediterranean aquaculture. The gilthead sea bream, a non-model fish species of commercial and evolutionary interest, is distributed in the Atlantic Ocean and the Mediterranean Sea [19, 20] and represents the worldwide-distributed species rich family of Sparidae, within the Perciformes. Comparative mapping for the gilthead sea bream Sparus aurata is reported through a gene-based radiation hybrid map with 428 markers including candidate genes for QTL and 74 microsatellite markers integrated with the previously published map of .
Furthermore, the considerable potential of comparative mapping for transferring information from model species to non-model species is demonstrated by the exploitation of conserved synteny. This established syntenic relationship between sea bream and Tetraodon enables to virtually map on the RH map ESTs of gilthead sea bream that give a significant similarity hit to Tetraodon. The sea bream RH map facilitates the scanning for QTLs mainly controlling growth, disease resistance, sex determination and reversal, reproduction as well as environmental tolerance, all traits of great importance for aquaculture. It also contributes to the identification of regions of conserved synteny and thereby provides a resource for further comparative mapping analysis between fish species and pinpoints possible chromosomes splitting, chromosomes fusions and chromosomes rearrangements during evolution.
Number of BLAST matches (against ENSEMBL databases [v.38 – Apr2006]) and BLAT matches (against datasets from Genoscope [Tetraodon nigroviridis V7, February 2004] and the Wellcome Trust Sanger Institute [Danio rerio Zv6, March 2006] including un_random scaffolds) of 794 sea bream sequences mapped on the RH map.
BLAST search e<10-4
BLAT search score>80
Comparative mapping of Sparus against Danio with the BLAT web server gave only 90 hits, out of which 5 were not assigned to a chromosome (NA_random). Syntenic relationships between Sparus aurata and Danio were not as apparent as in Tetraodon.
The gilthead sea bream unlike the model organisms zebrafish and medaka, mostly used to study diseases and malfunctions, is a species of great commercial interest. Consequently, considerable information has been gathered on different aspects of its husbandry, physiology, biology and pathology, while a comprehensive genomic "tool box" has been created. The basis for sea bream genomics was recently established with the creation of a first generation linkage map  and radiation hybrid map . The power of the RH map is significantly increased in the present study with the mapping of ESTs and this will be an important resource for future QTL detection and identification of functional units. Moreover, the present RH map represents a significant tool for comparative mapping as the sea bream belongs to the successful order of Perciformes which underwent an explosive radiation 50–70 million years ago.
Comparison of the radiation hybrid map to the linkage map
BLAT search of 31,705 EST sequences generated by Marine Genomics. BLAT searching was performed using -q = dnax and -t = dnax as recommended for mapping ESTs to the genome across species.
In order to retrieve information by comparative mapping two approaches were pursued which are described in more detail below. The first approach looked at the molecular markers mapped in sea bream to localize potential candidate genes in the Tetraodon genome. In the second approach candidate genes or ESTs available in sea bream were mapped on the Tetraodon genome (Table 2) to facilitate primer design in specific candidate regions for growth, disease resistance or sex determination and also to use them in further studies which aimed to result in higher resolution mapping of these radiation hybrid groups.
The standard approach to find a gene in classical genetics is to specify a gene product and then to try to identify the gene. In the field of molecular genetics the reverse approach is applied; genes are identified purely on the basis of their position in the genome through so-called reverse genetics or positional cloning. In the present study in silico RH mapping is demonstrated to identify candidate genes, first by localizing specific functional groups of interest in Tetraodon chromosomes, and subsequently to identify the corresponding RH groups in sea bream and to corroborate the findings by in vitro RH mapping. Three examples, namely DMRT1, gonadal P450 aromatase and cytochrome P450 aromatase are described below for which first in silico positioning was performed and then confirmed by RH mapping with primers designed within the exons of those genes. DMRT1 belongs to the highly conserved group of genes containing the DM domain, which may be involved in sex determination . In Teleostei although at least six genes containing the DM domain are found their function is still unknown . Looking at those genes we found that they are localized in chromosome 12 and 1 of Tetraodon and chromosome 5 in zebrafish; both Tetraodon chromosome 12 and Danio chromosome 5 correspond to RH group 16, suggesting that this RH group could be of interest for mapping of QTLs related to sex determination.
The second and third example for in silico mapping is positioned in the sex-determining region of Tilapia that was mapped to linkage group 1 in Tilapia [26, 27]. Linkage group 1 of Tilapia corresponds to Tetraodon chromosome 5 and Sparus RH group 18 (Figure 6). The gene order between Sparus RH group 18 and Tetraodon chromosome 5 is particularly well conserved compared to the other RH groups and their corresponding Tetraodon chromosomes, suggesting another specific region for QTL mapping. In this particularly well conserved region of Tetraodon chromosome 5 we found the gene for gonadal P450 aromatase, a neural marker of estrogen effect known to be involved in sex differentiation [28, 29] as well as cytochrome P450 aromatase, which catalyzes the key step in estrogen biosynthesis [30, 31] and is a neural marker of estrogen effect in teleosts.
The in vitro mapping of DM domain genes (DMRT 1 and 2), gonadal P450 aromatase and cytochrome P450 aromatase to Tetraodon assigned the DM domain genes to Tetraodon chromosome 12 and the two P450 aromatases genes to Tetraodon chromosome 5. Chromosome 12 and chromosome 5 are the homologues to RH group 16 and RH 18 respectively. In silico mapping corroborated these findings allocating the DM domain genes to RH group 16 and the two P450 aromatases genes to RH group 18. In this way the correspondence between Sparus aurata and Tetraodon can facilitate the identification of genes corresponding to QTLs.
Finally, by mapping gene-based markers, potential functional units were identified mapping in radiation hybrid groups 16 and 24: on RH16 the Sparus aurata prolactin receptor , growth hormone receptor  and the homologue of osteoclast-stimulating factor and on RH 24 the Sparus aurata growth hormone gene , prolactin (PRL)  and osteocalcin gene , all of which are candidate genes for growth-related QTLs of potential economic interest.
The RH panel used in the present study has been previously described . Amplification of the RH panel was perfomed four times in parallel using the GenomiPhi Kit (Amersham-Biosciences). Prior to pooling the four amplification reactions each panel was tested with two primer pairs in order to verify the absence of contamination.
Development of markers
Oligonucleotide primers were designed from sea bream cDNA sequences generated out of five cDNA libraries: mixed embryonic and early larvae library, liver library , kidney , pituitary , 20–135 days post hatch larvae , using Primer 3 software . When seabream cDNA aligned to the Genome of Tetraodon nigroviridis primer were designed within exons using the Spidey software .
PCR reactions were set up by a Biomek 2000 robot (Beckman) in 96 well microtiter plates. Each PCR reaction had a final volume of 10 μl containing 0.4 μl of forward and reverse primer (20 μM), 1 μl of 10 × PCR buffer (10× PCR buffer contained 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2 and 0.1% (w/v) gelatin), 0.02 μl each of 100 mM dATP, dCTP, dGTP and dTTP, 5.82 μl water, 0.1 μl Taq polymerase (5 U/μl, GenAxon) and 2.5 μl of radiation hybrid DNA (approx. 50 ng/μl). After the first denaturation step of 8 min at 94°C, PCR was performed for 20 cycles: 30 s at 94°C, 30 s at the appropriate annealing temperature × (-0,5°C/cycle) for a given primer set and 30 s at 73°C, following those 20 cycles final 15 cycles were performed: 30 s at 94°C, 30 s at × – 10°C and 72 for 30°C. The concluding elongation step was for 5 min at 73°C. The PCR reactions were performed using MWG PCR machines.
Gel electrophoresis and analysis
PCR products were separated on 2% TBE agarose (Qualex Gold) gels containing 0.01% (v/v) of ethidium bromide solution (10 mg/ml). The gels were poured into gel trays containing 16 combs with 30 wells. The gel run was performed at 200 V (5 V/cm) for 25 min. The gel images were captured by NIH Image 1.61  on a Power Macintosh 8500/120. The macros for gel capturing and semi-automatic analysis was developed by R. Geisler .
Construction of the radiation hybrid map
Bands were scored manually as present (1), absent (0) or unclear (2). In total 960 molecular markers were genotyped. We rejected those markers with no PCR product, or where sea bream and hamster band were not clearly distinguishable. The radiation hybrid analysis was performed for 1,171 molecular markers in total including previously published vectors of  using the TSP approach implemented in the rh_tsp_map2 software package in conjunction with the CONCORDE package . Radiation hybrid groups were generated by calculating the pairlods with retention set to the arithmetic mean of pair and all, with an initial LOD score of 3 which was then raised to 6. The resulting data were subsequently analysed by single-linkage clustering in order to obtain radiation groups .
BLAT searching was performed using -q = dnax and -t= dnax with a score above 80 and an alignment length of more than 50 bp as recommended for mapping ESTs to the genome across species . Sequences submitted to BLAT searching came from the 937 radiation hybrid mapped ESTs and microsatellites produced within the European project BRIDGE-MAP, (present study and ) in addition to 31,705 EST sequences generated by the Marine Genomics Europe network and sequences of selected genes such as genes with a putative role in sex determination downloaded from the NCBI database. BLAST searches were performed using a significance threshold of an alignment length of >50 bp and an e-value of <10-4 (Additional file 3).
This work was supported by the European Commission's 5th Framework Programme (Contract No. QLRT-CT-2000-01797, BRIDGE-MAP). The authors would like to acknowledge Marine Genomics Europe Network for providing cDNA sequences and Dimitrios Chatziplis for discussions concerning the comparison of the genetic linkage map to the radiation hybrid map. Furthermore, they thank Silke Rudolph-Geiger for mapping part of the loci and Ramona Doll for helping loading agarose gels.
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