Costas S. Tsigenopoulos

A Genetic Linkage Map of the Hermaphrodite Teleost Fish Sparus aurata L.

The gilthead sea bream (Sparus aurata L.) is a marine fish of great importance for fisheries and aquaculture. It has also a peculiar sex-determination system, being a protandrous hermaphrodite. Here we report the construction of a first-generation genetic linkage map for S. aurata, based on 204 microsatellite markers. Twenty-six linkage groups (LG) were found. The total map length was 1241.9 cM. The ratio between sex-specific map lengths was 1:1.2 (male:female). Comparison with a preliminary radiation hybrid (RH) map reveals a good concordance, as all markers located in a single LG are located in a single RH group, except for Ad-25 and CId-31. Comparison with the Tetraodon nigroviridis genome revealed a considerable number of evolutionary conserved regions (ECRs) between the two species. The mean size of ECRs was 182 bp (sequence identity 60–90%). Forty-one ECRs have a known chromosomal location in the pufferfish genome. Despite the limited number of anchoring points, significant syntenic relationships were found. The linkage map presented here provides a robust comparative framework for QTL analysis in S. aurata and is a step toward the identification of genetic loci involved both in the determination of economically important traits and in the individual timing of sex reversal.

Quantitative Trait Loci Involved in Sex Determination and Body Growth in the Gilthead Sea Bream (Sparus aurata L.) through Targeted Genome Scan

Among vertebrates, teleost fish exhibit a considerably wide range of sex determination patterns that may be influenced by extrinsic parameters. However even for model fish species like the zebrafish Danio rerio the precise mechanisms involved in primary sex determination have not been studied extensively. The zebrafish, a gonochoristic species, is lacking discernible sex chromosomes and the sex of juvenile fish is difficult to determine. Sequential protandrous hermaphrodite species provide distinct determination of the gender and allow studying the sex determination process by looking at the mechanism of sex reversal. This is the first attempt to understand the genetic basis of phenotypic variation for sex determination and body weight in a sequential protandrous hermaphrodite species, the gilthead sea bream (Sparus aurata). This work demonstrates a fast and efficient strategy for Quantitative Trait Loci (QTL) detection in the gilthead sea bream, a non-model but target hermaphrodite fish species. Therefore a comparative mapping approach was performed to query syntenies against two other Perciformes, the European sea bass (Dicentrarchus labrax), a gonochoristic species and the Asian sea bass (Lates calcarifer) a protandrous hermaphrodite. In this manner two significant QTLs, one QTL affecting both body weight and sex and one QTL affecting sex, were detected on the same linkage group. The co-segregation of the two QTLs provides a genomic base to the observed genetic correlation between these two traits in sea bream as well as in other teleosts. The identification of QTLs linked to sex reversal and growth, will contribute significantly to a better understanding of the complex nature of sex determination in S. aurata where most individuals reverse to the female sex at the age of two years through development and maturation of the ovarian portion of the gonad and regression of the testicular area. [Genomic sequences reported in this manuscript have been submitted to GenBank under accession numbers HQ021443–HQ021749.]

A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L.

European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) sustains a regional fishery and is commonly farmed in the Mediterranean basin, but has not undergone much long-term genetic improvement. An updated genetic linkage map of the European sea bass was constructed using 190 microsatellites, 176 amplified fragment length polymorphisms and two single nucleotide polymorphisms. From the 45 new microsatellite markers (including 31 type I markers) reported in this study, 28 were mapped. A total of 368 markers were assembled into 35 linkage groups. Among these markers, 28 represented type I (coding) markers, including those located within the peptide Y, SOX10, PXN1, ERA and TCRB genes (linkage groups 1, 7, 16, 17 and 27 respectively). The sex-averaged map spanned 1373.1 centimorgans (cM) of the genome. The female map measured 1380.0 cM, whereas the male map measured 1046.9 cM, leading to a female-to-male (F:M) recombination rate ratio of 1.32:1. The intermarker spacing of the second-generation linkage map of the European sea bass was 3.67 cM, which is smaller than that of the first-generation linkage map (5.03 cM). Comparative mapping of microsatellite flanking regions was performed with five model teleosts and this revealed a high percentage (33.6%) of evolutionarily conserved regions with the three-spined stickleback.

Phylogenetic relationships of hexaploid large-sized barbs (genus Labeobarbus, Cyprinidae) based on mtDNA data.

The phylogenetic relationships among species of the Labeobarbus genus (Teleostei, Cyprinidae) which comprises large body-sized hexaploid taxa were inferred using complete cytochrome b mitochondrial gene sequences. Molecular data suggest two main evolutionary groups which roughly correspond to a Northern (Middle East and Northwest Africa) and a sub-Saharan lineage. The splitting of the African hexaploids from their Asian ancestors and their subsequent diversification on the African continent occurred in the Late Miocene, a period in which other cyprinins also invaded Africa and radiated in the Mediterranean region. Finally, systematic implications of these results to the taxonomic validity of genera or subgenera such as Varicorhinus, Kosswigobarbus, Carasobarbus and Capoeta are further discussed.

The sex-specific transcriptome of the hermaphrodite sparid sharpsnout seabream (Diplodus puntazzo)

BackgroundTeleosts are characterized by a remarkable breadth of sexual mechanisms including various forms of hermaphroditism. Sparidae is a fish family exhibiting gonochorism or hermaphroditism even in closely related species. The sparid Diplodus puntazzo (sharpsnout seabream), exhibits rudimentary hermaphroditism characterized by intersexual immature gonads but single-sex mature ones. Apart from the intriguing reproductive biology, it is economically important with a continuously growing aquaculture in the Mediterranean Sea, but limited available genetic resources. Our aim was to characterize the expressed transcriptome of gonads and brains through RNA-Sequencing and explore the properties of genes that exhibit sex-biased expression profiles.ResultsThrough RNA-Sequencing we obtained an assembled transcriptome of 82,331 loci. The expression analysis uncovered remarkable differences between male and female gonads, while male and female brains were almost identical. Focused search for known targets of sex determination and differentiation in vertebrates built the sex-specific expression profile of sharpsnout seabream. Finally, a thorough genetic marker discovery pipeline led to the retrieval of 85,189 SNPs and 29,076 microsatellites enriching the available genetic markers for this species.ConclusionsWe obtained a nearly complete source of transcriptomic sequence as well as marker information for sharpsnout seabream, laying the ground for understanding the complex process of sex differentiation of this economically valuable species. The genes involved include known candidates from other vertebrate species, suggesting a conservation of the toolkit between gonochorists and hermaphrodites.

Mitochondrial DNA reveals the genealogical history of the snake-eyed lizards (Ophisops elegans and O. occidentalis) (Sauria: Lacertidae)

The snake-eyed lizards of the genus Ophisops (Lacertidae) have been through a series of taxonomical revisions, but still their phylogenetic relationships remain uncertain. In the present study we estimate the phylogeographic structure of O. elegans across its distributional range and we evaluate the relationships between O. elegans and the sympatric, in North Africa, species O. occidentalis, using partial mtDNA sequences (16S rRNA, COI, and cyt b). All phylogenetic analyses produced topologically identical trees where extant populations of O. elegans and O. occidentalis were found polyphyletic. Taking into account all the potential causes of polyphyly (introgressive hybridization, incomplete lineage sorting, and imperfect taxonomy) we suggest the inaccurate taxonomy as the most likely explanation for the observed pattern. Our results stress the need for re-evaluation of the current taxonomical status of these species and their subspecies. Furthermore, our biogeographic analyses and the estimated time of divergences suggest a late Miocene diversification within these species, where the present distribution of O. elegans and O. occidentalis was the result of several dispersal and vicariant events, which are associated with climatic oscillations (the late Miocene aridification of Asia and northern Africa) and paleogeographic barriers of late Miocene and Pliocene period.

Gilthead sea bream (Sparus auratus) and European sea bass (Dicentrarchus labrax) expressed sequence tags: Characterization, tissue-specific expression and gene markers.

The gilthead sea bream, Sparus auratus, and the European sea bass, Dicentrarchus labrax, are two of the most important marine species cultivated in Southern Europe. This study aimed at increasing genomic resources for the two species and produced and annotated two sets of 30,000 expressed sequence tags (EST) each from 14 normalized tissue-specific cDNA libraries from sea bream and sea bass. Clustering and assembly of the ESTs formed 5268 contigs and 12,928 singletons for sea bream and 4573 contigs and 13,143 singletons for sea bass, representing 18,196 and 17,716 putative unigenes, respectively. Assuming a similar number of genes in sea bass, sea bream and in the model fish Gasterosteus aculeatus genomes, it was estimated that approximately two thirds of the sea bream and the sea bass transcriptomes were covered by the unigene collections. BLAST sequence similarity searches (using a cut off of e-value <10(-5)) against fully the curated SwissProt (and TrEMBL) databases produced matches of 28%(37%) and 43%(53%) of the sea bream and sea bass unigene datasets respectively, allowing some putative designation of function. A comparative approach is described using human Ensembl peptide ID homologs for functional annotation, which increased the number of unigenes with GO terms assigned and resulted in more GO terms assigned per unigene. This allowed the identification of tissue-specific genes using enrichment analysis for GO pathways and protein domains. The comparative annotation approach represents a good strategy for transferring more relevant biological information from highly studied species to genomic resource poorer species. It was possible to confirm by interspecies mRNA-to-genomic alignments 25 and 21 alternative splice events in sea bream and sea bass genes, respectively. Even using normalized cDNA from relatively few pooled individuals it was possible to identify 1145 SNPs and 1748 microsatellites loci for genetic marker development. The EST data are being applied to a range of projects, including the development microarrays, genetic and radiation hybrid maps and QTL genome scans. This highlights the important role of ESTs for generating genetic and genomic resources of aquaculture species.

Quantitative trait loci for body growth and sex determination in the hermaphrodite teleost fish Sparus aurata L.

Gilthead sea bream (Sparus aurata L.) is an important marine fish in Mediterranean aquaculture. Sex determination by age and/or body weight is a critical life-history trait, the genetic basis for which is largely unknown in this sequential hermaphrodite species. Herein, we performed a partial genome scan to map quantitative trait loci (QTL) affecting body weight and sex using 74 informative microsatellite markers from 10 paternal half-sib families to construct nine linkage groups (LG). In total, four growth-related QTL (two chromosome-wide and two genome-wide) and six QTL related to sex determination (three pairs in three different LGs) were detected (two chromosome-wide and one genome-wide). The proportion of phenotypic variation explained by the body-weight QTL ranged from 9.3% to 17.2%, showing their potential for use in marker-assisted selection. The results obtained offer solid ground to investigate the structure and function of the genomic regions involved in the mechanisms of sex reversal.

Gene flow at major transitional areas in sea bass (Dicentrarchus labrax) and the possible emergence of a hybrid swarm.

The population genetic structure of sea bass (Dicentrarchus labrax) along a transect from the Atlantic Ocean (AO) to the Eastern Mediterranean (EM) Sea differs from that of most other marine taxa in this area. Three populations (AO, Western Mediterranean [WM], EM) are recognized today, which were originally two allopatric populations. How two ancestral genetic units have evolved into three distinct units has not been addressed yet. Therefore, to investigate mechanisms that lead to the emergence of the central WM population, its current status, and its connectivity with the two parental populations, we applied 20 nuclear loci that were either gene associated or gene independent. Results confirmed the existence of three distinct gene pools, with higher differentiation at two transitional areas, the Almeria-Oran Front (AOF) and of the Siculo-Tunisian Strait (STS), than within any population. Significant linkage disequilibrium and heterozygote excess indicated that the STS is probably another tension zone, as already described for the AOF. Neutrality tests fail to reveal marker loci that could be driven by selection within or among metapopulations, except for locus DLA0068. Collectively, results support that the central WM population arose by trapping two tensions zones at distinct geographic locations of limited connectivity. Population assignment further revealed that WM individuals were more introgressed than individuals from the other two metapopulations. This suggests that this population might result from hybrid swarming, and was or is still seeded by genes received through the filter of each tension zone.

NOBLAST and JAMBLAST

UNLABELLED NOBLAST (New Options for BLAST) is an open source program that provides a new user-friendly tabular output format for various NCBI BLAST programs (Blastn, Blastp, Blastx, Tblastn, Tblastx, Mega BLAST and Psi BLAST) without any use of a parser and provides E-value correction in case of use of segmented BLAST database. JAMBLAST using the NOBLAST output allows the user to manage, view and filter the BLAST hits using a number of selection criteria. AVAILABILITY A distribution package of NOBLAST and JAMBLAST including detailed installation procedure is freely available from http://sourceforge.net/projects/JAMBLAST/ and http://sourceforge.net/projects/NOBLAST. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.