A consensus microsatellite-based linkage map of the turbot (Scophthalmus maximus) was constructed from two unrelated families. The mapping panel was derived from a gynogenetic family of 96 haploid embryos and a biparental diploid family of 85 full-sib progeny with known linkage phase. A total of 242 microsatellites were mapped in 26 linkage groups, six markers remaining unlinked. The consensus map length was 1343.2 cM, with an average distance between markers of 6.5 6 0.5 cM. Similar length of female and male maps was evidenced. However, the mean recombination at common intervals throughout the genome revealed significant differences between sexes, $1.6 times higher in the female than in the male. The comparison of turbot microsatellite flanking sequences against the Tetraodon nigroviridis genome revealed 55 significant matches, with a mean length of 102 bp and high sequence similarity (81-100%). The comparative mapping revealed significant syntenic regions among fish species. This study represents the first linkage map in the turbot, one of the most important flatfish in European aquaculture. This map will be suitable for QTL identification of productive traits in this species and for further evolutionary studies in fish and vertebrate species.

BackgroundGene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industry.ResultsWe analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency.ConclusionOur results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency determination, respectively. We also recommend the use of UBQ and RPS4 for normalization of gonad development samples in turbot.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-648) contains supplementary material, which is available to authorized users.

Dead parts of salt-marsh plants form a considerable fraction of their annual average standing crop. A microbial assemblage living on and in the standing-dead leaves and stems of Spartina alterniflora and Juncus roemerianus responds to saltwater, freshwater or water-vapor wetting by immediately beginning to release CO. Water-saturated, standing-dead leaves and culms of S. alterniflora release CO at steady rates of as much as about 200 and 140 μg CO-C·g dry·h, respectively, at temperatures of 25-30°C, after an initial burst of higher rates. These CO-release rates are within the range of maximal rates reported for decaying terrestrial litter, and are as high as most rates reported for S. alterniflora decaying under continuously wetted or submerged conditions.

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Abstract. The Galician shelf off NW Spain (43N ø 9W ø) exhibits mesoscale spatial and temporal changes in biological productivity associated with upwelling. Spatial heterogeneity results from local geomorphic and land-sea interactions superimposed on the large scale atmospheric processes that produce upwelling. Wind-induced upwelling events, commonly of short (i.e., week) duration, are more common in the summer than in the winter. A series of cruises, including some time series sampling, and satellite imagery analysis showed that surface upwelling was more common and persistent on the northern coast compared with the western coast off the coastal embayments, the Rias Bajas. Nearshore off the rias, coastal runoff, which is greater in the rainy winter/spring versus the dry summer, affected upwelling. In early summer, upwelling less often reaches the surface because of increased water column stratification associated with lower surface salinities and thus upwelling is not detected by satellite imagery. Conversely, in late summer, upwelling more often reaches the surface because coastal runoff is reduced during the dry summer months and the water column tends to be less stratified. Plankton biomass and rate processes along the Galician shelf reflected both ambient hydrographic conditions as well as prior history of upwelling or downwelling. Phytoplankton and bacterioplankton were in greatest abundance during upwelling conditions (June through August); in contrast, both zooplankton and fish larvae exhibited highest abundances in March, when there were upwelling conditions prior to our cruise. Spatial differences in the duration and frequency of upwelling events, in combination with advection of water masses, are critical to the patterns of water column productivity and sardine fisheries production off the Galician coast. More persistent upwelling at this NW corner of the Iberian peninsula supports large sardine fisheries because zooplankton and larval fish populations have time to respond to the higher primary production. Farther down the western Galician coast, the episodic upwelling and resultant intermittent primary production does not support a stable food supply needed to support fisheries. Times series sampling revealed mean response times of bacteria, phytoplankton, and zooplankton to be on the order of a day, days, and weeks, respectively. Sardines showed no spawning response in the relatively short time series sampling. The observed distributional patterns of fish eggs and larvae showed some offshore transport of fish larvae that were spawned inshore during upwelling periods and aggregation of larvae in a convergence zone northwest of Cabo Villano.

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While flatfish in the wild exhibit a pronounced countershading of the dorso-ventral pigment pattern, malpigmentation is commonly observed in reared animals. In fish, the dorso-ventral pigment polarity is achieved because a melanization inhibition factor (MIF) inhibits melanoblast differentiation and encourages iridophore proliferation in the ventrum. A previous work of our group suggested that asip1 is the uncharacterized MIF concerned. In order to further support this hypothesis, we have characterized asip1 mRNAs in both turbot and sole and used deduced peptide alignments to analyze the evolutionary history of the agouti-family of peptides. The putative asip precursors have the characteristics of a secreted protein, displaying a putative hydrophobic signal. Processing of the potential signal peptide produces mature proteins that include an N-terminal region, a basic central domain with a high proportion of lysine residues as well as a proline-rich region that immediately precedes the C-terminal poly-cysteine domain. The expression of asip1 mRNA in the ventral area was significantly higher than in the dorsal region. Similarly, the expression of asip1 within the unpigmented patches in the dorsal skin of pseudoalbino fish was higher than in the pigmented dorsal regions but similar to those levels observed in the ventral skin. In addition, the injection/electroporation of asip1 capped mRNA in both species induced long term dorsal skin paling, suggesting the inhibition of the melanogenic pathways. The data suggest that fish asip1 is involved in the dorsal-ventral pigment patterning in adult fish, where it induces the regulatory asymmetry involved in precursor differentiation into mature chromatophore. Adult dorsal pseudoalbinism seems to be the consequence of the expression of normal developmental pathways in an inaccurate position that results in unbalanced asip1 production levels. This, in turn, generates a ventral-like differentiation environment in dorsal regions.

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