Analysis of qPCR reference genes stability determination methods and a practical approach for efficiency calculation on a turbot (Scphthalmus maximus) gonad dataset

Gene 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 industryVersión del edito

A single genomic region involving a putative chromosome rearrangement in flat oyster (Ostrea edulis) is associated with differential host resilience to the parasite Bonamia ostreae

European flat oyster (Ostrea edulis) is an ecologically and economically important marine bivalve, that has been severely affected by the intracellular parasite Bonamia ostreae. In this study, a flat oyster SNP array (~14,000 SNPs) was used to validate previously reported outlier loci for divergent selection associated with B. ostreae exposure in the Northeast Atlantic Area. A total of 134 wild and hatchery individuals from the North Sea, collected in naïve (NV) and long-term affected (LTA) areas, were analysed. Genetic diversity and differentiation were related to the sampling origin (wild vs. hatchery) when using neutral markers, and to bonamiosis status (NV vs. LTA) when using outlier loci for divergent selection. Two genetic clusters appeared intermingled in all sampling locations when using outlier loci, and their frequency was associated with their bonamiosis status. When both clusters were compared, outlier data sets showed high genetic divergence (FST > 0.25) unlike neutral loci (FST not ≠ 0). Moreover, the cluster associated with LTA samples showed much higher genetic diversity and significant heterozygote excess with outlier loci, but not with neutral data. Most outliers mapped on chromosome 8 (OE-C8) of the flat oyster genome, supporting a main genomic region underlying resilience to bonamiosis. Furthermore, differentially expressed genes previously reported between NV and LTA strains showed higher mapping density on OE-C8. A range of relevant immune functions were specifically enriched among genes annotated on OE-C8, providing hypotheses for resilience mechanisms to an intracellular parasite. The results suggest that marker-assisted selection could be applied to breed resilient strains of O. edulis to bonamiosis, if lower parasite load and/or higher viability of the LTA genetic cluster following B. ostreae infection is demonstratedBiotechnology and Biological Sciences Research Council. Grant Numbers: BBS/E/D/20002172, BB/S004181/1. Ministry of Agriculture, Nature and Food safety. Grant Numbers: BO43-18, KB33-004S

A single genomic region involving a putative chromosome rearrangement in flat oyster (Ostrea edulis) is associated with differential host resilience to the parasite Bonamia ostreae

European flat oyster (Ostrea edulis) is an ecologically and economically important marine bivalve, that has been severely affected by the intracellular parasite Bonamia ostreae. In this study, a flat oyster SNP array (~14,000 SNPs) was used to validate previously reported outlier loci for divergent selection associated with B. ostreae exposure in the Northeast Atlantic Area. A total of 134 wild and hatchery individuals from the North Sea, collected in naïve (NV) and long-term affected (LTA) areas, were analysed. Genetic diversity and differentiation were related to the sampling origin (wild vs. hatchery) when using neutral markers, and to bonamiosis status (NV vs. LTA) when using outlier loci for divergent selection. Two genetic clusters appeared intermingled in all sampling locations when using outlier loci, and their frequency was associated with their bonamiosis status. When both clusters were compared, outlier data sets showed high genetic divergence (FST > 0.25) unlike neutral loci (FST not ≠ 0). Moreover, the cluster associated with LTA samples showed much higher genetic diversity and significant heterozygote excess with outlier loci, but not with neutral data. Most outliers mapped on chromosome 8 (OE-C8) of the flat oyster genome, supporting a main genomic region underlying resilience to bonamiosis. Furthermore, differentially expressed genes previously reported between NV and LTA strains showed higher mapping density on OE-C8. A range of relevant immune functions were specifically enriched among genes annotated on OE-C8, providing hypotheses for resilience mechanisms to an intracellular parasite. The results suggest that marker-assisted selection could be applied to breed resilient strains of O. edulis to bonamiosis, if lower parasite load and/or higher viability of the LTA genetic cluster following B. ostreae infection is demonstratedBiotechnology and Biological Sciences Research Council. Grant Numbers: BBS/E/D/20002172, BB/S004181/1. Ministry of Agriculture, Nature and Food safety. Grant Numbers: BO43-18, KB33-004S

Signatures of selection for bonamiosis resistance in European flat oyster (Ostrea edulis): New genomic tools for breeding programs and management of natural resources

The European flat oyster (Ostrea edulis) is a highly appreciated mollusk with an important aquaculture production throughout the 20th century, in addition to playing an important role on coastal ecosystems. Overexploitation of natural beds, habitat degradation, introduction of non‐native species, and epidemic outbreaks have severely affected this important resource, particularly, the protozoan parasite Bonamia ostreae, which is the main concern affecting its production and conservation. In order to identify genomic regions and markers potentially associated with bonamiosis resistance, six oyster beds distributed throughout the European Atlantic coast were sampled. Three of them have been exposed to this parasite since the early 1980s and showed some degree of innate resistance (long‐term affected group, LTA), while the other three were free of B. ostreae at least until sampling date (naïve group, NV). A total of 14,065 SNPs were analyzed, including 37 markers from candidate genes and 14,028 from a medium‐density SNP array. Gene diversity was similar between LTA and NV groups suggesting no genetic erosion due to long‐term exposure to the parasite, and three population clusters were detected using the whole dataset. Tests for divergent selection between NV and LTA groups detected the presence of a very consistent set of 22 markers, located within a putative single genomic region, which suggests the presence of a major quantitative trait locus associated with B. ostreae resistance. Moreover, 324 outlier loci associated with factors other than bonamiosis were identified allowing fully discrimination of all the oyster beds. A practical tool which included the 84 highest discriminative markers for tracing O. edulis populations was developed and tested with empirical data. Results reported herein could assist the production of stocks with improved resistance to bonamiosis and facilitate the management of oyster beds for recovery production and ecosystem services provided by this species.This work was funded by the OYSTERECOVER project (FP7‐SME‐2008‐2‐243583) from the European Community's Seventh Framework Programme, the European Regional Development's funds (FEDER), and Xunta de Galicia local government (GRC2014/010, R2014/046). The development and provision of the medium‐density SNP array for oysters was supported by Biotechnology and Biological Sciences Research Council (BBSRC), and National Environment Research Council (NERC) grants (BB/M026140/1, NE/P010695/1), in addition to BBSRC Institute Strategic Program Grants (BBS/E/D/20002172 and BBS/E/D/30002275)S

Advancing fish breeding in aquaculture through genome functional annotation

Genomics is increasingly applied in breeding programmes for farmed fish and shellfish species around the world. However, current applications do not include information on genome functional activity, which can enhance opportunities to predict relationships between genotypes and phenotypes and hence increase the accuracy of selection. Here, we review prospects for improving aquaculture breeding practises through the uptake of functional genomics data in light of the EU Horizon 2020 project AQUA-FAANG: ‘Advancing European Aquaculture by Genome Functional Annotation’. This consortium targeted the six major farmed fish species in European aquaculture, producing thousands of functional genomic datasets from samples representing embryos to mature adults of both sexes, and following immunological stimulation. This data was used to catalogue functional activity across the genome of each species, revealing transcribed regions, distinct chromatin states and regulatory elements impacting gene expression. These functional annotations were shared as open data through the Ensembl genome browser using the latest reference genomes for each species. AQUA-FAANG data offers novel opportunities to identify and prioritize causative genetic variants responsible for diverse traits including disease resistance, which can be exploited to enhance selective breeding. Such knowledge and associated resources have the potential to improve sustainability and boost production in aquaculture by accelerating genetic gain for health and robustness to infection, whilst reducing the requirement for animal testing. We further outline directions to advance and leverage genome functional annotation beyond the AQUA-FAANG project. Given the diversity of aquaculture sectors and businesses, the incorporation of functional genomic information into breeding decisions will depend on technological readiness level and scale of operation, with cost-benefit analysis necessary to determine the most profitable approach for each species and production system

Ecological Factors and Diversification among Neotropical Characiforms

Morphological and DNA sequence data has been used to propose hypotheses of relationships within the Characiformes with minimal comparative discussion of causes underpinning the major intraordinal diversification patterns. We explore potential primary morphological factors controlling the early diversification process in some Neotropical characiforms as the first step to identifying factors contributing to the pronounced intraordinal morphological and species diversity. A phylogenetic reconstruction based on 16S rDNA (mitochondrial) and 18S rDNA (nuclear) genes provided the framework for the identification of the main morphological differences among the Acestrorhynchidae, Anostomidae, Characidae, Ctenoluciidae, Curimatidae, Cynodontidae, Gasteropelecidae, Prochilodontidae and Serrasalmidae. Results indicate an initial split into two major groupings: (i) species with long dorsal-fin bases relative to the size of other fins (Curimatidae, Prochilodontidae, Anostomidae, Serrasalmidae) which primarily inhabit lakes, swamps, and rivers (lineage I); and (ii) species with short dorsal-fin bases (Acestrorhynchidae, Gasteropelecidae, Characidae) which primarily inhabit creeks and streams (lineage II). The second diversification stage in lineage I involved substantial morphological diversification associated with trophic niche differences among the monophyletic families which range from detritivores to large item predators. Nonmonophyly of the Characidae complicated within lineage II analyzes but yielded groupings based on differences in pectoral and anal fin sizes correlated with life style differencesThis study was supported by the AMAPEZ project (CGL2006-02155 BOS) of the MEC (Ministry of Education and Science) under FEDER funds from the XUNTA de Galicia and by contributions from the Universidad de Vigo, the Universidad de Antioquia, and the Universidad Nacional de Colombia. A. Manjarrés-Hernández gratefully acknowledges a grant from COLCIENCIASS

Hematología de rodaballos (Scophthalmus maximus L.) triploides.

En los rodaballos (Scophthalmus maximus L.) triploides, tal como normalmente sucede en los peces a los que se ha inducido triploidía, el aumento de DNA provocó un incremento del volumen celular de sus eritrocitos. El objetivo de este trabajo fue determinar las características hematológicas de estos rodaballos triploides obtenidos por la aplicación de un choque frío

Resultados preliminares de la influencia de la temperatura de cultivo sobre la proporción de sexos en el rodaballo (Scophthalmus maximus L.)

Las larvas de tres familias de rodaballos se cultivaron a temperaturas de 15'C, 18'C y 22'C desde el día dos hasta día 90 de vida y a temperatura ambiente hasta el dia 210. En las familias 1 y 2 las proporciones sexuales, determinadas por el fenotipo, oscilaron en los tres grupos entre el 40%- 60%,y no se observaron diferencias siqníñcaffvas entre los diferentes grupos de temperatura (p>0.05). En la familia 3, el porcentaje de hembras fue mayor que el de machos en los tres grupos de temperatura, y además se observó diferencia significativa (p<0.05) entre el grupo de peces cultivados a temperatura fría con respecto a los cultivados en agua ambiente. En tas familias 1 y 2 el sexo genético coincidió en gran medida con~ sexo fenolípico, siendo la discrepancia menor del 10%. Sin embargo, en la familia 3 se observó que el 36,5% de los machos genéticos eran hembras a 15'C, el 29% a 18ºC y el 18% a 23'C. Los resultados sugieren la interacción temperatura-familia en la determinación sexual del rodaballo, que debe ser confirmada en un mayor número de familias.Larvae from three families of turbot were cultured at 15'C, 18'C and 22'C from 2 to 90 days old, and then at ambient temperature until210 daysold. Regarding families 1 and 2, \he sexual proportions determined by \he phenotype varied between 40%·60%, regardless of \he cunure temperature (p>O.05). Gontrary, in family 3, \he percentage of females was higher than for males in all three cunure temperatures. Furthermore, \here were differences (¡¡<O.05) between families cultured at 15'C and 18'C. For families 1 and 2, sex determined by genotype was similarto \hat detennined by the phenotype, wi\h differences <1 0%. Gontrary, for family 3,\he \he percentage of males determined by \he genotype \hat were phenotipycatt¡ females was 36, 29 and 18% for animals cunured at 15'C, 18'C and 22'C, respectively. Results suggest an interaction temperature-family in \he turbot sex determination which should be checked on a higher numberoffamilies

Morphological variation in a secondary contact between divergent lineages of brown trout (Salmo trutta) from the Iberian Peninsula

The aim of this study was to analyze the morphological variation of brown trout (Salmo trutta) in the Duero basin, an Atlantic river basin in the Iberian Peninsula, where a spatial segregation of two divergent lineages was previously reported, based on isozyme, microsatellite and mtDNA data. In these studies, two divergent pure regions (Pisuerga and Lower-course) and several hybrid populations between them were identified. Morphological variation was evaluated in 11 populations representative of the genetic differentiation previously observed in the Duero basin, using multivariate analysis on 12 morphometric and 4 meristic traits. A large differentiation between populations was observed (interpopulation component of variance: 41.8%), similar to that previously detected with allozymes and microsatellites. Morphometric differentiation was also reflected by the high classification success of pure and hybrid individuals to their respective populations, using multivariate discriminant functions (94.1% and 79.0%, respectively). All multivariate and clustering analyses performed demonstrated a strong differentiation between the pure regions. The hybrid populations, though showing large differentiation among them, evidenced an intermediate position between the pure samples. Head and body shape traits were the most discriminant among the morphometric characters, while pectoral rays and gillrakers were the most discriminant among the meristic traits. These results confirmed the high divergence of the brown trout from the Duero basin and suggest some traits on which selection could be acting to explain the spatial segregation observed

Revista de Vertebrados de la Estación Biológica de Doñana

Contribución al estudio de la bermejuela Rutilus arcasi, Steindachner, 1866 de la cuenca del Júcar (Osteichthyes: Cyprinidae)II. Edad y crecimientoSobre la taxonomía de Barbus comiza Steindachner, 1865 (Ostariophysi: Cyprinidae)Fenología de una comunidad de anfibios asociada a cursos fluviales temporales.Nueva especie para la ciencia de Anolis (Lacertilia: Iguanidae) de Cuba pertenecient eal complejo argillaceusSegregación ecológica en una comunidad de ofidios.El Aguila Imperial (Aquila adalberti): dispersión de los jóvenes, estructura de edades y mortalidaSobre diferencias individuales en la alimentación de Tyto albaInfluencia de las condiciones ambientales sobre la organización de la comunidad de aves invernantes en un bosque subalpino mediterráneoVariaciones en la agregación y distribución de la cabra montés (Capra pyrenaica Schinz,1838) detectadas con un muestreo de excrementosAlimentación del conejo (Oryctolagus cuniculus L. 1758) en Doñana. SO, EspañaSobre la distribución de Barbus meridionales Risso, 1826 (Ostariophysi: Cyprinidae) en la Península IbéricaSobre la distribución de Barbus meridionales Risso, 1826 (Ostariophysi: Cyprinidae) en la Península IbéricaNueva cita de Barbus microcephalus Almaça (Pisces, Cyprinidae) en España.Revisión taxonómica y distribución de Cobitis maroccana Pellegrin, 1929 (Osteichthyes, Cobitidae)Datos sobre una población de Lacerta viviparaSobre la presencia de Emys orbicularis en la provincia de León.Algunas observaciones sobre la captura de quirópteros por Falco subbuteo y Falco tinunculusNyctalus leisleri (Kuhk, 1818) (Mammalia: Chiroptera). Una nueva especie para las islas CanariaNuevos datos acerca de la distribución del topillo campesino Microtus arvalis, PALLAS 1778, en la Península IbéricaPeer reviewe