The use of genetic markers in the study of marine natural populations

Crassostrea angulata (Lamarck, 1819), known as the Portuguese oyster, is a species naturally located in the southwest Iberian Peninsula, mainly at river mouths and the heads of estuaries with suitable hard substrates. Genetic data on C. angulata are scarce, and Spanish natural populations (the main beds of the species) have not been studied previously. Electrophoresis is a good detector of variability, and isozyme polymorphisms have been widely used in genetic studies. The present paper reports on the electrophoretic examination of seven allozyme loci at 14 sites located between the mouth of the Guadiana River and the Straits of Gibraltar, which found a high level of genetic variability in terms of average number of alleles (min. of 2.71 and max. of 3.71), polymorphism (P₉₅ ≥ 71.43 %) and mean heterozygosities (H = 0.264). All populations showed a deficit of heterozygotes as well as the genes Lap, Mdh-1, Mdh-2, Me-2, Pgi and Xdh across populations. However, Pgm showed heterozygous excess. Null alleles appeared in Lap and Mdh loci, which could be responsible for this disequilibrium, at least in Mdh, because the increase in frequencies of null alleles was also related to the increase in heterozygote deficit. Values of genetic distance between populations have been used to cluster the populations, and the F-statistics showed a mean FST value across loci of 0.023 and Mdh-1 surveyed the highest value (0.046). At the same time, heterogeneity in allelic frequencies found in all genes indicated genetic differentiation between populations, in spite of the high dispersal of the species, probably due to local selective conditions.Crassostrea angulata (Lamarck, 1819), conocida como ostra portuguesa, es una especie que se encuentra localizada en el suroeste de la península Ibérica, principalmente en la desembocadura de los ríos y en la cabecera de estuarios con sustrato duro disponible. Los datos sobre la genética de C. angulata son escasos y las poblaciones naturales de España (la principal fuente de esta especie) no habían sido estudiadas. La electroforesis es un buen detector de la variabilidad, y los polimorfismos isoenzimáticos han sido ampliamente usados en estudios genéticos. Este trabajo está basado en el análisis electroforético de siete loci y catorce puntos de muestreo entre la desembocadura del río Guadiana y el estrecho de Gibraltar y se ha encontrado un alto nivel de variabilidad genética en términos de número medio de alelos (mínimo de 2,71 y máximo de 3,71), polimorfismos (P₉₅ ≥ 71,43 %) y heterocigosis media (H = 0,264). Todas las poblaciones mostraron un déficit de heterocigotos en los genes Lap, Mdh-1, Mdh-2, Me-2, Pgi y Xdh. En cambio, el sistema Pgm mostró un exceso de heterocigotos. Aparecieron alelos nulos en los loci Lap y Mdh que podrían ser los responsables del desequilibrio, al menos, en Mdh debido a que el incremento en la frecuencia de los alelos nulos estaba relacionado con un incremento en el déficit de heterocigotos. Las poblaciones se han agrupado según los valores de distancia genética obtenidos y también se han calculado los estadísticos F, obteniéndose un valor medio de FST en los loci de 0,023; Mdh mostró el valor mayor (0,046). Simultáneamente, la heterogeneidad en las frecuencias alélicas encontradas en todos los genes indicaban diferenciación genética entre las poblaciones, a pesar de la gran dispersión de la especie, probablemente debido a condiciones selectivas locales.Instituto Español de Oceanografí

Karyotype analysis and variability of Ag-NOR in Crassostrea angulata (Lamarck, 1819)

Genetic characterization of oyster species is the basis of future breeding programmes by chromosome manipulation. We determined the karyotype of Crassostrea angulata (Lamarck, 1819), and found some differences with regard to previously published reports. The nucleolus organizer regions (NOR), visualized using the silver stain method, were located on chromosome pair number 10, although NORs were occasionally detected on some other chromosomes, which had not been described before for this species. We also found a very high inter and intra-population variability in the number of chromosomes showing Ag-NOR activity.El estudio de la caracterización genética en las especies acuícolas de ostras es el preámbulo imprescindible de los futuros programas de mejora por manipulación cromosómica. Se ha realizado el cariotipo de Crassostrea angulata (Lamarck, 1819) y se han encontrado diferencias con respecto a otros trabajos publicados. Mediante tinción con plata, se analizó la localización y la variabilidad de las regiones organizadoras del nucleolo (Ag-NOR). Estas regiones se han visualizado en el par cromosómico número 10, aunque, esporádicamente, también se han observado NOR en otros cromosomas, lo que se describe aquí por primera vez para esta especie. Por otra parte, se ha observado una gran variabilidad, tanto entre individuos como dentro de ellos, en cuanto al número de cromosomas que presentan actividad Ag-NOR.Instituto Español de Oceanografí

Cytogenetic and isozymatic characterization of the sole Solea senegalensis Kaup, 1858

Se ha estudiado el lenguado Solea senegalensis Kaup, 1858 realizando el análisis de 12 loci alozímicos en 92 ejemplares cultivados en piscifactorías. De estos individuos, 30 se obtuvieron de la empresa Cultivos Piscícolas Marinos (Cupimar) y los restantes fueron proporcionados por la planta de cultivos marinos de la Facultad de Ciencias del Mar de la Universidad de Cádiz (Casem). Los loci estudiados fueron MDH-1, MDH-2, PGI-1, PGI-2, PGM, LDH, EM-1, EM-2, AAT-1, AAT-2, GPD, G-6PDH, y todos resultaron ser monomórficos, excepto el locus LDH en ambas poblaciones con el criterio del 95 % (P95) y el PGI-2 en la población del Casem (P99). En todos estos sistemas se ha encontrado variabilidad isoenzimática baja en cuanto al polimorfismo, la heterocigosis y el número medio de alelos. Las poblaciones analizadas se encuentran en equilibrio Hardy-Weingerg y la diferenciación genética de las mismas (FST) es muy baja. Además, se describe por primera vez el cariotipo del lenguado S. senegalensis, que muestra 21 pares cromosómicos y 48 brazos cromosómicos (NF = 48).We analysed 12 allozyme loci in 92 farm-grown specimens of Solea senegalensis Kaup, 1858, with 30 individuals from the Cupimar farm, and the remainder supplied by the Casem marine cultures laboratory. The loci studied were MDH-1, MDH-2, PGI-1, PGI-2, PGM, LDH, EM-1, EM-2, AAT-1, AAT-2, GPD and G-6PDH. All loci were monomorphic except for locus LDH in both populations, using the criterion of 95% (P95), and PGI-2 in the Casem specimens (P99). A low variability was found in all of these systems at both the polymorphism and heterozygosity levels, and also regarding the average number of alleles. The studied populations are under Hardy-Weinberg equilibrium, and the genetic differentiation between them (FST) is very low. In addition, the karyotype of S. senegalensis is here described for the first time, presenting 21 chromosome pairs and 48 chromosome arms (NF = 48).Instituto Español de Oceanografí

Identification of RFLP in growth-related genes in the gilthead seabream Sparus aurata L., 1758

The main objective of the present study was to analyse polymorphism of the growth hormone (GH) gene and of the prolactin (PRL) gene in the gilthead seabream Sparus aurata L., 1758, and to examine its possible association with quantitative characters, such as weight and size. In order to carry out this study, we used a total of 279 individuals (131 breeders and 148 offspring). Once the Restriction Fragment Length Polymorphisms of PCR products (PCR-RFLP) at the introns of both genes were analysed, we found a possible association between intron I of the GH and the quantitative characters studied.El objetivo de este trabajo fue analizar el polimorfismo de los genes de la hormona de crecimiento (GH) y de la prolactina (PRL) en dorada Sparus aurata L., 1758 y buscar su posible asociación con los caracteres cuantitativos peso y talla. Para llevar a cabo este estudio se utilizaron en total 279 individuos (131 reproductores y 148 descendientes). Una vez analizados los polimorfismos en la longitud de fragmentos de restricción de productos de PCR (PCR-RFLP) de los intrones en ambos genes, se ha encontrado una posible asociación entre el intrón I de la GH y los citados caracteres cuantitativos estudiados.Instituto Español de Oceanografí

Chromosome characterization of the toadfish Halobatrachus didactylus (Schneider, 1801) (Teleostei: Batrachoididae) by fluorescent in situ hybridization

Halobatrachus didactylus (Schneider, 1801) has a chromosome number of 2n = 46 (8 metacentrics, 12 submetacentrics and 26 acrocentrics). Specimens studied were collected from waters of the Bay of Cadiz. Chromosome preparations were made from the cephalic portion of the kidney. The telomeric probe hybridized on the terminal position of all toadfish chromosomes. (GATA)n hybridized preferentially on the subcentromeric region of only one submetacentric chromosome pair, although additional weak signals dispersed throughout the chromosomes were found. Major ribosomal genes were also located on a submetacentric pair, but at a telomeric position. Finally, the 5S rDNA probe produced a hybridization pattern similar to the GATA probe. These results provide interesting information which describes chromosomal markers that may be of utility for the management of natural populations of this species.Se ha realizado la caracterización cromosómica de Halobatrachus didactylus (Schneider, 1801) mediante la localización de diferentes secuencias repetidas en sus cromosomas. Los especímenes utilizados fueron capturados en aguas de la bahía de Cádiz. Las preparaciones cromosómicas se realizaron de la porción cefálica del riñón. La sonda telomérica hibridó en posición terminal de todos los cromosomas del pez sapo. (GATA)n hibridó preferentemente en la región subcentromérica de un par cromosómico submetacéntrico; se encontraron señales adicionales más débiles. Los genes ribosomales mayores también fueron localizados en un par submetacéntrico, pero en región telomérica. Finalmente, la sonda 5S rDNA presentó un patrón de hibridación similar a la sonda GATA. Los resultados aportan interesantes datos que describen marcadores cromosómicos que podrían ser de utilidad para la gestión de poblaciones naturales de esta especie.Instituto Español de Oceanografí

Immune function and dysfunction are determined by lymphoid tissue efficacy

Lymphoid tissue returns to a steady state once each immune response is resolved, and although this occurs multiple times throughout life, its structural integrity and functionality remain unaffected. Stromal cells orchestrate cellular interactions within lymphoid tissue, and any changes to the microenvironment can have detrimental outcomes and drive disease. A breakdown in lymphoid tissue homeostasis can lead to a loss of tissue structure and function that can cause aberrant immune responses. This Review highlights recent advances in our understanding of lymphoid tissue function and remodelling in adaptive immunity and in disease states. We discuss the functional role of lymphoid tissue in disease progression and explore the changes to lymphoid tissue structure and function driven by infection, chronic inflammatory conditions and cancer. Understanding the role of lymphoid tissues in immune responses to a wide range of pathologies allows us to take a fuller systemic view of disease progression

Two isoforms of vasa maternal factor in Senegalese sole: Biotechnological applications

Primordial Germ Cells (PGCs) identification and manipulation present considerable potential for hatchery practice and surrogate broodstocks. To carry out the PGCs characterization a specific molecular marker is required. The vasa gene is a good candidate to identify PGCs and others germinal cells (Nagasawa et al., 2009). The aim of this study was the cloning of the Solea senegalensis vasa cDNA and its expression pattern during early development and adulthood

Comparative gene expression of gonadotropins (FSH and LH) and peptide levels of gonadotropin-releasing hormones (GnRHs) in the pituitary of wild and cultured Senegalese sole (Solea senegalensis) broodstocks

12 páginas, 8 figuras, 2 tablas.The Senegalese sole (Solea senegalensis) is a valuable flatfish for aquaculture, but it presents important reproductive problems in captivity. Spawning is achieved by wild-caught breeders but cultured broodstocks fail to spawn spontaneously and, when they do, eggs are unfertilized. To gain knowledge on the physiological basis underlying this reproductive dysfunction, this study aimed at analyzing comparative hormone levels between wild and cultured broodstocks at the spawning season. The Senegalese sole gonadotropin (GTH) subunits, FSHβ, LHβ and GPα, were cloned and qualitative (in situ hybridization) and quantitative (real-time PCR) assays developed to analyze pituitary GTH gene expression. In females, FSHβ and GPα mRNA levels were higher in wild than in cultured broodstocks, whereas in males all three subunits were highest in cultured. By ELISA, three GnRH forms were detected in the pituitary, displaying a relative abundance of GnRH2 > GnRH1 > GnRH3. All GnRHs were slightly more abundant in wild than cultured females, whereas no differences were observed in males. Plasma levels of vitellogenin and sex steroids were also analyzed. Results showed endocrine differences between wild and cultured broodstocks at the spawning period, which could be related to the endocrine failure of the reproductive axis in cultured breeders.This research was funded by the Spanish Ministry of Education and Science (MEC) (AGL2006-13777-C01), the Ministry of Agriculture, Fisheries and Food (MAPA) (JACUMAR, II National Plan for the Cultivation of Sole) and the Regional Government of Galicia (PGIDIT06RMA004E). J.M. Guzmán received a FPI fellowship from the MEC. J.B. Ortiz-Delgado was supported by the “Ramón y Cajal” program (MEC, Spain).Peer reviewe

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 SNP variants in the follicle stimulating hormone receptor (fshr) consistent with an XX / XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. Fshr displayed differential gene expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 non-synonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.info:eu-repo/semantics/acceptedVersio

Molecular Cytogenetic Analysis of the European Hake Merluccius merluccius (Merlucciidae, Gadiformes): U1 and U2 snRNA Gene Clusters Map to the Same Location

The European hake (Merluccius merluccius) is a highly valuable and intensely fished species in which a long-term alive stock has been established in captivity for aquaculture purposes. Due to their huge economic importance, genetic studies on hakes were mostly focused on phylogenetic and phylogeographic aspects; however chromosome numbers are still not described for any of the fifteen species in the genus Merluccius. In this work we report a chromosome number of 2n = 42 and a karyotype composed of three meta/submetacentric and 18 subtelo/telocentric chromosome pairs. Telomeric sequences appear exclusively at both ends of every single chromosome. Concerning rRNA genes, this species show a single 45S rDNA cluster at an intercalary location on the long arm of subtelocentric chromosome pair 12; the single 5S rDNA cluster is also intercalary to the long arm of chromosome pair 4. While U2 snRNA gene clusters map to a single subcentromeric position on chromosome pair 13, U1 snRNA gene clusters seem to appear on almost all chromosome pairs, but showing bigger clusters on pairs 5, 13, 16, 17 and 19. The brightest signals on pair 13 are coincident with the single U2 snRNA gene cluster signals. Therefore, the use of these probes allows the unequivocal identification of at least 7 of the chromosome pairs that compose the karyotype of Merluccius merluccius thus opening the way to integrate molecular genetics and cytological data on the study of the genome of this important species.Versión del editor4,411