RESUMEN
The miR-430 microRNA family has been described in multiple fish species as one of the first microRNAs expressed by the zygote. It has been suggested that this family is implicated in maternal mRNA elimination, but may also play a role in steroidogenesis, sexual differentiation, and flatfish metamorphosis. The miR-430 sequences have been found in multiple-copy tandem clusters but evidence of their conservation outside of teleost fishes is scarce. In the present study, we have characterized the tandem repeats organization of these microRNAs in different fish species, both model and of interest in aquaculture. A phylogenetic analysis of this family has allowed us to identify that the miR-430 duplication, which took place before the Chondrostei and Neopterygii groups' divergence, has resulted in three variants ("a", "b", and "c"). According to our data, variant "b" is the most closely related to the ancestral sequence. Furthermore, we have detected isolated instances of the miR-430 repeat subunit in some species, which suggests that this microRNA family may be affected by DNA rearrangements. This study provides new data about the abundance, variability, and organization of the miR-430 family in fishes.
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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 single nucleotide polymorphism 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. The fshr gene displayed differential 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 nonsynonymous 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.
Asunto(s)
Peces Planos , Receptores de HFE , Femenino , Masculino , Animales , Receptores de HFE/genética , Receptores de HFE/metabolismo , Genoma/genética , Cromosomas , Peces Planos/genética , Hormonas/metabolismoRESUMEN
The Atlas Pistachio tree, Pistacia atlantica Desf., has great importance in the ecological landscape of North Africa, due to its adaptive plasticity, as well as its use as a rootstock in the cultivation of the economically important species, Pistacia vera L. The conservation and valuation of this species require sampling and an assessment of its genetic variability. For the first time in North Africa, the inter-simple sequence repeats (ISSR) molecular marker has been used in genetic-diversity assessment and in the population relationships of P. atlantica subsp. atlantica. The ISSR markers tested showed 74.1% polymorphism, while molecular variance (AMOVA) analysis revealed a high percentage of the total genetic diversity of 55.7% among the four populations studied. Cluster analysis with neighbor-joining (NJ) and principal coordinate analysis (PCO) divided the study sites into four distinct groups according to their geographical locations (Tiaret, Batna, Djelfa, and Bechar). Isolation by distance or Mantel test gave a positive correlation of r = 0.86 between geographical and genetic distances. The results in this study indicate an absence of gene flow, implying that conservation efforts should be taken separately for each population.
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Helicobacter pylori is a human pathogenic bacterium with a high genomic plasticity. Although the functional CRISPR-Cas system has not been found in its genome, CRISPR-like loci have been recently identified. In this work, 53 genomes from different geographical areas are analyzed for the search and analysis of variability of this type of structure. We confirm the presence of a locus that was previously described in the VlpC gene in al lgenomes, and we characterize new CRISPR-like loci in other genomic locations. By studying the variability and gene location of these loci, the evolution and the possible roles of these sequences are discussed. Additionally, the usefulness of this type of sequences as a phylogenetic marker has been demonstrated, associating the different strains by geographical area.
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Two new centromeric satellite DNAs in flatfish (Order Pleuronectiformes) have been characterized. The SacI-family from Hippoglossus hippoglossus, restricted to this species, had a monomeric size of 334 base pair (bp) and was located in most of the centromeres of its karyotype. The PvuII-family, with a monomeric size of 177 bp, was initially isolated from the genome of Solea senegalensis, and fluorescent in situ hybridization (FISH) localized the repeat to centromeres of most of the chromosomes. This family could only be amplified in 2 other species of the genus Solea (Solea solea and Solea lascaris). Molecular features and chromosomal location indicated a possible structural and/or functional role of these sequence repeats. The presence of species-specific satellite-DNA families in the centromeres and their possible role in the speciation processes in this group of fishes is discussed.
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Centrómero/genética , ADN Satélite/genética , Peces Planos/clasificación , Especiación Genética , Animales , Secuencia de Consenso , Hibridación Fluorescente in Situ , Cariotipificación , Especificidad de la EspecieRESUMEN
In order to determine if ostreid herpesvirus 1 (OsHV-1) can be vertically transmitted, 9 full-sib families of the Portuguese oyster Crassostrea angulata were produced using a factorial mating design with 3 males and 3 females. The parents were survivors from an OsHV-1 mortality outbreak. OsHV-1 DNA was not detected by conventional PCR in the mantle of parents, gametes or 3day-old larvae. However, viral DNA was detected by real-time PCR in all gametes and larvae samples. These results show that C. angulata that have survived an OsHV-1 mortality outbreak can carry the virus and vertically transmit it to their offspring.
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Crassostrea/virología , Infecciones por Herpesviridae/veterinaria , Animales , Femenino , Herpesviridae , Transmisión Vertical de Enfermedad Infecciosa , Larva/virología , Masculino , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Ostreid herpesvirus 1 (OsHV-1) infections have been reported in several bivalve species. Mortality of Pacific oyster Crassostrea gigas spat has increased considerably in Europe since 2008 linked to the spread of a variant of OsHV-1 called µvar. In the present study we demonstrated that O. edulis juveniles can be infected by OsHV-1µvar when administered as an intramuscular injection. Mortality in the oysters injected with OsHV-1µvar was first detected 4 days after injection and reached 25% mortality at day 10. Moreover, the high viral load observed and the detection of viral transcripts by in situ hybridization in several tissues of dying oysters suggested that OsHV-1µvar was the cause of mortality in the O. edulis juveniles. This is therefore the first study to provide evidence about the pathogenicity of OsHV-1µvar in a species that does not belong to the Crassostrea genus. Additionally, we present a novel method to detect OsHV-1 transcripts in infected individuals' using in situ hybridization.
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Herpesviridae/patogenicidad , Ostrea/virología , Animales , ADN Viral , Herpesviridae/ultraestructura , Hibridación in Situ , ARN Viral/análisis , Transcripción Genética , Carga ViralRESUMEN
This paper represents the first molecular cytogenetic characterization of the strictly dioecious pistachio tree (Pistacia vera L.). The karyotype was characterized by fluorescent in situ hybridization (FISH) with probes for 5S and 45S rDNAs, and the pistachio specific satellite DNAs PIVE-40, and PIVE-180, together with DAPI-staining. PIVE-180 has a monomeric unit of 176-178 bp and high sequence homology between family members; PIVE-40 has a 43 bp consensus monomeric unit, and is most likely arranged in higher order repeats (HORs) of two units. The P. vera genome is highly heterochromatic, and prominent DAPI positive blocks are detected in most chromosomes. Despite the difficulty in classifying chromosomes according to morphology, 10 out of 15 pairs (2n = 30) could be distinguished by their unique banding patterns using a combination of FISH probes. Significantly, the largest pair, designated HC1, is strongly heteropycnotic, shows differential condensation, and has massive enrichment in PIVE-40 repeats. There are two types of HC1 chromosomes (type-I and type-II) with differing PIVE-40 hybridization signal. Only type-I/II heterozygotes and type-I homozygotes individuals were found. We speculate that the differentiation between the two HC1 chromosomes is due to suppression of homologous recombination at meiosis, reinforced by the presence of PIVE-40 HORs and differences in PIVE-40 abundance. This would be compatible with a ZW sex-determination system in the pistachio tree.
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Cromosomas de las Plantas , Pistacia/genética , Recombinación Genética , Citogenética , Hibridación Fluorescente in Situ , CariotipificaciónRESUMEN
In the present study, Marteilia sp. was detected by histological examination and in situ hybridisation in Ostrea edulis and Ostrea stentina collected in southern Iberian Peninsula. Marteilia refringens DNA was detected by PCR in O. edulis (collected in southern Portugal) and O. stentina (collected in southern Spain and Portugal). Sequencing analysis revealed the presence of M. refringens type O in O. edulis, and type O and M in O. stentina. This is the first confirmed detection of M. refringens in Portugal and the first report on the occurrence of M. refringens infecting O. stentina in Europe.
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Cercozoos , Ostrea/parasitología , Animales , ADN Protozoario/análisis , Interacciones Huésped-Parásitos , Hibridación in Situ , Reacción en Cadena de la Polimerasa , Portugal , EspañaRESUMEN
In this study we have developed protocols for induced triploidy and gynogenesis of Senegalese sole (Solea senegalensis), a promising flatfish species for marine aquaculture, in order to: 1) identify the sex-determination mechanism; and 2) to improve its production by generating a) sterile fish, avoiding problems related with sexual maturation, and b) all-female stocks, of higher growth rate. Triploidy was induced by means of a cold shock. Gynogenesis was induced by activating eggs with UV-irradiated sperm, and to prompt diploid gynogenesis, a cold-shock step was also used. Ploidy of putative triploid larvae and gynogenetic embryos were determined by means of karyotyping and microsatellite analysis. Haploid gynogenetic embryos showed the typical "haploid syndrome". As expected, triploid and gynogenetic groups showed lower fertilization, hatching, and survival rates than in the diploid control group. Survival rate, calculated 49 days after hatching, for haploid and diploid gynogenetic groups was similar to those observed in other fish species (0% and 62.5%, respectively), whereas triploids showed worse values (45%). Sex was determined macroscopically and by histological procedures, revealing that all the diploid gynogenetic individuals were females. In conclusion, we have successfully applied chromosomal-manipulation techniques in the flatfish species Senegalese sole in order to produce triploid, haploid, and diploid gynogenetic progenies.
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Peces Planos/genética , Procesos de Determinación del Sexo , Triploidía , Animales , Cromosomas , Femenino , Haploidia , Cariotipificación , Masculino , Repeticiones de Microsatélite , Análisis para Determinación del Sexo , Espermatozoides/efectos de la radiaciónRESUMEN
The Senegalese sole (Solea senegalensis, Kaup 1858) is a flatfish species of great value for aquaculture. In this study, we develop the first linkage map in this species based on microsatellite markers characterized from genomic DNA libraries and EST databases of Senegalese sole and from other flatfish species. Three reference gynogenetic families were obtained by chromosome-manipulation techniques: two haploid gynogenetics, used to assign and order microsatellites to linkage groups and another diploid gynogenetic family, used for estimating marker-centromere distances. The consensus map consists of 129 microsatellites distributed in 27 linkage groups (LG), with an average density of 4.7 markers per LG and comprising 1,004 centimorgans (cM). Additionally, 15 markers remained unlinked. Through half-tetrad analysis, we were able to estimate the centromere distance for 81 markers belonging to 24 LG, representing an average of 3 markers per LG. Comparative mapping was performed between flatfish species LG and model fish species chromosomes (stickleback, Tetraodon, medaka, fugu and zebrafish). The usefulness of microsatellite markers and the genetic map as tools for comparative mapping and evolution studies is discussed.
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Mapeo Cromosómico/veterinaria , Peces Planos/genética , Repeticiones de Microsatélite/genética , Animales , Mapeo Cromosómico/métodos , Haploidia , Especificidad de la EspecieRESUMEN
One of the main objectives of genetic breeding programs in turbot industry is to reduce disease-related mortality. In the present study, a genome scan to detect quantitative trait loci (QTL) affecting resistance and survival to viral haemorrhagic septicaemia (VHS) was carried out. Three full-sib families with approximately 90 individuals each were genotyped and evaluated by linear regression and maximum likelihood approaches. In addition, a comparison between QTL detected for resistance and survival time to other important bacterial and parasite diseases affecting turbot (furunculosis and scuticociliatosis) was also carried out. Finally, the relationship between QTL affecting resistance/survival time to the virus and growth-related QTL was also evaluated. Several genomic regions controlling resistance and survival time to VHS were detected. Also significant associations between the evaluated traits and genotypes at particular markers were identified, explaining up to 14 % of the phenotypic variance. Several genomic regions controlling general and specific resistance to different diseases in turbot were detected. A preliminary gene mining approach identified candidate genes related to general or specific immunity. This information will be valuable to develop marker-assisted selection programs and to discover candidate genes related to disease resistance to improve turbot production.
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Acuicultura/métodos , Resistencia a la Enfermedad/genética , Enfermedades de los Peces/genética , Enfermedades de los Peces/virología , Peces Planos , Septicemia Hemorrágica Viral/genética , Sitios de Carácter Cuantitativo/genética , Animales , Cruzamiento/métodos , Infecciones por Cilióforos/genética , Infecciones por Cilióforos/veterinaria , Forunculosis/genética , Genotipo , Funciones de Verosimilitud , Modelos LinealesRESUMEN
BACKGROUND: The turbot (Scophthalmus maximus) is a relevant species in European aquaculture. The small turbot genome provides a source for genomics strategies to use in order to understand the genetic basis of productive traits, particularly those related to sex, growth and pathogen resistance. Genetic maps represent essential genomic screening tools allowing to localize quantitative trait loci (QTL) and to identify candidate genes through comparative mapping. This information is the backbone to develop marker-assisted selection (MAS) programs in aquaculture. Expressed sequenced tag (EST) resources have largely increased in turbot, thus supplying numerous type I markers suitable for extending the previous linkage map, which was mostly based on anonymous loci. The aim of this study was to construct a higher-resolution turbot genetic map using EST-linked markers, which will turn out to be useful for comparative mapping studies. RESULTS: A consensus gene-enriched genetic map of the turbot was constructed using 463 SNP and microsatellite markers in nine reference families. This map contains 438 markers, 180 EST-linked, clustered at 24 linkage groups. Linkage and comparative genomics evidences suggested additional linkage group fusions toward the consolidation of turbot map according to karyotype information. The linkage map showed a total length of 1402.7 cM with low average intermarker distance (3.7 cM; ~2 Mb). A global 1.6:1 female-to-male recombination frequency (RF) ratio was observed, although largely variable among linkage groups and chromosome regions. Comparative sequence analysis revealed large macrosyntenic patterns against model teleost genomes, significant hits decreasing from stickleback (54%) to zebrafish (20%). Comparative mapping supported particular chromosome rearrangements within Acanthopterygii and aided to assign unallocated markers to specific turbot linkage groups. CONCLUSIONS: The new gene-enriched high-resolution turbot map represents a useful genomic tool for QTL identification, positional cloning strategies, and future genome assembling. This map showed large synteny conservation against model teleost genomes. Comparative genomics and data mining from landmarks will provide straightforward access to candidate genes, which will be the basis for genetic breeding programs and evolutionary studies in this species.
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Mapeo Cromosómico/métodos , Etiquetas de Secuencia Expresada , Peces/genética , Peces Planos/genética , Animales , Femenino , Masculino , Repeticiones de Microsatélite , Sitios de Carácter Cuantitativo , SinteníaRESUMEN
Five Gram-negative bacterial isolates, recovered from an outbreak that occurred in March 2006 in Huelva, Spain, affecting adult diseased cultured wedge sole [Dicologlossa cuneata (Moreau)], were characterized phenotypically and genotypically in order to clarify their taxonomic position. On the basis of 16S rRNA gene sequence analysis, the isolates were included in the genus Pseudomonas, within the Pseudomonas fluorescens-related species group, their closest relatives being the Pseudomonas jessenii and Pseudomonas koreensis subgroups. The highest sequence similarities were recorded with the type strains of Pseudomonas reinekei, P. moorei, P. umsongensis, P. jessenii and P. mohnii (99.4-99.3â% similarity). Sequence analysis of the housekeeping genes gyrB and rpoD clearly differentiated the isolates from currently described Pseudomonas species, the highest sequence similarities recorded to type strains being below 95â% for both genes. Phylogenetic analysis using concatenated sequences of the three genes showed Pseudomonas moraviensis DSM 16007T and P. koreensis DSM 16610T as the closest reference strains. DNA-DNA hybridization assays with related strains confirmed that these isolates belong to a novel species of the genus Pseudomonas, for which the name Pseudomonas baetica sp. nov. is proposed. The type strain is strain a390T (=CECT 7720T=LMG 25716T). The novel species could be easily distinguished from phylogenetically related species by several phenotypic characteristics, including gelatin hydrolysis, acid production from glucose and growth at 6â% NaCl. Virulence assays revealed that the novel species is pathogenic for wedge sole.
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Peces Planos/microbiología , Filogenia , Pseudomonas/aislamiento & purificación , Animales , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Ácidos Grasos/química , Enfermedades de los Peces/microbiología , Genes Bacterianos , Hígado/microbiología , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Pseudomonas/clasificación , Pseudomonas/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , EspañaRESUMEN
The aims of this work were to characterize the 16S-23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T. soleae strains tested displayed a single internal spacer region class, containing tRNA(I) (le) and tRNA(A) (la) genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T. soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1 pg DNA per reaction (< 30 bacterial cells) when using pure cultures. The detection level in the presence of DNA from fish or other bacteria was lower; however, 10 pg were detected at a target/background ratio of 1 : 10(5) . The PCR assay proved to be more sensitive than agar cultivation for the detection of T. soleae from naturally diseased fish, offering a useful tool for diagnosis and for understanding the epidemiology of this pathogen.
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ADN Espaciador Ribosómico/genética , Enfermedades de los Peces/microbiología , Infecciones por Flavobacteriaceae/veterinaria , Reacción en Cadena de la Polimerasa/métodos , Tenacibaculum/genética , Tenacibaculum/aislamiento & purificación , Animales , Técnicas de Tipificación Bacteriana , Secuencia de Bases , ADN Bacteriano/genética , Peces/microbiología , Infecciones por Flavobacteriaceae/microbiología , Datos de Secuencia Molecular , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Tenacibaculum/clasificaciónRESUMEN
When genomic DNA from the European flat oyster Ostrea edulis L. was digested by BclI enzyme, a band of about 150 bp was observed in agarose gel. After cloning and sequencing this band and analysing their molecular characteristics and genomic organization by means of Southern blot, in situ hybridisation, and polymerase chain reaction (PCR) protocols, we concluded that this band is an interspersed highly repeated DNA element, which is related in sequence to the flanking regions of (CT)-microsatellite loci of the species O. edulis and Crassostrea gigas. Furthermore, we determined that this element forms part of a longer repetitive unit of 268 bp in length that, at least in some loci, is present in more than one copy. By Southern blot hybridisation and PCR amplifications-using primers designed for conserved regions of the 150-bp BclI clones of O. edulis-we determined that this repetitive DNA family is conserved in five other oyster species (O. stentina, C. angulata, C. gigas, C. ariakensis, and C. sikamea) while it is apparently absent in C. gasar. Finally, based on the analysis of the repetitive units in these oyster species, we discuss the slow degree of concerted evolution in this interspersed repetitive DNA family and its use for phylogenetic analysis.
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Crassostrea/genética , Elementos Transponibles de ADN , ADN/genética , Evolución Molecular , Ostrea/genética , Animales , Secuencia de Bases , Análisis Citogenético , Cartilla de ADN , Sitios Genéticos , Hibridación Fluorescente in Situ , Repeticiones de Microsatélite , Datos de Secuencia Molecular , FilogeniaRESUMEN
We report the first isolation of Vibrio harveyi from wedge sole Dicologoglossa cuneata. The pathogen was recovered from ulcers and internal organs of ailing cultured fish, from 7 different outbreaks between 2004 and 2006. The 15 isolates found were phenotypically characterized using biochemical tests and BIOLOG GN plates, which revealed high phenotypic diversity. Diagnosis was confirmed with PCR using V harveyi specific primers and partial 16S and 23S rRNA gene sequencing. A virulence evaluation of the isolates was also performed using fry and juvenile wedge sole. Significant mortalities were recorded by intraperitoneal injection; however, no mortalities were recorded by bath immersion.
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Enfermedades de los Peces/microbiología , Peces Planos/microbiología , Vibrio/aislamiento & purificación , Animales , Acuicultura , Filogenia , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Vibrio/clasificación , Vibrio/genética , Vibrio/patogenicidadRESUMEN
We report on the isolation and characterization of eight microsatellite markers from enriched libraries for the critically endangered Atropa baetica. These are the first microsatellite loci reported for Atropa species. The total number of alleles found was 18, the expected heterozygosity ranged from 0.198 to 0.505. These markers will be useful to establish the real census of individuals and the genetic diversity both within and among the different populations of A. baetica.
RESUMEN
In the course of a histopathological survey performed to discover the cause of mass mortality of the striped clam Chamelea gallina in the Balearic Islands (Spain, Mediterranean Sea), we detected a Marteilia-like parasite in 3 clams. Molecular methods were applied to identify the parasite. DNA extracted from a paraffin block was used to carry out a PCR assay for Marteilia refringens detection based on a rDNA sequence of the parasite (the intergenic spacer of ribosomal genes, IGS). The nucleotide sequence of the IGS amplified fragment and the positive signal obtained by in situ hybridisation analysis with a M. refringens-specific probe allowed us to confirm the presence of this parasite in the digestive gland tissue of C. gallina.
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Bivalvos/parasitología , Eucariontes/aislamiento & purificación , Hibridación in Situ/métodos , Reacción en Cadena de la Polimerasa/métodos , Animales , ADN Intergénico/genética , ADN Protozoario/genética , Eucariontes/genética , España/epidemiologíaRESUMEN
Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. It is believed to have a complex life-cycle involving several hosts. In this study, we applied molecular approaches to identify this parasite in samples of the razor clam Solen marginatus from the south west coast of Spain. We used a PCR assay to amplify a fragment of the IGS rDNA region. PCR products were sequenced and the phylogenetic affinity of the sequences was determined. In situ hybridization analysis showed tissue distribution and presence of different developmental stages of the parasite in the digestive diverticula epithelium, which suggested a true parasitism in these individuals. This is the first report of the occurrence of M. refringens in the razor clam S. marginatus in the south Atlantic. The methodology described herein may be useful for accurate identification of the parasite strain in different hosts and thus provide valuable information for marteiliosis control programmes.