Isolation and characterization of an RNA polymerase III encoded gene of Pinus radiata and its use in pine transformation

Several promoters such as the cauliflower mosaic virus 35S promoter (CaMV 35S) and its enhanced version, pEMU, the maize ubiquitin promoter, the alcohol dehydrogenase promoter (Adh) and rice actin promoter (Act1) are currently used in Pinus radiata (pine) transformation. These heterologous promoters were adopted for pine transformation for want of an endogenous promoter tailored specifically for the needs of pine. These promoters may not perform to the same extent in pine as in their homologous systems due to differences in quality and/or quantity of regulatory factors. Secondly, because of their heterologous origin, these promoters are open to silencing mechanisms that operate in plants against invasive DNA [Matzke & Birchler, 2005]. This could result in inactivation of these promoters at any time during the 30-year growth period of transformed pine which this poses a real threat to a forestry industry based on transgenic pine. A pine promoter on the other hand, being endogenous, is less prone to silencing. In addition, confidence in its longevity (continued expression) can be easily established even before using it in transformation. The aim of this study was to isolate and validate pine promoters that can be used in pine transformation. As only a few pine sequences were available in the public domain for gene discovery in pine (at the beginning of this study), heterologous sequence information was used to screen the pine genome or its transcriptome for orthologs with desirable expression features. The investigation proceeded along two lines. In the first approach, a putatively desirable gene was isolated and the expression profile of its promoter was then validated. This led to the characterization of 5Spr20, a pine SS rDNA paralog. 5Spr20 differs from all published SS rDNA sequence& dnd is therefore a novel pine gene. Analyses of its sequence using bioinformatics revealed that it is capable of initiating biologically active transcripts and 5Spr20 is therefore a functional gene. A recombinant 5Spr20 promoter consisting of the coding region and the immediately upstream region downregulated gus reporter activity by 90% by antisense activity in transient expression studies in pine embryogenic cells. In stable expression studies, a 5Spr20 promoter-driven shDNA construct targeting gus completely silenced reporter activity in the model plant Nicotiana benthamiana. The 5Spr20 promoter appears to hold great promise for use in pine functional genomics and in gene downregulation applications. In the second line of investigation, the expression profiles of pine orthologs of known heterologous genes were validated prior to gene isolation. Two pine genes that were identified as promising candidates are pine tDNAMet-l and an actin paralog pine, ActX. Both genes were strongly expressed in all vegetative tissues of pine. Several PCR-based methods were used to clone the upstream regions (containing putative promoter elements) but all attempts ended in failure, which is attributed to the presence of pseudogenes and regions homologous to walking/sequencing primers among paralogs. The pine transcriptome was also screened unsuccessfully for ortholgs of desirable heterologous candidate genes like the ribosomal protein genes MsRL5 of Medicago sativa and AtL 18 of Arabidopsis thaliana and genes for the second largest subunit of RNA polymerase II, gene T13794 and actin-2 of A. thaliana. Sequence heterogeneity, cell-specific expression and low transcript abundance are possible reasons for not being able to detect pine orthologs of these candidate genes in expression screens

Molecular biomarkers for fish welfare and species authentication.

Aquaculture is currently contributing almost half of fish consumed by the human population and it keeps growing more rapidly than other animal food production sectors. The introduction of molecular techniques, such as various genome projects, gene expression analysis and functional genomics, and the monitoring of stress levels through very early indicators such as molecular biomarkers, can bring considerable benefits to the quantity and quality of production and improve welfare of reared animals. Moreover in recent years, some teleost species have become model organisms, such as zebrafish and pufferfish, and the knowledge about them could be transferred to further improve reared animals and husbandry. Therefore, species of interest in aquaculture could, in turn, become new animal models. In this context, we looked for a new molecular biomarker for stress in Dicentrarchus labrax. Stress could involve alterations of brain functioning that may precipitate to mood disorders. The neurotrophin Brain Derived Neurotrophic Factor (BDNF) has recently been involved in stress-induced adaptation. BDNF is a key regulator of neuronal plasticity and adaptive processes. Regulation of BDNF is complex and may reflect not only stressspecific mechanisms, but also hormonal and emotional responses. For this reason, we used, as an animal model of stress, a fish, D. labrax, whose brain organization is very similar to that of higher vertebrates, but is generally considered free of emotional reactions. We provide, for the first time in a species of great interest in aquaculture, a comprehensive characterization of BDNF gene and its transcriptional, translational and post-translational regulation following acute stress. While total BDNF mRNA levels are unchanged, BDNF splicing variants 1c and 1d resulted down regulated after acute stress. Acute stress induces also a significant increase in proBDNF levels and reduction in mature BDNF suggesting altered regulation of proBDNF proteolytic processing. Notably, we provide here the first evidence that fishes possess a simplified proteolytic regulation of BDNF since the pro28kDa form, generated by the SKI-1 protease in mammals, is absent in fishes. The cleavage site, in fact, has first emerged in reptilians. Finally, we show that the proBDNF/totBDNF ratio is a highly predictive novel quantitative biomarker to detect stress in fishes with sensitivity = 100%, specificity = 87%, and Negative Predictive Value = 100%. The high predictivity of proBDNF/totBDNF ratio for stress in lower vertebrates indicates that processing of BDNF is a central mechanism in adaptation to stress and predicts that a similar regulation of pro/mature BDNF has likely been conserved throughout evolution of vertebrates from fish to man. The second part of this thesis is focused on the problem of seafood and fish species authentication. This is an important issue within the seafood industry to protect consumers from fraudulent practices, like species substitution, resulting from the increasingly wide diversification of species and globalization of fish trade. DNA-based methods for species identification are by far the best: they are generally based on PCR amplification of a target sequence, followed by a post-PCR analysis of amplified products, which could consist in sequencing or obtaining species-specific patterns of restriction fragments. The gene coding for the 5S ribosomal RNA is a suitable target for fish species identification because, for its particular sequence features, it does not require any further treatment after PCR. This gene consists of a small coding conserved region and a variable region of noncoding DNA, which is termed not transcribed spacer (NTS). Both regions are tandem repeated in the genome. The NTS, which is species-specific for length and sequence, has been used, here, to discriminate species subjected to substitution in the Italian fish market. Although preliminary, our results have demonstrated the value of this approach

Molecular biomarkers for fish welfare and species authentication.

Aquaculture is currently contributing almost half of fish consumed by the human population and it keeps growing more rapidly than other animal food production sectors. The introduction of molecular techniques, such as various genome projects, gene expression analysis and functional genomics, and the monitoring of stress levels through very early indicators such as molecular biomarkers, can bring considerable benefits to the quantity and quality of production and improve welfare of reared animals. Moreover in recent years, some teleost species have become model organisms, such as zebrafish and pufferfish, and the knowledge about them could be transferred to further improve reared animals and husbandry. Therefore, species of interest in aquaculture could, in turn, become new animal models. In this context, we looked for a new molecular biomarker for stress in Dicentrarchus labrax. Stress could involve alterations of brain functioning that may precipitate to mood disorders. The neurotrophin Brain Derived Neurotrophic Factor (BDNF) has recently been involved in stress-induced adaptation. BDNF is a key regulator of neuronal plasticity and adaptive processes. Regulation of BDNF is complex and may reflect not only stressspecific mechanisms, but also hormonal and emotional responses. For this reason, we used, as an animal model of stress, a fish, D. labrax, whose brain organization is very similar to that of higher vertebrates, but is generally considered free of emotional reactions. We provide, for the first time in a species of great interest in aquaculture, a comprehensive characterization of BDNF gene and its transcriptional, translational and post-translational regulation following acute stress. While total BDNF mRNA levels are unchanged, BDNF splicing variants 1c and 1d resulted down regulated after acute stress. Acute stress induces also a significant increase in proBDNF levels and reduction in mature BDNF suggesting altered regulation of proBDNF proteolytic processing. Notably, we provide here the first evidence that fishes possess a simplified proteolytic regulation of BDNF since the pro28kDa form, generated by the SKI-1 protease in mammals, is absent in fishes. The cleavage site, in fact, has first emerged in reptilians. Finally, we show that the proBDNF/totBDNF ratio is a highly predictive novel quantitative biomarker to detect stress in fishes with sensitivity = 100%, specificity = 87%, and Negative Predictive Value = 100%. The high predictivity of proBDNF/totBDNF ratio for stress in lower vertebrates indicates that processing of BDNF is a central mechanism in adaptation to stress and predicts that a similar regulation of pro/mature BDNF has likely been conserved throughout evolution of vertebrates from fish to man. The second part of this thesis is focused on the problem of seafood and fish species authentication. This is an important issue within the seafood industry to protect consumers from fraudulent practices, like species substitution, resulting from the increasingly wide diversification of species and globalization of fish trade. DNA-based methods for species identification are by far the best: they are generally based on PCR amplification of a target sequence, followed by a post-PCR analysis of amplified products, which could consist in sequencing or obtaining species-specific patterns of restriction fragments. The gene coding for the 5S ribosomal RNA is a suitable target for fish species identification because, for its particular sequence features, it does not require any further treatment after PCR. This gene consists of a small coding conserved region and a variable region of noncoding DNA, which is termed not transcribed spacer (NTS). Both regions are tandem repeated in the genome. The NTS, which is species-specific for length and sequence, has been used, here, to discriminate species subjected to substitution in the Italian fish market. Although preliminary, our results have demonstrated the value of this approach

The genome organization of the Bobwhite Quail (colinus virginianus), 1981

The DNA of the bobwhite quail (Colinus virginianus) was examined using isopycnic centrifugation in neutral CsCl, alkaline CsCl, Hg++-Cs2SOif and Ag+- CS2SO . When quail DNA was centrifuged in neutral CsCl, two low density minor bands (satellite DNA) were detected in addition to the main band DNA. Main band DNA had a buoyant density of 1.701 g/ml. The larger of the two minor bands had a buoyant density of 1.681 g/ml (I) while the smaller of the two minor bands had a buoyant density of 1.674 g/ml (II). The main band has a G + C content of 41.8% while the satellites, I and II, have G + C contents of 21.4% and 14.2%, respectively. Thermal denaturation studies were used to confirm the base composition data obtained from the buoyant density data. Restriction endonucleases (Hind 111, EcoR 1, EcoR 11, Hpa 1, Hpa 11, Bam 1, Bgl 11, Alu 1, Hae 111, Hha 1 and Mbo 1) were used to investigate the heterogeneity of repetitive DNA's in the total bobwhite quail genome. Bobwhite quail DNA consists of a heterogeneous population which is indicative of a genome in which most of the restriction endonuclease sites are randomly distributed. However, the Hind 111 sites are non-randomly distributed as indicated by the seven discrete fragments generated. The arrangement of repetitive and non-repetitive sequences was studied in the genomic DNA of the bobwhite quail (Colinus virginianus) and the Pharoah quail (Coturnix coturnix). The reassociation kinetics of short (450-500 nucleotide) and long (2000 nucleotide) DNA fragments were studied by the hydroxylapatite method. The data indicate that the bobwhite genome consists of three kinetic components; non-repetitive (Cot& pure = 70; 70%), moderately repetitive (Cotte pure = 0.50; 10%) and highly repetitve (Cotli pure = 0.01; 20%). The genome of the Pharoah quail is similar to the bobwhite with the non-repetitve sequences accounting for 68% of the genome (Cotl2 pure = 66.6); moderately repetitive sequences constitute 12% of the genome (Cotft pure = 0.65); and the highly repetitve sequences constitute about 20% of the genome (Cot& pure = 0.012). The lengths of the repetitve sequences obtained by reassociating total DNA to Cot 40 and digesting with SI nuclease were estimated by agarose gel electrophoresis and alkaline sucrose gradients. The repetitve sequences in both species of birds are approximately 1000 nucleotides long. The interspersion of repetitive and non-repetitive or single copy sequences have been examined. The results indicate that about 20-23% of the non-repetitve sequences in fragments 2000 nucleotides long are interspersed with long repetitive sequences in the bobwhite quail. In the Pharoah quail genome approximately 25- 27% of the non-repetitive sequences are interspersed. These data indicate that the bobwhite and Pharoah quail genomes contain some highly repetitive DNA sequences which are clustered and band as distinct satellites in neutral CsCl and some repetitive sequences which are intersperse

MOLECULAR RESOLUTION OF MARINE NEMATODES FOR IMPROVED ASSESSMENT OF BIODIVERSITY

Free-living nematodes are abundant in all marine habitats, highly diverse and can be important ecological indicators for monitoring anthropogenic impacts on the environment. Despite such attributes, nematode diagnostics has traditionally relied on detailed comparison of morphological characters which is often difficult and laborious, and as a result there is an increasing 'black hole' in faunal inventories where the biodiversity of groups such as nematodes is typically underestimated. Molecular methods offer a potentially efficient alternative approach to studying the biodiversity of marine nematode communities, and the main focus of this thesis was to apply molecular ecological tools for improved understanding of nematode diversity in marine and estuarine environments. Denaturing gradient gel electrophoresis (DGGE) has been evaluated as a novel tool for the identification of marine nematodes and for rapid assessment of their diversity based on amplification of the nuclear 18S rRNA gene. This approach successfully identified nematode taxa based on banding pattern and was also able to detect the most abundant taxa in samples from marine and estuarine environments. A DNA barcoding approach based on the 18S rRNA gene was applied for the first time in marine nematology, in an attempt to speed up the identification process. The success rate of this approach, across a range of nematode groups, was found to be close to 97%. A combined morphometrics and molecular approach was also undertaken to investigate cosmopolitanism and cryptic speciation by analysing populations of a cosmopolitan marine nematode, Terschellingia longicaudata, from different geographical regions. Results suggest that Terschellingia longicaudata is indeed truly cosmopolitan, with a wide geographic distribution. Two haplotypes that were divergent from most T. longicaudata were also identified in this study, indicating possible novel cryptic lineages or previously undescribed species of the genus. The final focus of this thesis was to develop methods for the molecular investigation of nematodes stored in formalin and other organic compounds. The effectiveness of formalin as a short term preservative was first evaluated, since this would allow morphological and molecular work to be conducted on the same specimen. Amplifiable DNA could be routinely obtained from specimens stored in formalin for periods of up to nine days. In addition the effectiveness of other organic solvents for the preservation of both molecular and morphological integrity of marine nematodes was investigated. The final part of this study developed and optimized a novel DNA extraction technique that could be employed to recover DNA from archived formalin fixed marine nematode specimens so as to carry out subsequent molecular analysis such as PCR amplification and sequencing.Plymouth Marine Laborator

Studies of the Relationship Between mRNA Stability and Gene Function in Saccharomyces cerevisiae

Twelve S. cerevisiae cDNAs, characterised by Santiago (1986) on the basis of the half-lifes of the respective mRNAs, have been partially or completely sequenced. To four have been assigned a definite function. cDNA10, which generates a long half-life mRNA, encodes the glucose-inducible form of the glycolytic enzyme, enolase. cDNAs 90, 39 and 13, each of which generates a short half-life mRNA, encode ribosomal proteins L3, L29 and YL6 respectively. cDNA46, which generates a long half-life mRNA, is related to a mouse gene of unknown function, MER5. In addition, there is circumstantial evidence that among the unidentified cDNAs that generate short half-life mRNAs are several that encode ribosomal proteins

Genetic analysis in various razor shell species of family pharidae, with focus on atlantic "Ensis"

[Resumen] En esta tesis se ha realizado un estudio genético de un grupo de bivalvos marinos de la familia Pharidae Adams y Adams, 1858, y principalmente de las navajas del género Ensis Schumacher, 1817. El estudio se desarrolla dentro de las áreas de la genética evolutiva, genética poblacional, citogenética, delimitación de especies y DNA barcoding. Las especies de navaja utilizadas fueron seleccionadas debido al interés comercial que muchas especies de Ensis tienen en varias regiones de Europa y América, incluyéndose también el litoral de Galicia. Además, su distribución a ambos lados del Atlántico y en zonas de la costa pacífica de los Estados Unidos, El Perú y Chile hace que sean de interés en estudios filogeográficos. Por último, los problemas en la identificación de las especies a partir de caracteres morfológicos hicieron de este grupo de organismos un buen candidato para el desarrollo de herramientas moleculares y citogenéticas que complementasen los estudios de morfometría. La metodología utilizada varía en cierto modo a lo largo de cada uno de los capítulos que conforman los resultados de la tesis, aunque puede resumirse como sigue: en el capítulo 4.1.1, el ADN ribosomal 5S y el ADN pequeño nuclear U1, que conforman familias génicas de copia múltiple, fueron estudiados mediante un procedimiento de amplificación por PCR, clonación y secuenciación. Todas estas secuencias obtenidas experimentalmente en el laboratorio fueron posteriormente analizadas mediante numerosas herramientas bioinformáticas. En el capítulo 4.1.2, en el que se estudiaron 97 especies de metazoos, la metodología utilizada fue diferente, ya que se utilizaron bases de datos de proyectos genoma y herramientas bioinformáticas de alta capacidad, algunas de las cuales fueron diseñadas expresamente para este trabajo. En el capítulo 4.2 se realizó un estudio citogenético en el que se utilizaron técnicas de microscopía en campo claro y fluorescente, así como la hibridación de sondas fluorescentes sobre los cromosomas de las navajas, que fueron utilizadas como marcadores cromosómicos. Se realizó también la medición y ordenación de los cromosomas por tamaño e índice centromérico, para elaborar los cariotipos. Por último, en los capítulos 4.3 y 4.4 se utilizaron como marcadores moleculares diferentes regiones de los genomas nuclear y mitocondrial, que han sido amplificadas mediante PCR, siendo a veces clonadas, previamente a su secuenciación. Estos marcadores moleculares son fragmentos de los genes mitocondriales citocromo oxidasa subunidad I y ADN ribosomal 16S; fragmentos de los genes nucleares adenina nucleótido translocasa y ADN ribosomal 18S; y la región nuclear que contiene los espaciadores ribosomales ITS1 e ITS2, y el gen ribosomal 5.8S. Algunos de ellos se han empleado solo a nivel poblacional, algunos solo a nivel de especie, mientras que otros se han empleado a ambos niveles. En el capítulo 4.1.1 se estudió el ligamiento entre el ADN ribosomal 5S y el ADN nuclear pequeño U1. Se describieron unidades de ligamiento entre ambas familias multigénicas en 10 especies (de cuatro géneros diferentes) de la familia Pharidae. Se obtuvo un número de clones que contenían repeticiones parciales o completas de ambos genes, en las que las regiones codificantes mostraron la misma orientación. Se obtuvo una completa colección de clones de ADN ribosomal 5S de varias especies de navajas, tanto ligados como no ligados al ADN nuclear pequeño U1. También se predijo la estructura secundaria de las regiones codificantes, y se caracterizó los elementos conservados de las regiones aguas arriba y aguas abajo de las mismas. El análisis de los espaciadores no transcritos (NTS) del ADN ribosomal 5S mostró que algunos de ellos estaban evolutivamente más relacionados con NTS de otras especies que con aquellos de la misma, sugiriendo polimorfismo ancestral y evolución a largo plazo mediante el proceso de birth-and-death. La conservación nucleotídica dentro de las regiones funcionales sugirió que la selección purificadora, además de los entrecruzamientos desiguales y las conversiones génicas, estuvieron implicados en la evolución de estas familias multigénicas. Considerando este estudio y otros previos, se discutieron los posibles mecanismos mediante los cuales ambas familias multigénicas han podido establecerse en unidades de ligamiento en el linaje de los Pharidae. La razón por la que el ADN ribosomal 5S se encuentra a menudo ligado a otras familias multigénicas parece ser el resultado de procesos estocásticos dentro de los genomas en el que su alto número de copia sería determinante. En el capítulo 4.1.2 se amplió el estudio anterior a 97 especies de metazoos, cuyos genomas estaban secuenciados y disponibles en bases de datos internacionales. Se analizaron y compararon sistemáticamente secuencias de ADN ribosomal 5S en especies de los principales clados de metazoos, usando métodos bioinformáticos. Tras haber realizado un filtrado de las secuencias candidatas, se obtuvieron 12766 copias putativamente funcionales que nos permitieron identificar algunas características generales del ADN ribosomal 5S de los animales. También se muestra que cada especie de mamífero analizada tiene una copia altamente conservada de ADN ribosomal 5S (que denominamos housekeeping) así como muchas otras copias más variables. Se analizó en detalle los NTS, la organización de esta familia multigénica en el genoma y su evolución. Nuestros resultados confirmaron la existencia de copias parálogas en 58 genomas. También fue evidente una organización flexible dentro del genoma de los animales. La existencia de agrupaciones heterogéneas de copias de ADN ribosomal 5S (compuestas de regiones codificantes similares y de NTS divergentes) en muchas especies apoya la hipótesis de un intercambio de ADN ribosomal 5S de un locus a otro del genoma. Además, obtuvimos un análisis detallado del grado de conservación evolutiva de las regiones promotoras internas, aguas arriba y aguas abajo en animales. Por último, describimos un método estadístico para analizar el ligamiento entre familias multigénicas codificadoras de ARN, que sin embargo no obtuvo ningún resultado de ligamiento estable entre el ADN ribosomal 5S y otras familias a lo largo de la evolución de los metazoos. En el siguiente capítulo (4.2) se realizó un estudio citogenético de la navaja europea Ensis minor (Chenu, 1843) y de la americana E. directus (Conrad, 1843). Se vio que ambas tienen un número cromosómico diploide de 38 y notables diferencias cariotípicas. E. minor tiene cuatro pares de cromosomas metacéntricos, uno metacéntrico-submetacéntrico, cinco submetacéntricos, uno subtelocéntrico y ocho telocéntricos. En cambio E. directus tiene tres pares metacéntricos, dos metacéntricos-submetacéntricos, seis submetacéntricos, seis subtelocéntricos y dos telocéntricos. La hibridación in situ fluorescente usando una sonda de genes ribosomales mayores localizó estos genes en un par submetacéntrico en ambas especies. La hibridación con ADN ribosomal 5S produjo una señal cromosómica débil en E. minor y ninguna en E. directus, apoyando una organización más dispersa de esta familia multigénica, comparada con la de los genes ribosomales mayores. La sonda telomérica de vertebrados (TTAGGG)n hibridó en ambos telómeros de cada cromosoma, sin señales intersticiales. Además, en este trabajo se realizó un estudio cariológico comparado de las cuatro Ensis analizadas hasta la fecha, las europeas E. minor, E. siliqua (Linné, 1758) y E. magnus Schumacher, 1817, y la americana E. directus. Las especies europeas mostraron más similitudes entre ellas que con E. directus. Además, se encontraron diferencias cariotípicas claras entre las especies morfológicamente similares E. minor y E. siliqua, en el número de pares cromosómicos telocéntricos y subtelocéntricos. En el capítulo 4.3 obtuvimos los niveles de variación genética actual de poblaciones de la navaja E. directus (Mollusca: Bivalvia: Pharidae) en los rangos de distribución nativo (América del Norte) e introducido (Europa) usando secuencias nucleares y mitocondriales. Esperábamos menor variación en el rango de distribución introducido, sobre todo considerando los frecuentes episodios de mortalidad en masa observados en Europa desde la introducción de la especie en 1978. Sin embargo, encontramos mayor variación en Europa. En este trabajo los resultados se comentaron a la luz de la posible influencia de incrementos o reducciones temporales de la variación genética, del efecto limitado de la deriva genética aleatoria y de posibles introducciones múltiples. Curiosamente, la hipótesis de las introducciones múltiples contrasta con la colonización gradual de la costa europea por parte de E. directus, pero es apoyada por la intensidad del tráfico transoceánico en el Atlántico. Por último, evidencias genéticas y morfométricas apoyaron claramente que los individuos de una población analizada de Terranova (Canadá) pertenecían a una especie nueva, desconocida hasta la fecha. Esta nueva Ensis se describió formalmente en este capítulo y fue denominada E. terranovensis n.sp. En el último capítulo (4.4) se realizó un trabajo de delimitación de especies y DNA barcoding en las Ensis del Atlántico, en el que se estudió si las morfoespecies actualmente descritas eran linajes evolutivos diferentes. En este trabajo estudiamos 109 especímenes pertenecientes a nueve especies de Ensis (todas las especies actuales del Atlántico) y en ellas analizamos la variación nucleotídica en cuatro regiones nucleares y en dos regiones mitocondriales. Los análisis filogenéticos realizados apoyan la monofilia recíproca de estas especies a cada lado del océano Atlántico. En Europa se encontraron cuatro linajes claramente diferenciados, que se correspondieron con las especies E. magnus, E. ensis (Linné, 1758), E. minor y E. siliqua, demostrándose, además, que E. minor y E. siliqua conviven en la costa NO de la Península Ibérica. Un grado de divergencia bastante relevante se apreció entre individuos de E. macha (Molina, 1792) muestreados en Chile y en Argentina, lo cual sugiere especiación incipiente. Además, se confirmó la presencia de E. directus al norte de Florida. De entre las regiones genómicas analizadas, se sugiere el fragmento de la citocromo oxidasa subunidad I para ser utilizada en identificación mediante DNA barcoding. Las contribuciones más relevantes de esta tesis son las siguientes: Se demostró que existen copias de ADN ribosomal 5S ligadas a copias de ADN pequeño nuclear U1 en los genomas de al menos 10 especies de la familia Pharidae, de cuatro géneros diferentes. Además de caracterizar, a nivel nucleotídico, ambas familias multigénicas, las secuencias obtenidas del ADN pequeño nuclear U1 son las primeras en la Clase Bivalvia. Se caracterizó por vez primera la diversidad del ADN ribosomal 5S en una escala evolutiva amplia, es decir, en 97 especies de metazoos. Se caracterizaron las especies E. minor y E. directus a nivel citogenético, obteniéndose conclusiones aplicadas a la taxonomía de estas especies, y a la organización genómica del ADN ribosomal 5S y de los genes ribosomales mayores. Se estudió la variación genética de poblaciones de E. directus en los rangos de distribución nativo e introducido, y se descrubrió y describió una nueva especie en Terranova, Canadá a la que se le llamó E. terranovensis. Se clarificó el estatus taxonómico de las especies actuales de Ensis en el Atlántico, y se definió que la región COI es adecuada para la identificación de estas especies mediante DNA barcoding