Identificación molecular y diversidad genética de las especies de nematodos Globodera rostochiensis y G. pallida en regiones productoras de papa de Guatemala / Molecular identification and genetic diversity of the species of nematodesGlobodera rostochiensisandG. pallidain potato growing regions of Guatemala

En Guatemala, la producción del cultivo de papa se ve afectada por los nematodos Globodera rostochiensis y Globo-dera pallida. La capacidad de ambas especies para formar quistes complica su control y provoca el aumento de sus poblaciones. En Guatemala se reporta la presencia de ambas especies de nematodos por identificación morfológica, sin embargo, no se ha realizado una confirmación molecular. Este es el primer estudio para validar la presencia de ambas especies de nematodos por PCR múltiple y la determinación de la diversidad y estructura genética de las poblaciones utilizando marcadores moleculares. Se realizaron muestreos en cuatro departamentos productores de papa del país. La identificación por PCR se realizó con el cebador común ITS5 y los cebadores PITSr3 específico para G. rostochiensisy PITSp4 para G. pallida. La caracterización molecular se realizó con el marcador AFLP. Se confirmó la presencia de las dos especies de nematodos en los cuatro departamentos. Los índices de diversidad Shannon y heterocigosidad esperada revelaron mayor diversidad genética en G. rostochiensis (H = 0.311, He = 0.301) que en G. pallida (H = 0.035, He = 0.223). Los métodos NJ, DAPC y PCA exhibieron una débil estructura entre las poblaciones de ambas especies de nematodos. Los resultados sugieren un patrón de dispersión desde Quetzaltenango hacia el resto del país, atribuido a la comercialización de semilla contaminada con nematodos. Se sugiere promover programas de socialización sobre los beneficios del uso de semilla certificada, además de constantes monitoreos moleculares para un diagnóstico certero de ambas especies de nematodos.

In Guatemala, potato crop production is affected by the nematodes Globodera rostochiensis and Globodera pallida. The ability of both species to form cysts complicates their control and causes an increase in their populations. In Guatemala, both species of nematodes have been reported by morphological identification; however, molecular confirmation has not been carried out. It is the first study to validate the presence of both nematode species by multiplex PCR and determine the diversity and genetic structure of the populations using molecular markers. Sampling was carried out in four pota-to-producing departments of the country. PCR identification was performed with the common primer ITS5 and the primers PITSr3 specific for G. rostochiensis and PITSp4 for G. pallida. We performed molecular characterization with the AFLP marker. We confirmed the presence of the two nematode species in the four departments. Shannon diversity and expected heterozygosity indices revealed higher genetic diversity in G. rostochiensis (H = 0.311, He = 0.301) than in G. pallida (H = 0.035, He = 0.223). The NJ, DAPC, and PCA methods exhibited weak structure among populations of both nematode species. The results suggest a dispersal pattern from Quetzaltenango to the rest of the country, attributed to the commer-cialization of seed contaminated with nematodes. We suggest promoting socialization programs on the benefits of using certified seeds and constant molecular monitoring for an accurate diagnosis of both species of nematodes.

Bases moleculares de la resistencia a meticilina en Staphylococcus aureus / Molecular basis of methicillin-resistance in Staphylococcus aureus

Resumen Desde el inicio de la era antimicrobiana se han ido seleccionando gradualmente cepas de Staphylococcus aureus resistentes a antimicrobianos de amplio uso clínico. Es así como en 1960 se describen en Inglaterra las primeras cepas resistentes a meticilina, y algunos años después son informadas en hospitales de Chile. Actualmente, S. aureus resistente a penicilinas antiestafilocóccicas es endémico en los hospitales de nuestro país y del mundo, siendo responsable de una alta morbimortalidad. La resistencia es mediada habitualmente por la síntesis de una nueva transpeptidasa, denominada PBP2a o PBP2' que posee menos afinidad por el β-lactámico, y es la que mantiene la síntesis de peptidoglicano en presencia del antimicrobiano. Esta nueva enzima se encuentra codificada en el gen mecA, a su vez inserto en un cassette cromosomal con estructura de isla genómica, de los cuales existen varios tipos y subtipos. La resistencia a meticilina se encuentra regulada, principalmente, por un mecanismo de inducción de la expresión del gen en presencia del β-lactámico, a través de un receptor de membrana y un represor de la expresión. Si bien se han descrito mecanismos generadores de resistencia a meticilina mec independientes, son categóricamente menos frecuentes.

Staphylococcus aureus isolates resistant to several antimicrobials have been gradually emerged since the beginning of the antibiotic era. Consequently, the first isolation of methicillin-resistant S. aureus occurred in 1960, which was described a few years later in Chile. Currently, S. aureus resistant to antistaphylococcal penicillins is endemic in Chilean hospitals and worldwide, being responsible for a high burden of morbidity and mortality. This resistance is mediated by the expression of a new transpeptidase, named PBP2a or PBP2', which possesses lower affinity for the β-lactam antibiotics, allowing the synthesis of peptidoglycan even in presence of these antimicrobial agents. This new enzyme is encoded by the mecA gene, itself embedded in a chromosomal cassette displaying a genomic island structure, of which there are several types and subtypes. Methicillin resistance is mainly regulated by an induction mechanism activated in the presence of β-lactams, through a membrane receptor and a repressor of the gene expression. Although mec-independent methicillin resistance mechanisms have been described, they are clearly infrequent.

Effect of clonal reproduction on genetic structure in Pentaclethra macroloba (Fabaceae: Mimosoideae) / Efecto de la reproducción clonal en la estructura genética de Pentaclethra macroloba (Fabaceae: Mimosoideae)

The existence of monodominant forests on well-drained soils in tropical regions has been widely reported. Such forests most likely result from a combination of both ecological and evolutionary factors. Under conditions of high seed and seedling mortality, vegetative reproduction could create a reproductive advantage leading to forest dominance, and profoundly affect the distribution of genetic variation in a clonal species. We investigated these effects in a low diversity forest site in Northeastern Costa Rica dominated by the species Pentaclethra macroloba, which sprouts from the root mass of fallen trees and from snapped trunks. We examined the population structure of juvenile P. macroloba growing in different soil types and across an elevational gradient. Using seven molecular markers, we genotyped 173 juvenile P. macroloba from 18 plots (six plots in seasonally inundated swamps, and 12 plots in upland non-swamp) spanning 50-300m in elevation at La Selva Biological Station and the adjacent Reserva Ecológica Bijagual in Northeastern Costa Rica. We answered two specific questions: (1) How extensive is clonal reproduction? and (2) what is the distribution of genetic diversity and structure?. We found that clonal reproduction occurred exclusively within inundated swamp areas. However, there was no significant difference between genetic diversity measures in swamp and non-swamp plots, which were both generally low when compared with other tropical forest species. Genetic structure was significant across all plots (F ST=0.109). However, genetic structure among swamp plots (F ST=0.128) was higher than among non-swamp upland plots (F ST=0.093). Additionally, spatial autocorrelation among individuals within non-swamp upland plots was significant from the 25 to 100m spatial scale, but not within swamp plots. The degree of overall genetic structure we found in P. macroloba is high for a tropical forest tree. The incidence of clonal reproduction is a contributing factor in genetic differentiation, but the high structure among plots without clonal reproduction indicates that other factors contribute as well.

La existencia de bosques monodominantes sobre suelos bien drenados en regiones tropicales ha sido ampliamente reportada. Investigaciones recientes han sugerido que tales bosques son probablemente resultado de una combinación de factores ecológicos y evolutivos. Bajo condiciones de alta mortalidad de semillas y plántulas, la reproducción vegetativa podría crear una ventaja reproductiva llevando a la dominancia del bosque, pero también podría afectar profundamente la distribución de la variación genética en especies clonales. Investigamos estos efectos en un sitio de bosque con baja diversidad de especies en el Noreste de Costa Rica que es ampliamente dominado por la especie Pentaclethra macroloba, la cual retoña de la masa de raíces de árboles caídos y de troncos partidos. Examinamos la estructura poblacional de individuos juveniles de P. macroloba creciendo en diferentes tipos de suelo y a través de un gradiente de altitud. Utilizamos siete marcadores moleculares, genotipamos 173 Pentaclethra macroloba de 18 parcelas (seis en ciénagas y 12 en ambientes no cenagosos) ubicados en un gradiente de elevación entre 50-300m en las reservas adyacentes: Reserva Biológica Bijagual y Estación Biológica La Selva, en el centro de Costa Rica. Abordamos dos preguntas específicas: (1) ¿Qué tan extensa es la reproducción clonal? y (2) ¿Cuál es la distribución de diversidad y estructura genética? Encontramos que la reproducción clonal ocurrió exclusivamente dentro de áreas cenagosas inundadas. La estructura genética fue significativa en todas las parcelas (F ST=0.109). Observamos una estructura genética más alta entre poblaciones juveniles dentro de las ciénagas (F ST=0.128) comparada con poblaciones no cenagosas en parcelas a mayor altura (F ST=0.093), con mayor autocorrelación espacial en sitios no cenagosos en el intervalo entre 25 y 100m. La presencia de reproducción clonal no afectó significativamente las medidas de diversidad entre las dos áreas, que fueron generalmente bajas comparadas con otras especies de bosque tropical. El alto grado de estructura genética en general es novedoso para un árbol de bosque tropical. La incidencia de reproducción clonal es un factor que contribuye en la diferenciación genética, pero la alta estructura en parcelas sin reproducción clonal indica que otros factores están contribuyendo también.

Diversidade genética de populações naturais de pariparoba [Pothomorphe umbellata (L. ) Miq.] por RAPD / Genetic diversity of natural populations of pariparoba [Pothomorphe umbellata (L. ) Miq.] according to RAPD analysis

O objetivo desse trabalho foi analisar a estrutura genética de populações de Pothomorphe umbellata (L.) Miq. com base em polimorfismos moleculares do tipo RAPD. Foram analisadas quatro populações naturais do estado de São Paulo (Jacareí, Jundiaí, Piquete e Ubatuba) e uma população do Paraná (Adrianópolis). Foram identificados 25 locos polimórficos (96,15%). Elevados índices de diversidade genética foram observados dentro das populações (Hs = 0,2220). Verificou-se que 65,33% da variabilidade genética total encontra-se dentro das populações e 34,67% entre as populações; índices estes, obtidos a partir do cálculo da divergência genética (G ST = 0,3467). Os resultados sugerem que essas populações possuem níveis elevados de variabilidade genética, a qual pode ser fortemente impactada pela ação humana.

The aim of this study was to analyze the genetic structure of populations of Pothomorphe umbellata (L.) Miq. based on RAPD molecular polymorphisms. Analysis included four natural populations from São Paulo State (Jacareí, Jundiaí, Piquete, Ubatuba) and one population from Paraná State (Adrianópolis). Twenty-five polymorphic loci (96.15%) were identified. There were high levels of genetic diversity within populations (Hs = 0.2220). Of the total genetic variability, 65.33% is within populations and 34.67% among populations (G ST = 0.3467). Results suggest that these populations have high levels of genetic variability, which can be strongly impacted by human action.

Estudo da função do gene kerV de Pseudomonas aeruginosa / Function of Pseudomonas aeruginosa kerV gene

P. aeruginosa PA14 é uma linhagem isolada de queimadura que apresenta vários fatores de patogenicidade comuns no quadro de infecção de hospedeiros filogeneticamente distintos (plantas, mamíferos ou invertebrados). O gene kerV foi revelado numa busca por mutantes atenuados em virulência em uma biblioteca de mutantes por transposons da linhagem PA14 (Rahme et al., 1997). A caracterização da linhagem D12, mutante em kerV, confirmou sua virulência atenuada (Apidianakis et al., 2005 e An et al., 2009) e resultados do transcriptoma mostraram alteração na expressão de mais de 500 genes, sendo alguns relacionados com o sistema de "quorum sensing" (Rahme et al, dados não publicados). O gene kerV está próximo à montante ao gene gloB, envolvido em detoxificação de metilglioxal, e à jusante aos genes rnhA e dnaQ, que codificam proteínas envolvidas na replicação e reparo do DNA. Este trabalho teve como objetivo estudar a função molecular do produto de kerV e a expressão dos genes do lócus kerV-rnhA-dnaQ. Análises de bioinformática indicam que a proteína KerV é uma metiltransferase dependente de S-adenosil-metionina (SAM), apresentando um domínio conservado de ligação a SAM e uma arquitetura de domínio compatível com a organização em fitas-beta e hélices-alfa alternadas descritas para a família das metiltransferases dependentes de SAM. Ela não apresenta outros domínios conservados que indiquem seu substrato de metilação. A expressão heteróloga desta proteína em E. coli, mostrou que ela é expressa de maneira parcialmente solúvel quando co-expressa com as chaperoninas GroEL/GroES em baixas temperaturas ou quando fusionada a MBP ou GST. A purificação desta proteína mostra que ela é co-eluída com a chaperonina GroEL sugerindo que para atingir sua conformação nativa ela necessita dessas proteínas acessórias. MBP-KerV purificado foi usado para ensaios "in vitro" de atividade de metiltransferase e ligação a SAM, que não foram conclusivos, pois não há certeza do seu ...

P. aeruginosa PA14 is a burn isolate multi-host pathogen strain. The screening for virulence attenuated mutants in a PA14 transposon mutant library revealed the kerV gene (Rahme et al., 1997). The characterization of D12 strain, a kerV mutant, confirmed the attenuated virulence phenotype (Apidianakis et al., 2005 and An et al., 2009) and transcriptome analysis showed the expression of more than 500 genes are affected in D12, some of these genes are related with quorum sensing (Rahme et al, unpublished data). kerV is upstream of the gloB gene, related with methylglioxal detoxification and downstream of the rnhA and dnaQ genes, both related with DNA replication and repair. The purpose of this work was to study the molecular function of KerV product and the expression of kerV-rnhA-dnaQ locus. Bioinformatics analysis indicated that KerV is a SAM dependent methyltransferase that have a conserved SAM binding domain with architecture compatible with classic alternating β-stranded and α-helical regions. KerV does not show any other conserved motif that could indicate its methylation substrate. Heterologous expression in E. coli showed that KerV is partially soluble only when co-expressed with GroeL/GroES chaperones at low temperatures or when KerV is in fusion with MBP or GST tag. During the purification process KerV was copurified with GroEL chaperone suggesting that this association may be required for the correct folding of KerV. Methyltransferase activity and SAM binding assays were done with purified MBPKerV and the results were not conclusive since the proper conformation of MBP-KerV cannot be verified. Yeast two-hybrid assays indicated that RNaseH and DnaQ are not interaction partners of KerV, suggesting that their functions are not directly related. The mutation frequency of D12 strain increased only about four times in relation to PA14, suggesting that KerV is not directly involved with DNA mismatch repair. The assays to detect methylation...

Genetic population structure of an anchialine shrimp, Metabetaeus lohena (Crustacea: Alpheidae), in the Hawaiian Islands

Anchialine habitats in the Hawaiian Islands, characterized as coastal bodies of land-locked salt or brackish water that fluctuate with the tides due to subterranean connections, are the only ecosystems of this type found within the United States. These habitats are currently subject to anthropogenic impacts tha t threaten their future existence. Previous research has shown strong genetic population structure of an endemic atyid shrimp, Halocaridina rubra, in these habitats. The native alpheid shrimp, Metabetaeus lohena, whose known range entirely overlaps that of H. rubra, has feeding and reproductive behaviors that are biologically distinct from H. rubra. Its historic scarcity and status as a candidate for the US Fish and Wildlife Department’s Endangered Species List, make M. lohena an ideal species to compare against the known genetic structure of H. rubra. We investigated the population structure of this native anchialine shrimp to test the hypothesis that genetic population structure differs between the two shrimp species and that M. lohena is genetically unstructured across its range. A survey of 605 bp of the mitochondrial cytochrome c oxidase subunit I (COI) gene from 127 individuals collected at 7 sites spanning the islands of O’ahu, Maui and Hawaii revealed 43 haplotypes. The most common haplotype was represented in similar proportions from all sites sampled, accounting for 44% of the surveyed sequences. Analyses of molecular variation (AMOVA), pairwise ΦST values, Bayesian estimates of migration (M), Mantel tests and Nested Clade Analyses (NCAs) all failed to reveal evidence of major barriers to gene flow among most populations separated by inter-island channels. This lack of genetic structure in M. lohena is found to be in stark contrast with the highly structured population of H. rubra, and may be attributed to oceanic dispersal strategies and/or a recent introduction to the Hawaiian Islands. Rev. Biol. Trop. 58 (1): 159-170. Epub 2010 March 01.

Los hábitats de los alfeidos de las islas de Hawaii, se caracterizan por ser zonas cerradas de aguas saladas o salobres, que fluctúan con las mareas, debido a las conexiones subterráneas, son los únicos ecosistemas de este tipo que se encuentran en Estados Unidos. Estos hábitats actualmente están sujetos a impactos antropogénicos que amenazan su existencia futura. La investigación anterior ha demostrado una fuerte estructura genética de una población de camarones atíidos endémicos, Halocaridina rubra, en estos hábitats. El camarón alfeido nativo, Metabetaeus lohena, cuya área de distribución conocida se superpone totalmente con la de H. rubra, tiene comportamientos alimenticios y reproductivos que son biológicamente diferentes a los de H. rubra. Su escasez histórica y su condición de candidato para aparecer en la Lista de Especies en Peligro del Departamento de Pesca y Vida Silvestre de Estados Unidos, hace de M. lohena una especie ideal para comparar su estructura genética con la de H. rubra. Se investigó la estructura de la población de este camarón alfeido nativo para probar la hipótesis que la estructura genética de la población difiere entre las dos especies y que la de M. lohena está genéticamente no estructurada en todo su ámbito. El análisis de 605 pb de la oxidasa mitocondrial citocromo c subunidad I (COI) de genes de 127 individuos recolectados en 7 sitios que abarcan las islas de Oahu, Maui y Hawaii reveló 43 haplotipos. El haplotipo más común fue representado en proporciones similares en todos los sitios incluidos en la muestra, de acuerdo al 44% de las secuencias estudiadas. El análisis de variación molecular (AMOVA), los valores de ΦST pareados, la estimación bayesiana de la migración (M), las pruebas de Mantel y los Análisis Cladísticos no pudieron revelar la existencia de importantes barreras al flujo genético entre las poblaciones más separadas por los canales entre las islas. La falta de estructura genética en M. lohena contrasta con la muy estructurada población de H. rubra, y puede ser atribuida a las estrategias de dispersión oceánica y/o una introducción reciente en las islas hawaianas.

Genetic structure of Aedes aegypti in the city of Córdoba (Argentina), a recently reinfested area

To understand the transmission of a vector-borne disease, knowledge of the magnitude of dispersal among vector populations is essential because of its influence on pathogen transfer. The principal vector of dengue, the most common arboviral disease in the world, is the mosquito Aedes aegypti (L.). This tropical and subtropical species is native to Africa but has dispersed worldwide since the XV century. In Argentina, the species was declared eradicated in 1963, but has reinfested the country in recent years. In the present work, we used RAPD-PCR markers to assess the levels of genetic variability and differentiation among populations of Ae. aegypti (the vector of dengue and yellow fever) in Córdoba, the second largest city in Argentina. We detected similar levels of genetic variability (He between 0.351-0.404) across samples and significant genetic differentiation between most population pairs within the city (F ST between 0.0013-0.0253). Genetic distances indicate that there are three distinct groups, formed predominantly by populations that are connected by, or near, main roads. This suggests that, in addition to other factors such as availability of oviposition sites or step-by-step migration, passive transport plays an important role in gene flow within the city.

The impact of local extinction on genetic structure of wild populations of lima beans (Phaseolus lunatus) in the Central Valley of Costa Rica: consequences for the conservation of plant genetic resources

Plant populations may experience local extinction and at the same time new populations may appear in nearby suitable locations. Species may also colonize the same site on multiple occasions. Here, we examined the impact of local extinction and recolonization on the genetic structure of wild populations of lima beans (Phaseolus lunatus) in the Central valley of Costa Rica. We compared genetic diversity from the samples taken from the populations before and after extinction at 13 locations using microsatellite markers. Locations were classified according to the occurrence of extinction episodes during the previous five years into three groups: 1) populations that experienced extinction for more than one year, and were later recolonized (recolonized), 2) populations that did not experience local extinction (control), and 3) populations that did not experience local extinction during the study, but were cut to experimentally simulate extinction (experimental). Our data did not show a clear tendency in variation in allele frequencies, expected heterozygosity, and effective number of alleles within and between groups of populations. However, we found that the level of genetic differentiation between samples collected at different times at the same location was different in the three groups of populations. Recolonized locations showed the highest level of genetic differentiation (mean Fst= 0.2769), followed by control locations (mean Fst= 0.0576) and experimental locations (mean Fst= 0.0189). Similar findings were observed for Nei’s genetic distance between samples (di,j= 0.1786, 0.0400, and 0.0037, respectively). Our results indicate that genetic change in lima beans depends on the duration and frequency of local extinction episodes. These findings also showed that control populations are not in equilibrium. Implications of these results for the establishment of conservation strategies of genetic resources of lima beans are discussed. Rev. Biol. Trop. 56 (3): 1023-1041. Epub 2008 September 30.

Las poblaciones de plantas pueden experimentar extinción local, y al mismo tiempo, pueden surgir a sus alrededores nuevas poblaciones. Algunas especies pueden colonizar el mismo sitio en múltiples ocasiones. Aquí examinamos el impacto de la extinción local y recolonización en la estructura genética de poblaciones silvestres del frijol lima (Phaseolus lunatus) en el valle Central de Costa Rica. Comparamos la diversidad genética de muestras tomadas en poblaciones, antes y después de la extinción, en 13 sitios, usando marcadores de microsatélite. Según los episodios de extinción durante los cinco años previos, clasificamos los sitios así: 1) poblaciones que han experimentado extinción por más de un año, y después han recolonizado (recolonizado), 2) poblaciones que no han experimentado extinción local (control), y 3) poblaciones que no han experimentado extinción local durante el estudio, pero fueron cortadas experimentalmente, simulando una extinción (experimental). Nuestros datos no mostraron una clara tendencia en la variación de las frecuencias alélicas, heterozigosidad, o número efectivo de alelos en y entre grupos de poblaciones. Los niveles de diferenciación genética entre muestras recolectadas en diferentes momentos en el mismo sitio fueron diferentes en los tres grupos de poblaciones. Los sitios recolonizados mostraron el mayor nivel de diferenciación genética (Fst = 0.2769), seguidos de los sitios control (Fst= 0.0576) y sitios experimentales (Fst= 0.0189). Obtuvimos resultados similares en la distancia genética Neis entre muestras (d i,j = 0.1786, 0.0400, y 0.0037, respectivamente). Los cambios genéticos en los frijoles lima dependen de la duración y frecuencia de los episodios de extinción local. Las poblaciones "control" no están en equilibrio. Las implicaciones de estos resultados para el establecimiento de estrategias de conservación de los recursos genéticos de habas se encuentran en discusión.

Estudo da subdivisão genética da raça Mangalarga Marchador / Study on genetic subdivision of the Mangalarga Marchador horse breed

Foram analisadas as informações de 233.214 animais inscritos no arquivo zootécnico da Associação Brasileira dos Criadores do Cavalo Mangalarga Marchador, descendentes de 16 ancestrais com contribuição genética mínima de até 1 por cento para a população atual. Análises de componentes principais foram feitas com o intuito de agrupar animais geneticamente semelhantes e o de avaliar a subdivisão da raça em famílias ou grupos genéticos distintos. A média do coeficiente de parentesco entre animais da atual população e os ancestrais de maior contribuição genética variou de 4,7 por cento, para a égua Herdade Alteza, a 0,7 por cento, para o garanhão Tabatinga Fanfarra. A atual população da raça Mangalarga Marchador é constituída, em ordem de importância, por cinco grupos genéticos descendentes dos animais Herdade Alteza e Seta Caxias, Providência Itu e Tabatinga Predileto, Abaíba Marengo, Tabatinga Cossaco e Angaí Miron.