Abstract The rhizoma peanut (Arachis glabrata Benth., section Rhizomatosae) is a tetraploid perennial legume. Although several A. glabrata cultivars have been developed as forage and ornamental turf, the origin and genomic constitution of this species are still unknown. In this study, we evaluated the affinity between the genomes of A. glabrata and the probable diploid donors of the sections Rhizomatosae, Arachis, Erectoides and Procumbentes by genomic in situ hybridization (GISH). Single GISH analyses detected that species of the sections Erectoides (E2 subgenome) and Procumbentes (E3 subgenome) were the diploid species with the highest degree of genomic affinity with A. glabrata. Based on single GISH experiments and DNA sequence similarity, three species -A. duranensis, A. paraguariensis subsp. capibarensis, and A. rigonii-, which showed the most uniform and brightest hybridization patterns and lowest genetic distance, were selected as probes for double GISH experiments. Double GISH experiments showed that A. glabrata is constituted by four identical or very similar chromosome complements. In these assays, A. paraguariensis subsp. capibarensis showed the highest brightness onto A. glabrata chromosomes. Thus, our results support the autopolyploid origin of A. glabrata and show that the species with E2 subgenome are the most probable ancestors of this polyploid legume forage.

Summary Background and aims : Zephyranthes brachyandra belongs to a tribe of ornamental Amaryllidaceae native of South America, whose genera circumscription and phylogenetic relationships are still unclear. Cytologically, Z. brachyandra is a tetraploid whose chromosomes are of similar size and morphology, hindering the identification of its 2n = 24 chromosomes. The aim of this study was to investigate the stability of the many CMA+ and DAPI+ bands and the occurrence of B chromosomes by a cytomolecular approach. M&M : For this investigation we conducted a cytomolecular analysis with CMA/DAPI staining and fluorescence in situ hybridization with 5S and 35S rDNA probes, and the TTTAGGG telomeric probe. Results : In the present work, a cytomolecular analysis of Z. brachyandra, revealed a large and variable number of CMA+ and DAPI+ heterochromatic bands and 5S and 35S rDNA sites, and a regular distribution of the TTTAGGG telomeric sequences. In addition, one individual was monotrisomic with 2n = 24, and another one had a B chromosome. Both numerical and structural chromosome alterations were clearly characterized by CMA/DAPI bands and rDNA sites. Conclusions : Comparing the present data with the cytological data for other species of Zephyranthes, it becomes clear that a cytomolecular approach is fundamental to the understanding of the chromosome variation and cytotaxonomy of the group.

Resumen Introducción y objetivos : Zephyranthes brachyandra pertenece a una tribu de Amaryllidaceae ornamentales nativa de América del Sur, cuya circunscripción de géneros y relaciones filogenéticas aún no están claras. Citológicamente, Z. brachyandra es un tetraploide cuyos cromosomas son de tamaño y morfología similar, lo que dificulta la identificación de sus 2n = 24 cromosomas. El objetivo de este estudio fue investigar la estabilidad de las numerosas bandas CMA+ y DAPI+ y la aparición de cromosomas B mediante un enfoque citomolecular. M&M : Para esta investigación realizamos un análisis citomolecular con tinción CMA/ DAPI e hibridación fluorescente in situ con sondas de ADNr 5S y 35S, y la sonda telomérica TTTAGGG. Resultados : En el presente trabajo se realizaron varios análisis citomoleculares de Z. brachyandra, que revelaron un número alto y variable de bandas heterocromáticas CMA+ y DAPI+ y de sitios de ADNr 5S y 35S, además de una distribución típica de las secuencias teloméricas TTTAGGG. Además, un individuo era monotrisómico con 2n = 24 y otro tenía un cromosoma B. Las alteraciones cromosómicas tanto numéricas como estructurales se caracterizaron claramente por bandas CMA / DAPI y sitios de ADNr. Conclusión : Al comparar los datos actuales con la literatura citológica de otras especies del género Zephyranthes, queda claro que un enfoque citomolecular es fundamental para la comprensión de la variación cromosómica y la citotaxonomía del grupo.

Summary: Great efforts have been done to collect germplasm of theArachisgenus in South America, however, many regions still remain underexplored. Under the hypothesis that these regions have new and diverse populations/species ofArachis, several expeditions were carried out since 2000 in Bolivia, to increase the documentation of the genus diversity. As a first result of these explorations, a new species of sectionArachiswith B genome is formally described.Arachis inflatais closely related toA. magnaandA. ipaënsis, but it can be clearly distinguished from them, and from any other species of the genus, for having a type of fruit with a completely distinct morphology. The fruit has a smooth epicarp, but shows a bullated aspect, due to the presence of air chambers in the mesocarp.

Resumen: Se han realizado grandes esfuerzos para coleccionar germoplasma del géneroArachisen Sudamérica, sin embargo, aún quedan muchas regiones subexploradas. Bajo la hipótesis de que estas tienen poblaciones/especies nuevas y diversas deArachis, se realizaron nuevas expediciones en Bolivia a partir del año 2000 con el objetivo de incrementar la documentación de la diversidad de este género. Como primer resultado de estas exploraciones, en este trabajo se describe formalmente una nueva especie de la secciónArachisperteneciente al genoma B.Arachis inflataes una especie afín aA. magnayA. ipaënsis, aunque se distingue claramente de ellas, y de todas las demás especies del género, por presentar un tipo de fruto distinto. El mismo presenta epicarpo liso, con aspecto ampollado, debido a la presencia de cámaras de aire en el mesocarpo.

Abstract Capsicum pubescens is a cultivated hot chili pepper, consumed in Latin American cuisine as a distinctive ingredient, and popularly known as “locoto” or “rocoto”. This taxon is also an outstanding source of resistance to biotic and abiotic stresses as well as other valuable fruit traits for breeding of the worldwidely cultivated C. annuum and related species. In this study, the chromosome complement of C. pubescens cv. “locoto” (2n = 24) was deeply characterized through a sequential combination of conventional and molecular cytogenetics approaches comprising: Ag-NOR staining, heterochromatic fluorescent C-DAPI, DAPI/AMD-CMA/DA bandings, fluorescence in situ hybridization (FISH) of Capsicum-derived probes of the 5S and 18S-25S rRNA genes and different regions of spacers of the ribosomal unit, as well as telomeric probe. The markers identified were systematically combined with morphological karyotype parameters - number, size, centromeres, satellites - to produce a physical map which allowed the identification of several landmarks in each individual chromosome. The reference chromosomal map of C. pubescens here presented is the most comprehensively developed in Capsicum so far. It is envisioned that this chromosomal map will serve as a reference framework for the upcoming sequencing projects and as starting point to assist future genetic mapping of important agronomic traits.

Resumen Capsicum pubescens es un ají picante cultivado, consumido en Latinoamérica como ingrediente distintivo y conocido como “locoto” o “rocoto”. Este taxón es un recurso sobresaliente por sus características de resistencia y tolerancia a estrés biótico/abiótico, y del fruto, que pueden ser introgresadas en programas de mejoramiento de C. annuum y especies relacionadas. En este estudio, se caracterizó detalladamente el complemento cromosómico de C. pubescens cv. “locoto” (2n = 24) a través de una combinación secuencial de herramientas citogenéticas convencionales y moleculares que incluyeron: tinción Ag-NOR, bandeos heterocromáticos fluorescentes C-DAPI, DAPI/AMD-CMA/DA, e hibridación in situ fluorescente (FISH) con sondas de genes de rRNA 5S y 18S-25S y diferentes regiones espaciadoras de la unidad ribosomal derivadas de Capsicum, y sonda telomérica. Los marcadores identificados se combinaron sistemáticamente con parámetros morfométricos del cariotipo - número, tamaño, centrómeros, satélites - para producir un mapa físico que permitió la identificación de varios caracteres de referencia en cada cromosoma individual. El mapa cromosómico de referencia de C. pubescens aquí presentado es el más completo hasta ahora desarrollado en Capsicum. Se prevé que este mapa cromosómico sirva de referencia para próximos proyectos de secuenciación y como punto de partida para asistir al mapeo genético de caracteres agronómicos importantes.

Abstract Peanut (Arachis hypogaea L.) is a tetraploid species with an A and B genome, while the majority of wild Arachis species are diploid with distinct genomes. In pre-breeding programs, one way to introgress interesting wild genes into peanut is by producing amphidiploids. This study aimed at the hybridization between distinct amphidiploids and their characterization, to combine high crossability with peanut, observed in some amphidiploids, with high pest and disease resistances observed in others. These new hybrids were called complex hybrids. Four amphidiploids previously obtained were crossed at four different combinations, and the derived complex hybrids were crossed with four peanut cultivars. Morphological, reproductive, chromosome complement, molecular markers for hybrid identification, phytopatological, and entomological characterizations were performed on the complex hybrids. All cross combinations resulted in complex hybrids. One complete complement of each diploid progenitor was confirmed in each hybrid. Plants of six distinct hybrid combinations were obtained between the complex hybrids and peanut. Based on morphological characterization, differences among progenies from distinct cross combinations were observed. Complex hybrids were considered more resistant to all diseases and pests than peanut cultivars. The simultaneous introgression of genes from four wild Arachis species into peanut was possible through the development of complex hybrids.

The cytogenetic characterization of Arachis species is useful for assessing the genomes present in this genus, for establishing the relationship among their representatives and for understanding the variability in the available germplasm. In this study, we used fluorescence in situ hybridization (FISH) to examine the distribution patterns of heterochromatin and rDNA genes in 12 Brazilian accessions of five species of the taxonomic section Arachis. The heterochromatic pattern varied considerably among the species: complements with centromeric bands in all of the chromosomes (A. hoehnei) and complements completely devoid of heterochromatin (A. gregoryi, A. magna) were observed. The number of 45S rDNA loci ranged from two (A. gregoryi) to eight (A. glandulifera), while the number of 5S rDNA loci was more conserved and varied from two (in most species) to four (A. hoehnei). In some species one pair of 5S rDNA loci was observed adjacent to 45S rDNA loci. The chromosomal markers revealed polymorphism in the three species with more than one accession (A. gregoryi, A. magna and A. valida) that were tested. The previous genome assignment for each of the species studied was confirmed, except for A. hoehnei. The intraspecific variability observed here suggests that an exhaustive cytogenetic and taxonomic analysis is still needed for some Arachis species.

Saint Louis encephalitis virus (SLE) is a mosquito borne disease. Only a small proportion of cases progress to severe clinical forms. There have been few reports on HIV-infected patients and the relationship between immunodeficiency and the course of the disease remains unclear. Herein we describe two cases of SLE in HIV-1-infected patients in Buenos Aires city.

We describe the epidemiological and clinical findings of 13 patients with diagnosis of infection by St. Louis encephalitis virus, transmitted between January and March 2010 in Buenos Aires Metropolitan Area (AMBA). Thirteen patients, average age 38 years, had an acute onset with hyperthermia and headache. Between days two and ten of the onset of symptoms, 7/13 patients had signs and symptoms of neurological involvement. This was characterized by meningitis without encephalic sings in 1/7 and in 6/7 the most frequent findings were: stiff neck, disorientation, photophobia, confusion and language impairment. Two MR and one EEG revealed signs of involvement of temporal lobes. The cerebrospinal fluid showed pleocytosis with predominance of mononuclear cells, normal glucose and moderately elevated protein. There were no fatalities cases. In 6/13 patients the initial clinical suspicion was dengue. As epidemic virus circulation had not been previously reported in the AMBA it can be considered an outbreak of St. Luis encephalitis for the spatial and temporal clustering of cases.

Se describen los hallazgos epidemiológicos y clínicos de 13 enfermos con diagnóstico de infección por virus de la encefalitis de San Luis, con transmisión entre enero y marzo de 2010, en el Area Metropolitana Buenos Aires (AMBA). Los 13 enfermos, promedio de edad 38 años, tuvieron un comienzo agudo caracterizado por hipertermia y cefalea. Entre los días dos y diez de iniciados los síntomas, 7/13 enfermos tuvieron signos y síntomas de compromiso neurológico caracterizado por meningitis sin signos encefálicos en 1/7. En 6/7 los hallazgos más frecuentes fueron: rigidez de nuca, desorientación temporoespacial, fotofobia, confusión y alteración del lenguaje. Dos resonancias magnéticas y un electroencefalograma presentaron signos de afectación de lóbulos temporales. El líquido cefalorraquídeo se caracterizó por pleocitosis con predominio de células mononucleadas, glucorraquia normal y discreto aumento de proteínas. No hubo casos fatales. En 6/13 pacientes la sospecha clínica inicial fue dengue. Por la agrupación espacial y temporal de los casos puede considerarse un brote epidémico, el primero conocido en el AMBA, ya que no se había notificado previamente la circulación epidémica del virus.

The karyotype structure of Arachis trinitensis was studied by conventional Feulgen staining, CMA/DAPI banding and rDNA loci detection by fluorescence in situ hybridization (FISH) in order to establish its genome status and test the hypothesis that this species is a genome donor of cultivated peanut. Conventional staining revealed that the karyotype lacked the small "A chromosomes" characteristic of the A genome. In agreement with this, chromosomal banding showed that none of the chromosomes had the large centromeric bands expected for A chromosomes. FISH revealed one pair each of 5S and 45S rDNA loci, located in different medium-sized metacentric chromosomes. Collectively, these results suggest that A. trinitensis should be removed from the A genome and be considered as a B or non-A genome species. The pattern of heterochromatic bands and rDNA loci of A. trinitensis differ markedly from any of the complements of A. hypogaea, suggesting that the former species is unlikely to be one of the wild diploid progenitors of the latter.

Turnera sidoides is a polyploid complex (x= 7) of dystilous perennial rhizomatous herbs that includes five subspecies. Owing to the high frequency of polyploids within the complex, ongoing studies in T. sidoides are focused on the understanding of the mechanisms involved in the origin and the establishment of polyploids in order to provide evidences of the evolutionary patterns in T. sidoides. In this report we investigate the occurrence and frequency of unreduced microspores in diploid plants from nine natural populations of four subspecies of T. sidoides by analyzing the range of variation of pollen volume and the constitution of the sporads. The results obtained showed that almost all the populations studied produced 2x and 4x microspores, both in short and long-styled floral morphs. Furthermore, the analysis performed provided evidences that support the hypothesis of sexual polyploidization as one of the mechanisms involved in the origin of polyploids within this species complex. The role of unreduced pollen in the establishment and persistence of newly formed polyploids within diploid populations of T. sidoides is also discussed.

Turnera sidoides es un complejo poliploide (x= 7) de hierbas rizomatosas, perennes y distilas, que incluye cinco subespecies. Debido a la alta frecuencia de poliploides en el complejo, los estudios que se llevan a cabo actualmente están orientados a la comprensión de los mecanismos involucrados en el origen y establecimiento de los mismos a fin de proveer evidencias sobre los patrones evolutivos que operan en T. sidoides. En este trabajo se investiga la producción y frecuencia de microsporas no reducidas en plantas diploides provenientes de nueve poblaciones naturales pertenecientes a cuatro subespecies de T. sidoides, mediante el análisis del rango de variación del tamaño del polen y de la constitución de las esporadas. Los resultados obtenidos mostraron que casi todas las poblaciones estudiadas producen microsporas 2x y 4x, tanto en las flores brevistilas como en las longistilas. Asimismo, el análisis realizado aportó evidencias que sustentan la poliploidización sexual como uno de los mecanismos involucrados en el origen de los poliploides dentro del complejo. Se discute, además, el papel del polen no reducido en el establecimiento y la persistencia de los poliploides formados de novo en las poblaciones diploides de la especie.

Chromosome markers were developed for Arachis glandulifera using fluorescence in situ hybridization (FISH) of the 5S and 45S rRNA genes and heterochromatic 4'-6-diamidino-2-phenylindole (DAPI) positive bands. We used chromosome landmarks identified by these markers to construct the first Arachis species ideogram in which all the homologous chromosomes were precisely identified. The comparison of this ideogram with those published for other Arachis species revealed very poor homeologies with all A and B genome taxa, supporting the special genome constitution (D genome) of A. glandulifera. Genomic affinities were further investigated by dot blot hybridization of biotinylated A. glandulifera total DNA to DNA from several Arachis species, the results indicating that the D genome is positioned between the A and B genomes.

No presente estudo, a PCR foi utilizada para detectar leptospiras em urina humana. Diversas abordagens para processamento de amostra foram avaliadas para otimizar a detecção de leptospiras em urina misturada com esta bactéria. Além disso, algumas mudanças na composição da mistura de reação foram analisadas. Não se observou amplificação em urina ácida, conseqüentemente, a neutralização da amostra imediatamente após a coleta é fortemente recomendada. PBS apresentou melhores resultados que Tris ou NaOH como reagentes neutralizadores. Congelamento e descongelamento de amostras antes do processamento produziram resultados negativos. Eliminação de células epiteliais, leucócitos e cristais por centrifugação a 3.000rpm, à temperatura ambiente, aumentou a sensibilidade. Ademais, ambas, a etapa de lavagem após a coleta de leptospiras por centrifugação e a inclusão de albumina de soro bovino a 0,1% na mistura de reação minimizaram a interferência de outros compostos inibidores. Essas modificações contribuíram para melhorar a detecção de Leptospira em urina através da PCR.

In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.

Foram realizadas mudanças nas relações hídricas celulares durante a fase de precultura afim de se entender os mecanismos de endurecimento celular pela vitrificação durante a criopreservação. Suspensões celulares de laranja e cenoura foram cultivadas em meio contendo 0,4 M sacarose (psiw = -1,45 MPa) e 5 mgL-1 de ABA (MT psiw = -0,73 MPa e MS psiw = -0,48 MPa). A precultura nesse meio não promoveu um significante decréscimo na viabilidade celular, entretanto, aumentou a sobrevivência das células a exposição a PVS2 e a nitrogênio líquido. Quando as células foram incubadas em meio contendo 0,4 M de sacarose, o psiw celular decresceu rapidamente igualando-se ao psiw do meio, enquanto o turgor foi também severamente reduzido ou desapareceu. Ao mesmo tempo o psis começou a decrescer e após 48h de tratamento em meio com baixo psiw , o turgor foi recuperado pela osmoregulação até atingir o mesmo valor que as células tinham no início do experimento. A precultura em meio contendo ABA também causou um decréscimo no psiw celular em menor intensidade que aquela verificada quando a precultura foi realizada em meio com baixo psiw. O aumento na sobrevivência celular ao choque osmótico e vitrificação não pode ser atribuído unicamente ao acúmulo quantitativo de solutos uma vez que a precultura aumentou significativamente a sobrevivência com o pré-tramento com PVS2 e exposição a nitrogênio líquido. Estes resultados sugerem que o acúmulo qualitativo de determinados solutos poderia ser o evento responsável pela aumento a tolerância a exposição ao PVS2 e ao nitrogênio líquido.

Changes in cell water relations during precultures were followed in an attempt to understand the mechanism of cell hardening for cryopreservation by vitrification. Medium containing 0.4 M sucrose (psiw=-1.45 MPa) and containing 5 mg L-1 of ABA (MT psiw=-0.73 MPa and MS psiw=-0.48 MPa) were used to harden cell suspensions of orange and carrot. Preculture in these medium did not cause a significant decrease of cell viability, however, it improved the cell survival to PVS2 and liquid nitrogen expositions. When cells were inoculated into medium containing 0.4 M sucrose, the cell psiw decreased rapidly until reach the medium psiw , and turgor was also severely reduced or disappeared. Subsequently, cell psis began to decrease and after 48 h of treatment in low water potential medium, turgor was recovered by osmoregulation to almost the same values that cells had at the beginning of the experiment. Preculture in medium containing ABA also caused a decrease of cell psis but in much less extent than the preculture in low water potential medium. Since both precultures improved significantly the survival to PVS2 pretreatment and to LN exposure, this increment of survival to osmotic shock and vitrification can not be attribute solely to quantitative solute accumulation. The results suggest that qualitative accumulation of determined solutes would be the events which contributed for the improvement of tolerance to PVS2 exposure and thence tolerance to liquid nitrogen.