Caracterización y evaluación preliminar de quince clones de Ñame dioscorea sp.

Se caracterizaron y evaluaron quince accesiones de Ñame (Dioscorea sp) provenientes de ocho países de la cuenca del Caribe. El ensayo se estableció en el programa de Recursos Genéticos Nicaragüenses (REGEN). Del Instituto Superior de Ciencias Agropecuarias (ISCA), Managua. Utilizando un arreglo fr bloques al azar con dos repeticiones. Se observaron 89 descriptores, entre los cuales se seleccionaron para análisis 42 cualitativos y 23 cuantitativos. Los datos se sometieron a análisis de agrupamiento por encadenamiento simple: El fonograma de descriptores cualitativos determino la experiencia de dos especies: D alata y D. Trífida mientras que dos clones que se sospecha eran duplicados por cercanía geográfica, presentan alta diferenciación: Del análisis muy similar al patrón presentado por el total de diez descriptores de color: sugiriendo la importancia de los primeros en la diferenciación de accesiones. El fonograma de descriptores cualitativos resulto diferente en conformación e integrantes de los grupos, que los obtenidos del análisis de descriptores cualitativos: los datos de rendimiento se procesaron de la misma manera, sometidos a un análisis de varianza que arrojo diferentes pruebas de medidas de Duncan se obtuvieron dos niveles de diferenciación a y b. concordante con rendimiento presento estrecha concordancia con el de los descriptores cuantitativos. De 23 descriptores cuantitativos se seleccionaron trece, que sometidos a prueba de t. determinó nueve capaces de encontrar diferencias significativas entre pares de grupos de accesiones. De la relación entre el color y rendimiento, se desprende que los clones con subcuticula morada. Blanco amarillenta y amarillo pálido, con pulpa morada o blanca amarillento. Tienen tendencia a generar altos rendimientos. Se elaboró un catálogo de características morfológicas de las accesiones estudiadas.

Caracterización y evaluación preliminar de cincuenta clones de yuca (Manihot esculenta crantz)

Este trabajo se inició con la búsqueda de material criollo y/o introducido en todas las regiones del país, con el objetivo de concentrar la mayor variabilidad posible, de la especie. Posteriormente se preparó una guía preliminar de descriptores para efectuar la caracterización (se caracterizaron 50 clones). Esta consta de 55 descriptores, la que permite hacer una descripción completa de la colección. Para poder validar se realizaron dos siembras; una en la “Hacienda Las Mercedes y otra en los campos experimentales del programa de Recursos Genéticos Nicaragüenses, Ambos en la localidad del Rodeo km 12 ½ carretera Norte, Managua, Managua. Esta investigación permitió establecer una guía de descriptores definitiva >(consta de 31 descriptores) en la que se eliminan aquellos que no definen clones, a la vez se proponen descriptores que no deben excluirse cuando se realizan trabajos de este tipo. Por otra parte se seleccionaron los mejores clones de acuerdo a: Rendimiento promedio de raíces, siendo los más sobresalientes: yuca Sutre, Ingram, cubana; CM-91-3, White Joe, M-Col -673, Yuca blanca, yuca plátano, yuca ceiba. Producción de follaje: Smalling santa Cruz, Chilkena , CMC16, Yuca Blanca, Agria, Valencia, White Joe, Yuca Batata, Yuca Sutre valencia, Colorado, Turrucares 1. Además, se proponen algunas características morfológicas (prominencia de la base, longitud de entrenudos, textura de la superficie de la raíz) para colectar germoplasma de yuca Manihot esculents Crantz en Nicaragua, con el fin de conseguir clones sobresalientes.

Evaluación agronómica de veintidos clones de cacao (Theobroma cacao L.) en la Estación Experimental El Recreo

Se realizó una evaluación agronómica de 22 clones de Theobroma cacao L. de origen Criollos y Trinitarios en el Banco de Germoplasma de la Estación Experimental El Recreo, ubicada en el municipio de El Rama, Región Autónoma Atlántico Sur. La plantación se estableció en 1982 y su caracterización se realizó entre Diciembre 1992 y Junio 1994. Se utilizaron parámetros de evaluación como promedios y coeficientes de variación, según metodología del CATIE para la caracterización de clones de cacao. Se encontró que los materiales genéticos más promisorios lo conforman los clones ICS-6, ICS-8, RIM-9, RIM-48, RIM-52, ICS-39, RIM-117, ICS-16 Y RIM-15. Respecto a la tolerancia a Phytophthora palmivora L., por su época de producción, la mayoría de los clones demostró un efecto de escape natural, exceptuando al clon RIM-52 que demostró ser susceptible a este hongo. Los clones criollos RIM-52, RIM-9, RlM-15 Y RIM-44 presentaron mayor productividad comparado con los tratamientos restantes aunque la productividad de todos los clones fue afectada por el Huracán Juana en 1988 y posteriores inundaciones del Río Mico. El clon ICS-84 fue confirmado como autoincompatible, los genotipos restantes pueden clasificarse como autocompatibles

Análisis bioinformático de los genes de lacasas YfiH en clones virulentos de Acinetobacter baumannii

[ES] Acinetobacter baumannii es una bacteria Gram negativa, patógena y multirresistente. Su alta capacidad de supervivencia en hospitales y su resistencia a químicos puede deberse a la producción de lacasas. Estas enzimas son capaces de oxidar un sinfín de compuestos como los fenoles utilizados en hospitales para la desinfección de superficies. En este estudio se ha realizado un análisis de actividad lacasa en aislamientos altamente virulentos de los clones internaciones I y II, observando que estas cepas presentan actividad lacasa. Paralelamente, se ha realizado un análisis bioinformático con el que se ha determinado la similitud de los genes de estas lacasas con las ya descritas de la familia “YfiH” y con otras enzimas procedentes de otras especies, demostrando su similitud de secuencia con la lacasa RL5, procedente de una muestra de rumen bovino. Estos hechos suponen un avance en el estudio de lacasas bacterianas en Acinetobacter baumannii cuya caracterización podría desembocar en nuevas líneas de lucha contra dicho patógeno.

Utilização de técnica de análise de risco numa unidade de produção de proteínas recombinantes: estudo de caso da ferramenta de análise de risco - HAZOP

Em novembro de 2005, com o guia de Gestão de Riscos à Qualidade (Q9) - a Conferência Internacional de Harmonização (ICH), em conjunto com as agências regulatórias dos Estados Unidos, Japão e Europa, passaram a recomendar que seja aplicado o gerenciamento de riscos para regulação da indústria farmacêutica. Em concordância, a Agência Nacional de Vigilância Sanitária (ANVISA) publicou a Resolução de Diretoria Colegiada - RDC 17/2010 que dispõe sobre as Boas Práticas de Fabricação de Medicamentos que possibilita a comercialização de produtos farmacêuticos. Esta resolução preconiza que a validação de um processo produtivo seja efetuada com base em uma análise de risco. Seguindo as orientações da RDC este trabalho se propôs a aplicar a ferramenta de análise de risco de Estudos de Perigos e Operabilidade HAZOP num sistema de biorreação bacteriana para produção de proteínas recombinantes instalado no Instituto de Tecnologia em Imunobiológicos Bio-Manguinhos/Fiocruz. Este sistema é formado por fermentadores de 100 (FE01) e 600 (FE02) litros, um tanque de colheita de 600 litros (HT01) e um tanque de preparo de meios de cultura de 600 litros (MT01). Através da aplicação desta ferramenta de análise de riscos foi possível classificar os riscos dos sistemas identificando os nós, palavras-guia primárias (parâmetros) e secundárias (desvios), assim como a severidade e frequência dos eventos. Foram identificados 82 riscos associados aos fermentadores FE01 e FE02, sendo 8,5% riscos insignificantes, 65,9% riscos aceitáveis e 25,6% riscos não desejáveis. No tanque de colheita HT01 foram identificados 55 riscos, dos quais 14,5% são insignificantes, 67,3% são aceitáveis e 18,2% não desejáveis. Para o tanque de preparo de meios MT01 foram identificados 66 riscos que estão divididos em 9% de riscos insignificantes, 69,7% de riscos aceitáveis e 21,3% de riscos não desejáveis. Foi percebido que não houve riscos catastróficos que pudessem comprometer os equipamentos fabricados, porém somente com utilização dos mesmos na rotina de produção e o ciclo de melhoria continua dos equipamentos será possível validar este estudo prospectivo

Induction of mitotic and meiotic gynogenesis and production of genetic clones in rohu, Labeo rohita Ham.

Studies were undertaken to produce genetic clones derived from all homozygous mitotic gynogenetic individuals in rohu, Labeo rohita Ham. ln view of this, attempts were made to interfere with the normal functioning of the spindle apparatus during the first mitotic cell division of developing eggs using heat shocks, there by leading to the induction of mitotic gynogenetic diploids in the F1 generation. Afterwards, viable mitotic gynogenetic alevins were reared and a selected mature female fish was used to obtain ovulated eggs which were fertilized later with UV-irradiated milt. Milt was diluted with Cortland’s solution and the sperm concentration was maintained at 10⁸/ml. The UV-irradiation was carried out for 2 minutes at the intensity of 200 to 250 µW/cm² at 28± 1°C. The optimal heat shock of 40°C for 2 minutes applied at 25 to 30 minutes a.f. was used to induce mitotic gynogenesis in first (F1) generation and at 3 to 5 minutes a.f. to induce meiotic gynogenesis in the second (F2) generation. The results obtained are presented and the light they shed on the timing of the mitotic and meiotic cell division in this species is discussed.

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.

Two unisexual artificial polyploid clones constructed by genome addition of common carp (Cyprinus carp) and crucian carp (Carassius auratus)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.

Genetic heterogeneity and ploidy level analysis among different gynogenetic clones of the polyploid gibel carp

Background: Some triploid and tetraploid clones have been identified in the gynogenetic gibel carp, Carassius auratus gibelio Bloch, by karyotypic and cytologic analyses over many years. Further, 5-20% males and karyotypic diversity have been found among their natural and artificial populations. However, the DNA contents and the relation to their ploidy level and chromosome numbers have not been ascertained, and whether normal meiosis occurs in spermatogenesis needs to be determined in the different clones. Methods: The sampled blood cells or sperms were mixed with blood cells from chicken or individual gibel carp and fixed in 70% pre-cooled ethanol overnight at 4degreesC. The mixed cell pellets were washed 2-3 times in 1x phosphate buffered saline and then resuspended in the solution containing 0.5% pepsin and 0.1 M HCl. DNA was stained with propidium iodide solution (40 mug/mL) containing 4 kU/ml RNase. The measurements of DNA contents were performed with Phoenix Flow Systems. Results: Triploid clones A, E, F, and P had almost equal DNA content, but triploid clone D had greater DNA content than did the other four triploid clones. DNA content of clone M (7.01 +/- 0.15 pg/nucleus) was almost equal to the DNA content of clone D (5-38 +/- 0.06 pg/nucleus) plus the DNA content of common carp sperm (1.64 +/- 0.02 pg/nucleus). The DNA contents of sperms from clones A, P, and D were half of their blood cells, suggesting that normal meiosis occurs in spermatogenesis. Conclusions: Flow cytometry is a powerful method to analyze genetic heterogeneity and ploidy level among different gynogenetic clones of polyploid gibel carp. Through this study, four questions have been answered. (a) The DNA content correlation among the five triploid clones and one multiple tetraploid clone was revealed in the gibel carp, and the contents increased with not only the ploidy level but also the chromosome number. (b) Mean DNA content was 0.052 pg in six extra chromosomes of clone D, which was higher than that of each chromosome in clones A, E, F, and P (about 0.032 pg/ chromosome). This means that the six extra chromosomes are larger chromosomes. (c) Normal meiosis occurred during spermatogenesis of the gibel carp, because DNA contents of the sperms from clones A, P, and D were almost half of that in their blood cells. (d) Multiple tetraploid clone M (7.01 +/- 0.15 pg/nucleus) contained the complete genome of clone D (5.38 +/- 0.06 pg/nucleus) and the genome of common carp sperm (1.64 +/- 0.02 pg/nucleus). Cytometry Part A 56A:46-52, 2003. (C) 2003 Wiley-Liss, Inc.

Molecular analysis of silver crucian carp (Carassius auratus gibelio Bloch) clones by SCAR markers

Random amplified polymorphic DNA (RAPD) molecular markers specific for one, two or three clones have been identified from five gynogenetic clones of silver crucian carp (Carassius auratus gibelio Bloch) using RAPD markers developed earlier. In this study, three RAPD markers (RA1-PA, RA2-EF and RA4-D) produced by Opj-1, and two RAPD DNA fragments (RA3-PAD and RA5-D) produced by Opj-7, were selected for molecular cloning and sequencing. Sequence data indicated that there were identical 801-bp nucleotide sequences in the shared marker RA1-PA cloned respectively from clones P and A, and the shared marker RA2-EF (which was cloned from clones E and F), were also of identical 958-by nucleotide sequences. The nucleotide sequences of the shared marker RA3-PAD fragments were also similar for 1181 by among clones P, A and D. The specific fragment RA4-D was composed of 628 bp, and the fragment RA5-D from clone D contained 385 nucleotides. According to the nucleotide sequences, we designed and synthesized five pairs of sequence characterized amplified regions (SCAR) primers to identify the specific fragments in these gynogenetic clones of silver crucian carp. Only individuals from clones P and A amplified a specific band using a pair of SCI-PA primers synthesized according to the marker RA1-PA sequences, whereas no products were detected in individuals from clones D, E and F. The PCR products amplified using SC2-EF and SC3-PAD primers were as expected. Furthermore, the pair of SC4-D primers amplified specific bands only in individuals from clone D, although weak bands could be produced in all individuals of the five clones when lower annealing temperatures were used. However, an additional pair of SC5-D primers designed from the RA5-D marker sequences could amplify a DNA band in individuals from clones P, A and D, and the same weak band was produced in clone E, whereas no products were detected in individuals from clone F. Searches in GenBank revealed that the 385-bp DNA fragment from RA5-D was homologous to the 5' end of gonadotropin I beta subunit 2 gene and growth hormone gene. No homologous sequences were found for other markers in GenBank. The SCAR markers identified in this study will offer a powerful, easy, and rapid method for discrimination of different clones and for genetic analyses that examine their origins and unique reproductive modes in crucian carp. Furthermore, they will likely benefit future selective breeding programs as reliable and reproducible molecular markers. (C) 2001 Elsevier Science B.V. All rights reserved.

Genetic diversity among different clones of the gynogenetic silver crucian carp, Carassius auratus gibelio, revealed by transferrin and isozyme markers

Genetic diversity among four clones (A, D, E, F) of gynogenetic silver crucian carp was studied using transferrin and isozymes in the blood as markers. Of the five proteins investigated, three (transferrin, esterase and superoxide dismutase) indicated polymorphism and eight polymorphic loci were detected. These loci were probably encoded by codominant alleles and their inheritance patterns were analyzed. Intraclonal homogeneity and interclonal heterogeneity were observed in these clones, which allowed us to infer the clonal nature and evolutionary relationship between them. Clonal diversity in this population of silver crucian carp in China was also compared with data reported from gynogenetic crucian carp in Germany.

Assessing genetic identity of sporophytic offspring of the brown alga Undaria pinnatifida derived from mono-crossing of gametophyte clones by use of amplified fragment length polymorphism and microsatellite markers

The haploid stage of gametophytes of the subtidal brown alga Undaria pinnatifida can be vegetatively propagated under favorable conditions. This unique characteristic makes it possible to establish independent gametophyte cell lines that are zoospore-derived. Sporophytic offspring can be generated through hybridizing the male and female gametophytes, which are derived from different cell lines. Accumulated experiences in this and other species in Laminariales demonstrated the applicability of this novel way to breed desired strains for open-sea cultivation. Sporophytic offspring originated from mono-crossing of male and female gametophyte clones were shown to have similar morphological characteristics under identical ambient conditions. However, there has been no report to relate this similarity on molecular levels. In this report, amplified fragment length polymorphism (AFLP) and microsatellite markers were used to analyze the genetic identity of sporophytic offspring of U. pinnatifida originated from two mono-crossing lines (M1 and M2), two self-breeding lines (S1 and S2) and one wild population (W). Totally 318 AFLP loci were revealed by use of 11 primer sets, of which 4.7%, 0.3%, 17.9%, 16.4% and 36.5% were polymorphic in M1, M2, S1, S2 and W, respectively. The pairwise genetic identity among the individuals of the same line was assessed. It was shown that offspring from mono-crossing lines had a higher degree of identity (95.6-100%) than self-breeding lines (87.7-98.4%) and the wild population (81.5-92.1%). Analysis by use of six microsatellite loci also revealed a higher genetic identity among individuals of the mono-crossing line, further confirming the results of AFLP analysis. Results from this investigation support, on molecular levels, the novel way to produce and maintain strains in U. pinnatifida by use of different gametophyte cell lines.

Effect of light period on egg-discharge of gametophyte clones of Undaria pinnatifida (Phaeophyta)

A sporeling culture method using gametophyte clones developed in the early 1990s led to egg discharge occurring in the dark 5 min after the start of the dark period under growth under a 11:13 L-D photoperiod. The course of egg discharge could be disturbed by light, with irradiance as low as 5-6 mu mol photon m(-2) s(-1) causing 75-80% of the discharged eggs to detach from the oogonia and consequently to die within several hours. In order to enhance outgrowth rate of young sporophytes, a study was conducted to test the effect of controlling darkness in the period 2-3 h after dusk. When the slides were transferred from the standard 11:13 L-D regime to continuous light, eggs were discharged 5 min after the end of the light phase and peaked 5-l5 min later on first day after transfer, indicating that the female gametes "remember" the light-dark regime. This suggests the existence of an endogenous circadian rhythm. During the second and third days, very few eggs were discharged throughout the 11 h of the normal light phase of the L-D regime, indicating the inhibitory effect of continuous light and that the rhtyhm is easily damped by light.