Data mining of metagenomes to find novel enzymes: a non-computationally intensive method.

Currently, there is a need of non-computationally-intensive bioinformatics tools to cope with the increase of large datasets produced by Next Generation Sequencing technologies. We present a simple and robust bioinformatics pipeline to search for novel enzymes in metagenomic sequences. The strategy is based on pattern searching using as reference conserved motifs coded as regular expressions. As a case study, we applied this scheme to search for novel proteases S8A in a publicly available metagenome. Briefly, (1) the metagenome was assembled and translated into amino acids; (2) patterns were matched using regular expressions; (3) retrieved sequences were annotated; and (4) diversity analyses were conducted. Following this pipeline, we were able to identify nine sequences containing an S8 catalytic triad, starting from a metagenome containing 9,921,136 Illumina reads. Identity of these nine sequences was confirmed by BLASTp against databases at NCBI and MEROPS. Identities ranged from 62 to 89% to their respective nearest ortholog, which belonged to phyla Proteobacteria, Actinobacteria, Planctomycetes, Bacterioidetes, and Cyanobacteria, consistent with the most abundant phyla reported for this metagenome. All these results support the idea that they all are novel S8 sequences and strongly suggest that our methodology is robust and suitable to detect novel enzymes.

Biological responses of shoal flounder (Syacium gunteri) to toxic environmental pollutants from the southern Gulf of Mexico.

The Gulf of Mexico (GoM) is exposed to a diversity of contaminants, such as hydrocarbons and heavy metal(oid)s, either from natural sources or as a result of uncontrolled coastal urbanisation and industrialisation. To determine the effect of these contaminants on the marine biota along the Mexican GoM, the biological responses of the shoal flounder Syacium gunteri, naturally exposed, were studied. The study area included all the Mexican GoM, which was divided into three areas: West-southwest (WSW), South-southwest (SSW) and South-southeast (SSE). The biological responses included the global DNA methylation levels, the expression of biomarker genes related to contaminants (cytochrome P450 1A, glutathione S-transferase, glutathione reductase, glutathione peroxidase, catalase, and vitellogenin), histopathological lesions and PAH metabolites in bile (hydroxynaphthalene, hydroxyphenanthrene, hydroxypyrene and Benzo[a]pyrene). The correlation between the biological responses and the concentration of contaminants (hydrocarbons and metal(oid)s), present in both sediments and organisms, were studied. The shoal flounders in WSW and SSW areas presented higher DNA hypomethylation, less antioxidative response and biotransformation gene expression and a higher concentration of PAH metabolites in bile than SSE area; those responses were associated with total hydrocarbons and metals such as chromium (Cr). SSE biological responses were mainly associated with the presence of metals, such as cadmium (Cd) and copper (Cu), in the tissue of shoal flounders. The results obtained on the physiological response of the shoal flounder can be used as part of a permanent active environmental surveillance program to watch the ecosystem health of the Mexican GoM.

Using Flow Cytometry Analysis in Plant Tissue Culture Derived Plants.

Somaclonal variation (SC) in plants regenerated from tissue culture, via organogenesis or somatic embryogenesis, is frequently associated with abnormalities in the content of deoxyribonucleic acid (DNA), viz., aneuploidy and polyploidy. Flow cytometry (FCM) using the nucleic acid-specific fluorochrome propidium iodide has proven to be a rapid, simple, and reproducible technique for assessment of DNA content and ploidy variation occurring in plant tissue cultures. Here an outline of the sample preparation of suspension with intact nuclei by the two-step standard method, and FCM analysis of DNA ploidy stability in plants regenerated from embryogenic cell suspension (ECS) of banana Musa acuminata, AAA, cv. Grand Naine (Cavendish subgroup) using an internal standard is described.

A quantitative PCR approach for determining the ribosomal DNA copy number in the genome of Agave tequila Weber

Background: Agave tequilana has a great economic importance in Mexico in order to produce alcoholic beverages and bioenergy. However, in this species the structure and organization of the rDNAs in the genome are limited, and it represents an obstacle both in their genetic research and improvement as well. rDNA copy number variations per eukaryotic genome have been considered as a source of genetic rearrangements. In this study, the copy number of 18S and 5S rDNAs in the A. tequilana genome was estimated, and an absolute quantitative qPCR assay and genome size was used. In addition, an association between the rDNAs copy number and physical mapping was performed to confirm our results. Results: The analysis were successfully applied to determine copy number of 18S and 5S rDNAs in A. tequilana genome, showing high reproducibility with coefficient of variation (CV) values of 0.014-0.0129%, respectively. A variation of 51 times in the copy number the 18s regarding 5s rDNA was found, thus contributing to genome size of 1.47 and 8.38 x 10-3%, respectively. Similarly, data show a linear relationship (R [2] = 0.992) between rDNA copy number and the detected signals for each of the loci by FISH. The comparison of the rDNA copy number of agave showed differential relationship with other organisms and it may be due to evolutionary ecology.Conclusions: Results show that the proposed method a) can correctly detect the rDNA copy number, b) could be used as species-specific markers and c) might help in understanding the genetic diversity, genome organization and evolution of this species.

Primer reporte de empleo de marcadores AFLP en Asteraceae en Cuba / First report of the employment of AFLP markers in Asteraceae in Cuba

Rhodogeron coronopifolius Griseb., es una especie vegetal de la familia Asteraceae, que se encuentra en peligro crítico de extinción. Es endémico de la provincia Villa Clara en la región central de Cuba. Habita en el matorral xeromorfo sub espinoso sobre serpentina. Existen solo cinco poblaciones naturales dentro de un área protegida, la principal causa de amenaza es la fragmentación de su hábitat por acciones antrópicas. Debido a su situación de conservación, se hace necesario realizar estudios de la diversidad genética de las poblaciones naturales para así generar información básica y diseñar una estrategia de conservación. El objetivo de este trabajo fue analizar de manera preliminar la diversidad genética de cuatro poblaciones de esta especie utilizando marcadores AFLP (Polimorfismo de Longitud de Fragmentos Amplificados). Se emplearon dos combinaciones de iniciadores y se evaluó el porcentaje de polimorfismo así como la similitud entre los individuos. Se obtuvieron 165 loci de los cuales el 78,7 % fueron polimórficos. La población de mayor polimorfismo fue Corojito con 85,2%, de manera general el polimorfismo fue alto con valores entre 75 y 87%. La similitud entre los individuos también fue alta con un promedio de 0,74. El agrupamiento genético fue independiente a la población de procedencia, lo que sugiere que existe intercambio genético entre las poblaciones y que estas comparten más del 80 % de los alelos que fueron analizados. Los resultados obtenidos son importantes para el mantenimiento in situ de la especie y para tomar decisiones en aras de su conservación.

Rhodogeron coronopifolius Griseb., is a specie of Asteraceae family, in critical danger of extinction. It is an endemic of Villa Clara city in the central region of Cuba. Its inhabits sub thorny xeromorphic heath on serpentine soil. Only five natural populations exist included in a protected area, the main threat cause is the fragmentation of its habitat for antropics activities. Due to their conservation status, it becomes necessary to analyze the genetic diversity of the natural populations in order to generate basic information usefull to apply a conservation strategy. The main goal of this work was to evaluate in a preliminary manner the genetic diversity of four populations of this specie using AFLP markers (Amplified Fragment Length Polymorphism). Two primer combinations were used and the polymorphism percentage was evaluated as well as the similarity among the individuals. A total of 165 loci were obtained of which 78,7% were polymorphic. The population with higher polymorphism was Corojito with 85,2%. High level of polymorphism was observed among population showing values between 75 and 87%. The similarity among the individuals was also high with an average of 0,74. The genetic grouping was independent to the origin of the population. This suggesting that gene flow exists among the populations, those which share more than 80% of the alleles analyzed. This is important for the in situ maintenance of the specie and to take decisions for their conservation.

Plant tissue culture and molecular markers.

Tissue culture can be used to propagate elite material or to generate new variability by employing somaclonal variation. Genetic stability of the process must be evaluated analyzing DNA profiles by the use of molecular markers. Several techniques have been reported for the screening of genetic variation on tissue culture derived material; however, a highly informative and good relation among the time-cost-information is obtained using Amplified Fragment Length Polymorphism (AFLP) in automatic sequencer. This technique involves a double-digestion of DNA with restriction enzymes, ligation of adapters at both extremities of the restriction fragments, and finally, selective polymerase chain reaction (PCR) amplification of the fragments. A semiautomatic process for the analysis could be used, but several considerations must be taken into account before such a use.

Primer reporte del empleo de marcadores ISTR en cactaceae (pilosocereus sp) / First report of the employment of ISTR markers in cactaceae (pilosocereus sp)

Pilosocereus sp es una especie en peligro crítico de extinción, la única población conocida se encuentra en una mina de mármol verde, hoy abandonada, en la que su explotación produjo la disminución del 80% de la población en 3 años; en la actualidad quedan 28 ejemplares, de ellos unos pocos son adultos, de los cuales solo dos producen frutos. Una de las etapas necesaria para su recuperación es la producción de plántulas para realizar el reforzamiento de la población natural. Como las plantas obtenidas serán plantadas en condiciones naturales, donde se enfrentarán a diversas situaciones ambientales, es conveniente realizar un estudio de diversidad genética. El objetivo de este trabajo fue estimar la variabilidad genética de plántulas de Pilosocereus sp empleando la técnica Inverse Sequence Tagged Repeat (ISTR). Se realizó la germinación in vitro de semillas y se determinó la variabilidad genética de las plántulas obtenidas. Con el análisis molecular se detectaron un total de 97 bandas, de ellas el 62,8% fueron polimórficas. El mayor porcentaje de bandas polimórficas (85,7%) se obtuvo con la combinación de oligonucleótidos F6/B6. Con las combinaciones de oligonucleótidos empleados se detectaron de 4 a 6 patrones de banda diferentes. La heterocigosidad media esperada fue de 0,39.

Pilosocereus sp is a species in critical extinction danger, the only known population is in a mine of green marble, abandoned today, but it exploitation produced the decrease of the population's 80% in 3 years, at the present time they are 28 individuals, of them some few ones are mature, of those which alone two produce fruits. One of the necessary stages for their recovery is the seedlings production to carry out the natural population's reinforcement. As the obtained plants they will be planted under natural conditions, where they will face diverse environmental situations, it is convenient to carry out a study of genetic diversity. The objective of this work was to estimate the genetic variability using the technical Inverse Sequence Tagged Repeat (ISTR). In vitro germination of seeds of Pilosocereus sp and the genetic variability of the obtained seedlings was determined. With the molecular analysis a total of 97 bands were detected, of them 62.8% were polymorphic. The biggest percentage of polymorphism (85.7%) was obtained with the primer combination F6/B6. With the primer combinations employed were detected from 4 to 6 different band patterns. The heterocigocity hoped was 0.39.

Caracterización de accesiones de papaya (Carica papaya L)a través de marcadores AFLP en Cuba / Characterising Cuban papaya accessions (Carica papaya L) by AFLP markers

Los marcadores moleculares son herramientas valiosas en los estudios genéticos en plantas, y están siendo empleados exitosamente en programas de mejoramiento principalmente en la elección de progenitores y en la selección. El polimorfismo observado mediante la técnica molecular AFLP (Amplified Fragment Length Polymorphism) ha sido de utilidad para estudios de diversidad genética en frutales. En el presente trabajo se realizó la caracterización molecular de 12 accesiones de papaya (Carica papaya L.) del banco de germoplasma del Instituto de Investigaciones en Fruticultura Tropical (IIFT), empleando la técnica AFLP. Se evaluaronseis combinaciones de iniciadores para la amplificación selectiva, las cuales amplificaron un total de 431 bandascon 73,3% de polimorfismo. El número total de patrones de bandas identificados fue igual en todas las combinaciones utilizadas, con un porcentaje de identificación alto, lo que sugiere que dichas combinaciones pudieran ser empleadas para estudios de variabilidad genética en papaya. En general, los resultados presentados demuestran que existe diversidad genética entre las accesiones evaluadas, lo cual constituye un reflejo del origen que presentan los genotipos analizados a partir de la introducción de materiales foráneos y la polinización abierta de un grupo de materiales selectos. Por tanto, se recomienda retomar la prospección y selecciónde accesiones locales, así como la introducción de nuevos genotipos foráneos, como dos vías fundamentalespara aumentar la diversidad genética presente en el banco de germoplasma de papaya de Cuba.

Molecular markers are valuable tools for genetic studies in plants and they are often used successfully in genetic breeding, mainly for choosing progenitors and selection. Polymorphism observed by amplified fragment length polymorphism (AFLP) has been useful for genetic diversity studies in fruit trees. Twelve papaya accessions from the Tropical Fruit Crop Research Institute germplasm bank were molecularly characterised by AFLP. 431 bands having 73.3% polymorphism were obtained using 6 primer combinations. The total number of band patterns identified was the same in all combinations assayed with a high percentage of identification, suggesting that such primer combinations could be used for genetic variability studies in papaya.The results demonstrated genetic diversity among the papaya accessions evaluated, indicating the origin ofthe analysed genotypes from exogenous material and open pollination of a selected group of material. It is thus recommended that local accessions and their selection be monitored as well as the introduction of new foreign genotypes as two ways of increasing the genetic diversity of the Cuban papaya germplasm bank.

Cell viability and leakage of electrolytes in Avicennia germinans exposed to heavy metals.

The effect of heavy metal stress on the cell viability and leakage of electrolytes of Avicennia germinans leaf discs was investigated by the tissue tolerance test. Foliar discs were incubated with different Cd2+ or CU2+ concentrations for 24 h; thereafter, the cell membrane stability of the tissue was assayed by the cell viability Evans blue and leakage electrolytes methods. The results indicated that electrolyte leakage of the leaf discs increased 24 h after exposure to heavy metal stress, as shown by a reduction of the cell viability by 30% in discs exposed to higher doses of Cd2+ (0.546 M) and Cu2+ (0.7 M), respectively. Additionally, the histological analysis of the leaf discs exposed to heavy metal stress revealed that at higher Cd2+ and/or Cu2+ concentrations an increase in the intercellular spaces and destruction of mesophyll cells was observed 24 h after exposure. In summary, the biochemical and structural changes observed in foliar tissues of A. germinans suggest that higher cadmium and copper concentrations may result in structural changes and altered physiological characters in leaves.