Characterization and transferability of microsatellite markers of the cultivated peanut (Arachis hypogaea)

BACKGROUND: The genus Arachis includes Arachis hypogaea (cultivated peanut) and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species. RESULTS: Thirteen loci were isolated and characterized using 16 accessions of A. hypogaea. The level of variation found in A. hypogaea using microsatellites was higher than with other markers. Cross-transferability of the markers was also high. Sequencing of the fragments amplified using the primer pair Ah11 from 17 wild Arachis species showed that almost all wild species had similar repeated sequence to the one observed in A. hypogaea. Sequence data suggested that there is no correlation between taxonomic relationship of a wild species to A. hypogaea and the number of repeats found in its microsatellite loci. CONCLUSION: These results show that microsatellite primer pairs from A. hypogaea have multiple uses. A higher level of variation among A. hypogaea accessions can be detected using microsatellite markers in comparison to other markers, such as RFLP, RAPD and AFLP. The microsatellite primers of A. hypogaea showed a very high rate of transferability to other species of the genus. These primer pairs provide important tools to evaluate the genetic variability and to assess the mating system in Arachis species

Secondary Metabolites

Plants are the main source for obtaining secondary metabolites that are used in the pharmaceutical, cosmetic and food industries. In nature, the performance of bioactive compounds is affected by biotic and abiotic factors, an alternative to overcome this adversity is in vitro plant cultures and particularly plant cell culture that has multiple advantages, highlighting the possibility of controlling variables to increase the content. of compounds of interest. The objective of this research was to determine and optimize the effect of some elicitors on the production of secondary flavonoid metabolites in suspension cell cultures of Thevetia peruviana on a shake flask scale. The experimental part was performed from cell cultures in suspension cells of T. peruviana maintained by the Bioconversion Laboratory of the Universidad Nacional de Colombia, sede Medellin. Firstly, methyl jasmonate (MeJa) 3 µM and salicylic acid (SA) 300 µM were added to two los of cell cultures, that circumstantially they differed in their time suspension state lot 1 (9 months) and lot 2 (3 months). Secondly, certain were evaluated, in order to be optimized; the operational parameters cosidered were: day of addition, concentration and hours of harvest. Finally, the combined effect of MeJa (0.3 µM) and SA (100 µM) was determined in different proportions, the day of elicitation and the hours of harvest were constant. Flavonoid content was quantified by UV-vis spectrophotometry usinfg the AlCl3 complexation method and was evaluated at extracellular and intracellular level. In the first part, at intracellular level, SA generated 14.6 % and 9.56 % more flavonoid content than MeJa in lots 1 and 2, respectively. In the second part, 4.14 mg EQ/g DW were generated with MeJa 0.3 µM, elicitation day 5 and harvest at 90 h; 3.75 mg EQ/g DW were generated with 100 µM SA, elicitation day 0 and harvests at 96 h. In the third part, 4.62 mg EQ / g DW were generated with the combination of MeJa (0.3 µM) – SA (100 µM) in a ratio of 20-80, elicitation on day 0 and harvest at 96 h. Eliciting cell cultures of Thevetia peruviana with MeJa and SA under optimal conditions of concentration, day of addition and hours of harvest increased the content of flavonoid compounds. The results obtained could serve as a basis for the development of investigations at the bioreactor scale.Las plantas son la principal fuente para la obtención de metabolitos secundarios que se usan en la industria farmacéutica, cosmética y alimentaria. En la naturaleza el rendimiento de compuestos bioactivos se ve afectado por factores bióticos y abióticos, una alternativa para superar esta adversidad son los cultivos vegetales in vitro y particularmente el cultivo de células en suspensión que presenta múltiples ventajas destacándose la posibilidad de controlar variables para aumentar el contenido de compuestos de interés. El objetivo del presente trabajo de investigación fue determinar y optimizar el efecto de algunos elicitores sobre la producción de metabolitos secundarios tipo flavonoides en cultivos de células en suspensión de Thevetia peruviana a escala de matraz agitado. La parte experimental se realizó a partir de cultivos de células en células en suspensión de T. peruviana que mantenía el laboratorio de bioconversiones de la Universidad Nacional de Colombia sede Medellín. Primeramente, metil jasmonato (MeJa) 3 μM y ácido salicílico (AS) 300 μM se adicionaron a dos lotes de cultivos de células; que circunstancialmente diferían en su tiempo en estado de suspensión lote 1 (9 meses) y lote 2 (3 meses). Segundamente, determinadas condiciones fueron evaluadas, con el fin de ser optimizadas; los parámetros operacionales que se consideraron fueron: día de adición, concentración y las horas de cosecha. Por último, el efecto combinado de MeJa (0,3 μM) – AS (100 μM) se determinó en diferentes proporciones, el día de elicitación y las horas de cosecha fueron constantes. El contenido de flavonoides se cuantificó por espectrofotometría UV – vis por el método de complejación de AlCl3 y se evaluaron a nivel extracelular e intracelular. En la primera parte, a nivel intracelular AS generaron 14,6 % y 9,56 % más contenido de flavonoides que MeJa en lotes 1 y 2, respectivamente. En la segunda parte, 4,14 mg EQ/g MS fueron generados con MeJa 0,3 μM, elicitación día 5 y cosecha a 90 h; por otro lado, 3,75 mg EQ/g MS fueron generados con AS 100 μM, elicitación día 0 y cosecha a 96 h. En la tercera parte, 4,62 mg EQ/g MS fueron generados con la combinación de MeJa (0,3 μM) – AS (100 μM) en proporción 20 – 80, elicitación el día 0 y cosecha a las 96 h. Elicitar cultivos celulares de Thevetia peruviana con MeJa y AS en condiciones óptimas de concentración, día de adición y horas de cosecha incrementaron el contenido de compuestos flavonoides. Los resultados obtenidos podrían servir como base para el desarrollo de investigaciones a escala de biorreactor.Maestrí

In silico evaluation of the Eucalyptus transcriptome

The expressed sequence tags (ESTs) produced in the Forests project provide an invaluable opportunity to assess the Eucalyptus transcriptome. Besides providing information on the different proteins produced by this plant, it is possible to infer gene expression profiles because non-normalized cDNA libraries were used. The EST frequency from any gene is correlated to the transcript levels in the tissues from which the cDNA libraries were constructed. The goal of this work was to identify Eucalyptus genes that showed either differential expression pattern or were ubiquitously expressed in the tissues sampled in the Forests project. Six robust statistical tests and very restrictive rules were applied to gain confidence in the in silico data aiming to avoid false positives. Several genes with interesting expression profiles were identified and some of them were validated by RT-PCR.487495Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

A linkage map for the B-genome of Arachis (Fabaceae) and its synteny to the A-genome

<p>Abstract</p> <p>Background</p> <p><it>Arachis hypogaea </it>(peanut) is an important crop worldwide, being mostly used for edible oil production, direct consumption and animal feed. Cultivated peanut is an allotetraploid species with two different genome components, A and B. Genetic linkage maps can greatly assist molecular breeding and genomic studies. However, the development of linkage maps for <it>A. hypogaea </it>is difficult because it has very low levels of polymorphism. This can be overcome by the utilization of wild species of <it>Arachis</it>, which present the A- and B-genomes in the diploid state, and show high levels of genetic variability.</p> <p>Results</p> <p>In this work, we constructed a B-genome linkage map, which will complement the previously published map for the A-genome of <it>Arachis</it>, and produced an entire framework for the tetraploid genome. This map is based on an F₂population of 93 individuals obtained from the cross between the diploid <it>A. ipaënsis </it>(K30076) and the closely related <it>A. magna </it>(K30097), the former species being the most probable B genome donor to cultivated peanut. In spite of being classified as different species, the parents showed high crossability and relatively low polymorphism (22.3%), compared to other interspecific crosses. The map has 10 linkage groups, with 149 loci spanning a total map distance of 1,294 cM. The microsatellite markers utilized, developed for other <it>Arachis </it>species, showed high transferability (81.7%). Segregation distortion was 21.5%. This B-genome map was compared to the A-genome map using 51 common markers, revealing a high degree of synteny between both genomes.</p> <p>Conclusion</p> <p>The development of genetic maps for <it>Arachis </it>diploid wild species with A- and B-genomes effectively provides a genetic map for the tetraploid cultivated peanut in two separate diploid components and is a significant advance towards the construction of a transferable reference map for <it>Arachis</it>. Additionally, we were able to identify affinities of some <it>Arachis </it>linkage groups with <it>Medicago truncatula</it>, which will allow the transfer of information from the nearly-complete genome sequences of this model legume to the peanut crop.</p

Comet and cytogenetic tests as tools for evaluating genomic instability in seeds of Oryza sativa L. and Phaseolus vulgaris L. from gene banks

This study aimed to assess the feasibility of comet and cytogenetic tests as tools for evaluating genomic instability in seeds of Oryza sativa L. (rice) and Phaseolus vulgaris (beans) L. from gene banks. Rice and beans were exposed to methyl methanesulfonate (MMS) as a reference DNA damaging agent. Seeds of two accessions of rice and beans were obtained from Embrapa Rice and Beans - Brazil. Seed groups were imbibed in three concentrations of MMS for three periods of time to carry out cytogenetic tests, and for one period for the comet test. At concentrations of 10 and 15 mg/L, MMS induced cytotoxic and/or mutagenic effects in the meristematic cells of roots from all the accessions of both species. In the comet test, MMS induced genotoxic effects at all the concentrations in the evaluated accessions of rice and beans, except in one accession of beans at the lowest concentration (5 mg/L). Both species showed sensitivity to MMS. The comet test can be proposed for the measurement of genomic instability in accessions of rice and beans in gene banks, as being more sensitive than the cytogenetic tests used

Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines

Recently, the cytotoxic effects of apigenin (4′,5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes

Genetic diversity analysis in the section Caulorrhizae (genus Arachis) using microsatellite markers

Diversity in 26 microsatellite loci from section Caulorrhizae germplasm was evaluated by using 33 accessions of A. pintoi Krapov. & W.C. Gregory and ten accessions of Arachis repens Handro. Twenty loci proved to be polymorphic and a total of 196 alleles were detected with an average of 9.8 alleles per locus. The variability found in those loci was greater than the variability found using morphological characters, seed storage proteins and RAPD markers previously used in this germplasm. The high potential of these markers to detect species-specific alleles and discriminate among accessions was demonstrated. The set of microsatellite primer pairs developed by our group for A. pintoi are useful molecular tools for evaluating Section Caulorrhizae germplasm, as well as that of species belonging to other Arachis sections