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Santo Domingo de los Colorados, Ecuador

Martinez-Marquez A.,University of Alicante | Morante-Carriel J.,University of Alicante | Morante-Carriel J.,Technical State University of Quevedo | Selles-Marchart S.,University of Alicante | And 4 more authors.
Journal of Proteome Research | Year: 2013

Multiple reaction monitoring (MRM) is emerging as a promising technique for the detection and quantification of protein biomarkers in complex biological samples. Compared to Western blotting or enzyme assays, its high sensitivity, specificity, accuracy, assay speed, and sample throughput represent a clear advantage for being the approach of choice for the analysis of proteins. MRM assays are capable of detecting and quantifying proteolytic peptides differing in mass unique to particular proteins, that is, proteotypic peptides, through which different protein isoforms can be distinguished. We have focused on polyphenol oxidase (PPO), a plant conspicuous enzyme encoded by a multigenic family in loquat (Eriobotrya japonica Lindl.) and other related species. PPO is responsible for both the protection of plants from biotic stress as a feeding deterrent for herbivore insects and the enzymatic browning of fruits and vegetables. The latter makes fruit more attractive to seed dispersal agents but is also a major cause of important economic losses in agriculture and food industry. An adequate management of PPO at plant breeding level would maximize the benefits and minimize the disadvantages of this enzyme, but it would require a precise knowledge of the biological role played by each isoform in the plant. Thus, for the functional study of the PPOs, we have cloned and overexpressed fragments of three PPO isoforms from loquat to develop MRM-based methods for the quantification of each isoform. The method was developed using an ion trap instrument and validated in a QQQ instrument. It resulted in the selection of at least two peptides for each isoform that can be monitored by at least three transitions. A combination of SDS-PAGE and MRM lead to detect two out of three monitored isoforms in different gel bands corresponding to different processing stages of PPO. The method was applied to determine the amount of the PPO2 isoform in protein extracts from fruit samples using external calibrants. © 2013 American Chemical Society.

Rivano F.,CIRAD - Agricultural Research for Development | Mattos C.R.R.,Michelin | Cardoso S.E.A.,Michelin | Martinez M.,Technical State University of Quevedo | And 3 more authors.
Industrial Crops and Products | Year: 2013

The CIRAD-Michelin-Brazil (CMB) breeding program was set up in 1992 and has produced several genotypes as alternative varieties for growing in suboptimal regions and areas affected by South American Leaf Blight (SALB). From a large parent population of more than 113 clones, the program developed CMB genotypes evaluated in large-scale clone trials. Based on accurate knowledge of the parents' agronomic potential, the CMB breeding program combined family and individual selection in the seedling evaluation trials. The segregation ratios of the SALB resistance traits in the progeny were used to identify and reject parents whose resistance was determined by a small number of genes, easily overcome by Microcyclus ulei strains. After evaluating the germplasm, 13 genotypes were selected for evaluation of their resistance, girth and rubber production in a trial network covering eight sites in Brazil and Ecuador. There were significant differences between clones, sites and clone-site interactions. The resistance of the clones to SALB was confirmed for all sites, both for conidial and sexual fungal stages. The growth rate in Ecuador was always higher than in Brazil with the exception of one clone. Data from previous years of production for a few clones was used to estimate the potential yield of these clones compared to clones usually planted in Latin America. Simultaneous selection for SALB resistance, yield and growth resulted in promising genotypes which need to be tested in areas with different environments. © 2012 Elsevier B.V.

Triazol and their mixtures with strobylurins are the main fungicides used to control Asian rust on soybeans. Shifting in fungal sensibility to fungicides demands continuous monitoring on the efficacy of such compounds. In this research a comparative study on rust control was carried out with the fungicide tebuconazol (triazol) and the mixture of epoxiconazol + pyraclostrobin (triazol + strobylurin), which were sprayed once, twice or three times from different plant growth stages (V9, R4, or R5.3). A total of 64 randomized plots of Nidera 5909 AG soybeans were assessed for number of lesions and uredia, later converted into percent severity for each third part of the plant (lower, medium, and upper). Rust severity was over 40% on non-treated plants. Disease progress was higher at the lower third. Spray applications began at V9 (main stem with nine trifolia) resulted in better rust control. The mix of triazol + strobylurin was more efficacious than the triazol alone. Disease increase was mainly driven by the amount of lesions, since the number of uredia per lesion did not vary significantly among treatments.

Morante-Carriel J.,University of Alicante | Morante-Carriel J.,Technical State University of Quevedo | Selles-Marchart S.,University of Alicante | Martinez-Marquez A.,University of Alicante | And 3 more authors.
Analytical Biochemistry | Year: 2014

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A260/A280 ratios ranged from 1.90 to 2.04 and A 260/A230 ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function. © 2014 Elsevier Inc. All rights reserved.

Zambrano-Vega C.,Technical State University of Quevedo | Nebro A.J.,University of Malaga | Aldana-Montes J.F.,University of Malaga
Methods in Ecology and Evolution | Year: 2016

Phylogenetic inference is the process of searching and reconstructing the best phylogenetic tree that explains the evolution of species from a given data set. It is considered as an NP-hard problem due to the computational complexity required to find the optimal phylogenetic trees in the space of all the possible topologies. We have developed MO-Phylogenetics, a software tool to infer phylogenetic trees optimizing two reconstruction criteria simultaneously, integrating a framework for multi-objective optimization with two phylogenetic software packages. As a result, researchers in life sciences have at their disposal a high-performance tool including a number of multi-objective metaheuristics that can be applied to phylogenetic inference using the maximum parsimony and maximum likelihood as objectives to be optimized at the same time. © 2016 British Ecological Society.

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