A PCR-Based Diagnostic System for Differentiating Two Weevil Species (Coleoptera: Curculionidae) of Economic Importance to the Chilean Citrus Industry.

A PCR-based method was developed to identify Naupactus cervinus (Boheman) and Naupactus xanthographus (Germar), two curculionids affecting the citrus industry in Chile. The quarantine status of these two species depends on the country to which fruits are exported. This identification method was developed because it is not possible to discriminate between these two species at the egg stage. The method is based on the species-specific amplification of sequences of internal transcribed spacers, for which we cloned and sequenced these genome fragments from each species. We designed an identification system based on two duplex-PCR reactions. Each one contains the species-specific primer set and a second generic primer set that amplify a short 18S region common to coleopterans, to avoid false negatives. The marker system is able to differentiate each Naupactus species at any life stage, and with a diagnostic sensitivity to 0.045 ng of genomic DNA. This PCR kit was validated by samples collected from different citrus production areas throughout Chile and showed 100% accuracy in differentiating the two Naupactus species.

Heritability and identification of QTLs and underlying candidate genes associated with the architecture of the grapevine cluster (Vitis vinifera L.).

KEY MESSAGE: We have identified 19 QTLs for rachis architecture, a key and complex trait for grapevine production. Fifty out of 1,173 genes underlying these QTLs are candidates to be further explored. In the table grape industry, the rachis architecture has economic and management implications. Therefore, understanding the genetics of this trait is key for its breeding. The aim of this work was to identify genetic determinants of traits associated with the cluster architecture. Characterisations of eight traits was performed on a 'Ruby Seedless' × 'Sultanina' crossing (F1: n = 137) during three seasons, with and without gibberellic acid (GA3) applications. The genotypic effects and the genotype × GA3 interactions were significant for several traits. Rachis length (rl), lateral shoulder length and node number along the central axis were the most prominent traits. On average, the heritability of these traits was ~71 %, with heritability of rl being 76 % as estimated under different seasons. Quantitative trait loci (QTLs) analyses showed that linkage group 5 (LG5) and LG18 harboured the largest number of QTLs for these traits. According to the variance explained, the main QTL (corresponding to rl) was found on LG9. These QTLs were supported mainly by a paternal additive effect and revealed possible pleiotropic effects. Based on the grapevine reference genome, we identified 1,173 genes located under these QTL confidence intervals. Fifty of the 891 annotated genes of this list were selected for their further characterisation because of their possible participation in the rachis architecture. In conclusion, the QTLs detected indicate that these traits and their GA3 responsiveness have a clear genetic basis. Due to the percentage of the total variance explained, they are good candidates to participate in the genetic determination of the cluster architecture.

Cloning and sequencing of the gene encoding benzaldehyde lyase from Pseudomonas fluorescens biovar I.

The gene (bzl) encoding benzaldehyde lyase (BL) from Pseudomonas fluorescens biovar I has been cloned and characterized. The nucleotide sequence contains an open reading frame encoding a protein of 563 amino acids. The deduced BL protein shares little homology with proteins contained in the NBRF-PIR data bank. However, the higher homologies (up to about 28%) were obtained with enzymes that also utilize thiamine pyrophosphate as a cofactor.

Isolation and Molecular Characterization of a Chilean Isolate of Zucchini yellow mosaic virus.

Zucchini yellow mosaic virus (ZYMV) was described in 1981 affecting squash, melon, and other cultivated cucurbits with severe stunting and yellowing symptoms. It was reported to be present in most countries where cucurbits are grown, and in Chile since 1995, from surveys using enzyme-linked immunosorbent assay (ELISA) but without further characterization. A potyvirus was isolated from ELISA-positive symptomatic plants. The results indicate that this virus is ZYMV based on symptoms on herbaceous indicators, immunospecific electron microscopy of the purified particle, and sequencing of 395 bases of the 3' end of the coat protein gene. The virus was detected in melon, watermelon, and squash plants. In agreement with previous descriptions for ZYMV, the Chilean isolate is a flexuous filamentous particle 740 nm long with one main protein of approximately 36 kDa. Nucleotide sequence comparisons of the 3' portion of the coat protein gene revealed a high similarity to the Connecticut and California strains.

Proteomic analysis of grapevine (Vitis vinifera L.) leaf changes induced by transition to autotrophy and exposure to high light irradiance.

Using a proteomics approach, we evaluated the response of heterotrophic and autotrophic leaves of grapevine when exposed to high light irradiation. From a total of 572 protein spots detected on two-dimensional gels, 143 spots showed significant variation caused by changes in the trophic state. High light treatment caused variation in 90 spots, and 51 spots showed variation caused by the interaction between both factors. Regarding the trophic state of the leaf, most of the proteins detected in the heterotrophic stage decreased in abundance when the leaf reached the autotrophic stage. Major differences induced by high light were detected in autotrophic leaves. In the high-light-treated autotrophic leaves several proteins involved in the oxidative stress response were up-regulated. This pattern was not observed in the high-light-treated heterotrophic leaves. This indicates that in these types of leaves other mechanisms different to the protein antioxidant system are acting to protect young leaves against the excess of light. This also suggests that these protective mechanisms rely on other sets of proteins or non-enzymatic molecules, or that differences in protein dynamics between the heterotrophic and autotrophic stages makes the autotrophic leaves more prone to the accumulation of oxidative stress response proteins. BIOLOGICAL SIGNIFICANCE: Transition from a heterotrophic to an autotrophic state is a key period during which the anatomical, physiological and molecular characteristics of a leaf are defined. In many aspects the right functioning of a leaf at its mature stage depends on the conditions under what this transition occurs. This because apart of the genetic control, environmental factors like mineral nutrition, temperature, water supply, light etc. are also important in its control. Many anatomical and physiological changes have been described in several plant species, however in grapevine molecular data regarding changes triggered by this transition or by light stress are still scarce. In this study, we identify that the transition from heterotrophic to autotrophic state in grapevine triggers major changes in the leaf proteome, which are mainly related to processes such as protein synthesis, protein folding and degradation, photosynthesis and chloroplast development. With the exception of proteins involved in carbon fixation, that increased in abundance, most of the proteins detected during the heterotrophic stage decreased in abundance when the leaf reached its autotrophic stage. This is most likely because leaves have reached their full size and from now they have to work as a carbon source for sink organs located in other parts of the plant. Despite the potential control of this transition by light, to date, no studies using a proteomics approach have been conducted to gain a broader view of the effects of short-term high light stress. Our results indicate that short-term high light exposure has a major impact on the proteome of the autotrophic leaves, and trigger a differential accumulation of several proteins involved in the oxidative stress response. Surprisingly, heterotrophic leaves do not display this pattern which can be attributed to a lower sensitivity of these leaves to high light stimulus. In fact we discovered that heterotrophic leaves are more tolerant to light stress than autotrophic leaves. This finding is of high biological significance because it helps to understand how young leaves are able to evolve to autotrophy in areas where high light intensities are predominant. This also reveals in this type of leaves the existence of alternative mechanisms to address this stressful condition. These observations provide new insights into the molecular changes occurring during transition of leaves to autotrophy particularly when this transition occurs under high light intensities. This for example occurs during the springtime when the grapevine buds burst and the young leaves are suddenly exposed to high light intensities.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2011-30 September 2011.

This article documents the addition of 299 microsatellite marker loci and nine pairs of single-nucleotide polymorphism (SNP) EPIC primers to the Molecular Ecology Resources (MER) Database. Loci were developed for the following species: Alosa pseudoharengus, Alosa aestivalis, Aphis spiraecola, Argopecten purpuratus, Coreoleuciscus splendidus, Garra gotyla, Hippodamia convergens, Linnaea borealis, Menippe mercenaria, Menippe adina, Parus major, Pinus densiflora, Portunus trituberculatus, Procontarinia mangiferae, Rhynchophorus ferrugineus, Schizothorax richardsonii, Scophthalmus rhombus, Tetraponera aethiops, Thaumetopoea pityocampa, Tuta absoluta and Ugni molinae. These loci were cross-tested on the following species: Barilius bendelisis, Chiromantes haematocheir, Eriocheir sinensis, Eucalyptus camaldulensis, Eucalyptus cladocalix, Eucalyptus globulus, Garra litaninsis vishwanath, Garra para lissorhynchus, Guindilla trinervis, Hemigrapsus sanguineus, Luma chequen. Guayaba, Myrceugenia colchagüensis, Myrceugenia correifolia, Myrceugenia exsucca, Parasesarma plicatum, Parus major, Portunus pelagicus, Psidium guayaba, Schizothorax richardsonii, Scophthalmus maximus, Tetraponera latifrons, Thaumetopoea bonjeani, Thaumetopoea ispartensis, Thaumetopoea libanotica, Thaumetopoea pinivora, Thaumetopoea pityocampa ena clade, Thaumetopoea solitaria, Thaumetopoea wilkinsoni and Tor putitora. This article also documents the addition of nine EPIC primer pairs for Euphaea decorata, Euphaea formosa, Euphaea ornata and Euphaea yayeyamana.

Possible initial steps in the catabolism of 1,2-diphenylethanone (deoxybenzoin) by Pseudomonas fluorescens DB-5.

A natural bacterial strain, identified as Pseudomonas fluorescens DB-5, was isolated in enrichment cultures containing 1,2-diphenylethanone as the only source of carbon and energy. On the basis of characteristic features observed in the mass spectra of degradation intermediates, it is proposed that metabolism of 1,2-diphenylethanone is initiated by two hydroxylations on the benzyl ring. Phenol, presumably arising from the benzoyl ring, was transiently detected as a catabolic intermediate.

Coronary artery plaque rapidly induced by local electromagnetic stimulation in the baboon.

A reliable method has been developed to produce stenosis of the right coronary artery of baboons as a consequence of electrostimulation of the vessel at a chosen position. At that site a pair of electrodes were implanted and activated with a train of 9-volt pulses (length: 10 ms, separation: 100 ms) for 30 min, 5 days/week, up to 6 weeks. 13 animals were included in the experiment, 2 of those served as controls. Pathohistologically the structure of the artificially produced constrictions is similar to atherosclerotic lesions. On average the stenoses occupied 55% of the available lumen; total occlusion and no stenosis were observed in 1 case each.

Yeast pyruvate kinase: essential lysine residues in the active site.

1. Yeast pyruvate kinase was purified to near homogeneity and subjected to chemical modification by trinitrobenzenesulfonate and by P1, P2-bis (5' pyridoxal) diphosphate. 2. Labeled peptides were isolated and their amino acid composition was determined. 3. The results suggest that yeast pyruvate kinase has an essential lysine residue, and that this residue is in a location equivalent to an essential lysine described in the muscle enzyme. 4. Protection experiments indicate that this lysine is located at the nucleotide binding site.