A new set of microsatellite markers for Fragaria species and their application in linkage analysis

Summary

This study reports the development of 68 new microsatellite markers. Of these, 45 were obtained, together with 20 others already published, from an AC-enriched genomic library of the wild strawberry Fragaria vesca. The 68 markers were tested for transportability to the cultivated strawberry F. ananassa ‘Miss’ and 83% gave positive amplifications. Twenty pairs of primers were selected and tested for their transportability to 16 Fragaria taxa and eight species of Rosaceae (peach, almond, apricot, European and Sino-Japanese plums, sweet and sour cherry, apple). The average proportion of primers amplifying loci in Fragaria was 69%, while the transportability to Rosaceae was very low and resulted in null amplification for 80% of the primer pairs. In addition, 23 microsatellite markers were developed from F. ananassa ‘expressed sequence tags’ databases. A total of 141 primer pairs from these and published primers, were tested for polymorphism in the two parents (91.333.2 and ‘Snovit’; both belonging to F. vesca) of a full sib population of 46 individuals. Fifty-eight percent of the primers were discarded because they were monomorphic, or were difficult to interpret, or their allelic conformation was not useful for mapping. The segregation of 73 primers was tested in the progeny and a partial map of the female parent was constructed, based on the segregation of 66 useful markers that were ordered into eight linkage groups of which four had from seven to 14 markers.     

Spread of levan-positive populations of <Emphasis Type="Italic">Pseudomonas savastanoi</Emphasis> pv. <Emphasis Type="Italic">savastanoi</Emphasis>, the causal agent of olive knot,in central Italy

Mixtures of wet vegetable wastes (Brassica, carrot or onion) and dry onion waste were composted at 50 °C for 7 days. The incorporation of the raw or composted vegetable waste mixtures into sandy loam, silt and peat soils reduced the viability of sclerotia of S. cepivorum in glasshouse pot bioassays. The reduction in viability was dependent on waste type, rate of incorporation, duration of exposure and soil type. Onion waste was the most effective waste type in reducing sclerotia viability in all three soils. The Brassica and carrot wastes were as effective as the onion waste in silt soil but less effective in sandy loam and peat soil. A 50% w/w incorporation rate of the wastes gave the largest reduction in viability, with an increase in reduction over time. Composted onion waste reduced sclerotia viability under glasshouse and field conditions although the effect was smaller in the field. Composted onion waste incorporated into soil at 50% w/w reduced the incidence of Allium white rot on onion seedlings in glasshouse pot tests. Incidence and control of the disease differed with soil type. The most consistent control was achieved in peat soil whereas no control was observed in silt soil. Incorporation of the waste 2 months prior to sowing or transplanting reduced seedling emergence in sandy loam soil and growth in all three soil types. The potential for field application of composted vegetable wastes as a sustainable method for control of Allium white rot and waste disposal is discussed.     

Multicolor and electron microscopic imaging of connexin trafficking

Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.     

Bioactive constituent production in St. John's Wort in vitro hairy roots. Regenerated plant lines

A wild strain of Agrobacterium rhizogenes was used to regenerate twelve in vitro plant lines from different hairy roots of H. perforatum (St. John's Wort). The production of the main bioactive constituents was observed even though their yields varied in the different plant lines. Two lines were selected for the hyperoside production (4.9-4.6 mg/gdw) while nine were characterized by significant yields of chlorogenic acid (ranged from 0.47 to 1.09 mg/gdw). Furthermore, one out of twelve lines showed a 10-fold higher hypericin content (0.25 mg/gdw) than that reported for the in vitro shoots in the literature. Morphological and phytochemical features were determined in order to select H. perforatum genotypes enriched in valuable bioactive compounds.     

Treatment of cereal products with a tailored preparation of trichoderma enzymes increases the amount of soluble dietary fiber

Nutritionists recommend increasing the intake of soluble dietary fiber (SDF), which is very low in most cereal-based products. Conversion of insoluble DF (IDF) into SDF can be achieved by chemical treatments, but this affects the sensorial properties of the products. In this study, the possibility of getting a substantial increase of SDF from cereal products using a tailored preparation of Trichoderma enzymes is reported. Enzymes were produced cultivating Trichoderma using durum wheat fiber (DWF) and barley spent grain (BSG) as unique carbon sources. Many Trichoderma strains were screened, and the hydrolysis conditions able to increase by enzymatic treatment the amount of SDF in DWF and BSG were determined. Results demonstrate in both products that it is possible to triple the amount of SDF without a marked decrease of total DF. The enzymatic treatment also causes the release of hydroxycinnamic acids, mainly ferulic acid, that are linked to the polysaccharides chains. This increases the free phenolic concentration, the water-soluble antioxidant activity, and, in turn, the phenol compounds bioavailability.     

Ferulic acid from aleurone determines the antioxidant potency of wheat grain (Triticum aestivum L.)

Grain is an important source of phytochemicals, which have potent antioxidant capacity. They have been implicated in the beneficial health effect of whole grains in reducing cardiovascular disease and type 2 diabetes. The aim of the present study was to identify the most important antioxidant fractions of wheat grain. It was found that the aleurone content of these fractions was highly correlated with the antioxidant capacity of the fractions (r = 0.96, p < 0.0001). Ferulic acid appeared to be the major contributor to the antioxidant capacity in fractions with higher antioxidant capacity. The contribution of protein was rather limited. It was concluded that the antioxidant potency of wheat grain fractions is predominantly determined by aleurone content, which can be attributed to the presence of relatively large amounts of phenolic compounds, primarily ferulic acid.     

Evaluation of antibody-ELISA and real-time RT-PCR for the diagnosis and profiling of bluetongue virus serotype 8 during the epidemic in Belgium in 2006

In 2006 bluetongue (BT) emerged for the first time in North-Western Europe. Reliable diagnostic tools are essential in controlling BT but data on the diagnostic sensitivity (Se) and specificity (Sp) are often missing. This paper aims to describe and analyse the results obtained with the diagnostics used in Belgium during the 2006 BT crisis. The diagnosis was based on a combination of antibody detection (competitive ELISA, cELISA) and viral RNA detection by real-time RT-PCR (RT-qPCR). The performance of the cELISA as a diagnostic tool was assessed on field results obtained during the epidemic and previous surveillance campaigns. As the infectious status of the animals is unknown during an epidemic, a Bayesian analysis was performed. Both assays were found to be equally specific (RT-qPCR: 98.5%; cELISA: 98.2%) while the diagnostic sensitivity of the RT-qPCR (99.5%) was superior to that of the cELISA (87.8%). The assumption of RT-qPCR as standard of comparison during the bluetongue virus (BTV) epidemic proved valid based on the results of the Bayesian analysis. A ROC analysis of the cELISA, using RT-qPCR as standard of comparison, showed that the cut-off point with the highest accuracy occurred at a percentage negativity of 66, which is markedly higher than the cut-off proposed by the manufacturer. The analysis of the results was further extended to serological and molecular profiling and the possible use of profiling as a rapid epidemiological marker of the BTV in-field situation was assessed. A comparison of the serological profiles obtained before, during and at the end of the Belgian epidemic clearly showed the existence of an intermediate zone which appears soon after BTV (re)enters the population. The appearance or disappearance of this intermediate zone is correlated with virus circulation and provides valuable information, which would be entirely overlooked if only positive and negative results were considered.