Molecular Characterization of the USDA White Clover (Trifolium repens L.) Core Collection by RAPD Markers

White clover is one of the most important forage legume species worldwide, playing an important role in Southern Brazil temperate cultivated pastures. This work was aimed to characterize the genetic variability of the USDA white clover core collection formed by 78 accessions representing 50 countries, together with two very well known cultivars (Huia and Ladino Regal), using RAPD (Random Amplified Polymorphic DNA) markers to produce genetic fingerprints. There were used DNA bulks formed by the extraction and mixture of 20 random individuals from each accession. Twenty four primers were used, which revealed from 3 to 29 bands, forming a total of 371 polymorphic bans and only one monomorphic, ranging from 50 to 3098 bp. The results showed a genetic similarity among the accessions, ranging from 0.18 to 0.58 (Jaccard’s index), with an average of 0.24, allowing the identification of each individual accession using just three primers. The results also showed a large genetic variability within the white clover core collection, probably due to its reproduction mode and ploidy level, which could be used in plant breeding program.     

Real-time polymerase chain reaction based assays for quantitative detection of barley, rice, sunflower, and wheat

Quality assurance is a major issue in the food industry. The authenticity of food ingredients and their traceability are required by consumers and authorities. Plant species such as barley (Hordeum vulgare), rice (Oryza sativa), sunflower (Helianthus annuus), and wheat (Triticum aestivum) are very common among the ingredients of many processed food products; therefore the development of specific assays for their specific detection and quantification are needed. Furthermore, the production and trade of genetically modified lines from an increasing number of plant species brings about the need for control within research, environmental risk assessment, labeling/legal, and consumers' information purposes. We report here the development of four independent real-time polymerase chain reaction (PCR) assays suitable for identification and quantification of four plant species (barley, rice, sunflower, and wheat). These assays target gamma-hordein, gos9, helianthinin, and acetyl-CoA carboxylase sequences, respectively, and were able to specifically detect and quantify DNA from the target plant species. In addition, the simultaneous amplification of RALyase allowed bread from durum wheat to be distinguished. Limits of detection were 1 genome copy for barley, sunflower, and wheat and 3.3 copies for rice real-time PCR systems, whereas limits of quantification were 10 genome copies for barley, sunflower, or wheat and approximately 100 haploid genomes for rice real-time PCR systems. Real-time PCR cycling conditions of the four assays were stated as standard to facilitate their use in routine laboratory analyses. The assays were finally adapted to conventional PCR for detection purposes, with the exception of the wheat assay, which detects rye simultaneously with similar sensitivity in an agarose gel.     

Comparison between phenetic characterisation using RAPD and ISSR markers and phenotypic data of cultivated chestnut (Castanea sativa Mill.)

Patterns of phenotypic and phenetic variability in sixPortuguese cultivars of chestnut (Castanea sativa Mill.) are evaluated. Morphological characterisationwas based on the quantification of seventeen traits. Varianceanalysis showed significant differences among cultivars, and cultivar× year for all the traits studied, and trees within cultivarsshowed also some significant differences for some of themorphological variables. A significant correlation was obtainedbetween length of the leaf blade and the percentage of unisexual andandrogynic inflorescence with the effective thermal index,accumulated rainfall from April to October and from July to October,or the accumulated temperature below seven during the dormant period.Principal Component and cluster analysis were performed to group thecultivars, according to their similarity coefficients. For molecularcharacterisation, 125 RAPD and 157 ISSR polymorphic markers wereamplified using 28 and 7 primers respectively. High level ofcongruence among the two marker systems (r =90.5%) was obtained from comparison of pheneticsimilarities based on the percentage of shared fragments. ISSRmarkers revealed important advantages over RAPDs, due to a higheffective multiplex ratio (12.5 for ISSR compared with 2.2 forRAPD analysis) and reproducibility. Although morphological andmolecular results are comparable, slight differences are showed incluster analysis UPGMA dendrograms. Molecular analysis explainedhomonym situations among 'Martainha' and'Longal' cultivars in Portugal.     

Metschnikowia andauensis as a new biocontrol agent of fruit postharvest diseases

Potential antagonists were isolated from the epiphytic flora associated with oranges and pome fruit. A total of 1465 microorganisms were tested in a preliminary screening against blue and green moulds on pome and citrus fruit, respectively. Among them, approximately 3% reduced incidence and severity by more than 50% and 4 microorganisms fulfilled the selection criteria of reduction in severity and incidence by 75%. The most effective was a yeast identified as Metschnikowia andauensis, strain NCYC 3728 (PBC-2), isolated from the surface of ‘Bravo de Esmolfe’ apple fruit cultivated in North Portugal. The biocontrol activity of M. andauensis PBC-2 was dependent on its applied concentration. At 5 × 10⁶ cfu/mL incidence (% of infected wounds) and severity (lesion diameter) were reduced by 62 and 70%, respectively and at 1 × 10⁷ cfu/mL, the greatest reduction was achieved, 90% of incidence and 95% of severity. The broad spectrum of action of M. andauensis PBC-2 was evaluated with effective control being achieved against Rhizopus stolonifer, Penicillium expansum and Botritys cinerea, on ‘Rocha’ pears and on different apple cultivars and against Penicillium digitatum and Penicillium italicum on mandarins and oranges. In semi-commercial trials in cold storage, the reduction of blue mould was 90%. Rapid colonization of fresh apple fruit wounds was observed during the first 24 h of cold storage, followed by a significant population increase during the first 15 days of storage and then the population remained stable until the end of storage.     

Enantioselective organocatalysis using SOMO activation

The asymmetric alpha-addition of relatively nonpolar hydrocarbon substrates, such as allyl and aryl groups, to aldehydes and ketones remains a largely unsolved problem in organic synthesis, despite the wide potential utility of direct routes to such products. We reasoned that well-established chiral amine catalysis, which activates aldehydes toward electrophile addition by enamine formation, could be expanded to this important reaction class by applying a single-electron oxidant to create a transient radical species from the enamine. We demonstrated the concept of singly occupied molecular orbital (SOMO) activation with a highly selective alpha-allylation of aldehydes, and we here present preliminary results for enantioselective heteroarylations and cyclization/halogenation cascades.     

Control of crystal size and lattice formation by starmaker in otolith biomineralization

The stone-like otoliths from the ears of teleost fishes are involved in balance and hearing and consist of calcium carbonate crystallites embedded in a protein framework. We report that a previously unknown gene, starmaker, is required in zebrafish for otolith morphogenesis. Reduction of starmaker activity by injection of modified antisense oligonucleotides causes a change in the crystal lattice structure and thus a change in otolith morphology. The expression pattern of starmaker, along with the presence of the protein on the growing otolith, suggest that the expression levels of starmaker control the shape of the otoliths.     

Beef authentication and retrospective dietary verification using stable isotope ratio analysis of bovine muscle and tail hair

Stable isotope ratio analysis (SIRA) was used as an analytical tool to verify the preslaughter diet of beef cattle. Muscle and tail hair samples were collected from animals fed either pasture (P), a barley-based concentrate (C), silage followed by pasture (SiP), or silage followed by pasture with concentrate (SiPC) for 1 year (n = 25 animals per treatment). The (13)C/(12)C, (15)N/(14)N, (2)H/(1)H, and (34)S/(32)S isotope ratios in muscle clearly reflected those of the diets consumed by the animals. By applying a stepwise canonical discriminant analysis, a good discrimination of bovine meat according to dietary regimen was obtained. On the basis of the classification success rate, the (13)C/(12)C and (34)S/(32)S ratios in muscle were the best indicators for authentication of beef from animals consuming the different diets. Analysis of (13)C/(12)C and (15)N/(14)N in tail hair sections provided an archival record of changes to the diet of the cattle for periods of over 1 year preslaughter.     

Identification of the molecular make-up of the Potato virus Y strain PVY(Z): genetic typing of PVY(Z)-NTN

Potato virus Y (PVY) strains were originally defined by interactions with different resistance genes in standard potato cultivars. Five distinct strain groups are defined that cause local or systemic hypersensitive responses (HRs) in genetic background with a corresponding N gene: PVY(O), PVY(N), PVY(C), PVY(Z), and PVY(E). The nucleotide sequences of multiple isolates of PVY(O) and PVY(N) differ from each other by ≈8% along their genomes. Additionally, complete genome sequences of multiple recombinant isolates are composed of segments of parental PVY(O) and PVY(N) sequences. Here, we report that recombinant isolate PVY-L26 induces an HR in potato 'Maris Bard' carrying the putative Nz gene, and is not recognized by two other resistance genes, Nc and Ny(tbr). These genetic responses in potato, combined with the inability of PVY-L26 to induce vein necrosis in tobacco, clearly define it as an isolate from the PVY(Z) strain group and provide the first information on genome structure and sequence of PVY(Z). The genome of PVY-L26 displays typical features of European NTN-type isolates with three recombinant junctions (PVY(EU-NTN)), and the PVY-L26 is named PVY(Z)-NTN. Three typical PVY(NTN) isolates and two PVY(N) isolates, all inducing vein necrosis in tobacco, were compared with PVY-L26. One PVY(NTN) isolate elicited HR reactions in Maris Bard, similar to PVY-L26, while two induced a severe systemic HR-like reaction quite different from the quasi-symptomless reaction induced by two PVY(N) isolates. 'Yukon Gold' potato from North America produced HR against several PVY(NTN) isolates, including PVY-L26, but only late and limited systemic necrosis against one PVY(N) isolate. Consequently, according to symptoms in potato indicators, both PVY(Z) and PVY(NTN) isolates appeared biologically very close and clearly distinct from PVY(O) and PVY(N) strain groups.