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Tomosynthesis (DBT) has proven to be more sensitive than digital mammography, but it requires longer reading time. We retrospectively compared accuracy and reading times of a simplified protocol with 1-cm-thick slabs versus a standard protocol of slabs + 1-mm-spaced planes, both integrated with synthetic 2D. We randomly selected 894 DBTs (including 12 cancers) from the experimental arm of the RETomo trial. DBTs were read by two radiologists to estimate specificity. A second set of 24 cancers (8 also present in the first set) mixed within 276 negative DBTs was read by two radiologists. In total, 28 cancers with 64 readings were used to estimate sensitivity. Radiologists read with both protocols separated by a 3-month washout. Only women that were positive at the screening reading were assessed. Variance was estimated taking into account repeated measures. Sensitivity was 82.8% (53/64, 95% confidence interval (95% CI) 67.2–92.2) and 90.6% (95% CI 80.2–95.8) with simplified and standard protocols, respectively. In the random screening setting, specificity was 97.9% (1727/1764, 95% CI 97.1–98.5) and 96.3% (95% CI 95.3–97.1), respectively. Inter-reader agreement was 0.68 and 0.54 with simplified and standard protocols, respectively. Median reading times with simplified protocol were 20% to 30% shorter than with standard protocol. A simplified protocol reduced reading time and false positives but may have a negative impact on sensitivity. • The adoption of digital breast tomosynthesis (DBT) in screening, more sensitive than mammography, could be limited by its potential effect on the radiologists’ workload, i.e., increased reading time and fatigue. • A DBT simplified protocol with slab only, compared to a standard protocol (slab plus planes) both integrated with synthetic 2D, reduced time and false positives but had a negative impact on sensitivity.

The DNA polymorphism diffusely present in the introns of the members of the Eukaryotic beta-tubulin gene families, can be conveniently used to establish a DNA barcoding method, named tubulin-based polymorphism (TBP), that can reliably assign specific genomic fingerprintings to any plant or/and animal species. Similarly, many plant varieties can also be barcoded by TBP. The method is based on a simple cell biology concept that finds a conveniently exploitable molecular basis. It does not depend on DNA sequencing as the most classically established DNA barcode strategies. Successful applications, diversified for the different target sequences or experimental purposes, have been reported in many different plant species and, of late, a new a version applicable to animal species, including fishes, has been developed. Also, the TBP method is currently used for the genetic authentication of plant material and derived food products. Due to the use of a couple of universal primer pairs, specific for plant and animal organisms, respectively, it is effective in metabarcoding a complex matrix allowing an easy and rapid recognition of the different species present in a mixture. A simple, dedicated database made up by the genomic profile of reference materials is also part of the analytical procedure. Here we will provide some example of the TBP application and will discuss its features and uses in comparison with the DNA sequencing-based methods.

Traceability of processed food and feed products has been gaining importance due to the impact that those products can have on human/animal health and to the associated economic and legal concerns, often related to adulterations and frauds as it can be the case for meat and milk. Despite mandatory traceability requirements for the analysis of feed composition, few reliable and accurate methods are presently available to enforce the legislative frame and allow the authentication of animal feeds. In this study, nine sensitive and speciesspecific real-time PCR TaqMan MGB assays are described for plant species detection in animal feed samples. The method is based on selective real-time qPCR (RT-qPCR) amplification of target genes belonging to the alternative oxidase (AOX) gene family. The plant species selected for detection in feed samples were wheat, maize, barley, soybean, rice and sunflower as common components of feeds, and cotton, flax and peanut as possible undesirable contaminants. The obtained results were compared with end-point PCR methodology. The applicability of the AOX TaqMan assays was evaluated through the screening of commercial feed samples, and by the analysis of plant mixtures with known composition. The RT-qPCR methodology allowed the detection of the most abundant species in feeds but also the identification of contaminant species present in lower amounts, down to 1% w/ w. AOX-based methodology provides a suitable molecular marker approach to ascertain plant species composition of animal feed samples, thus supporting feed control and enforcement of the feed sector and animal production.

Wild grasses represent a source of genetic biodiversity for the introgression of agronomical traits of interest into their cultivated relatives, and contribute a relevant part of livestock diets. Characterization of their genetic material is often difficult due to poor knowledge of genomes, complex taxonomic relationships and difficulty in correct classification due to elevated phenotypic variation. A suitable universal marker for plants is still lacking and genomic barcoding according to CBOL relies on rbcL plus matK gene sequencing. TBP is a PCR-based ILP molecular marker, targeting introns of the beta-tubulin gene family. Amplicons are separated by PAGE or by capillary electrophoresis giving reproducible fingerprinting. This codominant marker is suitable for fingerprinting at the species level, but is also useful for lower taxonomic levels and for elucidating genotype relationships. A case study of genetic diversity among 17 different Phalaris species is shown. CE-TBP has also been successfully applied to the authentication of forage composition within the FILAGRO project. TBP profiles from temporary grasslands (first and second cut) report, in a single analysis, the sum of specie-specific fingerprinting. Analytical results were further confirmed using the trnL intron as a marker.

Il genere Passiflora (Famiglia Passifloraceae) comprende più di 560 specie di piante rampicanti perenni, originarie della fascia intertropicale del continente americano (90% delle specie). Nei paesi a clima tropicale le passiflore sono coltivate per la commestibilità dei frutti (Passiflora edulis Sims.) o per proprietà terapeutiche (P. incarnata L.), mentre in Europa sono diffusi ibridi pregiati di valore ornamentale con grandi fiori dai colori sgargianti e più resistenti a temperature medio-basse (P. 'Guglielmo Betto' M. Vecchia). L'ibridazione interspecifica è spesso limitata da fenomeni di incompatibilità e da una bassa germinabilità del seme. L'obiettivo di questo lavoro è stato quello di ottimizzare la tecnica dell'ibridazione interspecifica nelle passiflore ornamentali. A tale scopo è stata valutata l'attitudine combinatoria di specie ed ibridi, dei subgeneri Passiflora e Decaloba (sensu Feuillet and MacDougal 2004), appartenenti alla collezione di passiflore ornamentali del CRA-FSO. La vitalità del polline fresco di 22 specie e 14 ibridi è stata determinata mediante colorazione con fluoresceina diacetato e osservazione al microscopio ottico. I genotipi con una vitalità dei granuli pollinici superiore all'80% sono stati utilizzati come genitore maschile nelle successive ibridazioni. Venti genotipi (55% del totale) hanno sviluppato frutti maturi a seguito di impollinazione manuale. Solo le combinazioni di incroci interspecifici effettuate fra specie ed ibridi del subgenere Passiflora (8 combinazioni) e fra specie ed ibridi del subgenere Decaloba (2 combinazioni) hanno prodotto semi in grado di germinare in condizioni controllate (pretrattamento in GA3 2 mg/l per 24 ore, incisione del tegumento, semina alla temperatura di 28°C). Il valore ornamentale degli ibridi interspecifici è in corso di valutazione con descrittori morfologici mirati. Protocolli di coltura in vitro sono stati utilizzati per il mantenimento e la propagazione dei nuovi genotipi.

We have developed a new version of the cTBP (combinatorial tubulin-based polymorphism) method, a previously described approach based on intron-length polymorphism (ILP), to rapidly characterize the ²-tubulin gene family of Camelina sativa (L.) Crantz, a plant species of importance for oil production but still largely unexplored at genomic level. The method, named h-TBP, allows the rapid cloning of the ²-tubulin genomic sequences that encompass the two introns, invariantly present at fixed positions within the coding region of the vast majority of the plant species. The ²-tubulin sequences cloned by h-TBP also comprise part of exon1 and exon3 and the whole sequence of exon2. The h-TBP method has then been used to isolate, clone and characterize the ²-tubulin gene family of C. sativa, composed of at least 20 different ²-tubulin isotypes, named CsTUB1 through CsTUB20. The relatively high number of ²-tubulin genes has been further substantiated by Southern-blot analysis. Comparison of the ²-tubulin exon sequences of C. sativa with those of Arabidopsis thaliana, the closest relative among crucifers, defines distinct groups of putative orthologous genes, identified by a UPMGA cluster analysis. Analysis of the C. sativa ²-tubulin intron sequences reveals some molecular features that can provide the first hints for the understanding of intron plasticity and evolution. From a more immediate perspective, these data provide the first substantial contribution to the characterization of the largely unexplored genome of C. sativa, and the tools for assisting programmes of breeding and selection of the most productive plants.

There seem to be a certain degree of reluctance in accepting ILP-based methods as part of the range of molecular markers that are classically used for plant genotyping. Indeed, since DNA polymorphism results from difference in length of fragments amplified from specific gene loci, not anonymous sequences, the number of markers that can be generated is sometime inadequate for classical phylogeny studies. Yet, ILP-based markers have many other useful advantages that should not go neglected. We support this statement by presenting a large variety of data we have been collecting for a long while regarding the use of cTBP, an ILP marker based on difference in length of the introns present within the members of the plant beta-tubulin gene family.

One of the main task in plant breeding is the identification of germplasm containing genes that could improve the performance of currently established cultivars. In the past few years, molecular markers, in association with more powerful statistical models, have been applied to genetic analysis and breeding of several crops. Moreover, advances in molecular biology have allowed the development of rapid, sensitive and specific screening methods to study genetic diversity and relatedness among plant species and varieties. One recently developed, useful genotyping method is based on intron length polymorphisms present in members of the plant ?-tubulin gene family (cTBP). The genomic organization of these genes, that encode proteins of relevance for growth, is such to allow a multiple approach for detection of genetic diversity. The cTBP method for plant genotyping is fast, reproducible, transferable among species, capable of recognizing single components in heterogeneous mixtures and doesn't require preliminary knowledge of the target genome. In addition to the classic cTBP method, we are now introducing a new approach that uncovers gene-sequence variability in the 5'upstream region of the beta-tubulin genes. Applied on different cultivated plants, this AFLP-like technique holds the potential to become a new powerful DNA fingerprinting tool for studying genetic relationships and diversity.

Jatropha curcas L., a multipurpose shrub, originated in Central America, is present worldwide throughout tropical and subtropical regions. In India, J. curcas had recently been promoted as a potential source to reduce dependence on crude oil. However, our knowledge concerning genotype, phenotype and environmental interaction is limited. In the present study the magnitude of phenotypic growth, oil yield and quality of promising jatropha sources from India have been evaluated at Jorhat in Assam. The molecular basis of the phenotypic diversity present in different accessions predominantly recovered from different locations in India was also verified. After 36 months of field planting, significant differences were noticed among all accessions tested for agronomical and physiological parameters. Free fatty acids, triglyceride acid composition and the presence of phorbol esters and tocopherols have been studied as they influence oil quality. Integration of biochemical parameters with physiological and agronomical data shows that, under the Jorhat environment, accessions expressing the best performance in the field are also the best in oil yield and quality. Genetic diversity among all 31 jatropha accessions has also been studied using different molecular markers. Our results suggest that phenotypic diversity does not seem to rely on polymorphic genomic DNA traits as inferred by the use of the Tubulin Based Polymorphism and the Random Amplified Polymorphic DNA molecular markers.