Tubulin-basedpolymorphism(TBP)isanintronlengthpolymorphism(ILP)methodwidelyapplicabletoanyplantspeciesandparticularlysuitableforafirstandrapidclassificationofanyplantgenome.Itisbasedontheselective,polymerasechainreaction(PCR)-basedamplificationofthetwointronspresentatconservedpositionswithinthecodingsequencesofplant?-tubulingenes.Amplificationreleasesasimpleyetdistinctivegenomicprofile.

An original decision support Web service is described, designed to aid biologists in identifying the animal and plant species of raw materials present in food products, based on the automatic analysis of their DNA bar-codes. This service allows to speed up a rather burden and meticulous task of biologists as well as standardizing the recognition so as to increase their confidence on the results whose objectives are assessing the quality of food products.

The simple and straightforward recognition of Triticum species is not an easy task due to their complex genetic origins. To provide a recommendation, we have compared the performance of different PCR-based methods relying on the discrimination ability of the Q- and g-gliadin (GAG56D) genes, as well as TBP (Tubulin-Based Polymorphism), a method based on the multiple amplification of genes of the -tubulin family. Among these approaches, the PCR-RFLP (Restriction Fragment Length Polymorphism) assay based on a single-nucleotide polymorphism (SNP) present in the Q gene is the only one capable of fully discerning hexaploid spelt and common wheat species, while both g-gliadin and TBP fail with similar error frequencies. The Q-locus assay results in the attainment of either a single fragment or a doublet, depending on the presence of a suitable restriction site, which is affected by the mutation. This dual pattern of resolution limits both the diagnostic effectiveness, when additional Triticum species are assayed and compared to each other, and its usefulness, when commercially available flours are analyzed. These limitations are overtaken by flanking the Q-locus assay with the TBP analysis. In this way, almost all of the Triticum species can be accurately identified.

Duckweeds have been increasingly studied in recent years, both as model plants and in view of their potential applications as a new crop in a circular bioeconomy perspective. In order to select species and clones with the desired attributes, the correct identification of the species is fundamental. Molecular methods have recently provided a more solid base for taxonomy and yielded a consensus phylogenetic tree, although some points remain to be elucidated. The duckweed genus Lemna L. comprises twelve species, grouped in four sections, which include very similar sister species. The least taxonomically resolved is sect. Lemna, presenting difficulties in species delimitation using morphological and even barcoding molecular markers. Ambiguous species boundaries between Lemna minor L. and Lemna japonica Landolt have been clarified by Tubulin Based Polymorphism (TBP), with the discovery of interspecific hybrids. In the present work, we extended TBP profiling to a larger number of clones in sect. Lemna, previously classified using only morphological features, in order to test that classification, and to investigate the possible existence of other hybrids in this section. The analysis revealed several misidentifications of clones, in particular among the species L. minor, L. japonica and Lemna gibba L., and identified six putative 'L. gibba' clones as interspecific hybrids between L. minor and L. gibba.

Duckweeds (Lemnaceae) are the smallest and fastest-growing angiosperms. This feature, together with high starch production and good nutritional properties, makes them suitable for several applications, including wastewater treatment, bioenergy production, or feed and food supplement. Due to their reduced morphology and great similarity between diverse species, taxonomic identification of duckweeds is a challenging issue even for experts. Among molecular genotyping methods, DNA barcoding is the most useful tool for species identification without a need for cluster analysis. The combination of two plastid barcoding loci is now considered the gold standard for duckweed classification. However, not all species can be defined with confidence by these markers, and a fast identification method able to solve doubtful cases is missing. Here we show the potential of tubulin-based polymorphism (TBP), a molecular marker based on the intron length polymorphisms of b-tubulin loci, in the genomic profiling of the genera Spirodela, Landoltia, and Lemna. Ninety-four clones were analyzed, including at least two representatives of each species of the three genera, with a special focus on the very heterogeneous species Lemna minor. We showed that a single PCR amplification with universal primers, followed by agarose gel analysis, was able to provide distinctive fingerprinting profiles for 10 out of 15 species. Cluster analysis of capillary electrophoresis-TBP data provided good separation for the remaining species, although the relationship between L. minor and Lemna japonica was not fully resolved. However, an accurate comparison of TBP profiles provided evidence for the unexpected existence of intraspecific hybrids between Lemna turionifera and L. minor, as further confirmed by amplified fragment length polymorphism and sequence analysis of a specific b-tubulin locus. Such hybrids could possibly correspond to L. japonica, as originally suggested by E. Landolt. The discovery of interspecific hybrids opens a new perspective to understand the speciation mechanisms in the family of duckweeds.

Transgenerational epigenetics inheritance refers to the possibility to inherit epigenetic-based information acquired from previous generations. The design of experiments that can measure this phenomenon presents complexities. These are related mainly to difficulties in the identification of epigenetic variation components that are independent from genetic variation, and to difficulties related to the time needed to expose genetically stable plants to different environments for several generations. In this study we propose an experimental approach that takes advantage of seed material routinely produced by seedbanks in order to circumvent the abovementioned issues. By taking advantage of this freely available seed material, it is possible to evaluate epigenetic differences induced by the environment experienced by previous generations and to evaluate both the extent and the impact of epigenetic variation in crops. Seeds of two barley (Hordeum vulgare L.) cultivars, which were previously multiplied in six different seedbanks located worldwide, were grown in a common environment and the plants were evaluated at a genetic, epigenetic, and phenotypic level. Our results indicate that barley accessions multiplied in different environments displayed a comparable level of epigenetic variation, which was, however, greater than the genetic variation. Cluster analysis of methylation differences, likewise DNA differences, split the two cultivars in two distinct groups evidencing a clear link between genetic and epigenetic variation. Nevertheless, the analysis of phenotypic traits and of the CCGG sites that are variable in both cultivars suggests that there might be a possible link between previous cultivation environment and induced epigenetic changes at specific DNA regions.

Animal Tubulin-Based-Polymorphism (aTBP), an intron length polymorphism method recently developed for vertebrate genotyping, has been successfully applied to the identification of several fish species. Here, we report data that demonstrate the ability of the aTBP method to assign a specific profile to fish species, each characterized by the presence of commonly shared amplicons together with additional intraspecific polymorphisms. Within each aTBP profile, some fragments are also recognized that can be attributed to taxonomic ranks higher than species, e.g. genus and family. Versatility of application across different taxonomic ranks combined with the presence of a significant number of DNA polymorphisms, makes the aTBP method an additional and useful tool for fish genotyping, suitable for different purposes such as species authentication, parental recognition and detection of allele variations in response to environmental changes.

Background: Plant discrimination is of relevance for taxonomic, evolutionary, breeding and nutritional studies. To this purpose, evidence is reported to demonstrate TBP (Tubulin-Based-Polymorphism) as a DNA-based method suitable for assessing plant diversity. Results: Exploiting one of the most valuable features of TBP, that is the convenient and immediate application of the assay to groups of individuals that may belong to different taxa, we show that the TBP method can successfully discriminate different agricultural species and their crop wild relatives within the Papilionoideae subfamily. Detection of intraspecific variability is demonstrated by the genotyping of 27 different accessions of Phaseolus vulgaris. Conclusions: These data illustrate TBP as a useful and versatile tool for plant genotyping. Since its potential has not yet been fully appreciated by the scientific community, we carefully report all the experimental details of a successful TBP protocol, while describing different applications, so that the method can be replicated in other laboratories. Keywords: Genotyping, Legumes, ?-Tubulins, Intron Length Polymorphism, TBP (Tubulin Based-Polymorphism)

We present a new method for the authentication of the biological identity of raw meat and processed meat products that is based on length polymorphism found in the introns of the members of the animal beta-tubulin gene family. The method, denominated aTBP for animal TubulinBased-Polymorphism, is shown to be capable of assigning an exclusive genomic fingerprinting to ten different animal species, eight of which are largely consumed as food products. Besides an exclusive DNA profiling, each species is characterized by the presence of specific diagnostic fragments, that assist their selective recognition in admixtures and products sold in the market. The aTBP method is also shown to be effective in both DNA/DNA and weight/weight mixtures where the presence of the low abundance species can be detected at the level of 0.5% and 1% respectively. Detection by aTBP genome profiling is also obtained from either frozen/thawed or cooked samples. The composition of 25 market products made by meat was also assessed with respect to what declared in the label. The results are discussed with reference to biosurveillance and disclosure of frauds and contaminations in comparison with other DNA-based diagnostic methods currently used.

Registrazione presso l'Ufficio Italiano Brevetti e Marchi del marchio DNA tested per la certificazione genetica dei prodotti dell'agroalimentare

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.

New food commodities, particularly pasta, bread and cookies, made with mixed flours containing ancient wheat species and other cereals, have become popular in recent years. This calls for analytical methods able to determine authenticity of these products. Most DNA-based methods for the authentication of foodstuff rely on qPCR assays specifically targeting each plant species, not allowing the identification of unsearched ingredients. Moreover, the discrimination among closely related plant species, particularly congeneric ones like Triticum spp, remains a challenging task. DNA fingerprinting through tubulin-based polymorphism (TBP) and a new assay, TBP light, have been optimized for the authentication of different wheat and farro species and other cereals and tested on a set of commercial food products. The assay has a sensitivity of 0.5-1% w/w in binary mixtures of durum wheat in einkorn or emmer flour and was able to authenticate the composition of test food samples and to detect possible adulterations.

Si tratta di un metodo di riconoscimento genetico della identità di specie animali in cibo e preparati alimentari

Il TBP è un nuovo metodo di veloce caratterizzazione genetica dei vitigni, in grado di assegnare ad ognuno il suo specifi co DNA barcode. Rispetto ad analisi molecolari correnti, basate essenzialmente sui microsatelliti, è più rapido, meno laborioso, più intuitivo, applicabile con successo a viti coltivate e selvatiche. È particolarmente idoneo per la caratterizzazione di collezioni vitivinicole e l'autenticazione delle barbatelle.

BACKGROUND: Microtubules, polymerized from alpha and beta-tubulin monomers, play a fundamental role in plant morphogenesis, determining the cell division plane, the direction of cell expansion and the deposition of cell wall material. During polarized pollen tube elongation, microtubules serve as tracks for vesicular transport and deposition of proteins/lipids at the tip membrane. Such functions are controlled by cortical microtubule arrays. Aim of this study was to first characterize the flax ?-tubulin family by sequence and phylogenetic analysis and to investigate differential expression of ?-tubulin genes possibly related to fibre elongation and to flower development. RESULTS: We report the cloning and characterization of the complete flax ?-tubulin gene family: exon-intron organization, duplicated gene comparison, phylogenetic analysis and expression pattern during stem and hypocotyl elongation and during flower development. Sequence analysis of the fourteen expressed ?-tubulin genes revealed that the recent whole genome duplication of the flax genome was followed by massive retention of duplicated tubulin genes. Expression analysis showed that ?-tubulin mRNA profiles gradually changed along with phloem fibre development in both the stem and hypocotyl. In flowers, changes in relative tubulin transcript levels took place at anthesis in anthers, but not in carpels. CONCLUSIONS: Phylogenetic analysis supports the origin of extant plant ?-tubulin genes from four ancestral genes pre-dating angiosperm separation. Expression analysis suggests that particular tubulin subpopulations are more suitable to sustain different microtubule functions such as cell elongation, cell wall thickening or pollen tube growth. Tubulin genes possibly related to different microtubule functions were identified as candidate for more detailed studies.

A consortium of European enterprises and research institutions has been engaged in the Feed-Code Project withthe aim of addressing the requirements stated in European Union Regulation No. 767/2009, concerning market placement and use of feed of known and ascertained botanical composition. Accordingly, an interlaboratory trial was set up to compare the performance of different assays based either on optical microscope or DNA analysis for the qualitative and quantitative identification of the composition of compound animal feeds. A tubulin-based polymorphism method, on which the Feed-Code platform was developed, provided the most accurate results. The present study highlights the need for the performance of ring trials for the determination of the botanical composition of animal feeds and raises an alarm on the actual status of analytical inaccuracy.

An autochthonous starter culture was used in the production of Protected Designation of Origin (PDO) Silter raw milk cheese, and compared to a control cheese in order to study its influence on microbial population dynamics during ripening. Curd and cheese at different ripening periods (0, 30, 60 and 200 days) from two independent dairies were analysed combining culture-based microbiological analysis, Length Heterogeneity-PCR and 16S microbiome profiling. The autochthonous starter determined higher lactic acid bacteria (LAB) levels for the first 30 days of ripening, without interfering with secondary microbiota that determines the tipicality of this cheese. Only a few genera and species persisted in the cheese despite the microbial richness of the curd. In addition, the high levels of different LAB reduced harmful microorganisms. The various analytic methods used resulted in discrepancies in the proportions of Enterococcus, Lactococcus and Lactobacillus spp., but the pivotal role of Streptococcus and Lactococcus genera was evident. The dominant species included those selected to formulate the starter (St. thermophilus, Lc. lactis, Ln. mesenteroides) as well as Lb. paracasei. The addition of autochthonous starter proved to be effective in controlling the first phases of the cheese-making, without compromising cheese typicality

Feed for producing animals represents the first ring of the human food chain. EU Regulations apply to feed the same labeling requirements as for food, but the only accreditated method for the determination of feed composition is based on light microscopy and microdissection. In the framework of the EU project Feed-code, we developed and validated a modular method for the authentication of feed botanical composition.

Within the framework of the EU Project Feed-code, we developed and pre-validated a DNAbased protocol for the qualitative and quantitative determination of the botanical composition of dairy cow feed. Due to the biological complexity and intrinsic variability of the animal feed matrixes, we were confronted with several problematic issues. Hereby we present our current scheme and solutions, successfully tested on feed samples and raw materials