Floriana Gavazzi

Technical improvement of the TBP (tubulin-based polymorphism) method for plant species detection, based on capillary electrophoresis.

Nowadays, feed and food safety and traceability are of primary importance. Hence, a correct labeling of the different products is highly desirable in general, but mandatory for those people who are suffering from eating disorders and food allergies. Among the technologies that have been developed for feed and food analysis, the patented tubulin‐based polymorphism (TBP) method emerges as an easy, versatile, and inexpensive diagnostic tool. Initially used to fingerprint different plant species and varieties, TBP was then successfully applied to trace species in mixtures of plant origin such as commercial feeds. TBP is a DNA‐based molecular marker, that makes use of PCR for the selective amplification of plant β‐tubulin introns. Amplified fragments are then separated by PAGE and visualized by silver staining. We have now developed an improved version of TBP. Based on capillary electrophoresis and fluorescence detection, it makes the method automatic, more sensible, reproducible, and faster. Compared to the classic TBP, this new version allows to obtain a better data resolution and an easier interpretation of the results, clearing the way to large‐scale feed/food diagnostics.

TBP-assisted species and hybrid identification in the genus Passiflora

We used the tubulin-based-polymorphism (TBP) method to analyze 45 different species and interspecific hybrids of the genus Passiflora. With regard to genetic characterization, the TBP method is a convenient, fast, simple and reliable tool for assigning a specific polymorphic pattern (i.e., molecular fingerprint) to each of the species and hybrids analyzed. In the case of hybrids, an accurate, easy and immediate recognition of parental contribution is an additional benefit of the TBP method. Molecular data collected using the TBP marker system resulted in a classification of the species of Passiflora that is fully consistent with previous studies performed with a variety of nuclear and chloroplast markers. Similar to those molecular studies, the TBP phylogenetic classification of the various species differs from that obtained using the more restricted number of morphological markers. Overall, this outcome indicates the limited value of these descriptors with respect to genomic diversity when discriminating among different species of Passiflora.

Development and validation of the modular Feed-code method for qualitative and quantitative determination of feed botanical composition

AbstractThe analysis of feed composition in terms of ingredients is addressed by Regulation (EC) 767/2009 and is important for detecting economic fraud and for monitoring feed safety. Within the framework of the EU project Feed-code, we developed and internally validated a modular assay, relying on intron polymorphism, for the complete qualitative analysis of the botanical composition of feed and the quantitative determination of six target plant species. Main performance parameters of each module, such as applicability, repeatability, specificity, and limit of detection, were evaluated. The whole assay was applied to a set of feed-like samples and results were in agreement with the expected composition. Application to a large set of compound feed and individual raw materials revealed the occurrence of botanical impurities. When compared with microscopic analysis, the proposed method gave more reliable results. We conclude that the Feed-code prototype, readily upgradable to include more plant species, is worthy of consideration for a full validation through a collaborative trial. Graphical AbstractThe modular Feed-code method for the authentication of feed botanical composition

Evolutionary characterization and transcript profiling of β-tubulin genes in flax (Linum usitatissimum L.) during plant development

BackgroundMicrotubules, 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 β-tubulin family by sequence and phylogenetic analysis and to investigate differential expression of β-tubulin genes possibly related to fibre elongation and to flower development.ResultsWe report the cloning and characterization of the complete flax β-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 β-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 β-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.ConclusionsPhylogenetic analysis supports the origin of extant plant β-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.

Untargeted DNA-based methods for the authentication of wheat species and related cereals in food products

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 sample and to detect possible adulterations.

Interlaboratory Comparison of Methods Determining the Botanical Composition of Animal Feed

A consortium of European enterprises and research institutions has been engaged in the Feed-Code Project with the 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.