Characterization of the cryptic interspecific hybrid Lemna×mediterranea by an integrated approach provides new insights into duckweed diversity.

Lemnaceae taxonomy is challenged by the particular morphology of these tiny free-floating angiosperms. Although molecular taxonomy has helped clarify the phylogenetic history of this family, some inconsistency with morphological data leads to frequent misclassifications in the genus Lemna. Recently, the finding that Lemna japonica is an interspecific hybrid between Lemna minor and Lemna turionifera provided a clear explanation for one such taxonomic question. Here we demonstrated that L. minor is also capable of hybridizing with Lemna gibba, generating a cryptic but widespread taxon in the Mediterranean area. The nothotaxon Lemna ×mediterranea is described and compared with clones of the putative parental species L. minor and L. gibba. Genetic analysis by nuclear and plastid markers, as well as genome size measurement, revealed that two different cytotypes, diploid and triploid, originated by at least two independent hybridization events. Despite high overall similarity, morphometrical, physiological, and biochemical analyses showed an intermediate position of L. ×mediterranea between its parental species in most qualitative and quantitative characters, and also separation of the two hybrid cytotypes by some criteria. These data provide evidence that hybridization and polyploidization, driving forces of terrestrial plant evolution, contribute to duckweed genetic diversity and may have shaped the phylogenetic history of these mainly asexual, aquatic plants.

Tubulin-Based Polymorphism (TBP) in Plant Genotyping.

Tubulin-based polymorphism (TBP) is an intron length polymorphism (ILP) method widely applicable to any plant species and particularly suitable for a first and rapid classification of any plant genome. It is based on the selective, polymerase chain reaction (PCR)-based amplification of the two introns present at conserved positions within the coding sequences of plant ß-tubulin genes. Amplification releases a simple yet distinctive genomic profile.

New Insights into Interspecific Hybridization in Lemna L. Sect. Lemna (Lemnaceae Martinov).

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.

aTBP: A versatile tool for fish genotyping.

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.

On the applicability of the Tubulin-Based Polymorphism (TBP) genotyping method: a comprehensive guide illustrated through the application on different genetic resources in the legume family.

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.

Tubulin-Based DNA Barcode: Principle and Applications to Complex Food Matrices.

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.

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.

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

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 ß-tubulin family by sequence and phylogenetic analysis and to investigate differential expression of ß-tubulin genes possibly related to fibre elongation and to flower development. RESULTS: We report the cloning and characterization of the complete flax ß-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 ß-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 ß-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 ß-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.

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

The 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 Abstract The modular Feed-code method for the authentication of feed botanical composition.

The Tubulin-Based-Polymorphism Method Provides a Simple and Effective Alternative to the Genomic Profiling of Grape.

The TBP (Tubulin-Based-Polymorphism) method, based on a nuclear ILP (Intron-Length-Polymorphism) molecular marker, has been used for genotyping 37 accessions of the genus Vitis inclusive of different species, rootstocks, wild and cultivated subspecies. A distinct DNA barcode made up by a different number of amplicons, was attributed to each of the different accessions. TBP data were compared with those obtained, with the use of an internationally validated set of six SSR markers. Genetic relationships among the different accessions, dendrogram distributions, correlation values and polymorphic index values (PICs) were definitively comparable when not in favor of TBP. Such an experimental consistency is based upon a genomic organization of the multiple members of the ß-tubulin gene family, the targets of TBP-mediated amplification, that is conserved in Vitis as in any other plant species. The TBP amplicons can actually be used as a useful source of sequence polymorphisms for generating primer pairs capable of identifying specific cultivars in a simple assay. An example for the identification of the 'Sangiovese' cv. is reported. More generally, these data are discussed in terms of the actual advantages that the introduction of the TBP method in the field of grape characterization and genotyping can provide.

Traceback identification of plant components in commercial compound feed through an oligonucleotide microarray based on tubulin intron polymorphism.

According to EU Regulations, all components of commercial compound feed need to be declared on the label. Effective protection against fraud requires severe controls based on accurate analytical methods to ascertain what is declared by the producers. The aim of this work was to develop an oligonucleotide microarray for the molecular recognition of multiple plant components in commercial feeds. We tested the potential of the highly polymorphic first intron sequences from members of the plant ß-tubulin gene family as a target for plant DNA identification. 23 oligonucleotide capture probes, targeting species-specific intron sequences, were assembled within a low density microarray for the identification of 10 plant species, selected from among those most commonly used in cattle feed formulation. The ability of the array to detect specific components in complex flour blends and in compound feed was evaluated.

A multiplex, bead-based array for profiling plant-derived components in complex food matrixes.

Authentication of processed food ingredients is becoming an important issue for customers, and some DNA-based analytical methods have been developed, especially for animal products. As food products typically contain several different ingredients, a current challenge is to increase the multiplexing capacity of DNA-based methods, to develop "all-in-one" assays. Oligonucleotide-coupled, bead-based suspension arrays are sensitive and reproducible multiplex analytical tools. We applied the Multi-Analyte Profile (xMAP™) technology to develop an assay able to concurrently detect five different plant components in mixed flours and in processed feed and food. Capture probes were targeted to species-specific DNA polymorphisms present within the first intron of plant ß-tubulin genes, which can be amplified by the tubulin-based polymorphism-amplification method (TBP-PCR). The workflow is very simple and straightforward, consisting of a PCR amplification step with universal primers, followed by the direct hybridization assay. Results are highly reproducible. For each single plant species, the absolute detection limit was as low as one target DNA copy. In complex mixtures of flours derived from seeds or from commercial dry "pasta," relative limits of detection ranged, in weight, from 2% for soybean to less than 0.5% for wheat. The specificity of the capture probes and the high sensitivity of the method allowed the successful determination of the analytical composition of three feeds as well as eleven food samples, such as snacks, biscuits, and pasta. The multiplexing ability of the assay (up to 100 different analytes) provides scalability and flexibility, in response to specific needs.

Multiple tubulins: evolutionary aspects and biological implications.

Plant tubulin is a dimeric protein that contributes to formation of microtubules, major intracellular structures that are involved in the control of fundamental processes such as cell division, polarity of growth, cell-wall deposition, intracellular trafficking and communications. Because it is a structural protein whose function is confined to the role of microtubule formation, tubulin may be perceived as an uninteresting gene product, but such a perception is incorrect. In fact, tubulin represents a key molecule for studying fundamental biological issues such as (i) microtubule evolution (also with reference to prokaryotic precursors and the formation of cytomotive filaments), (ii) protein structure with reference to the various biochemical features of members of the FstZ/tubulin superfamily, (iii) isoform variations contributed by the existence of multi-gene families and various kinds of post-translational modifications, (iv) anti-mitotic drug interactions and mode of action, (v) plant and cell symmetry, as determined using a series of tubulin mutants, (vi) multiple and sophisticated mechanisms of gene regulation, and (vii) intron molecular evolution. In this review, we present and discuss many of these issues, and offer an updated interpretation of the multi-tubulin hypothesis.

Two anti-microtubular drugs for two differential responses: a rice cell line resistant to EPC remains susceptible to oryzalin.

Sensitivity to the two anti-microtubular drugs oryzalin and EPC (ethyl-N-phenylcarbamate) is shown to be uncoupled in the rice EPC-resistant ER31d cell line, derived from the corresponding ER31 mutant. The ER31d cell line grows in the presence of EPC but it remains susceptible to oryzalin. In the presence of concentrations of EPC up to 0.4 mM, ER31d cells remain viable maintaining cell anisotropy and detectable cortical microtubule array. The amount of α- and ß-tubulin is also maintained high through a regulatory mechanism that operates at post-transcriptional level. In contrast, all these cellular and molecular parameters are heavily affected by the addition of 1 µM oryzalin. Also, the pattern of post-translationally modified α-tubulins changes in the ER31d cells compared to that of their Nihon-Masari wild type line of reference. The different response elicited by the two herbicides is discussed in relation to a possible differential sensitivity of the cortical MT array, that may in turn relate to their different tubulin-binding specificities and chemical structure.

Testing the IMEter on rice introns and other aspects of intron-mediated enhancement of gene expression.

In many eukaryotes, spliceosomal introns are able to influence the level and site of gene expression. The mechanism of this Intron Mediated Enhancement (IME) has not yet been elucidated, but regulation of gene expression is likely to occur at several steps during and after transcription. Different introns have different intrinsic enhancing properties, but the determinants of these differences remain unknown. Recently, an algorithm called IMEter, which is able to predict the IME potential of introns without direct testing, has been proposed. A computer program was developed for Arabidopsis thaliana and rice (Oryza sativa L.), but was only tested experimentally in Arabidopsis by measuring the enhancement effect on GUS expression of different introns inserted within otherwise identical plasmids. To test the IMEter potential in rice, a vector bearing the upstream regulatory sequence of a rice ß-tubulin gene (OsTub6) fused to the GUS reporter gene was used. The enhancing intron interrupting the OsTub6 5'-UTR was precisely replaced by seven other introns carrying different features. GUS expression level in transiently transformed rice calli does not significantly correlate with the calculated IMEter score. It was also found that enhanced GUS expression was mainly due to a strong increase in the mRNA steady-state level and that mutations at the splice recognition sites almost completely abolished the enhancing effect. Splicing also appeared to be required for IME in Arabidopsis cell cultures, where failure of the OsTub6 5' region to drive high level gene expression could be rescued by replacing the poorly spliced rice intron with one from Arabidopsis.

Traceability of plant diet contents in raw cow milk samples.

The use of molecular marker in the dairy sector is gaining large acceptance as a reliable diagnostic approach for food authenticity and traceability. Using a PCR approach, the rbcL marker, a chloroplast-based gene, was selected to amplify plant DNA fragments in raw cow milk samples collected from stock farms or bought on the Italian market. rbcL-specific DNA fragments could be found in total milk, as well as in the skimmed and the cream fractions. When the PCR amplified fragments were sent to sequence, the nucleotide composition of the chromatogram reflected the multiple contents of the polyphytic diet.

In trangenic rice, alpha- and beta-tubulin regulatory sequences control GUS amount and distribution through intron mediated enhancement and intron dependent spatial expression.

The genomic upstream sequence of the rice tubulin gene OsTub6 has been cloned, sequenced and characterized. The 5'UTR sequence is interrupted by a 446 bp long leader intron. This feature is shared with two other rice beta-tubulin genes (OsTub4 and OsTub1) that, together with OsTub6, group in the same clade in the evolutionary phylogenetic tree of plant beta-tubulins. Similarly to OsTub4, the leader intron of OsTub6 is capable of sustaining intron mediated enhancement (IME) of gene expression, in transient expression assays. A general picture is drawn for three rice alpha-tubulin and two rice beta-tubulin genes in which the first intron of the coding sequence for the formers and the intron present in the 5'UTR for the latters, are important elements for controlling gene expression. We used OsTua2:GUS, OsTua3:GUS, OsTub4:GUS and OsTub6:GUS chimeric constructs to investigate the in vivo pattern of beta-glucuronidase (GUS) expression in transgenic rice plants. The influence of the regulatory introns on expression patterns was evaluated for two of them, OsTua2 and OsTub4. We have thus characterized distinct patterns of expression attributable to each tubulin isotype and we have shown that the presence of the regulatory intron can greatly influence both the amount and the actual site of expression. We propose the term Intron Dependent Spatial Expression (IDSE) to highlight this latter effect.

Plant tubulin intronics.

Introns of plant tubulin genes are useful molecular tools to study IME (Intron Mediated Enhancement of gene expression) and to define plant genetic and evolutionary relationships through ILP (Intron Length Polymorphism). Here we show that the intron present within the 5'UTR sequence of some rice beta-tubulin genes can sustain IME in rice transgenic plants and that degenerated oligonucleotide mixtures designed to amplify the first and the second intron present within the coding sequence of plant beta-tubulin genes can successfully detect ILPs among different bean varieties.

Tubulin-based polymorphism (TBP): a new tool, based on functionally relevant sequences, to assess genetic diversity in plant species.

TBP (tubulin-based polymorphism) is a new molecular marker based tool that relies on the presence of intron-specific DNA polymorphisms of the plant beta-tubulin gene family. The multifunctional and essential role of the tubulin proteins is reflected in the conservation of regions within their primary amino acid sequence. The ubiquitous nature of this gene family can be exploited using primers that amplify the first intron of different beta-tubulin isotypes, revealing specific fingerprints. The method is rapid, simple, and reliable and does not require preliminary sequence information of the plant genome of interest. The ability of TBP to discriminate between accessions and species in oilseed rape, coffee, and lotus is shown. In all cases, TBP was able to detect specific genetic polymorphisms in the context of a simplified and readily appreciable pattern of DNA amplification. The application of TBP for assessing genetic diversity and genome origins in disseminated plant landraces rather than in highly inbred cultivated species is also discussed.