Validation of a simplified small-scale DNA extraction protocol from wine by quantitative real-time PCR.

In the present study, we compared a simplified small-scale purification protocol to obtain DNA admixtures out of wine, with our large-scale published method. The extraction methods must provide DNA free of PCR inhibitors, that can interfere with DNA amplification. To evaluate the efficiency of grapevine's nuclear DNA extraction from wine, the new protocol was also compared in terms of purity and yield to the DNA obtained out of grapevine's (Vitis vinifera) leaf tissue, using a commercial kit. Two single-copy nuclear genes, nine-cis-epoxy carotenoid dioxygenase 2 (NCED2), and prefoldin subunit 5-like (PS5) were amplified in DNA extracted from wine and grapevine by real-time TaqMan PCR to determine the presence of inhibitors in relation to the diversity of starting biological matrix. This study showed that the small-scale, simpler method for extracting DNA from wine produced effective results in terms of inhibitor presence and purity. Furthermore, even though the initial biological matrix was more complicated, the grapevine nuclear DNA that was removed from wine was qualitatively equivalent to the DNA that was isolated from the leaves. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03992-x.

Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth.

Pollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identify Nicotiana tabacum Differentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC-ESI-MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

How to integrate wet lab and bioinformatics procedures for wine DNA admixture analysis and compositional profiling: Case studies and perspectives.

The varietal authentication of wines is fundamental for assessing wine quality, and it is part of its compositional profiling. The availability of historical, cultural and chemical composition information is extremely important for quality evaluation. DNA-based techniques are a powerful tool for proving the varietal composition of a wine. SSR-amplification of genomic residual Vitis vinifera DNA, namely Wine DNA Fingerprinting (WDF) is able to produce strong, analytical evidence concerning the monovarietal nature of a wine, and for blended wines by generating the probability of the presence/absence of a certain variety, all in association with a dedicated bioinformatics elaboration of genotypes associated with possible varietal candidates. Together with WDF we could exploit Bioinformatics techniques, due to the number of grape genomes grown. In this paper, the use of WDF and the development of a bioinformatics tool for allelic data validation, retrieved from the amplification of 7 to 10 SSRs markers in the Vitis vinifera genome, are reported. The wines were chosen based on increasing complexity; from monovarietal, experimental ones, to commercial monovarietals, to blended commercial wines. The results demonstrate that WDF, after calculation of different distance matrices and Neighbor-Joining input data, followed by Principal Component Analysis (PCA) can effectively describe the varietal nature of wines. In the unknown blended wines the WDF profiles were compared to possible varietal candidates (Merlot, Pinot Noir, Cabernet Sauvignon and Zinfandel), and the output graphs show the most probable varieties used in the blend as closeness to the tested wine. This pioneering work should be meant as to favor in perspective the multidisciplinary building-up of on-line databanks and bioinformatics toolkits on wine. The paper concludes with a discussion on an integrated decision support system based on bioinformatics, chemistry and cultural data to assess wine quality.

Distribution and Characterization of the Vitis vinifera L. subsp sylvestris in Southern Tuscany.

BACKGROUND: The Eurasian grapevine is the most widespread fruit crop in the world. Numerous studies have focused on clarifying the events of domestication and the geographical areas in which they occurred. OBJECTIVES: In order to add upon information on the process of grapevine domestication, the distribution and genetic diversity of a small, wild population localized in Poggio Ghiaccio Forte, an archaeological site in Maremma (Southern Tuscany), were assessed. In a preliminary survey the wild forms of Vitis vinifera L. were identified based on morphological traits. METHOD: Fourty-two accessions of Vitis vinifera ssp. sylvestris were collected near the Albegna river and its secondary conduits. As a control, four non-vinifera grapevines (Vitis berlandieri, Vitis riparia Fabre, rupestris Constantia, rupestris H. Goethe) and ten varieties of Vitis vinifera ssp. sativa characterizing the Tuscan grapevine germplasm (Sangiovese, Ciliegiolo, Aleatico, Ansonica, Canaiolo Nero, Trebbiano Toscano, Barsaglina, Malvasia Lunga, Moscato Bianco, Abrusco) were added to the wild population. All fifty-six vines were treated as one population and tested by 10 SSR-based genotyping. RESULTS: According to SSR analysis, the wild population seems to be characterized by a systematic reduction of observed compared to expected heterozygosity due to the tendency of inbreeding and genetic trait fixation. There are a lot of registered patents about different applications involving Vitis vinifera mostly relating to disease resistance, grapevine fitness and novel combinations of antioxidants useful in therapeutic, foodstuff and cosmetic fields.

Archaeobotanical reconstructions of vegetation and report of mummified apple seeds found in the cellar of a first-century Roman villa on Elba Island.

In the late Roman Republic period (2nd-1st century BC), in the area of San Giovanni on Elba Island, previously subject to intense extraction of iron ore, a rustic villa was established by Marco Valerio Messalla, a supreme Roman magistrate. The foundations of the walls were discovered and excavated by an archaeological mission. Palaeobotanical analysis of a set of stratigraphic layers was performed. Palynological slides showed remains of palynomorphic and non-pollen objects, while data combined with anthracological investigations confirmed the hypothesis that in the 1st century AD the villa was destroyed by a fire that created a compact crust under which were discovered four broken Roman amphorae containing about five hundred apple seeds. Comparisons of archaeological and fresh seeds from reference collections showed discontinuous morphology except for one group of archaeological samples. DNA was isolated from seeds that had well-preserved embryos in all groups. DNA extracts from archaeological, wild and modern domestic seeds (controls) were amplified by PCR and tested with SSR molecular markers, followed by genome analysis.

Genetic differentiation between cinta senese and commercial pig breeds using microsatellite

Background: Cinta Senese (CS) is an autochthonous Tuscan breed, which risked extinction since the ‘60s. Results: Monitoring the genetic variability of the actual population by use of DNA molecular markers is essential to address a correct breeding policy, finalized to obtain the race preservation and its fitness in the future. 17 SSRs autosomal markers and 1 associated to the X chromosome were used to genotype 86 individuals belonging to the CS and 12 belonging to two main white races Landrace (L), Large White (LW) and crosses between LW and L and L and CS widespread in Tuscany and used in the recent past to obtain hybrids with the CS. Conclusions: A dendrogram of similarity measures the relative genetic distance between individuals in the population. Data show that CS pigs have a distinct genotype from L, LW, LW x L and L x CS.

Pomology observations, morphometric analysis, ultrastructural study and allelic profiles of "olivastra Seggianese" endocarps from ancient olive trees (Olea europaea L.).

Preliminary studies of historical sources and remote sensing were used to identify ancient olive trees near archaeological sites and heritage buildings in the Orcia Valley (Siena, Italy). Distinctive characters were assessed by traditional pomological observation. Trees with similar characters were selected on the basis of the features of endocarps, the only structure that survives aerobic deterioration and conserves useful botanical information for centuries. Non-invasive morphometric analysis of endocarp size and shape established morphological variations in individuals of different populations. Plastid organization in the endocarp and location of DNA in the endocarp tegument were detected by morphological and ultrastructural observations using light and electron microscopy. Cytoplasmic markers with high polymorphism were used to test similarity of endocarp and leaf DNA within individuals and to confirm low variability and minimal divergence between individuals. The ancient trees studied showed the same allelic profiles and therefore belonged to a distinct cultivar. The traditional pomological descriptions of the trees, leaves and fruits, morphometric analysis of size, and shape elliptic Fourier analysis of endocarp outline, ultrastructural observations and allelic profiles of endocarp tegument delineated the general species-specific qualities of the cultivar "olivastra Seggianese" of the Orcia Valley.

The SSR-based molecular profile of 1005 grapevine (Vitis vinifera L.) accessions uncovers new synonymy and parentages, and reveals a large admixture amongst varieties of different geographic origin.

A collection of 1005 grapevine accessions was genotyped at 34 microsatellite loci (SSR) with the aim of analysing genetic diversity and exploring parentages. The comparison of molecular profiles revealed 200 groups of synonymy. The removal of perfect synonyms reduced the database to 745 unique genotypes, on which population genetic parameters were calculated. The analysis of kinship uncovered 74 complete pedigrees, with both parents identified. Many of these parentages were not previously known and are of considerable historical interest, e.g. Chenin blanc (Sauvignon × Traminer rot), Covè (Harslevelu selfed), Incrocio Manzoni 2-14 and 2-15 (Cabernet franc × Prosecco), Lagrein (Schiava gentile × Teroldego), Malvasia nera of Bolzano (Perera × Schiava gentile), Manzoni moscato (Raboso veronese × Moscato d'Amburgo), Moscato violetto (Moscato bianco × Duraguzza), Muscat of Alexandria (Muscat blanc à petit grain × Axina de tres bias) and others. Statistical robustness of unexpected pedigrees was reinforced with the analysis of an additional 7-30 SSRs. Grouping the accessions by profile resulted in a weak correlation with their geographical origin and/or current area of cultivation, revealing a large admixture of local varieties with those most widely cultivated, as a result of ancient commerce and population flow. The SSRs with tri- to penta-nucleotide repeats adopted for the present study showed a great capacity for discriminating amongst accessions, with probabilities of identity by chance as low as 1.45 × 10(-27) and 9.35 × 10(-12) for unrelated and full sib individuals, respectively. A database of allele frequencies and SSR profiles of 32 reference cultivars are provided.

Ultrastructural study of archaeological Vitis vinifera L. seeds using rapid-freeze fixation and substitution.

The ultrastructure of Vitis vinifera seeds from different archaeological sites was studied. Preservation status differed between sites. Preliminary investigations of grape seeds from Poggio Bacherina (Chianciano Terme, Siena) and Miranduolo (Chiusdino, Siena) showed collapsed or charred tegument, making this material suitable for morphometric studies only. Rapid-freeze fixation and substitution of grape seeds from Shahr-I Sokhta in Iran and via De' Castellani in Florence revealed well preserved tegument suitable for chemical and cytochemical analysis. Energy dispersive X-ray microanalysis was used to determine chemical composition. Cytochemical analysis based on fluorescent staining with DAPI suggested the presence of cytoplasm residues.

Polymorphism of some native Sistan grapes assessed by long and short primers for RAPD markers.

Grapevines have Bronze ages archive in Sistan area of Iran. In order to study the genetic variation and taxonomic relationships between 6 cultivars of the Sistan grapevines (Vitis vinifera L.) at molecular level, random amplified polymorphic DNA (RAPD) markers were used. The data were subjected to statistical analyses and genetic resemblance was calculated using Dice similarity index. The grapevines related to the different geographic areas of Sistan were assessed by 50 short (10 mer) and long (15-21 mer) primers. Out of 50 primers which were tested, 21 primers gave reproducible results. Selected primers created 497 bands. Resulting profiles showed that the produced bands varied in size from 300 to 3500 base pairs. The numbers of reliable polymorphic fragments for short and long primers were 86 and 334 bands, respectively. In multiplication reaction the items in the size area of 564 to 1904 base pair resulted for short primers and 564 to 4277 base pair for long primers. From the bands calculated a matrix that was analyzed by the unweighted pair group method on arithmetic averages to draw a dendrogram. The population was classified in 4 main groups in which Red Yaghooti and White Yaghooti had the maximum and Red Yaghooti and Laal had the minimum similarity coefficients. In our study, by comparing the results gained from technique long and short primers in RAPD, the potential value of long primers for the production ofpolymorphism in grapes was identified.

A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis.

A simple and universally applicable protocol for extracting high-quality proteins from recalcitrant plant tissues is described. We have used the protocol with no modification, for a wide range of leaves and fruits. In all cases, this protocol allows to obtain good electrophoretic separation of proteins. As the protocol is rapid, universal, and compatible with silver staining, it could be used for routine protein extraction from recalcitrant plant tissues for proteomic analysis.

A methionine synthase homolog is associated with secretory vesicles in tobacco pollen tubes.

Seven isoforms of 85 kDa polypeptides (p85) were identified as methionine synthase (MetE) homologs by partial aminoacid sequencing in tobacco pollen tube extracts. Immunocytochemistry data showed a localization of the antigen on the surface of tip-focussed post-Golgi secretory vesicles (SVs), that appear to be partially associated with microtubules (Mts). The chemical dissection of pollen tube high speed supernatant (HSS) showed that two distinct pools of MetE are present in pollen tubes, one being the more acidic isoforms sedimenting at 15S and the remaining at 4S after zonal centrifugation through a sucrose density gradient. The identification of the MetE within the pollen tube and its possible participation as methyl donor in a wide range of metabolic reactions, makes it a good subject for studies on pollen tube growth regulation.

Post-translational modifications of alpha-tubulin in Zea mays L are highly tissue specific.

To further understand post-translational modifications (PTMs) of plant alpha-tubulin, post-translationally modified alpha-tubulin isoforms from selected tissues of Zea mays L. were examined using two-dimensional electrophoresis and immunoblotting. Except for polyglycylated tubulin, tyrosinated, detyrosinated, acetylated and polyglutamylated alpha-tubulin isoforms were all present in maize tissues. Tyrosinated alpha-tubulin was the predominant variant in all cases, with isoforms alpha1-alpha4 (alpha5) being the most common components. Leaves exhibited a striking difference in PTM patterns of alpha-tubulin isoforms compared to other tissues examined. In leaves, several major specific isoforms were highly modified by detyrosination, acetylation and polyglutamylation. In pollen and anthers, only the most abundant isoform alpha3 was acetylated to an appreciable extent, and no acetylated isoform was found in roots. Similarly, in pollen, anthers and roots, only alpha3 was appreciably polyglutamylated. Additionally, a detyrosinated isoform alpha6 was present in anthers and in leaves, while the tyrosinated isoform alpha6 seemed to be pollen specific. These results indicate that certain types of PTM of plant alpha-tubulin preferentially occur in a tissue-specific way.

Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds.

The purpose of this research is to establish a routine procedure for the application of proteomic analysis to olive tree. Olive leaf tissue is notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds. We developed a protocol for isolating proteins suitable for two-dimensional electrophoresis (2-DE) from olive leaf. The remarkable characteristics of the protocol include: (i) additional grinding dry acetone powder of leaf tissue to a finer extent, (ii) after extensive organic solvent washes to remove pigments, lipids etc., using aqueous tricholoroacetic acid washes to remove water-soluble contaminants, and (iii) phenol extraction of proteins in the presence of sodium dodecyl sulfate. The final protein preparation is free of interfering compounds based on its well-resolved 2-DE patterns. The protocol can be completed within 3 h, and protein yield is approximately 2.49 mg.g(-1) of aged leaf. We also evaluated the protocol by immunoblotting with anti-tyrosinate alpha-tubulin antibody. To our knowledge, this is the first time that a protocol for protein extraction from olive leaf appears to give satisfactory and reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues and could be of interest to laboratories involved in plant proteomics.