Paula Martins-Lopes

DNA Markers for Portuguese Olive Oil Fingerprinting

The certification of olive oil has led to the definition of Protected Denomination of Origin (PDO) producing regions in European countries. PDO products should be protected, and a solution could be by using DNA fingerprinting. In this work we evaluate the efficiency of RAPD, ISSR, and SSR molecular markers for olive oil varietal identification and their possible use in certification purposes. Twenty-three Portuguese olive oil samples (11 obtained monovarietal and 12 purchased commercial oils) were screened by means of two RAPD, four ISSR, and four SSR markers. The quality of amplified products was used to evaluate the reproducibility and the level of polymorphism. Principal component analysis was performed with DCENTER using unweighted pair group mathematical average (UPGMA) that allowed group formation according to olive oil varietal geographic origin.

RAPD and ISSR molecular markers in Olea europaea L.: Genetic variability and molecular cultivar identification

Thirty Portuguese and eight foreign olive (Olea europaea L.) cultivars were screened using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers. Twenty RAPD primers amplified 301 reproducible bands of which 262 were polymorphic; and 17 ISSR primers amplified 204 bands of which 180 were polymorphic. The percentage of polymorphic bands detected by ISSR and RAPD was similar (88 and 87%, respectively). The genetic variability observed was similar in the Portuguese and foreign olive cultivars. Seven ISSR and 12 RAPD primers were able to distinguish individually all 38 olive cultivars. Twenty specific molecular markers are now available to be converted into Sequence Characterised Amplified Region (SCAR) markers. Relationships among Portuguese and foreign cultivars is discussed.

Cationic solid lipid nanoparticles (cSLN): Structure, stability and DNA binding capacity correlation studies

Cationic solid lipid nanoparticles (cSLN) are promising lipid nanocarriers for intracellular gene delivery based on well-known and widely accepted materials. cSLN containing single-chained cationic lipid cetyltrimethylammonium bromide were produced by high pressure homogenization and characterized in terms of (a) particle size distribution by photon correlation spectroscopy (PCS) and laser diffractometry (LD), (b) thermal behaviour using differential scanning calorimetry (DSC) and (c) the presence of various polymorphic phases was confirmed by X-ray diffraction (WAXD). SLN composed of Imwitor 900P (IMW) showed different pDNA stability and binding capacity in comparison to those of Compritol 888 ATO (COM). IMW-SLN, having z-ave=138-157 nm and d(0.5)=0.15-0.158 μm could maintain this size for 14 days at room temperature. COM-SLN had z-ave=334 nm and d(0.5)=0.42 μm on the day of production and could maintain similar size during 90 days. IMW-SLN revealed improved pDNA binding capacity. We attempted to explain these differences by different interactions between the solid lipid and the tested cationic lipid.

Assessing Genetic Diversity in Olea europaea L. Using ISSR and SSR Markers

Olea europaea L. is one of the most economically important crops in the Mediterranean area, and known for having large genetic variability. In order to assess the genetic diversity, DNA from 41 olive cultivars, present in the protected denomination of origin (PDO) region of Trás-os-Montes, was screened using inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. Eleven ISSR primers amplified 135 reproducible bands of which 108 were polymorphic. The percentage of polymorphic bands detected by ISSR was 79%. The highest number of polymorphic bands was obtained by the use of primers UBC807 (15) and UBC809 (16). A total of 67 alleles were detected by six SSR primers, with an average of 11 alleles per primer. The number of alleles per locus ranged from five (ssrOeUaDCA05) to 15 (ssrOeUaDCA03). The observed heterozygosity ranged from 0.219 (ssrOeUaDCA05) to 0.900 (ssrOeUaDCA04), while the expected heterozygosity varied between 0.426 (ssrOeUaDCA05) and 0.887 (ssrOeUaDCA03). The polymorphism information content (PIC) values ranged from 0.392 (ssrOeUaDCA05) to 0.863 (ssrOeUaDCA03). The collection of primers selected gave a reasonable number of amplification products for the genetic diversity analysis. Based on the results, the genetic diversity among 41 olive cultivars is discussed. This study reveals the great importance of guaranteeing the differentiation of olive cultivars and their application for certification purposes.

Molecular characterization of TaSTOP1 homoeologues and their response to aluminium and proton (H+) toxicity in bread wheat (Triticum aestivum L.)

BackgroundAluminium (Al) toxicity is considered to be one of the major constraints affecting crop productivity on acid soils. Being a trait governed by multiple genes, the identification and characterization of novel transcription factors (TFs) regulating the expression of entire response networks is a very promising approach. Therefore, the aim of the present study was to clone, localize, and characterize the TaSTOP1 gene, which belongs to the zinc finger family (Cys2His2 type) transcription factor, at molecular level in bread wheat.ResultsTaSTOP1 loci were cloned and localized on the long arm of homoeologous group 3 chromosomes [3AL (TaSTOP1-A), 3BL (TaSTOP1-B) and 3DL (TaSTOP1-D)] in bread wheat. TaSTOP1 showed four potential zinc finger domains and the homoeologue TaSTOP1-A exhibited transactivation activity in yeast. Expression profiling of TaSTOP1 transcripts identified the predominance of homoeologue TaSTOP1-A followed by TaSTOP1-D over TaSTOP1-B in root and only predominance of TaSTOP1-A in shoot tissues of two diverse bread wheat genotypes. Al and proton (H+) stress appeared to slightly modulate the transcript of TaSTOP1 homoeologues expression in both genotypes of bread wheat.ConclusionsPhysical localization of TaSTOP1 results indicated the presence of a single copy of TaSTOP1 on homoeologous group 3 chromosomes in bread wheat. The three homoeologues of TaSTOP1 have similar genomic structures, but showed biased transcript expression and different response to Al and proton (H+) toxicity. These results indicate that TaSTOP1 homoeologues may differentially contribute under Al or proton (H+) toxicity in bread wheat. Moreover, it seems that TaSTOP1-A transactivation potential is constitutive and may not depend on the presence/absence of Al at least in yeast. Finally, the localization of TaSTOP1 on long arm of homoeologous group 3 chromosomes and the previously reported major loci associated with Al resistance at chromosome 3BL, through QTL and genome wide association mapping studies suggests that TaSTOP1 could be a potential candidate gene for genomic assisted breeding for Al tolerance in bread wheat.

A novel lipid nanocarrier for insulin delivery: production, characterization and toxicity testing.

A novel nanocarrier based on solid lipid nanoparticles (SLNs) was developed for insulin delivery using a novel double emulsion method. Physical stability of particles was assessed by size analysis using dynamic light scattering (DLS), matrix crystallinity by differential scanning calorimetry (DSC) and toxicity analysis by Drosophila melanogaster testing. Insulin-SLNs were composed of Softisan®100 1.25% wt, Lutrol®F68 1% wt, soybean lecithin 0.125% wt, and loaded with 0.73–0.58 mg/mL peptide. Placebo-SLNs (insulin-free) also contained 0.025% wt Tween®80. Mean particle sizes of placebo-SLN and insulin-SLN were 958 ± 9.5 and 978 ± 8.3 nm, respectively. The polydispersity index (PI) was 0.28 ± 0.018 and 0.29 ± 0.013, respectively. Polarized light microscopy analysis depicted no aggregation of developed particles. DSC analysis allowed characterizing SLN with 43–51% matrix crystallinity. Using Drosophila melanogaster test, no toxicity was reported for SLN and for the bulk lipid. This study shows that SLNs are promising and helpful to overcome conventional insulin therapy, in particular for their lack of toxicity for oral delivery.

Molecular characterization of the citrate transporter gene TaMATE1 and expression analysis of upstream genes involved in organic acid transport under Al stress in bread wheat (Triticum aestivum)

In bread wheat, besides malate, the importance of citrate efflux for Al tolerance has also been reported. For better understanding the Al tolerance mechanism in bread wheat, here, we performed both a molecular characterization of the citrate transporter gene TaMATE1 and an investigation on the upstream variations in citrate and malate transporter genes. TaMATE1 belong to multidrug transporter protein family, which are located on the long arm of homoeologous group 4 chromosomes (TaMATE1-4A, TaMATE1-4B TaMATE1-4D). TaMATE1 homoeologues transcript expression study exhibited the preponderance of homoeologue TaMATE1-4B followed by TaMATE1-4D whereas homoeologue TaMATE1-4A seemed to be silenced. TaMATE1, particularly homoeologue TaMATE1-4B and TaALMT1 transcripts were much more expressed in the root apices than in shoots of Al tolerant genotype Barbela 7/72/92 under both control and Al stress conditions. In addition, in both tissues of Barbela 7/72/92, higher basal levels of these gene transcripts were observed than in Anahuac (Al sensitive). Noticeably, the presence of a transposon in the upstream of TaMATE1-4B in Barbela 7/72/92 seems to be responsible for its higher transcript expression where it may confer citrate efflux. Thus, promoter variations (transposon in TaMATE1-4B upstream and type VI promoter in TaALMT1) associated with higher basal transcript expression of TaMATE1-4B and TaALMT1 clearly show how different mechanisms for Al tolerance operate simultaneously in a single genotype. In conclusion, our results demonstrate that Barbela 7/72/92 has favorable alleles for these organic acids transporter genes which could be utilized through genomic assisted selection to develop improved cultivars for acidic soils.

Modified Rose Bengal assay for surface hydrophobicity evaluation of cationic solid lipid nanoparticles (cSLN)

Surface hydrophobicity of nanocarriers influences protein binding and subsequently fate of nanoparticles in blood circulation. Therefore, characterization of surface hydrophobicity of nanocarriers provides important preclinical information. Here, a modified classical adsorption method for the needs of characterization of cationic solid lipid nanoparticles (cSLN) was developed. We have identified possible method limitations that should be considered when performing the analysis, i.e. the problems associated with particle separation from the dispersion and their own absorbance in visible spectrum. We propose two modified methods for performing the assay overcoming the stated limitations. We also discuss here evaluation by different approaches (calculation of binding constants or partitioning quotient) and their suitability for the prepared cSLN formulation. Overall, we confirmed that our modified adsorption method can provide useful information about surface properties of (cationic) SLN, however, performing and evaluation of the assay need special attention in order to obtain the desired results.

An Enhanced Method for Vitis vinifera L. DNA Extraction from Wines

Wine quality and value largely depend on grape variety, which is of primary importance in wine identification. The aim of the present work was to enhance a wine DNA extraction protocol and, subsequently, grapevine variety identification. This enhanced method is an outcome from several previously developed extraction methods and effectively allows obtaining large amounts of high-quality DNA exhibiting an optimal 260/280 ratio. Grapevine variety DNA extracted from wine was amplifiable with a specific SSR primer. This procedure was applicable for monovarietal and older commercial red and white wines. The potential of this enhanced method relies on its use for traceability as part of protecting both consumer and producer interests.

Biosensor for label-free DNA quantification based on functionalized LPGs.

A label-free fiber optic biosensor based on a long period grating (LPG) and a basic optical interrogation scheme using off the shelf components is used for the detection of in-situ DNA hybridization. A new methodology is proposed for the determination of the spectral position of the LPG mode resonance. The experimental limit of detection obtained for the DNA was 62±2nM and the limit of quantification was 209±7nM. The sample specificity was experimentally demonstrated using DNA targets with different base mismatches relatively to the probe and was found that the system has a single base mismatch selectivity.