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Backcrossing between Sikitita and its male parent Arbequina, offers the possibility to check the suitability of different self-incompatibility models proposed for olive. To determine Sikitita’s response to self- and cross-pollination treatments, including pollination with its father Arbequina, we compared the parameters following pollen–pistil interaction, the resulting initial and final fruit set, and the paternity of the seeds produced under different crosses. The results showed that Sikitita behaves as a self-incompatible cultivar due to the inhibition of pollen tube growth in the pistil of self-pollinated flowers. This incompatibility reaction led to a significant reduction of self-fertilization and fruit set. Seed paternity analyses confirmed the self-incompatibility response of Sikitita. A similar incompatibility response was observed in Sikitita flowers when hand-pollinated with pollen of Arbequina and Koroneiki. On the contrary, cross-pollination with Arbosana gave excellent results, with analyses showing that pollen of Arbosana is largely preferred by Sikitita to father its seeds more than the pollen of other cultivars presented in the orchard. The backcross failure of Sikitita with Arbequina pollen suggests that the self-incompatibility system in olives is not of the gametophytic type. In contrast, pollination tests fit features of previously reported sporophytic self-incompatibility systems. However, some amendments are proposed, among them the incompatibility groups for Sikitita and Koroneiki.

Helichrysum italicum (Roth) G. Don is a Mediterranean medicinal plant with great potential in the cosmetics, culinary and pharmaceutical fields due to its unique bioactive compounds. Its recent introduction into agroecosystems has enhanced the exploitation of genetic diversity in natural populations, although limited molecular markers have made this challenging. In the present study, primers were designed for all 43 SSRs (72.1% mononucleotide, 21% dinucleotide and 6.9% trinucleotide repeats) identified in the chloroplast genome. Populations from Cape Kamenjak (Croatia) and Corsica (France) were analyzed with ten carefully selected cpSSR markers. From the initial set of 16 cpSSRs amplified in all samples, 6 cpSSR markers were removed due to low-length polymorphisms, size homoplasy and nucleotide polymorphisms that could not be detected with allele length. Of the 38 haplotypes detected, 32 were unique to their geographic origin. The highest number of private haplotypes was observed in the Cape Kamenjak population (seven out of nine detected). Based on clustering analyses, the Kamenjak population was the most similar to the Capo Pertusato (south Corsica) population, although only one sub-haplotype was shared. Other Corsican populations were more similar to each other. A cross-species transferability test with Helichrysum litoreum Guss. and Helichrysum arenarium (L.) Moench was successfully conducted and private alleles were identified.

Population aging has led to an increased interest in various dietary supplements in order to preserve cognitive function. The aim of our study was to examine the effects of Hericium erinaceus supplementation (HE) on cognitive function and serum levels of Brain-Derived Neurotrophic Factor (BDNF) and Neuropeptide Y (NPY). An 8-week double-blind comparative study involved 33 subjects, randomly assigned to a HE group and a placebo (P) group. Anthropometric measurements and analyses of selected biochemical parameters were performed before and after the intervention. Simultaneously, faecal samples were collected to determine gut microbiota and chitinase 3-like-1 (CHI3L1) levels. Cognitive function was assessed using two non-verbal speed tests. In the HE group, a significant improvement in cognitive ability was observed when the corresponding cognitive scores at baseline, CHI3L1 level, age, and gender were considered. Moreover, in the HE group there was a significant increase in gut microbiota diversity that was positively correlated to NPY levels. The mechanism that could help to explain the results on the relationship between the active ingredients of the H. erinaceus and the cognitive parameters could point to the CHI3L1 activity that may enhance bioavailability of the bioactive ingredients. Supplementing the diet with HE was recognised as a safe and well-tolerated intervention with a neurocognitive benefit.

Trendy high-density olive fields are often monovarietal orchards, mostly using the cultivar Arbequina. However, Arbequina shows a strong self-incompatibility response, and its yields depend on wind cross-pollination, which is not always available. With the aim of finding suitable self-compatible cultivars that can replace Arbequina, we evaluated pollen–pistil interaction, fruit set and seed paternity in Arbosana under different pollination treatments: self-pollination, open-pollination and three cross-pollination treatments: × Arbequina, × Sikitita and × Koroneiki. All these cultivars are low-vigor cultivars suitable for high-density orchards, making them potential pollinizers for Arbosana. The results show that Arbosana is also self-incompatible with a strong reduction in fruit set due to a lower fertilization level caused by a strong inhibition of pollen tube growth in self-pollinated flowers. Seed-paternity analyses confirmed the self-incompatibility response of Arbosana and suggest that some fruit obtained in bagged shoots under self-pollination were, in fact, a product of cross-fertilization. In conclusion, we recommend against the use of Arbosana in large monovarietal orchards. On the contrary, good results were obtained under cross-pollination with Sikitita, Arbequina and Koroneiki pollen, allowing us to recommend them as pollinizers for Arbosana in appropriate pollination designs. This is the first time Arbosana self-incompatibility has been reported.

Abstract Helichrysum arenarium (L.) Moench (Asteraceae) is a self‐compatible, insect‐pollinated herb occurring in sand grasslands, and is declining and endangered in many parts of its European distribution range. A recovery plan of H. arenarium has been conducted in southern Belgium, involving plant translocations. We developed multiplex genotyping protocol for nine microsatellite markers previously published for Helichrysum italicum and two newly developed microsatellite markers for H. arenarium. Eleven polymorphic loci were associated (pooled) in two multiplex panels, to assess the genetic status of the only small remaining population in Belgium and of three large German populations used as seed source for propagating transplants. The small Belgian population was characterized by high clonality, with only two, however heterozygous, genets detected. The three large German populations showed high genetic diversity (He ranging from 0.635 to 0.670) and no significant inbreeding coefficient values, despite expectations of geitonogamous selfing. Management practices (grazing livestock) increasing seed dispersal distances, inbreeding depression at early stages of development, and mechanisms preventing or delaying selfing might be hypothesized to explain the observed patterns. The two Belgian genotypes remained within genetic variation range of German populations so that the high genetic differentiation between Belgian and German populations (FST values ranging from 0.186 to 0.206) likely resulted from genetic drift effects and small sample size. Transplants obtained from seeds sampled from the three large source populations from Germany constitute a highly diverse, noninbred gene pool, and are thus of high genetic quality for plant translocations.

The aim of this study was to assess changes in mental health, gut microbiota composition, and stress marker serum cortisol due to COVID-19 lockdown in asymptomatic individuals. Healthy adults participated in anthropometric measurements, blood and stool sample collection pre-lockdown and post-lockdown (n = 38, 63.2% females), lifestyle and psychological questionnaires were included in pre-lockdown measurement and lockdown survey (n = 46, 67.4% females). Subjects reported significantly higher body dissatisfaction (p = 0.007) and anxiety (p = 0.002), and significantly lower positive affect (p = 0.001) during lockdown compared with pre-lockdown. According to perceived stress, 51.6% of females and 20% of males experienced moderate to high stress. This was reflected in serum cortisol levels that significantly increased only in females (p = 0.006) post-lockdown and correlated with perceived stress (p = 0.037) and anxiety (p = 0.031). In addition to psychological measures, changes in gut microbiota composition were observed. Gut microbial alpha diversity significantly decreased (p = 0.033), whereas relative abundance of Proteobacteria significantly increased (p = 0.043) post-lockdown. Depression during lockdown was moderately positively correlated with changes in Bacteroidetes abundance (p = 0.015) and negatively with changes in Firmicutes abundance (p = 0.008). Alistipes abundance post-lockdown was moderately positively correlated with anxiety (p = 0.004) and negative affect (p = 0.005) during lockdown. Despite a small sample size and not being able to perform objective measurements during lockdown, the results confirm the effect of lockdown on mental health and gut microbiota composition that could have a great impact on our health (ClinicalTrials identifier: NCT04347213).

Helichrysum italicum (Roth) G. Don (HI) and Helichrysum arenarium (L.) Moench (HA) are rich in polyphenols and their infusions have beneficial effects for patients with metabolic syndrome. To investigate whether these effects are mediated by the gut microbiota, we analysed the effects of daily consumption of HI or HA infusion on the composition of gut microbiota, inflammatory status, and zonulin, a marker of gut barrier permeability. The study was a randomized, double-blind comparative trial. Thirty participants were randomly assigned to two groups and received either HA or HI tea filter bags, each containing 1 g of dried plant material, for daily consumption lasting 4 weeks. The results show that consumption of both infusions resulted in a reduction of some genera belonging to Firmicutes and in a slight but significant reduction in Shannon diversity index. Consumption of HI infusion significantly reduced serum levels of proinflammatory markers and zonulin alongside with the observed trend of Proteobacteria reduction. It can therefore be concluded that the HI and HA infusions could act as prebiotics and thus improve the intestinal environment. In addition, HI infusion has a positive impact on microbial dysbiosis and gut barrier dysfunction that occur in obesity and metabolic syndrome.

Jujube (Ziziphus jujuba Mill.) has favourable horticultural properties including adaptation to arid conditions, abiotic and biotic stresses, as well as positive impact on human health. The present study describes the characterization of genetic diversity of the germplasm of jujube from the Istrian peninsula, the determination of important chemical compounds, antioxidative properties in relation to antibacterial and antifungal activities of jujube fruit extracts, and the determination of nutritional properties of jujube fruit. The results of the genetic analysis showed that most of the samples from the Istrian peninsula belong to two recently introduced varieties, 'Li' and 'Lang', and the most widespread local variety 'Navadna žižola'. The local variety has smaller fruit than the ‘Li’ and ‘Lang’ varieties, with thick and fleshy mesocarp. Chemical analysis indicated that fruits of the local variety contained a valuable source of dietary fibre ((9.7±0.6) g/100 g) and were rich in minerals such as (in g/100 g dry mass): potassium (829±51), calcium (177±11) and phosphorus (129±19). Aqueous extracts showed slight antibacterial activity, while ethanol extracts had higher mass fractions of phenolic compounds (expressed as gallic acid equivalents (GAE), 5.8-8.7 mg/g) than the aqueous extracts, but did not show antimicrobial activity. Compounds other than phenolic compounds in jujube fruit may be more biologically active. Based on the results of these analyses, the local Istrian jujube variety is a promising candidate for cultivation potential.

The ‘Žutica’ represents the most common Montenegrin olive varieties mainly used for the production of olive oil and green and black fruit canning. Traditionally, the olive plants have been propagated vegetatively, and a small level of genetic polymorphism is expected among clones of the same variety. This topic was only partially studied in the Montenegrin olive ‘Žutica’. Therefore, this study aimed to determine intra-varietal genetic variability in twenty-three ‘Žutica’ trees selected in situ, analyzing the variability of morphological traits and microsatellites. The Principal Component Analyses (PCA) with six axes explains the total cumulative variance of 91.3 %, with fruit and endocarp traits in the first three PC. The unweighted pair group method with arithmetic mean of twenty morphological traits grouped ‘Žutica’ trees into two clusters and five independent trees. Nine microsatellite primers amplified 31 fragments of which 22 were polymorphic and enabled the detection of nine different microsatellite profiles (potential different clones). A comparison of dendrogram groups based on morphological and microsatellite markers showed low cophenetic values in the determination of intra-varietal variability. The results showed that the old variety ‘Žutica’, from a relatively small geographic region, has a variable genetic base, which could be used in the selection of superior clones.

Chili and its main active ingredient capsaicin have been shown to induce several favorable biological activities. In the present study we have tested the effect of three chili sauces mimicking popular commercially available chili sauces with an increasing amount of capsaicinoids on a broad range of serum biochemical markers in thirty healthy subjects. We show that in the region where daily use of chili is traditionally low, moderate consumption of 4.4 mg of capsaicinoids per day has beneficial health effects, such as decrease in glucose level, LDL cholesterol and C-reactive protein. However, when a product with higher daily dose of capsaicinoids (16.7 mg/day) was consumed, those effects were lost. Considering that biochemical marker levels returned to the initial levels after a week-long wash-out period, we suggest that regular intake of moderate amounts of chili product would be most profitable.

The olive tree (Olea europaea L.) is a wind-pollinated crop that exhibits an extreme alternate bearing habit. To improve fruit set, several methods have been used to determine the most successful compatible combinations of cultivars. In this study, priority is given to seed paternity analysis based on simple sequence repeats (SSRs), microsatellite markers used for the identification of potential pollen donors of cultivar ‘Oblica’ in a mixed olive orchard during two consecutive years. Seven microsatellite primers were successfully used to examine the paternity of olive embryos from ‘Oblica’ mother trees. Embryos were considered as a product of self-fertilization if only maternal alleles were present, but not a single case of self-fertilization was found among all the embryos analyzed. Two dominant pollen donors were not the closest nor the cultivars with the highest number of trees in the orchard, suggesting that cross-compatibility may have a key role in determining pollen donor success. In our earlier studies, pollen tube growth and fertilization success correlated with fruit set when controlled crosses between cultivars were performed; however, some discrepancy might appear compared to paternity analyses when mother trees have a free choice among different pollen sources from cultivars growing in their surroundings.

Helichrysum italicum is an aromatic plant with promising pharmacological activities. Bioactive compounds found in plants represent an important alternative treatment for weight loss and an infusion of H. italicum contains compounds which could have such effect. Our aim was, therefore, to investigate its acute effects on resting energy expenditure (REE) and possible differences in substrate oxidation in a pilot study. A dried, ground plant material of H. italicum ssp. italicum was infused with hot water and chemically characterized with HPLC-MS analysis. Sensory evaluation of herbal tea was performed. A randomized, crossover, controlled pilot study was then conducted on eleven healthy male subjects. The REE and substrate oxidation were measured by indirect calorimetry at baseline and 30 and 120 min after ingestion of infusion or hot water. The expression of genes involved in lipid metabolism was examined in H. italicum infusion-treated hepatocytes. Several phenolic classes were identified in the infusion, caffeoylquinic acids were the most abundant, followed by pyrones and flavonols. A single ingestion of H. italicum infusion significantly increased REE by 4% and fat oxidation by 12% compared to hot water ingestion. A significant 2-fold up-regulation of β-oxidation-related genes in HepG2 cells, exposed to H. italicum infusion, was detected. This pilot study suggests that H. italicum infusion possesses bioactive substances with potential application in obesity prevention, which could, with additional studies, become an economically interesting novel application of the plant. Clinical trial registration number: NCT04818905

This study characterises the genetic variability of local pomegranate (Punica granatum L.) germplasm from the Slovenian and Croatian areas of Istria. The bioactive components and antioxidant and antimicrobial properties of ethanol and water extracts of different parts of pomegranate fruit were also determined, along with their preliminary nutritional characterisation. Twenty-six different genotypes identified with microsatellite analysis indicate the great diversity of pomegranate in Istria. The pomegranate fruit ethanol extracts represent rich sources of phenolic compounds (mean value of the mass fraction in exocarp and mesocarp expressed as gallic acid is 23 and 16 mg/g, respectively). The ethanol extracts of pomegranate exocarp and mesocarp showed the greatest antimicrobial activity against Candida albicans, Candida parapsilosis, Rhodotorula mucilaginosa, Exophiala dermatitidis and Staphylococcus aureus, and the same water extracts against S. aureus and Escherichia coli. To the best of our knowledge, this study represents the first report of the characterisation of pomegranate genetic resources from Istria at different levels, including the molecular, chemical, antimicrobial and nutritional properties.

Helichrysum arenarium (L.) Moench (abbrev. as HA) has a long tradition in European ethnomedicine and its inflorescences are approved as a herbal medicinal product. In the Mediterranean part of Europe, Helichrysum italicum (Roth) G. Don (abbrev. as HI) is more common. Since infusions from both plants are traditionally used, we aimed to compare their antioxidative potential using in vitro assays. Two morphologically distinct HI plants, HIa and HIb, were compared to a commercially available HA product. Genetic analysis using microsatellites confirmed a clear differentiation between HI and HA and suggested that HIb was a hybrid resulting from spontaneous hybridization from unknown HI subspecies. High-performance liquid chromatography–mass spectrometry analysis showed the highest amounts of hydroxycinnamic acids and total arzanol derivatives in HIa, whereas HIb was richest in monohydroxybenzoic acids, caffeic acids, and coumarins, and HA contained the highest amounts of flavonoids, especially flavanones. HIa exhibited the highest radical scavenging activity; it was more efficient in protecting different cell lines from induced oxidative stress and in inducing oxidative stress-related genes superoxide dismutase 1, catalase, and glutathione reductase 1. The antioxidative potential of HI was not only dependent on the morphological type of the plant but also on the harvest date, revealing important information for obtaining the best possible product. Considering the superior properties of HI compared to HA, the evaluation of HI as a medicinal plant could be recommended.

Mediterranean plant Helichrysum italicum represents a rich source of versatile bioactive compounds with potential benefits for human health. Despite extensive research on the plant’s active constituents, little attention has yet been paid to characterizing the relationship between its intra-specific genetic diversity and metabolite profile. The study aimed to determine metabolic profile of H. italicum ssp. italicum (HII) and ssp. tyrrhenicum (HIT) cultivated on the experimental plantation in Slovenia and to compare the chemical composition of extracts regarding the solvent extraction process. Extracts were prepared upon conventional extract preparation procedures: maceration with 50% methanol or ethanol and cold or hot water infusion and analyzed using High Performance Liquid Chromatography-Diode Array Detection-Electrospray Ionization-Quadrupole Time-of-Flight-Mass Spectrometry (HPLC-DAD-ESI-QTOF-MS). One hundred compounds were identified in the samples, among them several isomers and derivatives were reported for the first time, while caffeoylquinic acids and pyrones were the most abundant. Semi-quantitative comparison revealed that the extraction procedure had a greater impact on the chemical profile than genetic variability. All HIT extracts showed a higher total phenolic content compared to HII, while the antioxidant potential evaluated by 1,1-diphenyl-2-picrylhydrazil test was not proportionally higher. In addition, hot water extracts proved to be comparably active as alcoholic ones, confirming high commercial potential of Helichrysum italicum as herbal functional beverages.

Microsatellites have been identified as the marker of choice in plant genotyping projects. However, due to length discrepancies obtained between different laboratories for the same allele, interlaboratory comparison of fingerprinting results is often a difficult task. The objectives of this study were to compare genotyping results of two laboratories, to evaluate genetic parameters of microsatellite markers and to determine reference allele sizes for fig cultivars from the Istrian peninsula. Genotyping results of ninety fig (Ficus carica L.) accessions were comparable between the laboratories despite differences observed when comparing electropherograms of different capillary electrophoresis systems. Differences in lengths of the same alleles were detected due to different PCR methods and laboratory equipment, but the distances between alleles of the same locus were preserved. However, locus FSYC01 exhibited one allele dropout which led to misidentification of 28 heterozygotes as homozygote individuals suggesting this locus as unreliable. Allele dropout was assigned to the tail PCR technology or to a touchdown PCR protocol. Genotypes of twenty-four reference cultivars from the Istrian peninsula were confirmed by both laboratories. These results will contribute to the usage of markers with greater reliability, discrimination power and consequently, to more reliable standardization with other fig genotyping projects.

The aromatic plant immortelle contains valuable bioactive molecules and a unique fragrance. These key factors contribute to its attractiveness and implementation in agricultural ecosystems as well as applications in the pharmaceutical and cosmetic industries. In this study, we have characterized the complete chloroplast genome sequence of Helichrysum italicum subsp. italicum from the North Adriatic region. The total genome size was 152,431 bp in length, containing 85 protein-coding genes, 36 transfer RNA genes, 8 ribosomal RNA genes, and 2 partial genes (ycf1 and rps19). Based on the available chloroplast genomes from Gnaphalieae tribe, the closest relationship was identified with Anaphalis sinica.

The genetic diversity and ampelographic variability of autochthonous red wine cultivar ‘Refošk’ (Vitis vinifera L.) grown in Slovenia were evaluated with AFLP molecular markers and OIV descriptors, respectively. SSR molecular markers were employed to confirm cultivar identity of analysed samples. Eight AFLP primer combinations, one was monomorphic, produced 16 polymorphic markers in 41 out of 113 samples, what classified samples into monomorphic and polymorphic group. Dendrogram constructed with simple matching coefficient and unweighted pair-group method analysis presented genetic diversity within polymorphic group. Refošk biotypes from monomorphic and polymorphic groups were evaluated with 22 OIV descriptors related to bunch, berry and must, but on the basis of ampelographic characterization samples were not differentiated among two major groups obtained with AFLP analysis. Results of genetic analysis indicated that ‘Refošk’ originated from closely related plants that are phenotypically very similar. With regard to low observed genetic diversity more attention should be dedicated to the selection in order to conserve remaining genetic diversity.

Rugose wood disease complex is one of the most important graft-transmissible grapevine diseases and it is considered to be a viral disease. With the aim to obtain more information about appearance of rugose wood disease observed on cultivar ‘Refošk’, ‘Refošk’ vines from collection vineyard in Komen were used for green grafting on SO4 rootstock and autografts for control were made as well. Rugose wood symptoms were observed on grafts of two ‘Refošk’ biotypes, which confirmed graft transmissibility. Appearance of rugose wood symptoms on autografts excluded the impact of incompatibility in rugose wood disease, but at the same time it could be proposed that stress caused by grafting has an important role.

The objective of the research is to determine the leading microorganisms in spontaneous fermentations of table olives in Slovenian Istria. The influence of traditional regional and modified Spanish style technology on yeast and lactic acid bacteria population dynamics and on the quality of Istrska belica and Storta table olive varieties was studied during 180 days of fermentation. Apart from that, pH of the brine during fermentation and total biophenols in olive fruits before processing and after 60 and 180 days of fermentation were determined. The quality of the final product was determined with sensory analysis. Table olive fermentation was carried out by yeasts. Aureobasidium pullulans, Cryptococcus adeliensis, Metschnikowia pulcherrima, Rhodotorula mucilaginosa, Pichia anomala and Candida oleophila were isolated and identified using PCR-RFLP analysis of ITS regions and traditional phenotypic tests. High initial amount of total biophenols in olive fruits and their better preservation during traditional processing influenced microbial population dynamics and quality characteristics of table olives. The modified Spanish style technology was not confirmed as suitable for retaining positive characteristics of the product of traditional regional technology in Slovenian Istria.

The olive tree has anatomical-morphological and physiological adaptations which enable it to cope well with dry conditions and water deficits. However, if water shortage occurs during the development phases (shoot growth, flower bud development, bloom, fruit set, cell division and enlargement and oil accumulation), which are the most susceptible to stress, it can also have a negative effect on the growth and productivity of olive trees. The Slovenian Istria is facing with increased risk of drought. Due to increased occurrence and intensity of agricultural droughts controlled deficit irrigation will become an inevitable element of agricultural practice in Slovene Istria.

Microsatellites combine several features of an ultimate molecular marker and they are used increasingly in various plant genetic studies and applications. In this work we report on the utilisation of fourteen previously developed olive microsatellite markers for the identification and differentiation of a set of nineteen olive varieties. All analysed microsatellite markers revealed a high level of polymorphism that allowed unique genotyping of the examined varieties. Ninety-six alleles were detected at all 14 loci, which multiplied into a large number of observed genotypes, giving high discrimination value for varietal identification. A minimum number of three microsatellite markers was chosen for the rapid and unambiguous varietal identification of nineteen olive varieties and only two markers were sufficient for differentiation of five local varieties. DNA fingerprints of olive cultivars by means of microsatellites provided meaningful data, which can be extended by additional olive varieties or new microsatellites and used for accurate inter-laboratory comparison. The data obtained can be used for the varietal survey and construction of a database of all olive varieties grown in Slovenia providing also additional genetic information on the agronomic and quality characteristics of the olive varieties.

The leading olive variety in Slovenia is “Istrska belica” (Olea europaea L.), which currently represents 70% of all olive trees in productive orchards. Paternity analysis based on microsatellite markers was used for genotyping and identification of the potential pollen donors of “Istrska belica” and for assessing the proportion of self-fertilization in monovarietal olive orchards in the Slovene Istria. Seven microsatellite loci were used for genotyping thirty-one olive embryos from “Istrska belica” trees and for all potential pollen donor varieties, which are grown in the region and could participate as pollinators. Genotyping results and allele identification were performed using the FaMoz software. The most probable pollen donor was assigned to 39% of all analyzed embryos. Among all analyzed embryos no single case of self-fertilization was confirmed. According to the present results, the variety “Istrska belica” was in all cases fertilized by foreign pollen. The results will contribute to defining the new guidelines for farmers regarding the proper management and growing practice in monovarietal olive groves.

A novel method for the economic fluorescent labelling of PCR products and its features are described. To investigate the efficiency of the method for amplification of olive microsatellite markers, PCR conditions of three loci were optimised. The results showed that the method can be successfully applied in genotyping studies of olive varieties.

The AFLP (amplified fragment length polymorphism) technique using fluorescence-labelled primers and automated detection is described and some features of this method are discussed. The modified AFLP technique is suitable for efficient fingerprinting analysis and gives a much higher resolution than silver staining detection systems. With the aid of an automated ALFexpress sequencer and computer software, the AFLP method is much considerably easier to perform and the total time required is reduced.

RAPD markers were applied to assess molecular polymorphisms within olive cultivars held in a collection orchard and to develop a system of a minimum number of primers for cultivar identification. Random Amplified Polymorphic DNA of fifteen cultivars ('Istrska belica', 'Štorta', 'Buga', 'Črnica', ‘Unknown 2’, 'Leccino', 'Nocelara del belice', 'Frantoio', 'Cipressino', 'Santa Caterina', 'Pendolino', 'Arbequina', 'Maurino', 'Picholine', 'Unknown 1') was analysed, using twelve different arbitrary primers: OPA-1, OPA-4, OPA-8, OPX-2, OPX-3, OPX-4, OPX-8, OPX-11, OPX-12, OPX-13, OPX-14, OPX-19. The results confirmed identity within trees of thirteen cultivars in the collection orchard while mislabeling was found in two cultivars. The high polymorphism detected by RAPD markers was successfully used for the development of an identification key for rapid olive cultivar determination.

ABSTRACT: The olive (Olea europaea L.) is a leading oil crop in the Mediterranean area. Limited information on the inheritance of agronomic significant traits hinders progress in olive breeding programs, which encourages the development of markers linked to the traits. In this study, we report on the development of 46 olive simple sequence repeat (SSR) markers, obtained from 577,025 expressed sequence tags (ESTs) in developing olive fruits generated in the framework of the Slovenian national olive transcriptome project. Sequences were de novo assembled into 98,924 unigenes, which were then used as a source for microsatellites searching. We identified 923 unigenes that contained 984 SSRs among which dinucleotide SSRs (36 %) were the most abundant, followed by tri- (33 %) and hexa- (21 %) nucleotides. Microsatellite repeat motif GA (37 %) was the most common among dinucleotides, while microsatellite repeat motif GAA was the most abundant trinucleotide SSR motif (16 %). Gene ontology annotations could be assigned to 27 % of the unigenes. A hundred and ten expressed sequence tag-derived-simple sequence repeats (EST-SSRs) with annotated genes were selected for primer designing and finally, 46 (42 %) polymorphic EST-SSRs were successfully amplified and used to validate genetic diversity among 24 olive varieties. The average number of alleles per locus, observed heterozygosity, expected heterozygosity, and polymorphic information content were 4.5, 0.649, 0.604 and 0.539, respectively. Twenty-seven EST-SSRs showed good diversity properties and were recommended for further olive genome investigation.