Određivanje DNA otiska različitih sorta masline pomoću mikrosatelitskih markera

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.U različitim genetičkim istraživanjima biljaka sve se više koriste mikrosateliti kao najpogodniji molekularni marker. U četrnaest analiziranih lokusa otkriveno je ukupno 96 alela, što omogućava vrlo veliku razlikovnost pri identifikaciji pojedinih sorta. Tipiziranjem genoma različitih sorta masline pomoću mikrosatelita dobivene su vrlo korisne informacije koje se mogu dopuniti uporabom dodatnih markera. Tom se metodologijom mogu karakterizirati i nove sorte maslina, a i obavljati pouzdane međulaboratorijske usporedbe rezultata. Dobiveni podaci mogu se koristiti pri izradbi baze podataka za identifikaciju pojedinih sorta i njihovu rasprostranjenost na području Slovenije. Oni također omogućavaju da se agronomske značajke i kakvoća različitih vrsta maslina dopune dodatnim genetičkim informacijama

Complete mitochondrial genome of the Verticillium-wilt causing plant pathogen Verticillium nonalfalfae

Verticillium nonalfalfae is a fungal plant pathogen that causes wilt disease by colonizing the vascular tissues of host plants. The disease induced by hop isolates of V. nonalfalfae manifests in two different forms, ranging from mild symptoms to complete plant dieback, caused by mild and lethal pathotypes, respectively. Pathogenicity variations between the causal strains have been attributed to differences in genomic sequences and perhaps also to differences in their mitochondrial genomes. We used data from our recent Illumina NGS-based project of genome sequencing V. nonalfalfae to study the mitochondrial genomes of its different strains. The aim of the research was to prepare a V. nonalfalfae reference mitochondrial genome and to determine its phylogenetic placement in the fungal kingdom. The resulting 26,139 bp circular DNA molecule contains a full complement of the 14 "standard" fungal mitochondrial protein-coding genes of the electron transport chain and ATP synthase subunits, together with a small rRNA subunit, a large rRNA subunit, which contains ribosomal protein S3 encoded within a type IA-intron and 26 tRNAs. Phylogenetic analysis of this mitochondrial genome placed it in the Verticillium spp. lineage in the Glomerellales group, which is also supported by previous phylogenetic studies based on nuclear markers. The clustering with the closely related Verticillium dahliae mitochondrial genome showed a very conserved synteny and a high sequence similarity. Two distinguishing mitochondrial genome features were also found-a potential long non-coding RNA (orf414) contained only in the Verticillium spp. of the fungal kingdom, and a specific fragment length polymorphism observed only in V. dahliae and V. nubilum of all the Verticillium spp., thus showing potential as a species specific biomarker

Identification and validation of novel EST-SSR markers in olives

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

Paternity Analysis of the Olive Variety “Istrska Belica” and Identification of Pollen Donors by Microsatellite Markers

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

Effects of an Ad Libitum Consumed Low-Fat Plant-Based Diet Supplemented with Plant-Based Meal Replacements on Body Composition Indices

Objective. To document the effect of a diet free from animal-sourced nutrients on body composition indices. Methods. This was a nonrandomized interventional ( = 241)-control ( = 84) trial with a 10-week, low-fat, plant-based diet supplemented with two daily meal replacements. The meals were allowed to be eaten to full satiety without prespecified calorie restrictions. Control subjects received weekly lectures on the rationale and expected benefits of plant-based nutrition. Body composition indices were measured with bioimpedance analysis. Results. Relative to controls, in cases, postintervention body fat percentage was reduced by 4.3 (95% CI 4.1-4.6)% points (a relative decrement of −13.4%), visceral fat by 1.6 (95% CI 1.5-1.7) fat cross-sectional surface units, and weight by 5.6 kg (95% CI 5.2-6), while muscle mass was reduced by 0.3 kg (95% CI 0.06-0.5) with a relative increase of muscle mass percentage of 4.2 (3.9-4.4)% points. Analysis of covariance showed significantly larger adjusted fat reductions in cases compared to controls. Late follow-up revealed further weight loss in 60% of cases and no significant change in controls. Conclusions. Low-fat, plant-based diet in free-living nonresidential conditions eaten ad libitum enables significant and meaningful body fat reductions with relative preservation of muscle mass. This trial is registered with NCT02906072, ClinicalTrials.gov

The oldest macroremains of Vitis from Slovenia

Abstract The oldest Vitis vinifera ssp. L. (grape) pips (seeds) from Slovenian archaeological sites were found at the late Neolithic (Copper Age) pile-dwelling settlement of Hočevarica on the Ljubljansko barje and date to the 37th/ 36th century B.C. Various biometric studies were carried out to establish whether the grapes were wild or cultivated. A comparison of several morphological characteristics of grape pip samples was performed. Neolithic pips from Hočevarica, Roman pips from Vrhnika (1st century A.D.) and recent Slovenian cultivated grape pips were included in the study. According to the standard indices, the Neolithic Vitis pips were wild. In the case of the Roman pips, it was impossible to determine whether they were wild or cultivated, although they had most probably been cultivated. Since the morphology of grape pips varies considerably (as it did in the Neolithic, as well), the attempted identification of the subspecies Vitis vinifera ssp. sylvestris/vinifera, based on morphological characteristics alone, was unsatisfactory. Therefore, an attempt was made to analyse the DNA, which could help to resolve this question. Initial experiments on extraction of archaeological DNA and amplification of the chloroplast region were performed, but further optimization steps are needed to achieve successful amplification

Ampelographic and molecular characterisation of grapevine varieties in the gene bank of the experimental vineyard ‘Radmilovac’ – Serbia

Characterisations of thirty grapevine varieties (Vitis vinifera L.) from the experimental vineyard ‘Radmilovac’ were conducted using a large number of OIV descriptors and eight highly polymorphic microsatellite loci. The ampelographic description contained 45 features. Molecular characterisation of selected microsatellite loci was performed using capillary electrophoresis fragment analysis. Dendrograms based on ampelographic and genetic data resulted in three groups of varieties. Qualitative ampelographic characteristics tended to manifest significant differences. The most common deviation among varieties within the group was in the characteristic OIV 051 (colouration of the upper side of a young leaf). Genetic characterisation of SSR markers through analyses of a large number of varieties contributes to better organisation of grapevine collections and simpler identification of varieties, as well as data exchange. When identifying the varieties, the results of the DNA analysis should be combined with the ampelographic descriptors, in order to select grapevine varieties with desirable viticultural and oenological traits. Integration of the obtained genetic data with the ampelographic data is of utmost importance for accurate identification of the varieties and offers a significant means for the preservation and use of the varieties

Reference-Grade Genome and Large Linear Plasmid of Streptomyces rimosus : Pushing the Limits of Nanopore Sequencing

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters