1. Time-sampled population sequencing reveals the interplay of selection and genetic drift in experimental evolution of Potato virus YDenis Kutnjak, Santiago F. Elena, Maja Ravnikar, 2017, original scientific article Abstract: RNA viruses are one of the fastest-evolving biological entities. Within their hosts, they exist as genetically diverse populations (i.e., viral mutant swarms), which are sculpted by different evolutionary mechanisms, such as mutation, natural selection, and genetic drift, and also the interactions between genetic variants within the mutant swarms. To elucidate the mechanisms that modulate the population diversity of an important plant-pathogenic virus, we performed evolution experiments with Potato virus Y (PVY) in potato genotypes that differ in their defense response against the virus. Using deep sequencing of small RNAs, we followed the temporal dynamics of standing and newly generated variations in the evolving viral lineages. A time-sampled approach allowed us to (i) reconstruct theoretical haplotypes in the starting population by using clustering of single nucleotide polymorphisms' trajectories and (ii) use quantitative population genetics approaches to estimate the contribution of selection and genetic drift, and their interplay, to the evolution of the virus. We detected imprints of strong selective sweeps and narrow genetic bottlenecks, followed by the shift in frequency of selected haplotypes. Comparison of patterns of viral evolution in differently susceptible host genotypes indicated possible diversifying evolution of PVY in the less-susceptible host (efficient in the accumulation of salicylic acid).
IMPORTANCE High diversity of within-host populations of RNA viruses is an important aspect of their biology, since they represent a reservoir of genetic variants, which can enable quick adaptation of viruses to a changing environment. This study focuses on an important plant virus, Potato virus Y, and describes, at high resolution, temporal changes in the structure of viral populations within different potato genotypes. A novel and easy-to-implement computational approach was established to cluster single nucleotide polymorphisms into viral haplotypes from very short sequencing reads. During the experiment, a shift in the frequency of selected viral haplotypes was observed after a narrow genetic bottleneck, indicating an important role of the genetic drift in the evolution of the virus. On the other hand, a possible case of diversifying selection of the virus was observed in less susceptible host genotypes.
Keywords: Potato virus Y, experimental evolution, genetic drift, natural selection, sRNA deep sequencing
Published in DiRROS: 24.07.2024; Views: 13; Downloads: 5
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2. Cucujus cinnaberinus (Scopoli, 1763) at its terra typica in Slovenia : historical overview, distribution patterns and habitat selectionAl Vrezec, Špela Ambrožič Ergaver, Andrej Kobler, Andrej Kapla, Maarten De Groot, 2017, original scientific article Abstract: The saproxylic beetle, Cucujus cinnaberinus, has received increasing research attention in Europe since the adoption of the Habitats Directive and establishment of the Natura 2000 network. The history of the species has been investigated as well as the influence of abiotic and biotic variables on the distribution of C. cinnaberinus in Slovenia which is at the limit of its range and also terra typica for the species. The species was first described in 1763 by Joannes A. Scopoli in Carniola, a duchy of the Habsburg Monarchy. Today, most of the territory of Carniola is situated within Slovenia. C. cinnaberinus is particularly common in the eastern part of the country, but very scarce in the mountainous western part. According to historical and recent distribution patterns of C. cinnaberinus in the former Carniolan territory, the region of Ribnica-Kočevje in southern Slovenia is proposed as the most probable type locality of the species. Although the bulk of the C. cinnaberinus population in Slovenia is confined to the lowlands, the species has been found up to 1095 m a.s.l., albeit at a much lower abundance due to the influence of climate and forest structure. Although C. cinnaberinus is a quite an opportunistic species regarding host tree selection, it has been shown to exhibit a preference for Tilia, Populus and Robinia. It is suspected that the high abundance of C. cinnaberinus in lowland floodplain forests is due to the recent human-induced increase in preferred fast-growing and short-lived host trees, i.e. the planting of poplar trees and spread of invasive Black Locust (Robinia pseudoacacia) after the 1960s. In contrast, in montane forests, preferred host trees (e.g. Tilia) represent < 1 % of all growing stock. Although montane C. cinnaberinus populations are rare, they could still be important for the conservation of the species, since montane habitats cover the largest area within the species’ distribution range.
Keywords: saproxylic beetle, Natura 2000, type locality, Carniola, altitudinal distribution, host tree selection, macrohabitat, large-scale survey
Published in DiRROS: 24.07.2024; Views: 12; Downloads: 5
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3. Comparison between proteome and transcriptome response in potato (Solanum tuberosum L.) leaves following potato virus Y (PVY) infectionTjaša Stare, Katja Stare, Wolfram Weckwerth, Stefanie Wienkoop, Kristina Gruden, 2017, original scientific article Abstract: Plant diseases caused by viral infection are affecting all major crops. Being an obligate intracellular organisms, chemical control of these pathogens is so far not applied in the field except to control the insect vectors of the viruses. Understanding of molecular responses of plant immunity is therefore economically important, guiding the enforcement of crop resistance. To disentangle complex regulatory mechanisms of the plant immune responses, understanding system as a whole is a must. However, integrating data from different molecular analysis (transcriptomics, proteomics, metabolomics, smallRNA regulation etc.) is not straightforward. We evaluated the response of potato (Solanum tuberosum L.) following the infection with potato virus Y (PVY). The response has been analyzed on two molecular levels, with microarray transcriptome analysis and mass spectroscopy-based proteomics. Within this report, we performed detailed analysis of the results on both levels and compared two different approaches for analysis of proteomic data (spectral count versus MaxQuant). To link the data on different molecular levels, each protein was mapped to the corresponding potato transcript according to StNIB paralogue grouping. Only 33% of the proteins mapped to microarray probes in a one-to-one relation and additionally many showed discordance in detected levels of proteins with corresponding transcripts. We discussed functional importance of true biological differences between both levels and showed that the reason for the discordance between transcript and protein abundance lies partly in complexity and structure of biological regulation of proteome and transcriptome and partly in technical issues contributing to it.
Keywords: proteomics, transcriptomics, potato, potato virus Y
Published in DiRROS: 24.07.2024; Views: 11; Downloads: 5
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4. quantGenius : implementation of a decision support system for qPCR-based gene quantificationŠpela Baebler, Miha Svalina, Marko Petek, Katja Stare, Ana Rotter, Maruša Pompe Novak, Kristina Gruden, 2017, original scientific article Abstract: Background
Quantitative molecular biology remains a challenge for researchers due to inconsistent approaches for control of errors in the final results. Due to several factors that can influence the final result, quantitative analysis and interpretation of qPCR data are still not trivial. Together with the development of high-throughput qPCR platforms, there is a need for a tool allowing for robust, reliable and fast nucleic acid quantification.
Results
We have developed “quantGenius” (http://quantgenius.nib.si), an open-access web application for a reliable qPCR-based quantification of nucleic acids. The quantGenius workflow interactively guides the user through data import, quality control (QC) and calculation steps. The input is machine- and chemistry–independent. Quantification is performed using the standard curve approach, with normalization to one or several reference genes. The special feature of the application is the implementation of user-guided QC-based decision support system, based on qPCR standards, that takes into account pipetting errors, assay amplification efficiencies, limits of detection and quantification of the assays as well as the control of PCR inhibition in individual samples. The intermediate calculations and final results are exportable in a data matrix suitable for further statistical analysis or visualization. We additionally compare the most important features of quantGenius with similar advanced software tools and illustrate the importance of proper QC system in the analysis of qPCR data in two use cases.
Conclusions
To our knowledge, quantGenius is the only qPCR data analysis tool that integrates QC-based decision support and will help scientists to obtain reliable results which are the basis for biologically meaningful data interpretation.
Keywords: quantitative molecular biology, quantitative PCR, nucleic acid quantification, web application, decision support system
Published in DiRROS: 24.07.2024; Views: 4; Downloads: 3
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8. Zoantharians (Hexacorallia: Zoantharia) associated with cold-water corals in the Azores Region: new species and associations in the deep seaMarina Carreiro-Silva, Oscar Ocaňa, David Stanković, ĺris Sampaio, Filipe M. Porteiro, Marie-Claire Fabri, Sergio Stefanni, 2017, original scientific article Abstract: Zoantharians are a group of cnidarians that are often found in association with marine invertebrates, including corals, in shallow and deep-sea environments. However, little is known about deep-sea zoantharian taxonomy, specificity and nature of their associations with their coral hosts. In this study, analyses of molecular data (mtDNA COI, 16S, and 12S rDNA) coupled with ecological and morphological characteristics were used to examine zoantharian specimens associated with cold-water corals (CWC) at depths between 110 and 800 m from seamounts and island slopes in the Azores region. The zoantharians examined were found living in association with stylasterids, antipatharians and octocorals. From the collected specimens, four new species were identified: (1) Epizoanthus martinsae sp. n. associated with the antipatharian Leiopathes sp.; (2) Parazoanthus aliceae sp. n. associated with the stylasterid Errina dabneyi (Pourtalès, 1871); (3) Zibrowius alberti sp. n. associated with octocorals of the family Primnoidae [Paracalyptrophora josephinae (Lindström, 1877)] and the family Plexauridae (Dentomuricea aff. meteor Grasshoff, 1977); (4) Hurlizoanthus hirondelleae sp. n. associated with the primnoid octocoral Candidella imbricata (Johnson, 1862). In addition, based on newly collected material, morphological and molecular data and phylogenic reconstruction, the zoantharian Isozoanthus primnoidus Carreiro-Silva, Braga-Henriques, Sampaio, de Matos, Porteiro & Ocaña, 2011, associated with the primnoid octocoral Callogorgia verticillata (Pallas, 1766), was reclassified as Zibrowius primnoidus comb. nov. The zoantharians, Z. primnoidus comb. nov., Z. alberti sp. n., and H. hirondelleae sp. n. associated with octocorals showed evidence of a parasitic relationship, where the zoantharian progressively eliminates gorgonian tissue and uses the gorgonian axis for structure and support, and coral sclerites for protection. In contrast, the zoantharian P. aliceae sp. n. associated with the stylasterid E. dabneyi and the zoantharian E. martinsae sp. n. associated with the antipatharian Leiopathes sp., appear to use the coral host only as support with no visible damage to the host. The monophyly of octocoral-associated zoantharians suggests that substrate specificity is tightly linked to the evolution of zoantharians.
Keywords: antipatharians, octocorals, Azores, gorgonians, molecular taxonomy, phylogeny, stylasterids
Published in DiRROS: 24.07.2024; Views: 5; Downloads: 4
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10. Filling the gaps in diagnostics of Pepino mosaic virus and Potato spindle tuber viroid in water and tomato seeds and leavesNataša Mehle, Polona Kogovšek, Nejc Rački, Tjaša Jakomin, Ion Gutiérrez-Aguirre, Petra Kramberger, Maja Ravnikar, 2017, original scientific article Abstract: Waterborne and seedborne Pepino mosaic virus (PepMV) and Potato spindle tuber viroid (PSTVd) pose serious threats to tomato production due to seed transmission and mechanical transmission, coupled with their long-term stability outside the host plant. Therefore, rapid and sensitive diagnostic procedures are needed to prevent the spread of these quarantine pathogens. In particular, water and seed contamination are difficult to detect and confirm without efficient concentration methods. This study presents procedures that improve detection of PSTVd from tomato seeds and leaf tissue, and PepMV from water and tomato leaf tissue. For efficient concentration of PepMV from water samples, a procedure was optimized using convective interaction media monolithic chromatography columns, which provides concentration by three orders of magnitude. For concentration of PSTVd from seed extracts, an easy-to-use and efficient method was developed based on RNA binding to positively charged anion-exchange resin beads that provides up to 100-fold more sensitive detection in comparison with procedures without a concentration step. This thus allows confirmation of RT-qPCR results with sequencing of RT-PCR products in samples with low viroid levels. In addition, reverse-transcription loop-mediated isothermal amplification assays for detection of PSTVd and PepMV were optimized and adapted to both laboratory and on-site testing requirements. This allows rapid detection of these pathogens in crude leaf homogenates, in under 30 min. These procedures of concentration and detection are shown to be efficient and to fill the gaps in diagnostics of PepMV and PSTVd, especially when these pathogens are present at low levels in difficult matrices such as water and seeds.
Keywords: PSTVd, PepMV, seeds, water, concentration, loop-mediated isothermal amplification
Published in DiRROS: 24.07.2024; Views: 5; Downloads: 4
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