Metagenomic approaches used for virus diagnostics allow for rapid and accurate detection of all viral pathogens in the plants. In order to investigate the occurrence of viruses and virus-like ...organisms infecting grapevine from the Ampelographic collection Kromberk in Slovenia, we used Ion Torrent small RNA sequencing (sRNA-seq) and the VirusDetect pipeline to analyze the sRNA-seq data. The used method revealed the presence of:
(GLRaV-1),
(GLRaV-2),
(GLRaV-3),
(GRSPaV),
(GFLV) and its satellite RNA (satGFLV),
(GFkV),
(GRVFV),
(GPGV),
(GV-Sat),
(HSVd), and
(GYSVd-1). Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for validation of sRNA-seq predicted infections, including various combinations of viruses or viroids and satellite RNA. mRT-PCR could further be used for rapid and cost-effective routine molecular diagnosis, including widespread, emerging, and seemingly rare viruses, as well as viroids which testing is usually overlooked.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Testing inbred lines for their combining ability is, due to high numbers of line to line testing needed for determination of hybrid performance, the most limiting factor in the F
1
hybrid breeding ...procedure. We propose a novel method of F
1
hybrid breeding that enables evaluation of large number of line to line crosses for their hybrid performance. Inbred lines (preferably doubled haploid - DH) are produced from heterozygous populations, genotyped and maintained. A group of lines is inter-pollinated randomly and their progeny examined. To identify elite F
1
hybrids, these individual plants are selected by their superior phenotypic characteristics. Finally using paternity testing only of selected hybrids, the origin of paternal lines is revealed. To predict the number of F
1
offspring needed in relation to the number of inbred lines being inter-pollinated, a mathematical formula was developed. For instance, using this formula for the inter-pollination of 60 distinct lines, the probability of obtaining all descendants of paternal-parent lines in a maternal-parent row represented at least once is achieved with 420 F
1
plants in a row (p = 0.95). In a practical experiment with white cabbage, DH lines were produced using microspore culture; plants were grown to maturity and genotyped at eight polymorphic SSR loci. Two groups of lines (36 and 33 lines per group) were inter-pollinated by two methods, either using cage pollination with bumblebees or using open pollination in isolated field. A total of 9,858 F
1
plants were planted and based on their phenotypic characteristics 213 were selected as elite phenotypes. 99 of them were genetically diverse and 5 of them were selected as super elite. Selected plants were analysed by the same SSR markers and the paternal origin of selected F
1
plants was determined. Out of 213 selected elite plants 48 were reciprocals thus exhibiting power of selection based on single plant. We demonstrate that this new approach to hybrid development is efficient in white cabbage and we propose breeders to test it in various vegetable and crop species. Moreover, some other aspects of the proposed technique need to be tested and verified both for practical and economic criteria.
Hop plant (Humulus lupulus L.) is an important industrial crop, grown for harvesting hop cones however, it is a host to four different viroids as well. The nature of viroid infections is not entirely ...clarified. In our work, we focused on analyzing viroid accumulation and their strands polarity through RNA sequencing and reverse transcription polymerase chain reaction in real time. RNA-seq data indicate that viroids amplify until saturation further demonstrating plant's biological capacity. Negative fold changes in accumulation of individual viroids between hop samples with single and multiple infections are suggesting an antagonistic relationship amongst viroids, where citrus bark cracking viroid seems to be the least and hop stunt viroid the most sensitive to the other two. RNA-seq data also show that on average (−) viroid strand is dominating over (+), especially for the citrus bark cracking viroid. Reverse transcription polymerase chain reaction in real time results from strand polarity analysis seem to be less consistent between different combinations of infection but are showing level of conformity with RNA-seq in the case of citrus bark cracking viroid.
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.
Transposable elements (TE) are stretches of DNA that represent the greatest fraction of genomes, especially in plants. Because of their high copy numbers and ability to mobilize through genome, they ...are able to influence the phenotypic traits and evolution of plants and also plant adaptation to environmental stress. By genetic and epigenetic mechanisms, they change the gene structure, influence gene expression and create new regulatory networks. The fraction of genome that they represent and the influence they have is variable among species; however they were detected in practically every plant genome researched up to date. Deleterious mutations may be caused by their activity which is also another reason why their expression is tightly regulated by the host organism. Gaining knowledge of TE's mechanisms and research development in the future will allow us to use them, for example for crop improvement purposes, resistance development against diseases and pathogens and suppression of invasive species.
Identifikacija kultivarjev vinske trte z metodami, ki temeljijo na morfoloških razlikah med rastlinami, je lahko zaradi vpliva ekoloških dejavnikov nepravilna, zato so bile razvite metode, ki ...omogočajo analizo kultivarjev na nivoju genotipa. V zadnjih dvajsetih letih so se uveljavile različne tehnike za karakterizacijo kultivarjev, od izoencimskih analiz do analiz na nivoju DNA (RFLP, RAPD, AFLP, SCAR in SSR markerji). Med omenjenimi so se najbolj uveljavili mikrosateliti, ki kažejo največjo informacijsko vrednostjo polimorfizma in so večinoma zelo variabilni. Sestavljeni so iz kratkih, tandemsko ponovljivih motivov DNA, ki so prisotni pri večini organizmov. Glavni vzrok polimorfizma med posamezniki so spremembe v številu ponovitev osnovnega motiva mikrosatelita. Za izdelavo začetnih oligonukleotidov mikrosatelitskih lokusov so v začetnih raziskavah uporabili zaporedja obrobnih regij že znanih mikrosatelitov, ki so bila shranjena v podatkovnih bazah DNA. Kasneje pa se je strategija razvijala v smeri nastanka genomskih knjižnic obogatenih z mikrosateliti. Pri vinski trti je bilo razvitih več kot 500 mikrosatelitskih markerjev in njihov izredni potencial in uporabnost pri določanju kultivarjev vinske trte in podlag je bil dokazan v številnih raziskavah. Za genotipizacijo se večinoma uporablja set šestih oz. devetih mikrosatelitskih markerjev (označevalcev), ki so zelo polimorfni in najbolj primerni za ugotavljanje genetske variabilnosti med evropskimi kultivarji vinske trte. Uporaba enotnega seta markerjev ter vključevanje referenčnih kultivarjev v analize genotipizacije omogoča primerjave genotipov med različnimi raziskovalnimi skupinami, in tako lahko rešujemo številne dilem o sinonimih, homonimih ali o izvoru sort vinske trte.
Apple hammerhead viroid (AHVd, Pelamoviroid, Avsunviroidae) is one of the five viroids infecting apples. It has been identified on all continents except Australia since its viroid nature was ...confirmed (DiSerio et al. 2018; CABI and EPPO 2022). AHVd has been found in apple trees showing leaf mosaic, ringspot and dieback (Hamdi et al., 2021). Apple (Malus domestica Borkh.) and its wild relatives are traditionally grown in Montenegro. With an annual production of 7767 tons on 216 ha, it is the second most important fruit tree (after plum) in the country (Anonymous 2022). In a 2020-2022 survey, 29 apple trees exhibiting virus-like symptoms (e.g. mosaic, necrosis) were sampled throughout Montenegro, including 16 locations in eight municipalities (Podgorica, Danilovgrad, Niksic, Mojkovac, Bijelo Polje, Berane, Pljevlja and Savnik). Small RNAs were isolated using the mirVana miRNA Isolation Kit (Ambion, Life Technologies) and pooled into three bulk samples. Each bulk contained 9 to 10 samples. Libraries of sRNAs were constructed using the Ion Total RNA-Seq Kit v2 and barcoded using the Xpress RNA-Seq Barcode 1–16 Kit (Ion Torrent) according to the manufacturer's instructions. Small RNA library sequencing was performed on Illumina platform (Novogene Europe) yielding 9.9, 9.8 and 18.6 million reads in the three libraries. The CLC Genomics Workbench software was used to demultiplex the reads into pools using the 'Demultiplex Reads' tool. The online program VirusDetect (Zheng et al. 2017) was used for virus/viroid detection and identification. Besides viruses known to infect apple (apple stem grooving virus, apple stem pitting virus, apple mosaic virus), contigs mapping to AHVd were identified in all three bulks enabling full AHVd genomes reconstruction. To verify AHVd presence, all 29 apple samples were tested by reverse transcription-polymerase chain reaction (RT-PCR) using the AHVd PG13f/PG12r primers (Messmer et al. 2017). AHVd amplicons were obtained in three samples (30/21, 32/21 and 38/21) from bulk 1 and two samples (47/21 and 55/21) from bulk 2, while all samples from bulk 3 tested negative potentially due to the low titer of the pathogen or nucleotide mismatches at the 3’ end of the primers. The three amplicons from bulk 1 were Sanger sequenced and partial AHVd genomes over 200 nts were obtained from two of them (30/21 and 32/21) (GenBank acc. nos. OQ863319 and OR020603). Furthermore, three full consensus AHVd genomes were assembled in Geneious Prime by mapping Sanger sequences onto contigs from Virus Detect and named 30/21, 32/21 and 38/21 (acc. nos. PP133245, -46, and -47, respectively). All three genomes exhibited conserved hammerhead motifs (Messmer et al. 2017). In BLASTn analysis, the isolate 30/21 from Montenegro shared the highest nt identity (98.8%) with the isolate SA-36 (ON564299) from Czechia, while 32/21 and 38/21 showed the highest identities (95.4% and 92.3%) with isolates SD17_2-3 (MK188691) from Canada and JF2 (ON564298) from Czechia, respectively. To the best of our knowledge, this is the first report of AHVd infecting Malus domestica in Montenegro. The AHVd-positive samples 30/21 and 32/21 originated from at least two-decade-old apple trees from Niksic, whilst 38/21 came from a 40-year-old tree from Mojkovac district, suggesting that this viroid has long been present in different parts of the country. The AHVd discovery in Montenegro should be considered in any phytosanitary regulations and pome fruit certification program in the country.
Ten species (13 accessions) of the genus
Asparagus were analysed to reveal their genome size structure and genetic similarity. The choice of species was based on their possible application in ...crossbreeding with
Asparagus officinalis L. and their agricultural value
. Flow cytometric measurement of genome size revealed two distinctive groups, based on 1C values, the European dioecious species having on average twice the genome size of the South African hermaphrodite species. Variability among species was relatively high both as absolute values (from 1.43 to 7.87 pg per nucleus) or expressed as 1C values (0.67–1.71 pg/1C). Additionally,
Asparagus maritimus was determined as hexaploid in contrast to previous reports of it being tetraploid. Based on restriction fragment analysis of internal transcribed spacers (ITSs) of nuclear ribosomal DNA, the same two, distinctly distant groups of species were formed, which clearly separate species by their gender and geographic origin. The clustering obtained by the UPGMA method is in agreement with some botanical classifications. The genetic similarity and ploidy level results explain the successful interspecific hybridisation reported for
A. officinalis and
Asparagus tenuifolius and the failure of hybridisation between
A. officinalis and some other
Asparagus species.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Genska banka hmelja zagotavlja dolgoročno gensko pestrost in ohranitev biotske raznovrstnosti v kmetijstvu. Na Inštitutu za hmeljarstvo in pivovarstvo Slovenije predstavlja genska banka hmelja zbirko ...akcesij hmelja, nabranih na naravnih rastiščih hmelja v Sloveniji in na območju bivše Jugoslavije, ter slovenske sorte hmelja. V kolekciji avtohtonega hmelja ženskih rastlin, ki je bila nabrana v začetku sedemdesetih let na območju bivše Jugoslavije, imamo zbranih 25 različnih akcesij hmelja. Zbirko genskih virov hmelja vsako leto povečujemo z novo nabranim materialom. Pri žlahtnjenju novih sort hmelja je takšna kolekcija avtohtonega, genetsko raznolikega materiala izrednega pomena. V članku so predstavljeni rezultati vrednotenja divjih genotipov, pridobljeni z molekulskimi pristopi.
Asparagus officinalis is most extensively studied species within the genus Asparagus, which is well known as garden asparagus. This species is dioecious with unisexual flowers, which means that ...generative propagation gives roughly equal number of male and female plants. Male plants are high yielders and preferred commercially over female plants. Tissue culture techniques could efficiently promote vegetative propagation of male plants and pave the way for efficient plant breeding.This chapter describes an efficient micropropagation protocol for developing rapid growing in vitro Asparagus shoot cultures. The source of explants, inoculation, and shoot proliferation, followed by shoot propagation, rooting, and acclimatization is described. The optimal medium for Asparagus micropropagation described in this chapter is composed of MS macro- and microelements and a combination of auxins and cytokinins. Plant growth regulators NAA, kinetin, and BA were used in various concentrations. Three different media representing the whole micropropagation protocol of Asparagus are described; medium for shoot initiation, medium for shoot multiplication, and medium for root formation. By in vitro propagation of Asparagus, root initiation is difficult, but can be promoted by adding growth retardant ancymidol which also greatly promotes shoot development and suppresses callus formation.
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