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.
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.
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.
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.
Viroids, the smallest known pathogens, unable to encode any proteins, can cause severe diseases in their host plants. One of the proposed mechanisms of their pathogenicity includes silencing the ...host's genes via viroid-derived small RNAs, which are products of the host's immune response to the viroid's double stranded RNA. Humulus lupulus (hop) plants are hosts to several viroids; two of them, HLVd and CBCVd, are interesting models for studying host-viroid interactions, due to the symptomless infection of the former and severe stunting disease caused by the latter. To study these interactions, we constructed a deep hop NGS transcriptome based on 35 Gb paired-end sequencing data assembled into over 74 Mb of contigs. These transcripts were used for in-silico prediction of target transcripts of vd-sRNA of the two aforementioned viroids, using two different software tools. Prediction models revealed that 1062 and 1387 hop transcripts share nucleotide similarities with HLVd- and CBCVd-derived small RNAs, respectively, so they could be silenced in an RNA interference process. Furthermore, we selected 17 transcripts from 4 groups of targets involved in the metabolism of plant hormones, small RNA biogenesis, transcripts with high complementarity with viroid-derived small RNAs and transcripts targeted by CBCVd-derived small RNAs with high cellular concentrations. Their expression was monitored by reverse transcription quantitative PCR performed using leaf, flower and cone samples. Additionally, the expression of 5 pathogenesis related genes was monitored. Expression analysis confirmed high expression levels of four pathogenesis related genes in leaves of HLVd and CBCVd infected hop plants. Expression fluctuations were observed for the majority of targets, with possible evidence of downregulation of GATA transcription factor by CBCVd- and of linoleate 13S-lipoxygenase by HLVd-derived small RNAs. These results provide a deep transcriptome of hop and the first insights into complex viroid-hop plant interactions.
Hop plant (Humulus lupulus L.), cultivated primarily for its use in the brewing industry, is faced with a variety of diseases, including severe vascular diseases, such as Verticillium wilt, against ...which no effective protection is available. The understanding of disease resistance with tools such as differentially expressed gene studies is an important objective of plant defense mechanisms. In this study, we evaluated twenty-three reference genes for RT-qPCR expression studies on hop under biotic stress conditions. The candidate genes were validated on susceptible and resistant hop cultivars sampled at three different time points after infection with Verticillium albo-atrum. The stability of expression and the number of genes required for accurate normalization were assessed by three different Excel-based approaches (geNorm v.3.5 software, NormFinder, and RefFinder). High consistency was found among them, identifying the same six best reference genes (YLS8, DRH1, TIP41, CAC, POAC and SAND) and five least stably expressed genes (CYCL, UBQ11, POACT, GAPDH and NADH). The candidate genes in different experimental subsets/conditions resulted in different rankings. A combination of the two best reference genes, YLS8 and DRH1, was used for normalization of RT-qPCR data of the gene of interest (PR-1) implicated in biotic stress of hop. We outlined the differences between normalized and non-normalized values and the importance of RT-qPCR data normalization. The high correlation obtained among data standardized with different sets of reference genes confirms the suitability of the reference genes selected for normalization. Lower correlations between normalized and non-normalized data may reflect different quantity and/or quality of RNA samples used in RT-qPCR analyses.
Viroids are small non-capsidated, single-stranded, covalently-closed circular noncoding RNA replicons of 239-401 nucleotides that exploit host factors for their replication, and some cause disease in ...several economically important crop plants, while others appear to be benign. The proposed mechanisms of viroid pathogenesis include direct interaction of the genomic viroid RNA with host factors and post-transcriptional or transcriptional gene silencing via viroid-derived small RNAs (vd-sRNAs) generated by the host defensive machinery.
(hop) plants are hosts to several viroids among which
(HLVd) and
(CBCVd) are attractive model systems for the study of viroid-host interactions due to the symptomless infection of the former and severe symptoms induced by the latter in this indicator host. To better understand their interactions with hop plant, a comparative transcriptomic analysis based on RNA sequencing (RNA-seq) was performed to reveal the transcriptional alterations induced as a result of single HLVd and CBCVd infection in hop. Additionally, the effect of HLVd on the aggressiveness of CBCVd that underlies severe stunting in hop in a mixed infection was studied by transcriptomic analysis. Our analysis revealed that CBCVd infection resulted in dynamic changes in the activity of genes as compared to single HLVd infection and their mixed infection. The differentially expressed genes that are involved in defense, phytohormone signaling, photosynthesis and chloroplasts, RNA regulation, processing and binding; protein metabolism and modification; and other mechanisms were more modulated in the CBCVd infection of hop. Nevertheless, Gene Ontology (GO) classification and pathway enrichment analysis showed that the expression of genes involved in the proteolysis mechanism is more active in a mixed infection as compared to a single one, suggesting co-infecting viroids may result in interference with host factors more prominently. Collectively, our results provide a deep transcriptome of hop and insight into complex single HLVd, CBCVd, and their coinfection in hop-plant interactions.
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