INTRODUCTION
Background of Research
Viroids are the smallest infectious agents, consisting of highly structured, single-stranded circular RNA molecules that lack messenger RNA activity and exclusively infect plants Unlike viruses, which carry genetic information for replication, viroids are obligate parasites of the host's transcriptional machinery Among the nearly 30 known viroid species, four possess hammerhead ribozymes, indicating a potential common origin with hepatitis delta virus and viroid-like satellite RNAs Their replication occurs through a rolling-circle mechanism, leading to various insertion and deletion events Notably, the terminal domains of certain viroids, such as potato spindle tuber, exhibit significant sequence exchange and rearrangement Viroid populations often display a complex mix of sequence variants, with environmental stress contributing to increased sequence heterogeneity The emerging field of synthetic biology presents opportunities to explore the minimal size of a functional viroid genome, although significant challenges remain despite the availability of preliminary structural and functional data.
Viroids and viroid-like satellite RNAs were initially aligned using CLUSTAL-X, followed by manual editing to maintain local similarities Subsequently, these partial alignments were combined manually before employing CLUSTAL-X again to realign dissimilar regions, aiming to enhance overall similarity.
Virus and viroid diseases pose significant challenges to sustainable crop production in tropical countries Global climate change affects plants, vectors, and viruses, leading to heightened instability in virus-host ecosystems.
Viroid diseases, often less noticeable than those caused by fungi, bacteria, and nematodes, present challenges in accurately assessing global loss estimates The impact of viroids extends beyond direct and indirect damage, similar to the effects observed with plant virus infections.
Tropical regions face significant threats to food production from various economically important viral diseases Key diseases include tungro, yellow mottle, and hoja blanca affecting rice; mosaic viruses in sugarcane and cassava; tristeza in citrus; swollen shoot in cacao; sterility mosaic in pigeonpea; and multiple diseases in peanuts such as rosette, clump, and bud necrosis Additionally, necrosis impacts sunflower, legumes, vegetables, and ornamental crops, while yellow mosaic affects legumes, leaf curl targets cotton and tomato, and ring spot is a concern for papaya.
Key factors for emergence of new plant virus and virus-like disease include the intensification of agricultural trade (globalization), changes in cropping systems (crop diversification), and climate change.
Purposes of Research
Grape cultivation in Taiwan is a profitable agricultural business, driven by the high demand for fresh grape berries among local consumers and the value added through processed products In 2010, Taiwan's grape production reached 102,831 metric tons, with 99.5% sourced from Taichung City, Miaoli, Changhua, and Nantou Counties in central Taiwan Additionally, RT-PCR has emerged as an essential tool for the simultaneous detection and identification of viruses and viroids in grape crops.
-3- conserved primers are possible through RT-PCR, which has a higher sensitivity than that of molecular hybridization
This research aims to isolate and identify the causal agents of a suspected grapevine disease in Taiwan, while also determining their phylogenetic relationship to various HSVd and GYSVd-1 strains from different geographical regions.
Goals to Reach
The objectives of this study were as follows:
RT-PCR technique detect hop stunt viroid in grapevine (HSVd)
RT-PCR technique detect grapevine yellow speckle viroid 1 in grapevine
Multiplex RT-PCR technique simultaneously detect viroids in grapevine
Framework of Research
This research framework, illustrated in Figure 1-1, involves the analysis of HSVd and GYSVd-1 viroids using Clustal-W software A conserved region is identified for primer design, followed by the transfer of samples to a tube using an RT-PCR machine The process concludes with gel electrophoresis.
LITERATURE REVIEWS
Structure and Classification of Viroid
Viroids, similar to other RNA viruses affecting plants and animals, exist as complex populations of closely related sequence variants in living organisms Research has shown significant natural variability within these viroid populations, with the Subviral RNA Database housing complete sequences for over 1,100 viroid variants Notably, multiple sequence variants can often be isolated from a single infected plant.
Viroids are infectious agents composed of circular, single-strand RNA molecules that range from 250 to 400 nucleotides in length and do not code for proteins They can replicate and spread within infected plants, leading to serious diseases characterized by symptoms such as severe stunting, leaf necrosis, corky bark, leaf roll, and fruit deformation, which vary based on the specific host plant and viroid species.
The family Pospiviroidae features five structural regions: the left terminal region (T1), pathogenic region (P), central conserved region (C), variable region (V), and right terminal region (T2) All species within this family exhibit a rod-like secondary structure with five structural/functional domains and replicate in the nucleus In contrast, three of the four members of the Avsunviroidae possess a branched secondary structure and replicate in the chloroplast Notably, all Avsunviroidae members contain hammerhead ribozymes in both the infectious (+) strand and complementary (−) strand RNAs Figure 2-1 illustrates the secondary structures of PSTVd, highlighting its rod-like form.
Pospiviroidae) and Peach latent mosaic viroid (PLMVd; branched, Avsunviroidae)
With the possible exception of PLMVd, viroids do not appear to contain any modified
Nucleic acid extracts from infected leaf tissue reveal a range of viroid-related RNAs with both polarities, including longer molecules, particularly those with a complementary (−) strand polarity, compared to the infectious circular viroid (+) strand Northern analysis utilizing strand-specific probes and primer extension indicates that these longer RNAs are intermediates indicative of a "rolling-circle" replication mechanism.
Up to now, viroid species are classified into two families Pospiviroidae and Avsunviroidae, which are composed of five and two genera, respectively
Pospiviroidae possess a thermodynamically stable rod-like secondary structure with a CCR (central conserved region) and do not self-cleave, including the genera
Pospiviroid, Coleviroid, Hostuviroid, Cocadviroid and Apscaviroid Avsunviroidae do not possess a CCR and self-cleave via hammerhead ribozyme, with the genera
Nucleic acid extracts from infected leaf tissue reveal various viroid-related RNAs of both polarities, with some, particularly the complementary (−) strand molecules, being significantly longer than the infectious circular viroid (+) strand Northern analysis employing strand-specific probes and primer extension indicates that these longer RNAs are intermediates anticipated in the viroid replication process.
“rolling-circle” mechanism of replication.
Generation of Populations from Individual Viroid Variants
Several different approaches have been used to monitor the genetic stability of individual viroid sequence variants in vivo These include inoculation with
The introduction of recombinant plasmid DNAs, particularly through Agrobacterium-mediated methods, has significantly advanced plant transformation techniques Research highlights the effectiveness of using integrated transgenes for detecting rare events that may restore viroid infectivity, especially in highly debilitated variants Notably, Góra (1994) employed a reverse-transcription PCR strategy to successfully generate infectious full-length cDNAs from three distinct isolates of PSTVd in a single step.
“mild” isolate, only a single sequence variant was recovered “Intermediate” and
Severe isolates produced three to four variants, but not all variants caused severe symptoms in Rutgers tomato, suggesting that milder variants may be masked by more severe ones Follow-up studies by Góra-Sochacka (1997) indicated that many naturally occurring PSTVd sequence variants were unstable when inoculated alone, often disappearing within a 5-6 week passage in tomato This observation supports the quasispecies theory, which posits that mixtures of variants function as a single entity, akin to an individual with numerous alleles Typically, the newly detected variants exhibited less severe symptoms than the parent strain, with sequence changes primarily occurring in pathogenicity and variable domains, while only a few alterations were found in the terminal right domain.
Screening assays using mechanical inoculation of full-length viroid cDNAs or RNA transcripts offer the significant benefit of rapid results, typically within a few weeks However, it is important to note that certain point mutations in PSTVd and other viroids can render them non-infectious when subjected to mechanical inoculation.
-8- cases, these mutations have been shown to inhibit replication; in other cases, cell-to- cell or long-distance transport is disrupted(Qi Y 2004).
Origin and Evolution of Viroids
Several theories exist regarding the origins of viroids, which may be primitive ancestors or degenerate forms of conventional viruses However, their unique molecular structure and biological properties, along with a lack of sequence similarity, challenge this notion Additionally, some researchers propose that viroids may have evolved from transposable elements, plasmids, or introns Currently, the prevailing evidence indicates that viroids could represent a distinct evolutionary pathway.
Recent studies have focused on the "relics of precellular RNA evolution," highlighting the viroid origin in the RNA world RNA stands out as the only biological macromolecule that can serve as both genotype and phenotype, enabling evolution without DNA or protein Diener (1989) noted that a simple hammerhead ribozyme, akin to those in ASBVd and other Avsunviroidae members, can theoretically execute all necessary steps for viroid replication, including polymerization, cleavage, and ligation Additionally, the circular structure of the viroid genome and its rolling-circle replication mechanism allow for replication without a specific initiation site, while the polyploid nature of viroid genomes further supports this evolutionary framework.
1988) would have favored their survival under the error-prone conditions of the prebiotic world
The structure of the first intermediate in the PSTVd cleavage-ligation pathway, as illustrated in Figure 2-1, can be compared to those of the hammerhead and hairpin ribozymes Notably, the upper part of the pospiviroid's central conserved region features a short sequence motif (GAAA) that is also found in these ribozymes.
Hammerhead ribozymes, as noted by Diener in 1989, demonstrate a transition from RNA primary and secondary structures to tertiary structures, revealing a significant similarity with pospiviroids The hairpin ribozyme, found in the (-) strand satellite RNA of the Tobacco ringspot virus, comprises two domains that interact during the transition state, much like the central conserved region of pospiviroids, which also features a loop E motif Loop E motifs serve as crucial "organizers" for multi-helix loops in ribosomal RNAs, and in the hairpin ribozyme, a conformational change in this motif is vital for catalysis, as highlighted by Hampell in 2001 Beyond sequence-specific cleavage, the hairpin ribozyme also facilitates RNA ligation Recent findings by Burke in 2002 indicate that hammerhead and hairpin ribozymes share more similarities than previously recognized, reinforcing the notion that viroids may represent "relics of precellular evolution."
Pospiviroids and hammerhead ribozymes share a conserved GAAA sequence, indicating a potential common ancestor in the prebiotic RNA world, as suggested by Diener in 1989 Additionally, the presence of a loop E motif in the hairpin ribozyme further supports this evolutionary relationship However, the processing structure involved in the initial PSTVd cleavage lacks a loop E motif, with cleavage sites in the respective RNAs indicated by arrows.
Figure 2-1-Possible evolutionary relationships between viroids and ribozymes.
The Viroid Species Infect Grapevine
Five viroids, Hop stunt viroid (HSVd), Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2
Grapevines can be infected by GYSVd-2 and Citrus exports viroid (CEVd), as noted in research by Sano (1988) However, only GYSVd-1 and GYSVd-2 are known to cause yellow speckled symptoms, according to Kolunow (1988) In contrast, HSVd, AGVd, and CEVd do not exhibit any symptoms.
-11- obvious disease symptoms and infect in the grapevine unnoticed, acting as a symptomless reservoir, which represents a potential threat to other crops
The first viroid identified in grapevines was the Hop stunt viroid (HSVd) from Japan, classified within the Potato spindle tuber viroid group of the Pospiviroid family HSVd has a broad host range, infecting various plants including cucumber, citrus, and multiple Prunus species A total of 84 HSVd sequences are recorded in the subviral RNA database Both HSVd and Citrus exocortis viroid (CEVd) have been found in grapevines, necessitating the use of RT-PCR assays with specific primers to differentiate them Analysis of grapevine samples revealed the presence of both viroids, with phylogenetic studies indicating that Brazilian HSVd variants are distinct from citrus variants, while Brazilian CEVd variants clustered with other citrus and grapevine forms Additionally, molecular characterization of HSVd isolates from infected Prunus species identified five new sequence variants, enhancing the understanding of HSVd diversity.
Association of Hop stunt viroid (HSVd) with yellow corky vein disease of citrus
A study in India investigated the causal relationship between CYCVD and HSVd, revealing that HSVd consists of 295 nucleotides The analysis indicated that the isolates displayed nearly 100% nucleotide identity with six citrus cachexia isolates of HSVd This variant has been tentatively named Hop stunt viroid-ycv (Roy 2003).
Citrus exocortis viroid (CEVd) has been linked to CYCVD, with BLAST analysis indicating sequence alignment with various CEVd strains The isolates from CYCVD-infected plants have been tentatively identified as a variant of CEVd-ycv, which shares a close relationship with CEVd Gynura variants documented in Australia (Roy 2006).
Phylogenetic analyses revealed that one apricot isolate grouped with a recombinant cluster, while the remaining isolates—one apricot, two plum, and one peach—aligned with the hop-group, highlighting the genetic diversity of HSVd isolates Notably, the sequence variability was more closely associated with the geographical origins of the isolates than with their host species (Gazel 2008).
Grapevine yellow speckle disease, first identified in 1988, is caused by viroids, specifically GYSVd-1, which is responsible for the yellow speckle symptoms observed in infected grapevines These symptoms are characterized by yellowish spots or flecks that are often limited to the tissue along the major or minor veins of the leaves, as noted by Szychowski in 1998 GYSVd-1 is closely related to GYSVd-2, sharing significant sequence similarities, and both viroids can be associated with plant virus infections in the field, as previously mentioned by Woodham in 1972.
Australian grapevine viroid (AGVd) is a unique viroid that exhibits less than 50% sequence similarity with any known viroid Its sequence can be categorized into regions that show significant similarity to segments of citrus exocortis, potato spindle tuber, apple scar skin, and Grapevine yellow speckle viroids Notably, AGVd includes the complete central conserved region characteristic of the apple scar skin viroid group, suggesting its classification as a member of this group (Kolunow 1988).
CEVd exhibits a broad host range, infecting not only citrus and grapevine but also various species within the Compositae, Solanaceae, Leguminosae, Brassicaceae, and Moraceae families Notable hosts include chrysanthemum, tomato, and broad bean.
(Fagoaga 1995), eggplant, turnip carrot (Fagoaga 1996), and fig (Yakoubi 2007), respectively.
The Detection Techniques in Viroid Disease
Viroids are typically identified through various methods, including electron microscopy, biological characterization, and assessing host range Additional techniques for detection include bioassays, poly-acrylamide gel electrophoresis (PAGE), molecular hybridization, and advanced molecular methods such as RT-PCR, RT-PCR-enzyme-linked immunosorbent assay (RT-PCR-ELISA), and real-time PCR.
Real-time polymerase chain reaction (qPCR) is a molecular biology laboratory technique that enables the monitoring of targeted DNA molecule amplification during the polymerase chain reaction (PCR) This method allows for the observation of DNA amplification in real-time, providing valuable insights into genetic analysis and research applications.
Real-time PCR differs from conventional PCR by allowing for the monitoring of the amplification process as it occurs, rather than at the end This technique can be utilized for quantitative analysis, known as Quantitative Real-Time PCR, as well as for semi-quantitative assessments, which determine whether the DNA molecules are above or below a specified threshold, referred to as Semi-Quantitative Real-Time PCR.
Two common methods for the detection of PCR products in real-time PCR are:
(1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and
(2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence
The MIQE guidelines recommend using "qPCR" for quantitative real-time PCR and "RT-qPCR" for reverse transcription-qPCR, although the term "RT-PCR" often refers to reverse transcription polymerase chain reaction, leading to some inconsistencies among authors ELISA tests have proven valuable for differentiating strains of various viruses, including Tomato spotted wilt virus, Beet mild yellowing virus, Soybean mosaic virus, Potato virus Y, Apple chlorotic leaf spot virus, and Beet necrotic yellow vein virus In Potato virus Y, both the coat protein and a helper component protein (HC-Pro) are produced, with monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs) against HC-Pro aiding in strain differentiation Additionally, a MAb was utilized to distinguish Beet mild yellowing virus strains with varying host ranges from other prevalent BMYV strains in the field.
Monoclonal antibodies (MAbs) possess a high capacity for specificity, enabling them to interact with distinct epitopes on viral coat proteins or other associated proteins Plant viruses and their strains are categorized into various serotypes according to their reactivity with different MAbs, even though these serotypes may exhibit similarities.
Using the DAS-ELISA format with a combination of universal MAb5B and serotype-specific MAbs, 73 isolates of the West African Rice yellow mottle virus (RYMV) were categorized into three distinct serogroups These serogroups corresponded to two RYMV pathotypes, which were differentiated based on their reactions to a set of differential rice cultivars (Konaté G.; Traoré 1997).
A threshold of 90% amino acid sequence identity of the tospoviral nucleocapsid protein (NP), which encapsidates viral RNAs, is crucial for species designation (Goldbach 1996) The genus Tospovirus includes 16 official and tentative virus species, categorized into three major serogroups and four monospecies serotypes, based on serological relationships and phylogenetic analysis of NPs (Jan 2003) Tomato spotted wilt virus (TSWV) and Watermelon silver mottle virus (WSMoV) represent the TSWV and WSMoV serogroups, respectively Calla Lily chlorotic spot virus (CCSV), isolated from Taiwan, has been identified as a tospovirus that is serologically related but distantly linked to WSMoV This relationship was established through indirect ELISA and immunoblotting, utilizing polyclonal and monoclonal antibodies against WSMoV and CCSV NPs The monoclonal antibodies specifically reacted with homologous antigens in both ELISA and immunoblot analyses, demonstrating their specificity (Lin 2005).
Hybridization with cDNA or cRNA probes is an easy and powerful method for detecting differences located in any region of the genome (Rosner 1984, Rosner
The limitations of RNA purification and the safety hazards associated with radioactive probes have restricted the broader use of hybridization methods Recently, the availability of non-radioactive probes, such as digoxigenin (DIG)-labeled DNA and RNA probes, has expanded their applicability A novel non-isotopic hybridization procedure has been developed to differentiate isolates of Citrus Tristeza virus (CTV) using DIG-labeled cDNA probes with various target types.
DIG-labeled probes hybridized with purified dsRNA or concentrated total RNA extracts on nylon membranes can detect CTV nucleic acid in infected tissues at levels as low as 0.1–1.0 mg This sensitivity is comparable to or slightly better than that achieved with 32P-labeled probes Furthermore, under stringent conditions of 60°C and 50% formamide, hybridization of tissue prints with DIG-labeled probes effectively differentiates CTV isolates in citrus plants cultivated in both greenhouse and field settings (Narváez 2000).
A microarray system has been created to detect and differentiate plant viruses and their strains by utilizing amplicons from plant viral RNA This method involves hybridization with synthetic oligonucleotide probes organized in a two-dimensional array on a glass slide The Cucumber mosaic virus (CMV), recognized for its significant heterogeneity in coat protein (CP), serves as the model pathogen for this innovative detection approach.
The study successfully amplified CP genes from 14 different isolates using cy3-labeled species-specific primers and hybridized them with five serotype-specific 24-mer oligonucleotide probes on an aldehyde-coated glass slide This microarray technique effectively differentiated the isolates into serogroups 1 and 2, accurately classifying nine out of ten isolates into subgroups 1a and 1b, surpassing the capabilities of RFLP analysis with MspI The findings highlight the significant potential of oligonucleotide-based microarray technology for precise virus isolate detection and differentiation, marking the first development of a diagnostic chip for plant viruses.
However, these methods have limited practicality Bioassays are associated with constraints of time and space, while PAGE is restricted by the number of samples for
Quantitative PCR (qPCR) is an advanced form of standard PCR that allows for the detection and quantification of DNA in real-time using fluorescent dyes or tagged oligonucleotide probes This method offers faster results and reduced variability compared to traditional PCR, thanks to its sensitive fluorescent chemistry and the elimination of post-PCR detection steps qPCR is particularly effective for the specific detection and quantification of viruses and viroids.
Reverse transcription real-time PCR assays utilizing TaqMan® chemistry have been established for detecting and quantifying Cucumber vein yellowing virus (CVYV) and Cucurbit yellow stunting disorder virus (CYSDV) in individual adults of the whitefly vector Bemisia tabaci This method incorporates an internal control targeting a gene from B tabaci to account for variations in extraction efficiency The developed assays were employed to estimate the proportion of viruliferous whiteflies collected from commercial greenhouse crops in Spain.
A significant number of whiteflies were found to carry both CVYV and CYSDV viruses, with their quantities estimated to aid in assessing the risks these viruses pose to cucurbit crops This information is crucial for understanding the epidemiology and plant–virus–vector interactions associated with these diseases The Polymerase Chain Reaction (PCR) is a key biochemical technique used to amplify specific DNA sequences from minimal starting material, effectively mimicking natural DNA replication through the use of thermophilic DNA polymerase Conducted in a thermal cycler, PCR involves three main steps: melting, where the DNA duplex is denatured at high temperatures; annealing, where primers bind to the single-stranded target DNA; and elongation, where DNA polymerase adds nucleotides to extend the primers This cycle continues until the desired concentration of DNA is achieved.
Apple dimple fruit viroid (ADFVd) induces a serious fruit disorder that reduces the market quality of the apple fruits drastically An RT-PCR procedure was
-18- developed with differently labeled primers in conjunction with a universal primer for detection and differentiation of isolates of ADFVd and Apple scar skin viroid
Research on two ADFVd field isolates from commercial apple cultivars revealed five new sequence variants, highlighting the sequence variability of ADFVd Comparison of sequences identified nine polymorphic positions across different regions of the ADFVd molecule Given that ADFVd and ASSVd exhibit similar symptoms in apple fruits, it is crucial to determine the extent of their infection A multiplex detection method was developed, involving reverse transcription with primer ADA-36, followed by PCR with specific primers AD-38 rd and AS-3ft The resulting products were analyzed through agarose gel electrophoresis under UV light By leveraging the sequence conservation and divergence between ADFVd and ASSVd, viroid-specific primers labeled with distinct fluorescent dyes were designed for rapid detection and differentiation via RT-PCR amplification This method successfully amplified cDNA of the expected sizes (254-bp in red for ADFVd and 330-bp in green for ASSVd) from infected tissues, enabling differentiation even in cases of double infections where one viroid predominates.
Elimination of Viroids from Plants
Grape is one of the most popular in the world In Taiwan, the grape is the most economically valuable Nowadays, grapevine diseases are serious problems in
Grapevine cultivation faces significant challenges due to the presence of diseases and viruses that are hard to eradicate using traditional apical meristem culture methods Consequently, extensive efforts have been dedicated to developing grape plants that are entirely disease-free.
Extended low-temperature treatment is an effective method for generating grape plants, while an alternative approach involves using shoot tips cultured on a medium with anti-virus agents Advances in disease detection methods have led to the establishment of sensitive techniques, such as nested PCR and dot blot hybridization, specifically for viroid detection.
STUDY METHODS
Source of Plant Materials
In the summer of 2015, young leaves from 50 grapevine samples were collected from various vineyards in Changhua, Taiwan The leaves exhibited distinct symptoms, including yellow speckle or line patterns, while some plants appeared asymptomatic Samples were taken from four to five neighboring plants in every second row of the vineyard and were subsequently analyzed The collected leaves were stored at -80°C until further use, as illustrated in Figure 3-1.
Figure 3-1: Grapevine leaves collected from Changhua County, Taiwan in June 2015
(A) Hop stunt viroid (HSVd) and Grapevine yellow speckle 1 (GYSVd-1) were detected in the leaf samples; (B) Only GYSVd
Figure 3-1 (conti.): Grapevine leaves collected from Changhua County, Taiwan in
June 2015 (A) Hop stunt viroid (HSVd) and Grapevine yellow speckle 1 (GYSVd-1) were detected in the leaf samples; (B) Only GYSVd
Extraction of RNA
To extract RNA from grapevine leaves, first homogenize 100 mg of the leaves in a mortar with 450 µl of PRX buffer and 10 µl of β-mercaptoethanol After centrifuging at 13,000 x g for 2 minutes, transfer the flow-through sample to a new tube and add 230 µl of 98-100% ethanol, mixing thoroughly Then, apply 680 µl of the ethanol-treated sample to a Plant Total RNA Mini Column in a Collection Tube, close the cap, and centrifuge at 10,000 x g for 1 minute, discarding the filtrate Wash the column with 0.5 ml of WF buffer by centrifuging at full speed for 1 minute, discarding the filtrate, and perform two additional washes with 0.7 ml of WS buffer, centrifuging each time.
To prepare the sample, centrifuge at full speed for 1 minute and discard the filtrate Next, centrifuge again at full speed for 3 minutes to eliminate any remaining WS buffer traces Afterward, transfer the column to an RNase-free 1.5 ml elution tube, add 50 µl of RNase-free distilled water, and centrifuge at full speed to complete the elution process.
2 minutes to elute RNA The quality of the extracted RNA was measured using UV Spectrophotometer (Figure 3-2) with ratio 1.8~2.0
Designation of Viroid-Specific Primer
All available methods for analysis rely on approximations, leading to performance differences that are often based on average estimates Consequently, techniques effective for one gene or protein family may not yield the same results for another It is essential to employ multiple alignment approaches and parameter sets while meticulously examining the outcomes, as highlighted in previous reviews (Duret 2000, Notredame 2002) This article will provide a comprehensive review of these methodologies.
There are 24 different global alignment procedures used for multiple sequence alignment (MSA), which is a crucial yet often overlooked aspect of sequence analysis This article aims to elucidate the strategies behind some of the most widely used algorithms, highlighting their strengths and potential drawbacks.
Progressive alignment algorithms are the most popular choice due to their speed, simplicity, and efficiency These methods typically begin by estimating a tree, followed by constructing pairwise alignments of the subtrees at each internal node More advanced algorithms, such as iterative ones, often incorporate this basic strategy in their processes Among these, the Clustal-W algorithm, introduced by Thompson in 1994, and its graphical interface, Clustal-X, released in 1997, are the most widely utilized progressive algorithms.
With the increasing availability of viroid sequences in GenBank, it is now feasible to design specific primers for the detection of numerous viroids This analysis focuses on creating targeted primer pairs for effective viroid detection.
Hop stunt viroid (HSVd), Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2 (GYSVd-2), and Citrus exocortis viroid (CEVd) were synthesized following established protocols as detailed in previous research.
Four pairs of primers were designed for HSVd-DY (including HSVd-(O) Old and HSVd-New) and GYSVd-1-DY (including GYSVd-1-(O) Old and GYSVd-1-(N) New) These primers were developed based on sequences from the National Center for Biotechnology Information (NCBI) with specific accession numbers The HSVd-Old primers were created by Sano et al in 2001, while the new primers for GYSVd-1 were also designated with their respective accession numbers.
JF746193, JF746185, JF746182, JF746184, JF746189, JF746180, JF746183, JF746190), and GYSVd-1-O was designed by (Hajizadeh, Navarro et al 2012)
Complete sequence alignment was conducted by Clustal-W software (Figure 3-3 & 3-
4) and specific primers for RT-PCR were designed (Table 3-1)
Figure 3-3: Multiple sequence alignment of HSVd by CLUSTAL-W program for primer designing Complete sequence Hop stunt viroid was obtained from
GenBank The asterisk indicated conserved nucleotide Highline showed the position of the primers
Figure 3-4: Multiple sequence alignment of GYSVd-1 by CLUSTAL-W program for primer designing Complete sequence GYSVd-1 were obtained from GenBank
The asterisk indicated conserved nucleotide Highline shown the position of the primers
Table 3-1: Primers in this work
To design primers using Pick up Primer according to established standards, select a 500 bp subsequence from sequences longer than 1 Kb, focusing on conserved regions Set primer concentrations to 200 nM, increasing from 50 nM, and ensure that the % G/C content of the primers is as uniform as possible Additionally, aim for matching melting temperatures (Tms) of the primers, targeting around 59 °C.
Select 3-4 primers to test Check the sequence alignment to make sure the assay is in an acceptable region I designed my primers following the standards: HSVd-N-
-28- mF (GC content 55%, Tm(50mM Na+) is 53.8 oC), HSVd-N-mR (GC content 57.89%, Tm(50mM Na+) is 51.8 oC),GYSVd-1-N-mF (GC content 63.64%,
Tm(50mM Na+) is 60.4 oC) and GYSVd-1-N-mR (GC content 70%, Tm(50mM Na+) is 59.9 oC).
The Single RT-PCR Reaction
Viroids necessitate a reverse transcription (RT) step prior to the PCR amplification process, known as RT-PCR This technique involves the annealing and enzymatic extension of two oligonucleotide primers, typically ranging from 16 to 30 nucleotides in length, to target regions in duplex DNA A thermo-stable DNA polymerase is employed to ensure enzymatic activity is maintained even at elevated temperatures.
These three steps (denaturation, primer annealing and primer extension) which are carried out at discrete temperature ranges (for example, 94 oC to 98 oC, 37 oC to
A single PCR cycle involves temperatures of 65°C and 72°C, which are crucial for primer annealing and extension The specific temperatures can vary based on the DNA enzyme utilized and the unique sequence or length of the primers.
Multiplex RT-PCR (mRT-PCR)
The mRT-PCR was performed using 2 µl of cDNA with 0.2 µM of each primer, following a one-step PCR protocol The cycling parameters included an initial incubation at 50°C for 30 minutes, followed by inactivation at 94°C for 2 minutes This was succeeded by 32 cycles consisting of denaturation at 94°C for 1 minute, primer annealing at 53°C for 30 seconds, elongation at 72°C for 2 minutes, and a final extension at 72°C for 7 minutes.
We selected specific primer combinations to effectively separate and identify the anticipated amplified fragments on a 1% agarose gel Various primer concentration combinations were evaluated to ensure amplification of all target sequences The mRT-PCR products were subsequently analyzed through electrophoresis on a 1% agarose gel, stained with ethidium bromide, and visualized under UV light.
DNA Elution and Cloning
DNA was extracted from gel electrophoresis using the Micro-Elute DNA Clean extraction kit (GenMark) following the manufacturer's guidelines After electrophoresis in TBE buffer, the desired DNA band (≤500 mg) was cut out and transferred to a new micro-centrifuge tube, where 2 to 3 volumes of Binding Solution were added to the gel slice The silica matrix was resuspended to create a homogeneous mixture, followed by the addition of 15 µl of silica suspension and incubation at 60°C for 5-15 minutes to dissolve the agarose, with occasional vortexing After centrifugation, the supernatant was carefully removed, leaving 20 µl of solution with silica The mixture was applied to a Spin Filter in a Collection tube and centrifuged for 1 minute Following this, 700 µl of Wash Solution was added, and the centrifugation was repeated to remove any residual ethanol The Spin Filter was then transferred to a new micro-centrifuge tube, and 10-20 µl of Elution Solution or H2O (pH 7.0-8.5) was added before a final centrifugation at top speed for one minute The eluted DNA was stored at -20°C.
The PCR product was cloned into the yT&A cloning vector following the manufacturer's instructions, where DNA fragments were ligated with the TA vector and transformed into competent E coli XL-1 cells The reaction mixture of mixed DNA and TA vector was pipetted and incubated for 5 to 15 minutes at 22°C, followed by an overnight incubation at 4°C Recombinant clones were introduced into E coli XL-1 containing ampicillin and tetracycline, and the plates were incubated overnight at 37°C, as illustrated in Figure 3-6.
A single colony with inserted TA cloning was selected using a toothpick and introduced into 5 ml of L-Broth medium containing ampicillin This mixture was incubated overnight at 37°C with shaking Following incubation, the plasmid DNA was purified and digested with the enzyme HindIII to verify the correct size of the DNA fragment.
Phylogenetic Tree Construction
Phylogenetic approaches are crucial for understanding the diversity, origin, and distribution of plant viruses, as they reveal the evolutionary histories that enable pathogens to infect specific hosts These methods help identify pathogens, trace their origins, and analyze their evolution in response to eradication efforts Additionally, phylogenetic techniques are being refined to study population dynamics, differentiate historical impacts from current influences on population structure, and assess gene flow and phylogeographic relationships among distinct genotypes This chapter reviews fundamental methodologies, including population genetic approaches, to explore plant viral evolution.
A wide range of software is available for analyzing viral data, with 32 tools identified as particularly useful For an extensive overview of phylogenetic software, readers can visit Joseph Felsenstein's website, which provides a comprehensive summary and links to various software packages The phylogenetic tree was constructed using the complete genomic sequences of HSVd and GYSVd-1.
The sequences were aligned and analyzed using multiple sequence alignment in PHYLIP format Pairwise evolutionary distances for the nucleotide sequences were calculated with the DNADIST program from the PHYLIP software package version 3.69, utilizing the F84 substitution model Dendrograms illustrating the relationships were created using TreeView software, employing neighbor-joining (NJ) analyses with a bootstrap value of 100 replicates.
RESULTS AND DISCUSSION
Results
4.1.1 Yield and quality of RNA extract
In the summer season, 100 mg of leaf tissue was collected from symptomatic grapevine leaves (Figure 3-1) for analysis Total RNA was extracted using an Invitrogen RNA extraction kit, and its quantity and quality were assessed using a UV spectrophotometer The RNA purity was indicated by a 260/280 ratio of approximately 1.8-2.0.
4.1.2 Primer design for viroid detection
Seventeen complete sequences of HSVd and nineteen sequences of GYSVd-1 were retrieved from GenBank for the design of new primers Multiple sequence alignment was performed to identify conserved regions, leading to the synthesis of suitable primers for RT-PCR analysis.
4.1.3 Detection of Grapevine viroids by single RT-PCR reaction
Prior to conducting multiplex RT-PCR reactions, single RT-PCR was performed to assess the specificity of primers for detecting five grapevine viroids In a study of 50 field samples, HSVd was identified in all samples, while GYSVd-1 was detected in 8 samples, indicating a 16% infection rate However, AGVd, GYSVd-2, and CEVd were not found An amplicon from the RT-PCR using HSVd-specific primers was cloned and designated HSVd-DY, which included the complete genomic sequence of 301 nucleotides.
-34- fragment obtained from the GYSVd-1 specific primers was also clone and the whole genome contained 367 nt
Table 4-1: Results of HSVd & GYSVd-1 analysis from Changhua County, Taiwan by mRT-PCR (MP) and single RT-PCR (SP) from 2015-2016.
MP SP MP SP MP SP MP SP
(+) and (-) denotes infected and healthy status during two consecutive years, Italics highlights samples that tested positive to sRT-PCR, but negative to mRT-PCR
The agarose gel electrophoresis analysis presented in Figure 4-1 illustrates the results of single and multiplex RT-PCR reactions Lane 1 shows the amplification using specific primers for GYSVd-1, while Lane 2 displays the results for HSVd Lane 3 demonstrates the effectiveness of mixed primers, successfully detecting both GYSVd-1 and HSVd simultaneously.
Lane M, molecular weight markers (GENMARK, GM100)
4.1.4 Development of multiplex RT-PCR reaction
In multiplex RT-PCR assays, primer pairs specific to multiple viroids were combined in a single RT-PCR mixture, initially using a final concentration of 0.5 µM for all primers However, this resulted in a weak amplicon for GYSVd-1 An effective adjustment was made with a combination of 0.4 µM HSVd primer pairs and 0.5 µM GYSVd-1 primer pairs, successfully detecting both HSVd and GYSVd-1 in grapevine extracts exhibiting mixed infections of these two viroids.
Protocol for ligation using the yT&A® cloning vector
Centrifuge yT&A® cloning vector and PCR DNA tubes to collect contents at the bottom of the tubes
Vortex the ligation buffer vigorously before use
To prepare the ligation mixture, combine 1 µl of ligation buffer A, 1 µl of ligation buffer B, 2 µl of the yT&A cloning vector, 2 µl of the PCR product, and 1 µl of T4 DNA ligase Additionally, include control DNA and add deionized water to achieve a final volume of 10 µl.
Mix the reactions by pipetting
Incubate the reactions for 5 to 15 min at 22 oC Alternatively, if the maximum of transformants is required, incubate the reactions overnight at 4 oC
Competent cell transformation (Figure 4-2 and 4-3)
Protocol for colony PCR o Pick an isolated colony with a sterile toothpick, Use the colony as PCR template Inoculate 25 àl of PCR reaction buffer in a microfuge tube as
-36- described below: PCR Premixed buffer (O‟in1 DNA polymerase premix,
To set up the thermal cycling program, use YT005 (23àl), M13-F (10 μM) (1μl), and M13-R (10 μM) (1μl) After running the program, verify the results on a 1% agarose gel For instance, utilizing the control DNA provided in the yT&A® cloning vector kit as the insert DNA, the colony PCR results are displayed below.
Figure 4-2: Agarose gel electrophoresis analyze T&A cloning vector kit insert DNA, Lane 1,2,4,5 with specific primers for GYSVd-1, Lane 3,6 with specific primers for
HSVd Lane M, molecular weight markers (GENMARK, GM100)
-37- Figure 4-3: Restriction enzyme sites of yT&A® cloning vector
T&A cloning vector (25ng/àl), control insert DNA (10ng/àl), YEAST DNA ligase (2U/àl), 10x ligation buffer A, 10x ligation buffer B, forward primer (M13-F)(10àM), reverse primer (M13-R)(10 àM), storage condition: -20 oC
Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes by making single-use aliquots of Ligase Buffer
Pfu DNA polymerase exhibits proofreading activity but lacks the terminal transferase-like activity found in Taq DNA polymerase Consequently, ligation reactions using non-tailed amplified DNA did not yield any positive colonies.
Methods for increasing the ligation efficiency: o A-tailing: purified PCR product, 10X PCR buffer, 10mM dATP, Taq
Add deionized water to a final volume of 100 àl
To achieve optimal transformation efficiency, purify the A-tailed DNA and utilize it in the ligation reaction, incubating the reactions overnight at 4°C if a maximum number of transformants is desired It is recommended to use a 1:3 molar ratio of vector DNA to insert DNA for improved results Additionally, employing high-efficiency competent cells, such as the ECOSTM series with over 10^8 cfu/μg DNA, can enhance the transformation process.
Agarose gel electrophoresis was utilized to analyze the insert DNA from the T&A cloning vector kit in conjunction with the HindIII enzyme Specific primers for GYSVd-1 were used in lanes 1, 2, 4, and 5, while lanes 3 and 6 contained specific primers for HSVd Molecular weight markers from GENMARK were included in lane M for reference.
The phylogenetic tree was constructed using the complete genomic sequences of HSVd-DY and GYSVd-1-DY Analysis of the phylogenetic tree for HSVd-DY, which included 17 related HSVd sequences from GenBank, indicated a strong evolutionary relationship among them Similarly, the phylogenetic tree for GYSVd-1-DY, derived from 19 sequences from GenBank, also demonstrated close evolutionary ties.
Figure 4-5: Phylogenetic analysis of the complete sequence of HSVd-DY with other
Seventeen HSVd sequences were obtained from GenBank, and a phylogenetic tree was constructed using the PHYLIP software package (J 2005) The bootstrap confidence values for 1000 replicates from the neighbor-joining (NJ) analyses are indicated next to the nodes, with values below 75% omitted The scale bar represents nucleotide substitutions per site.
A phylogenetic analysis was conducted on the complete sequence of GYSVd-1-DY alongside 17 other HSVd sequences sourced from GenBank The phylogenetic tree, constructed with the PHYLIP software package, displays bootstrap confidence values for 1000 replicates derived from neighbor-joining analyses, with values below 75% omitted The scale bar indicates the nucleotide substitutions per site.
The sequence analysis of the detected HSVd-DY revealed a high sequence identity ranging from 85.79% to 98.7% when compared to 17 other HSVd strains from GenBank, which were sourced from various hosts and geographical regions In a similar vein, the GYSVd-1-DY exhibited a sequence identity ranging from 39.2% to 99.7% in comparison to other GYSVd-1 variants, with notable exceptions for specific strains.
Vitis vinifera of Australia (accession number XJ892929 and JX892932) shown low sequence identity (37,6% and 39.2%) with DY isolate (Table 4-2 and 4-3)
Table 4-2: The comparison of sequence identity between HSVd-DY and other HSVd from GenBank The accession number, infected host and isolated country are indicated
HSVd Host Country Da-Yeh
GQ995466 Vitis vinifera cultivar Pinot noir, clone ENTAV115 Italy 97.7 GQ995464 Vitis vinifera cultivar Pinot noir, clone ENTAV115 Hungary 98.7
FJ716190 citrus strain CC-D isolate 3 China 91.8
FJ716178 citrus strain CC-B isolate 3 China 92.7
JX418270 Citrus-Thomson navel sweet orange Iran 92.8
Y09352 Prunus persica cv Jeronimo J-16 Spain 96.3
HE575348 Humulus lupulus var Celeia Slovenia 85.7
JX401927 Malus sylvestris (wild apple) 82.9
DQ371446 grapevine cv Jingxiu China 99
Table 1-3: The comparison of sequence identity between GYSVd-1-DY and other GYSVd-1 from GenBank The accession number, infected host and isolated country are indicated
GYSVd-1 Host Country Da-Yeh
DQ371468 Vitis vinifera cv Jingchao China 86.2
DQ371469 Vitis vinifera cv Beiquan China 85.9
DQ371474 Vitis vinifera cv Zhiyuan 540 China 91.4
DQ371467 Vitis vinifera cv Shafu Seedless China 85.9
DQ371470 Vitis vinifera cv Thompson Seedless China 85.9
EU682453 Vitis vinifera cv Nebbiolo Italy 95.1
HQ447058 Vitis vinifera New Zealand 94.6
GQ995473 Vitis vinifera cultivar Pinot noir, clone ENTAV115 Hungary 94.9
AY639607 grape cultivar White Malaga Thai Land 95.1
AB028466 Japanese Campbell isolate Japan 99.2
JQ686713 isolate GYSVd-1-AO14#1 Iran 94.1
Brief Discussion of Results
The key viroids affecting grapevine, particularly linked to yellow speckle disease, can be reliably detected in young leaves during a specific physiological stage common to many plants Grapevine is abundant in phenolic compounds that increase with age, potentially complicating the extraction of high-quality RNA Additionally, the primary viruses associated with the leafroll disease complex are detectable in mature leaves, a stage characterized by heightened nuclease activities and reduced nucleic acid content.
This study developed five pairs of primers targeting HSVd, GYVd-1, GYSVd-2, CEVd, and AGVd for detecting viroid diseases in grapevines Using single-RT-PCR, HSVd and GYSVd-1 were successfully identified, with sequence analysis revealing high nucleotide identity to related viroids in GenBank Additionally, multiplex RT-PCR, utilizing mixed primers from HSVd and GYSVd-1, demonstrated effective amplification of DNA fragments specific to each viroid after gel electrophoresis This indicates that multiplex RT-PCR is a sensitive and rapid method for the simultaneous detection of various viroids in vineyards.
Fifty leaf samples exhibiting yellowing, mottling, or mosaic symptoms were collected from vineyards in Changhua County, Taiwan These mature leaves, selected from the third to fifth position on the grapevines, were chosen to minimize contamination from phenolic compounds, which can hinder high-quality RNA extraction Total RNA was extracted following the manufacturer’s instructions of the RNA extraction kit, and its quality was assessed using spectrophotometry.
The absorbance ratio of RNA at 260 nm/280 nm ranged from 1.8 to 2.0, indicating high purity To ensure optimal results in the RNA extraction process, it is crucial to thoroughly eliminate ethanol before eluting RNA from the column This step prevents organic compound contamination, which could negatively affect subsequent RT-PCR procedures.
The extraction procedure has been enhanced to improve the reliability of plant extracts for RT-PCR applications, as demonstrated by the developed control reactions RNA extraction involved the use of ethanol, which was subsequently eliminated through heat and water treatment.
Developing a reliable control for antibody preparations is challenging due to variations in antigen binding efficiency and titre It is essential to include a control in every assay, as improper storage or repeated freeze-thaw cycles can render an amplifiable extract un-amplifiable In mRT-PCR, we adjust the primer concentrations to a 1:1 ratio for GYSVd-1-N-DY and a 2:1 ratio for HSVd-N-DY during gel electrophoresis.
To accurately calculate the melting temperature, users must specify the buffer used for amplification, choosing between pre-set polymerase buffers or custom-buffer mode to define ionic content and algorithms The sequences for amplification should be submitted in a FASTA file format The software generates a results page displaying all primers, which can be downloaded as either a FASTA or CSV file Additionally, it includes a validation plot illustrating the melting temperature algorithm's performance on experimental data, along with program logs.
High-quality and abundant RNA is essential for creating cDNA libraries that accurately represent all expressed genes However, extracting RNA from plant tissues, especially from woody species like grape, apple, and citrus, poses challenges due to their high levels of extractable phenols and polysaccharides, often necessitating modifications to existing protocols or the development of new extraction methods.
Mature pre-climacteric and post-climacteric fruit tissues exhibit increased biosynthesis of potential interfering substances during maturation Attempts to extract high-quality total RNA from apple fruit and flower tissues for cDNA library construction have been unsuccessful A common issue in plant molecular biology is the presence of polysaccharides in extracted DNA; however, increased salt concentrations in the extraction buffer can mitigate this problem In cucumber tissues, polysaccharides were effectively removed, as indicated by unaffected OD280/OD260 ratios, while potato samples required higher salt concentrations to prevent polysaccharide contamination Primers designed from conserved sequences across multiple plants aimed to facilitate control reactions for various species, even those lacking sequence data Notably, amplification with HSVd and GYSVd-1 primers occurred in both infected and uninfected tissues, including dormant grapevine cane and bud samples, indicating their utility as internal controls for virus testing in imported and exported woody plant tissues.
In a study involving fifty field samples infected with viroids, one tube-one step RT-PCR successfully yielded expected product sizes for all tested isolates Notably, two distinct viroid bands were detected exclusively in the infected samples, with no presence in negative controls, confirming the effectiveness of the protocol for analyzing field samples.
All collected leaf samples were found to be infected by HSVd, which is widespread and likely prevalent in vineyards In contrast, GYSVd-1 showed a lower infection rate of 16% in the samples No other grapevine viroids, such as GYSVd-2, AGVd, or CEVd, were detected Notably, a DNA fragment obtained from RT-PCR analysis using GYSVd-2 primers revealed a sequence consistent with GYSVd.
1 GYSVd-1 and GYSVd-2 are very closely related and have high sequence identity to each other It is important to carefully check if the amplicon is the correct target when detected between these two viroids
CONCLUSIONS AND SUGGESTIONS
In designing primers for simultaneously detection viroids in plant samples, the following specifications needed to notice
1 The designed primers needed to be capable to specific amply a single viroid target
2 Primers sharing a similar melting temperature so as to facilitate setting the appropriate conditions for amplification of all the expected viroid target
3 The DNA products after amplified needed to show in different size from each other to easily identify each of them by standard agarous gel electrophorsis
4 When designed for the primers, it needs to consider the intraspecific sequence variability because of the quasispecies exist in the natural viroid population
We have developed primers designed to amplify various sizes of DNA fragments from different viroids, focusing on conserved regions across multiple plant sequences This approach aims to enable the use of the same primers for control reactions in diverse plants, including those lacking sequence data To ensure accurate melting temperature calculations, users must specify the buffer used for amplification, choosing between pre-set polymerase buffers or custom buffer settings The sequence library for amplification should be submitted as a FASTA file, and the software generates a results page displaying all primers, which can be downloaded in FASTA format.
The article discusses a CSV file containing a validation plot that illustrates the performance of the melting temperature algorithm applied to experimental data, along with program logs It highlights that the high concentration of certain viroids in the extracts may lead to the amplification of viroid-specific fragments overshadowing the control fragment during RT-PCR However, this situation is not a significant concern, as the control's primary function is to verify whether the extract can adequately support the RT-PCR reaction.
Prolonged storage of plant tissue and extracts at -80°C enables future testing for additional viruses as sequences become available and specific primers can be developed This study demonstrated the effectiveness of a multiplex RT-PCR protocol for the identification of two grapevine-infecting viroids The method was validated using naturally infected vines from Da-Yeh University in Taiwan, proving to be sensitive and reliable, regardless of the presence of the primer pair designed to amplify a host-derived internal control.
The formation of secondary structures in target sequences significantly influences their interaction with other molecules and their availability for hybridization (Murashige 1972) This study employs random amplification methods, which can produce non-targeted amplicons, potentially impacting the sensitivity and specificity of hybridization reactions.
The genomic sequences of tospoviruses were sourced from the National Center for Biotechnology Information (NCBI) databases To analyze these sequences, multiple sequence alignments were conducted, comparing newly determined sequences with reference sequences Additionally, nucleotide sequences were translated into amino acid residues using the Clustal-W, Bl2seq, and Six frame programs available in Biology Workbench.
The homologies of nt and aa sequences were calculated using the Gap program of SeqWeb Phylogenetic analysis was done using Philip 3.69 Bootstrapping was
To generate multiple data sets, the sequence "51-" was repeated 100 times, and these input data sets were resampled using the Seqboot program from Phylip 3.69 A distance matrix for the amino acid sequences was created with the dnadist.exe program, employing the PAM matrices based on the Dayhoff model The phylogenetic branches were established through the Neighbor program of Phylip 3.69, utilizing the neighbor-joining method Finally, phylogenetic trees were constructed using the Consense program from Phylip 3.69.
In our study, we compared HSVd and GYSVd-1 strains from grapevine samples collected in Dacun, Yuanlin, Taiwan, with previously published sequences available in GenBank The findings revealed mutations in the closest sequences, demonstrating a similarity range of 95% to 99%.
The HSVd-DY and GYSVd-1-DY sequences consist of 301 nt and 367 nt, respectively, exhibiting a high nucleotide identity of 95%-98% with sequences AB742224, AB742225, GQ995466, GQ995464, and Y14050 for HSVd, and 95%-99% identity with JF746190, JF746188, GU170805, AY639607, AB028466, and Z17225 for GYSVd-1 Phylogenetic analysis reveals that HSVd-DY is closely related to GQ995464, while GYSVd-1-DY shows a close relationship with JF746188, AB028466, and Z17225.
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The rod-shaped secondary structure of Potato Spindle Tuber Viroid features five distinct domains typical of the Pospiviroidae family: the terminal left (TL), pathogenicity (P), central (C), variable (V), and terminal right (TR).