Specific mRT-primer pairs for the detection of Hop stunt viroid HSVd, Australian grapevine viroid AGVd, Grapevine yellow speckle viroid-1 GYSVd-1, Grapevine yellow speckle viroid-2 GYSV
INTRODUCTION
Background of Research
Viroids are the smallest known agents of infectious disease – small, highly structured, single-stranded, circular RNA molecules that lack detectable messenger RNA activity Whereas viruses supply some or most of the genetic information required for their replication, viroids are regarded as “obligate parasites of the cell‟s transcriptional machinery” and infect only plants Four of the nearly 30 species of viroids described to date contain hammerhead ribozymes, and phylogenetic analysis suggests that viroids may share a common origin with hepatitis delta virus and several other viroid-like satellite RNAs Replication proceeds via a rolling-circle mechanism, and strand exchange can result in a variety of insertion/deletion events The terminal domains of potato spindle tuber and related viroids, in particular, appear to have undergone repeated sequence exchange and/or rearrangement Viroid populations often contain a complex mixture of sequence variants, and environmental stress (including transfer to different hosts) has been shown to result in a significant increase in sequence heterogeneity The new field of synthetic biology offers exciting opportunities to determine the minimal size of a fully functional viroid genome Much of the preliminary structural and functional information necessary is already available, but formidable obstacles still remain
Sequences of viroids and viroid-like satellite RNAs were aligned separately using CLUSTAL-X and then manually edited to preserve local similarities; these partial alignments were then manually aligned before CLUSTAL-X was used to realign dissimilar regions and maximize overall similarity
-2- Virus and viroid diseases have become increasingly important constrain to sustainable crop production in the tropical countries The climatic changes that are occurring throughout the world have an impact on plants, vectors, and viruses causing increasing instability within virus-host ecosystems
Viroid diseases are inconspicuous compared with disease caused by fungi, bacteria and nematodes, and loss identified in comparative trials on their effects do not necessarily translate to global estimates of loss Their effects also extend beyond direct and indirect damage associated with plant virus infection largely also applies to viroids
Some of the threatening and economically important virus diseases in tropical zone which affect the food production are tungro, yellow mottle, and hoja blanca in rice; mosaic in sugarcane, mosaic in cassava; tristeza in citrus; swollen shoot in cacao; sterility mosaic in pigeonpea; rosette, clump, and bud necrosis in peanut; necrosis in sunflower and legumes, vegetables, and ornamental crops; yellow mosaic in legumes; leaf curl in cotton and tomato; and ring spot in 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
In Taiwan, grape cultivation is a remunerative agri-business for the highly popular grape berries among local consumers in addition to the added values with the processed products The annual production of grapes was 102831 metric tons in 2010, and 99.5% was produced in Taichung City, Miaoli, Changhua and Nantou Counties in central Taiwan(Chiou-Chu 2013) RT-PCR has become crucial tools for detecting viroids Simultaneous detection and identification of viruses and viroids using
-3- conserved primers are possible through RT-PCR, which has a higher sensitivity than that of molecular hybridization
Our aim of this research is to isolate and identify the causal agents of the suspected disease in grapevines in Taiwan and to determine their phylogenetic relatedness to the other 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
The framework of this research is shown as Figure 1-1 HSVd and GYSVd-1 viroid are analyzed by Clustal-W solfware Also a conserved region is found to design primers, then transferring to tube by using RT-PCR machine Finally gel electrophoresis will be run
LITERATURE REVIEWS
Structure and Classification of Viroid
Like many RNA viruses that infect plants or animals, individual viroids exist as complex populations of often closely related sequence variants in vivo A number of studies have examined natural variability within viroid populations, and the Subviral RNA Database now contains the complete sequences of more than 1,100 viroid variants In many cases, multiple sequence variants have been isolated from a single infected plant
Viroids are circular RNAs, infectious agents with a small, single-strand RNA molecules, viroids do not code for proteins, the length of viroids is about 250 to 400 nucleotides Viroids are able to replicate and move through infected plants, which cause frequently severe diseases in plants to severe stunting, leaf necrosis, corky-bark, leaf-roll and fruit deformation depending on host plant and viroid species
They contain five structural regions: left terminal region (T1), pathogenic region (P), central conserved region (C), variable region (V) and right terminal region (T2)
All species in the family Pospiviroidae have a rod-like secondary structure that contains five structural/functional domains (Keese 1985) and replicates in the nucleus
Three of the four members of the Avsunviroidae have a branched secondary structure, and all replicate/accumulate in the chloroplast All members of the Avsunviroidae contain hammerhead ribozymes in both the infectious (+) strand and complementary (−) strand RNAs Figure 2-1 compares the secondary structures of PSTVd (rod-like,
Pospiviroidae) and Peach latent mosaic viroid (PLMVd; branched, Avsunviroidae)
With the possible exception of PLMVd, viroids do not appear to contain any modified
-6- nucleotides or unusual phosphodiester bonds Nucleic acid extracts from infected leaf tissue contain a variety of viroid-related RNAs of both polarities Some of these molecules – especially those having a complementary or (−) strand polarity – are considerably longer than the infectious circular viroid (+) strand Northern analysis using strand-specific probes and/or primer extension has shown that these molecules represent the intermediates expected for a “rolling-circle” mechanism of replication
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 contain a variety of viroid-related RNAs of both polarities Some of these molecules – especially those having a complementary or (−) strand polarity – are considerably longer than the infectious circular viroid (+) strand Northern analysis using strand-specific probes and/or primer extension has shown that these molecules represent the intermediates expected for a
“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
-7- recombinant plasmid DNAs (Góra-Sochacka 1997), Agrobacterium-mediated introduction of nondisarmed recombinant Ti plasmids (Hammond 1994), and Agrobacterium-mediated plant transformation (Wassenegger M 1994) When working with highly debilitated variants that are only weakly infectious, constitutive expression from an integrated transgene provides an effective means to detect the rare events that can restore viroid infectivity (Góra 1994) used a reverse-transcription PCR strategy to generate, in a single step, infectious full-length cDNAs from three phenotypically dissimilar isolates of PSTVd When this method was applied to a
“mild” isolate, only a single sequence variant was recovered “Intermediate” and
“severe” isolates yielded three and four variants, respectively Not all of the variants recovered from the severe isolate produced severe symptoms when inoculated onto Rutgers tomato; thus, the presence of milder variants in a mixed inoculum may be masked by variants that are more severe Follow-up studies of (Góra-Sochacka 1997) revealed that many of these naturally occurring PSTVd sequence variants were unstable when inoculated alone – sometimes disappearing within a single 5~6 week passage in tomato This finding supports one of the basic tenets of the quasis-pecies theory, that mixtures of variants can complement each other, and hence the whole population is in essence a single entity analogous to an individual with thousands of alleles rather than just two In most cases, the new variants detected induced symptoms that were less severe than those of the parent The number of sequence changes detected in both studies was relatively limited, confined almost exclusively to the pathogenicity and variable domains with only a few changes located in the terminal right domain
One important advantage of screening assays that involve mechanical inoculation of full-length viroid cDNAs or RNA transcripts is that the results are usually available within a few weeks Many point mutations in PSTVd and other viroids, however, appear to abolish infectivity via mechanical inoculation In some
-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 possible origins for viroids have been proposed Viroids could be primitive ancestors or highly degenerate derivatives of conventional viruses, but as discussed by (Diener 1989), their unusual molecular structure and biological properties together with a lack of sequence similarity Evolution argues against this possibility, of viroids from transposable elements, plasmids, or in-trons has also been proposed At present, the balance of evidence suggests that viroids could represent
“relics of precellular RNA evolution,” and several reviews exploring this area have been published (Diener 2003) In essence, the argument for viroid origin in the RNA world is straightforward: RNA is the only known biological macromolecule that can function as both genotype and phenotype, allowing evolution to occur in the absence of DNA or protein As described by (Diener 1989), a simple hammerhead ribozyme similar to those found in ASBVd and other members of the Avsunviroidae is theoretically capable of performing all the polymerization, cleavage, and ligation steps required for viroid replication The circular structure of the viroid genome and the rolling-circle mechanism of replication eliminate the need for replication to initiate at a specific site; likewise, the apparently polyploid nature of viroid genomes (Juhasz 1988) would have favored their survival under the error-prone conditions of the prebiotic world
It can be compared (shown as Figure 2-1) that the structure of the first intermediate in the PSTVd cleavage-ligation pathway (Baumstark 1997) with those of the hammerhead and hairpin ribozymes The upper portion of the pospiviroid central conserved region contains a short sequence motif (GAAA) that is also present in
-9- hammerhead ribozymes (Diener 1989) Moving from the level of RNA primary/secondary structure to tertiary structure However, one can see that pospiviroid share an even greater degree of similarity with ribozymes The hairpin ribozyme found in (-) strand satellite RNA of Tobacco ringspot virus contains two domains that interact in the transition state Like the central conserved region of pospiviroids, the loop B domain of the hairpin ribozyme also contains a loop E motif
Loop E motifs are found in many different contexts, often acting as “organizers” for multi helix loops in ribosomal RNAs; in the case of the hairpin ribozyme, a conformational change in the loop E motif accompanies domain docking and is essential for catalysis(Hampell 2001) In addition to sequence-specific cleavage, the hairpin ribozyme also catalyzes RNA ligation Recent experimental work with the hammerhead and hairpin ribozymes suggests that they have more similar than previously thought(Burke 2002), and the possibility that viroids are “relics of precellular evolution” continues to be very much alive
Pospiviroids and hammerhead ribozymes (lower left) both contain a short conserved GAAA (shaded) sequence, suggesting to (Diener 1989) the possible existence of a common ancestor in the prebiotic RNA world The presence of a loop E motif in the hairpin ribozyme (lower right) provides additional support for such a relationship The processing structure involved in the initial PSTVd cleavage (above) does not contain a loop E motif, however Cleavage sites in the respective RNAs are denoted by arrows
-10- 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
(GYSVd-2) and Citrus exports viroid (CEVd) have been reported to infect grapevines (Sano 1988) although only GYSVd-1 and 2 have been shown to induce yellow speckled symptom expression (Kolunow 1988) HSVd, AGVd, and CEVd produce no
-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 reported in grapevines was an isolate of Hop stunt viroid (HSVd) from Japan HSVd is a member of the Potato spindle tuber viroid group, belonging to the family Pospiviroid HSVd apparently has a wide host range and besides hopping it can propagate in cucumber, grapevine, citrus, plum, peach, pear (Teruo 1989), apricot and almond plants (Polivka 1996) Eighty-four HSVd sequences are present in the subviral RNA database (Pelchat 2003) Hop stunt viroid (HSVd) infects a large number of woody plant hosts such as Prunus spp., Citrus spp., and Vitis spp HSVd along with Citrus exocortis viroid (CEVd) has been detected in both citrus and grapevines Hence, to differentiate these two viroids, total RNA from leaves of grapevine Vitis vinifera „Cabernet Sauvignon‟ and V labrusca „Niagara Rosada‟ was used as a template for RT-PCR assay Primers specific for HSVd, CEVd, Grapevine speckle viroid 1 (GYSVd-1), Grapevine speckle viroid 2 (GYSVd-2) and Australian grapevine viroid (AGVd) were employed The PCR amplicons were cloned and sequenced The grapevine samples analyzed showed the presence of both HSVd and CEVd Phylogenetic analysis showed that Brazilian grapevine HSVd variants clustered with other grapevine HSVd variants, forming a specific group separated from citrus variants On the other hand, the Brazilian CEVd variants clustered with other citrus and grapevine variants (Eiras 2000) Molecular characterization of Hop stunt viroid (HSVd) isolates from different naturally infected Prunus sources including apricot, plum and peach were performed by determining the nucleotide sequences of eleven isolates Five new sequence variants of 296-nt (3 variants) or 297-nt (2 variants) comparable to the known HSVd isolates were identified
-12- Association of Hop stunt viroid (HSVd) with yellow corky vein disease of citrus
(CYCVD) occurring in India was investigated to establish its causal relationship with the disease In silico analysis showed that HSVd has 295 nucleotides and that the isolates exhibited nearly 100% nucleotide identity with six citrus cachexia isolates of HSVd This variant was tentatively designated Hop stunt viroid-ycv (Roy 2003) Later
Citrus exocortis viroid (CEVd) was also found to be associated with CYCVD
BLAST analysis revealed the alignment of the sequences with a different CEVd The isolates from CYCVD-infected plants was tentatively named as a variant of CEVd- ycv This variant showed close relationship with CEVd Gynura variants reported from Australia (Roy 2006)
Phylogenetic analyses indicated that one apricot isolate clustered with a recombinant group, whereas all others (one apricot, two plum and one peach isolate) clustered with the hop-group, confirming the genetic diversity of HSVd isolates The sequence variability appeared to be more related to the geographical origin of the isolates than to their hosts (Gazel 2008)
The first described Grapevine yellow speckle disease to be caused by viroids in grapevine to date(Kolunow 1988) Some of the infected plants developed yellow speckle symptoms indicating that both viroids can cause Grapevine yellow speckle disease GYSVd-1 is the causal agent of yellow speckle disease They are direct or indirect associated with plant virus infection in the field (Woodham.R.C 1972)
The foliar symptoms of yellow speckle are often absent in confined to a few yellowish spots or flecks scattered in tissue along major or minor veins of leaves (Szychowski 1998) GYSVd-1 is the most closely related viroid to GYSVd-2 with an overall sequence Similarly, that occurs between the two species
Australian grapevine viroid (AGVd) is a novel viroid with less than 50% sequence similarity with any known viroid Nevertheless, its entire sequence can be divided into regions, each with a high sequence similarity with segments from one of citrus exocortis, potato spindle tuber, apple scar skin, and Grapevine yellow speckle viroids AGVd contains the entire central conserved region of the apple scar skin viroid group and is proposed as a member of this group(Kolunow 1988)
CEVd has a wide host-range, that is, in addition to citrus (Fawcett 1948) and grapevine (García-Arenal 1987), it infects some species in the family Compositae (Niblett 1980), Solanaceae (Morris 1977) Leguminosae, Brassicaceae, and Moraceae, such as chrysanthemum (Niblett 1980), tomato (Mishra 1991), broad bean
(Fagoaga 1995), eggplant, turnip carrot (Fagoaga 1996), and fig (Yakoubi 2007), respectively.
The Detection Techniques in Viroid Disease
Viroids are commonly detected by electron microscopy and biological characterization, based on host range, bioassay, poly-acrylamide gel electrophoresis (PAGE), molecular hybridization, RT-PCR-enzyme-linked immune-sorbent assay (RT-PCR-ELISA), real-time PCR and RT-PCR
A real-time polymerase chain reaction (qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR) It monitors the amplification of a targeted DNA molecule during the PCR, i.e in real-
-14- time, and not at its end, as in conventional PCR Real-time PCR can be used quantitatively (Quantitative real-time PCR), and semi-quantitatively, i.e above/below a certain amount of DNA molecules (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 Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines proposes that the abbreviation qPCR be used for quantitative real-time PCR and that RT-qPCR is used for reverse transcription– qPCR The acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and not real-time PCR, but not all authors adhere to this convention The usefulness of ELISA tests for differentiation of strains of several viruses has been reported: Tomato spotted wilt virus (Sherwood 1989, De Ávila 1990), Beet mild yellowing virus (Stevens 1994, Smith 1996), Soybean mosaic virus (Bowers 1979), Potato virus Y (Canto 1995), Apple chlorotic leaf spot virus (Malinowski 1998) and Beet necrotic yellow vein virus (Mahmood 1999) In addition to the coat protein (CP) of Potato virus Y (PVY), a helper component protein (HC-Pro) that helps in the aphid transmission is also synthesized in the infected plants MAbs and PAbs generated against the HC-Pro could be used to differentiate the strains of PVY (Canto 1995, Blanco-Urgoiti 1998) A MAb was employed to differentiate Beet mild yellowing virus (BMYV) with a differing host range from other strains of BMYV commonly observed under field conditions (Smith 1996)
Monoclonal antibodies (MAbs) have the greater discriminating capacity as they can react with different specific epitopes present on the viral coat protein or other virus-associated protein Plant viruses and their strains have been assigned to different serotypes based on their reactivity to different MAbs The serotypes may have similar
-15- biological or structural characteristics in common By employing DAS-ELISA format using a combination of the universal MAb5B, and MAbs specific for serotypes, the West African Rice yellow mottle virus (RYMV) isolates (73) were grouped into three distinct serogroups These serogroups were correlated to two RYMV pathotypes that were differentiated based on their reaction on a set of differential rice cultivars (Konaté G.; Traoré 1997)
A threshold of 90% amino acid sequence identity of tospoviral nucleocapsid protein (NP) that Encapsidates viral RNAs is one of the important criteria for species designation (Goldbach 1996) Based on the serological relationships and phylogenetical analysis of NPs, the genus Tospovirus has been assigned with 16 official and tentative virus species that can be clustered into three major serogroups and four monospecies serotypes (Jan 2003) Tomato spotted wilt virus (TSWV), and Watermelon silver mottle virus (WSMoV) are the representatives of TSWV and WSMoV serogroups respectively Calla Lily chlorotic spot virus (CCSV) isolated from Taiwan was identified as a tospovirus serologically but distantly related to WSMoV based on serological relationship established by employing PAbs and MAbs to WSMoV NP and CCSV NP in indirect ELISA format and low-intensity banding in immunoblotting The MAbs produced against CCSV NP, or WSMoV NP reacted specifically with homologous antigens, but not with heterologous antigens in both ELISA and immune blot analyses (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 1986) However, the need for RNA purification and for radioactive probes which have a short life and safety hazards, limit the wider use of hybridization methods
Availability of non-radioactive probes like digoxigenin (DIG)-labeled DNA and RNA probes in the recent years, has enlarged the scope of their wider applicability A non- isotopic hybridization procedure was developed to differentiate isolates of Citrus Tristeza virus (CTV) using DIG-labeled cDNA probes and different kinds of target
-16- RNA Hybridization of DIG-labeled probes with purified dsRNA or concentrated total RNA extracts spotted on nylon membranes allowed detection of CTV nucleic acid equivalent to as little as 0.1–1.0 mg infected tissues Comparatively, the sensitivity level was similar or slightly better than that was obtained by hybridization with a 32P- labeled probe Hybridization of tissue prints with DIG- labeled probes under stringent conditions (60°C and 50% formamide) could differentiate CTV isolates in citrus plants grown either in the greenhouse or the field (Narváez 2000)
A system of microarrays has been developed for detecting and differentiating plant viruses and their strains The amplicons from plant viral RNA are used for their differentiation by hybridization to synthetic oligonucleotide probes arranged in a two- dimensional array on a glass slide Cucumber mosaic virus (CMV) known to be highly heterologous in its coat protein (CP) was used as the model pathogen
The CP genes of 14 different isolates were amplified using cy3-labeled generic, but species-specific primers These amplicons were hybridized against a set of five different serotype and subgroup-specific 24-mer oligonucleotides (probes) bound to an aldehyde-coated glass slide via an amino linker The probes targeted regions optimal for the differentiation between subgroups 1 and 2 or between subgroups 1a and 1b This microarray procedure allowed a clear differentiation of the 14 different CMV isolates into serogroup 1 and 2 and it was also able to assign nine out of ten different serogroups/isolates correctly into subgroup 1a and 1b Such a clear differentiation was not attainable using RFLP analysis using the restriction enzyme MspI The differentiation hybridization against five specifically selected nucleotides clearly demonstrates the high potential of oligonucleotide-based microarray technology for the virus isolate level detection and differentiation This report presents the development of a diagnostic chip for plant viruses for the first time (Deyong 2005)
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
-17- analysis Quantitative, or real-time, PCR (qPCR) is standard PCR with the advantage of detecting the amount of DNA formed after each cycle with either fluorescent dyes or fluorescently-tagged oligonucleotide probe qPCR results can be obtained faster and with less variability than standard PCR due to sensitive fluorescent chemistry and elimination of post-PCR detection procedures, Real-time PCR for specific detection and quantification of virus and viroid
Reverse transcription followed by real-time PCR assays based on TaqMan® chemistry have been developed for the detection and quantification of Cucumber vein yellowing virus (CVYV) and Cucurbit yellow stunting disorder virus (CYSDV) in individual adults of the whitefly vector Bemisia tabaci The method includes an internal control for the detection of a gene from B.tabaci to compensate for variations in extraction efficiency The assays designed were used to estimate proportions of viruliferous whiteflies collected from commercial greenhouse-grown crops in Spain
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
-20- grapevine cultivation Because these diseases and virus are difficult to eliminate from grape plants by conventional apical meristem culture methods, much effort has been made to generate grape plants completely free of diseases
A long period of low-temperature treatment is effective in generating grape plants Alternatively, grape plants can be generated from shoot tips cultured on a medium containing anti-virus agents Currently, methods of detecting various diseases have improved, and many sensitive methods, such as nested PCR or dot blot hybridization, have been established for viroid detection
STUDY METHODS
Source of Plant Materials
Young leaves of 50 grapevine samples were collected during the hotter months of summer from difference vineyards in Changhua, Taiwan during summer of 2015
Plant leaves showed either yellow speckle or line patterns or asymptomatic plants
Samples from four or five neighboring plants of every second row in the vineyard were analyzed Leaves were collected and stored at -80oC until use (shown as 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
-22- 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
Grapevine leaves (100 mg) were homogenized in a mortar with 450 àl PRX buffer (add ten àl β-mercaptoethanol) After centrifugation at full speed (13000 x g ) for 2 minutes, transfer flow-through sample from the Collection tube to a new tube, add 230 àl 98-100% ethanol to the clear lysate and mix by pipette, apply 680 àl of the ethanol added sample to a Plant Total RNA Mini Column sitting in a Collection Tube, close cap, centrifuge at 10000 x g for 1 minute, and discard the filtrate Wash the column once with 0.5 ml of WF buffer by centrifuging at full speed for 1 minute and discard the filtrate Wash the column twice with 0.7 ml of WS buffer by centrifuging
-23- at full speed for 1 minute and discard the filtrate then centrifuge at full speed for 3 minutes to remove traces of WS buffer Finally, transfer the column to an RNase-free
1.5 ml Elution tube, add 50 àl of RNase-free dd H2O, and centrifuge at full speed for 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
In fact, all of the available methods use approximations (heuristics) Moreover, observed performance differences in comparative analyses (see later) usually emerge as average estimates; hence, approaches that work well for a certain gene or protein family may not work as well for a different one Therefore, as a standard procedure, one should use multiple alignment approaches and parameter sets and carefully inspect the results (reviewed in (Duret 2000, Notredame 2002) Here we will review
-24- different global alignment procedures (i.e., for sequences related to their whole length) to perform MSA Alignment is one of the most important but ironically underappreciated and neglected aspects of sequence analysis (Crandall 2005, J 2005); hence, we will endeavor to explain here the strategies underlying some of the most commonly used algorithms, as well as their strengths and caveats
Progressive alignment algorithms are by far the most widely used because of their speed, simplicity, and efficiency The basic strategy of these methods is first to estimate a tree and then to construct a pairwise alignment of the subtrees found at each internal node More sophisticated algorithms (e.g., iterative algorithms) also use this basic strategy in the initial or final steps of their routines The most frequently used progressive algorithm is the one implemented in Clustal-W (Thompson 1994) and its window interface Clustal-X (Thompson 1997)
Since the sequences of an increasing number of viroids are available in Genbank on the world wide web, it is now possible to design primers specific for the detection of a large number of viroids In this analysis, specific primer pairs for the detection of
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 according to a previous report (Table
3-1) Four pairs of primers corresponding to HSVd-DY ( HSVd-(O) Old & HSVd- New) and GYSVd-1-DY( GYSVd-1-(O) Old & GYSVd-1-(N) New) were newly designed according to the sequences available at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) under the accession numbers: HSVd-New-DY (AB742225, HE575348, AB742224, JX401927, AY594202, FJ716178, JX418270, FJ716190, HQ386721, GU327606), HSVd-Old was designed by Sano et al (2001), GYSVd-1-New-DY (JF746188, JF746176,
JF746193, JF746185, JF746182, JF746184, JF746189, JF746180, JF746183, JF746190), and GYSVd-1-O was designed by (Hajizadeh, Navarro et al 2012)
-25- 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
-26- 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
-27- Table 3-1: Primers in this work
Using Pick up Primer to design Primers following the standards: For sequences over 1 Kb, use a 500 bp subsequence Choose the area based on a conserved region
Set the primer concentrations to 200 nM from 50 nM Keep the % G/C content of the primers as close as possible during selection Match the Tms of primers e.g., 59 oC, 55 oC, 53 oC
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 require the introduction of an RT step before the PCR amplification process (RT-PCR, shown as Figure 3-5) The PCR reaction is based on the annealing and enzymatic extension of two oligo-nucleotide primers (each usually 16 to 30 nucleotides in length), the target region in a duplex DNA using a „thermo-stable‟
DNA polymerase which will retain enzymatic activity at high 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 65 oC, and 72 oC respectively) represent a single PCR cycle The primer annealing and extension temperature may depend on the DNA enzyme used and on the specific sequence/length of the primers used
Multiplex RT-PCR (mRT-PCR)
The mRT-PCR was conducted with 2 àl of cDNA mixed 0.2 àM for each primer and set up one step PCR following cycling parameters: incubated at 50 oC for 30 min, inactivation at 94 oC for 2 min, followed by 32 cycles of denaturation (94 oC for 1 min), primer annealing for 30s at 53 oC for, elongation (72 oC for 2 min) and extension (72 oC for 7 min)
Primer combinations were chosen that would allow us to separate and distinguish the expected amplified fragments on a 1% agarose gel Several combinations of primer concentrations were tested to determine which gave amplification of all target sequences mRT-PCR products were analyzed by electrophoresis on 1% agarose gel, stained with ethidium bromide and visualized under UV light.
DNA Elution and Cloning
-30- DNA products was extracted from gel electrophoresis by Micro-Elute DNA Clean extraction kit (GenMark), according to the manufacturer‟s instructions: cut out the desired DNA band (≤500 mg) with a scalpel after electrophoresis in TBE buffer, transfer the gel slice into a 1.5 ml new micro-centrifuge tube and add 2 volumes (or 3 volumes if agarose gel >2%) of Binding Solution to the gel slice, resuspend the Silica Matrix until a homogeneous suspension is obtained Add 15 àl of silica suspension to the sample, incubate 5-15 min at 60 oC to dissolve agarose Vortex 2-3 times during incubation, centrifuge at top speed for 2 min and pipet out the supernatant carefully without disturbing the silica and leave 20 àl Solution with silica, insert the Spin Filter into a Collection tube, resuspend the DNA/silica matrix mixture by pipette, and apply the matrix solution into the center of the filter and centrifuge for 1 min at top speed, add 700 àl of Wash Solution and centrifuge for 2 min at top speed Discard the flow- through Repeat this step for one more time, discard the filtrate and centrifuge for 3-5 min at top speed to remove residual trace of ethanol Transfer the Spin Filter into a new micro-centrifuge tube, add 10-20 àl of Elution Solution or H 2 O (pH 7.0~8.5) into the center of the filter Centrifugation at top speed for one minute and store the eluted DNA at -20oC
The PCR product was cloned into the yT&A cloning vector (Invitrogen, Carlsbad, CA), according to manufacturer‟s instruction The DNA fragments were ligated with vector TA vector and transformed into E coli XL-1 as competent cells
Mixed DNA and TA vector in the reactions by pipetting then incubate the reactions for 5 to 15 min at 22oC and incubate the reactions overnight at 4oC The recombinants were introduced into Escherichia coli XL-1 containing ampicillin and tetracycline; concentration incubates the plates overnight at 37oC (shown as Figure 3-6)
A single colony containing inserted TA cloning was picked from a toothpick, introduced to 5 ml L-Broth (Tryptone, yeast) medium containing ampicillin, and incubated with shaking for overnight at 37 oC The plasmid DNA was purified and digested with enzyme HindIII to confirm the correct size of the DNA fragment
Phylogenetic Tree Construction
Phylogenetic approaches are essential for studying the diversity, origin, and distribution of plant viruses Phylogenies, or evolutionary histories, provide insights into key innovations that afford pathogens the ability to spread and attack particular hosts They are essential for determining what a particular pathogen might be, where it came from, and how it evolves to infect a particular host or escape attempts at eradication Phylogenetic approaches are also being developed to examine population dynamics, to partition historical effects from current effects on population structure, and to estimate gene flow, the directionality of migration, and phylogeographic relationships of unique genotypes These phylogenetic approaches are often complicated and are continually being revised and further developed In this chapter, we review some of the basic approaches, including some of the population genetic approaches to study plant viral evolution As we do so, we point the reader to some
-32- software that we have found useful in our analyses of viral data There is, of course, a plethora of software available For a comprehensive summary of most phylogenetic software utilities, we refer readers to the Web site of Joseph Felsenstein, who has diligently compiled such a summary with links to a wide variety of phylogenetic software packages: http://evolution.genetics.washington.edu/phylip/ software.html
The phylogenetic tree was constructed the by using the complete genomic sequence of HSVd and GYSVd-1, respectively
These sequences were aligned and compared using the multiple sequence alignment in Philip format The pairwise evolutionary distances for the nucleotide sequences were estimated using the DNADIST program of PHYLIP software package version 3.69 (J 2005) and the F84 method as the substitution model The Relationship dendrograms were drawn using the program TreeView (Page 1996) with the neighbor-joining (NJ) analyses, keeping the bootstrap value of 100 replicates
RESULTS AND DISCUSSION
Results
4.1.1 Yield and quality of RNA extract
A 100 mg of leaf tissue was obtained from symptomatic leaves (as shown in Figure 3-1) collected from grapevine farms during the summer season Total RNA was extracted using RNA extraction kit (Invitrogen, Carlsbad, CA) and the quantity and quality of the RNA were determined by UV spectrophotometer The ratio of 260/280 is about 1.8-2.0, indicated the purity of the RNA
4.1.2 Primer design for viroid detection
To design new primers for the detection of HSVd and GYSVd-1, seventeen HSVd complete sequences (Fig 3-3) and 19 GYSVd-1 sequences (Fig 3-4) were obtained from GenBank and conducted with multiple sequence alignment The conserved regions were found, and suitable primers were synthesized for RT-PCR (Table 3-1)
4.1.3 Detection of Grapevine viroids by single RT-PCR reaction
Before applying for multiplex RT-PCR reaction, single RT-PCR was carried out to evaluate the specificity of the primers in the detection of 5 grapevine viroids In the detection of 50 field samples, HSVd was found in all the tested samples (Fig 4-1, lane 2) and GYSVd-1 was found in 8 of the 50 samples (16% infectivity) (Fig 4-1, lane 1), while AGVd, GYSVd-2, and CEVd were not detected (Table 4-1) An amplicon derived from RT-PCR with HSVd specific primers was cloned and named-HSVd-DY which contained the complete genomic sequence of 301 nt DNA
-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
Figure 4-1: Agarose gel electrophoresis analyse of single and multiplex RT-PCR reaction Lane 1, with specific primers for GYSVd-1; Lane 2, with specific primers for HSVd; Lane 3, with mixed primers for the detection of both GYSVd-1 and HSVd
Lane M, molecular weight markers (GENMARK, GM100)
4.1.4 Development of multiplex RT-PCR reaction
For multiplex RT-PCR assays, the primer pairs specific for more than one viroid were added to the same RT-PCR mixture In the preliminary test, the final concentration of all primers was 0.5 àM However, it results that the amplicon of GYSVd-1 was weak An adjustment with a combination of 0.4 àM of HSVd primer pairs plus 0.5 àM of GYSVd-1 primer pairs was effective in the detection of HSVd and GYSVd-1 from grapevine extracts which shown mixed infection of these two viroids (Figure 4-1)
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
Set up the following items as described below: ligation buffer A(1àl), ligation buffer B(1àl), yT&Ađ cloning vector( 2àl), PCR product(2àl),
T4 DNA ligase(1àl), Control DNA and Add deionized water to 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,
YT005) (23àl), M13-F (10 μM) (1ul), M13-R (10 μM) (1ul) o Set up program of the thermal cycle o Check on 1% agarose gel o For example: Using the control DNA provide in the yT&A® cloning vector kit as insert DNA, the colony PCR result is showed as 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 possesses proofreading activity; it does not have the terminal transferase-like activity demonstrated by Taq DNA polymerase
Ligation reactions using non-tailed amplified DNA resulted in no 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
Purify the A-tailed DNA and use in the ligation reaction o If the maximum of transformants is required, incubate the reactions overnight at 4 oC o The optimized efficiency is using a 1:3 molar ratio of vector DNA to the insert DNA o Use higher efficiency competent cells e.g ECOSTM (>108cfu/μg DNA) series (Figure 4-4)
-39- Figure 4-4: Agarose gel electrophoresis analyzes T&A cloning vector kit insert DNA and HindIII enzyme 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,
The phylogenetic tree was constructed the by using the complete genomic sequence of HSVd-DY and GYSVd-1-DY, respectively (Figure 4-5 and 4-6) The phylogenetic tree derived from the complete sequence of HSVd-DY and other 17 HSVd from GenBank showed that they are highly related in evolutionary Similarly, tree obtained from GYSVd-1-DY and other 19 GYSVd-1-DY from GenBank reveal their close relations
-40- Figure 4-5: Phylogenetic analysis of the complete sequence of HSVd-DY with other
17 HSVd retrieved from GenBank In the phylogenetic tree constructed using the PHYLIP software package (J 2005), the values adjacent to the nodes indicate the bootstrap confidence values for 1000 replicates using neighbor-joining (NJ) analyses
Values below 75% are not given The units of the scale bar represent the nucleotide substitutions per site
-41- Figure 4-6: Phylogenetic analysis of the complete sequence of GYSVd-1-DY with other 17 HSVd retrieved from GenBank In the phylogenetic tree constructed using the PHYLIP software package (J 2005), the values adjacent to the nodes indicate the bootstrap confidence values for 1000 replicates using neighbor-joining (NJ) analyses
Values below 75% are not given The units of the scale bar represent the nucleotide substitutions per site
The sequence obtained from the detected HSVd-DY was compared to other 17 HSVd from GenBank The results shown HSVd-DY is highly sequence identity with the range of 98.7% to 85.79% to other HSVd which were collected from different hosts or different geographical regions Similarly, GYSVd-1-DY ranged 39.2% to 99.7% to of the sequence to other GYSVd-1, with the exception that GYSVd-1 from
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)
-43- 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
-44- 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 most important viroids of grapevine, especially those associated with yellow speckle disease, are reliably detectable in young leaves during a physiological stage that is characterized in most plants Grapevine is very rich in phenolic compounds, which accumulate more with age and can interfere with the process of extracting high- quality RNA(Newbury 1977) The most important viruses of grapevine, especially those associated with the leafroll disease complex, are reliably detectable in mature leaves during a physiological stage that is characterized in most plants by increased nuclease activities and decreased nucleic acid content(Malfitano 2003)
In this study, five pairs of primers specific to HSVd, GYVd-1, GYSVd-2, CEVd, and AGVd are designed for the detection of viroid diseases in grapevines Through single-RT-PCR, HSVd and GYSVd-1 are detected and the sequence results showed that they shared high nucleotide identity to the related viroids published in GenBank
When the primers derived from HSVd and GYSVd-1 sequences were mixed together and multiplex RT-PCR was conducted for the detection, the amplified DNA fragments corresponding to HSVd and GYSVd-1, respectively, were found after gel electrophoresis, indicated multiplex RT-PCR analyses possess a sensitivity, rapid strategy for simultaneous detection of difference viroids in vineyards
Fifty leaf samples were collected from vineyards in Changhua county of Taiwan
Those leaves were selected because of the yellowing, mottling, or mosaic symptoms appeared on the leaves Since grapevine is very rich in phenolic compounds, which accumulate more with age(Kobayashi 2005) The phenolic compounds may interfere with the process of extracting high quality RNA In our study, we picked the mature but not aged leaves from grapevines (usually located at the third to fifth position from the top), so as not contaminated with more phenolic compounds Total RNA was extracted according to the manufacturer‟s instructions of RNA extraction kit without further modification The quality of the RNA was determined by spectrophotometry
-46- and the ratio of absorbance at 260 nm/280 nm were between 1.8 to 2.0, indicating their purity of RNA During RNA extraction process, it is important to completely remove ethanol before wash out the RNA from column In this way, there will not have organic compound contamination in RNA solution which might interfere the following step of RT-PCR
The extraction procedure has been modified to increase the reliability of plant extracts for use in RT-PCR, as shown in the control reactions that were developed
The extraction of RNA contained ethanol, removed ethanol by heat-water
Such a control will be difficult to develop since antibody preparations differ from each other on antigen binding efficiency and titre We recommend that a control is included in every assay because an amplifiable extract might become un-amplifiable upon improper storage or repeated freeze-thawing For mRT-PCR, we also change concentration two pair primers with ratio 1àl for GYSVd-1-N-DY and 2 àl for HSVd- N-DY when we run gel electrophoresis
To calculate accurately the melting temperature, the users have to specify which buffer is used for the amplification They can choose between using pre-set polymerase buffers or to specify the ionic content of the buffer and the algorithms they want using the custom-buffer mode The library of sequences to amplify should be provided in the form of a FASTA file The software returns a result page in which all primers are shown and can be downloaded as a FASTA or a CSV file The validation plot showing the performance of the melting temperature algorithm used on experimental data and the logs of the program are also provided
Obtaining RNA of high quality and quantity is a prerequisite for constructing good-quality cDNA libraries with adequate representation of all expressed genes
However, extracting RNA from plant tissues can be difficult and often requires the modification of existing protocols or the development of new procedures This is particularly true for woody species such as grape, apple, and citrus because they contain high levels of extractable phenols and polysaccharides(Horst 1980) and for
-47- mature pre-climacteric and post-climacteric fruit tissues in which biosynthesis of potential interfering substances is known to increase during maturation and ripening
However, attempts to use these reagents to extract total RNA from apple fruit and flower tissues have failed to provide high enough quality and quantity of total RNA necessary for cDNA library construction The presence of polysaccharides in extracted plant DNA is a common concern for plant molecular biologists However, the data presented show that in many cases this can be averted with the use of increased salt concentrations in extraction buffer The fact that OD280/OD260 ratios were unaffected by the salt concentration in the extraction buffer when using cucumber suggests that polysaccharides were at a low enough concentration in that plant tissue that they were effectively removed by both buffers tested This was not so for the potato samples, where the use of a higher salt concentration in the extraction buffer was necessary to avoid polysaccharide carry over into nucleic acid samples
The design of the primers was based on conserved regions in the sequence from several plants, in the hope that the same primers could be used in control reactions for all those plants and even for plants for which currently no sequence data are available
However, amplification with HSVd and GYSVd-1 primers occurred in extracts from both uninfected and infected tissues from all these plants It is important to note that the tested tissues include cane and bud samples from the dormant grapevine because this means that the HSVd and GYSVd-1 primers detection can also serve as an internal control for virus testing of dormant woody plant tissues that are imported and exported
When fifty viroid-infected field samples were tested by one tube-one step RT- PCR, products of the expected sizes were obtained from all viroid isolates assayed
Two viroid bands of different sizes were observed only in infected samples but not in negative controls These findings showed the successful use of the protocol with field samples
-48- In our collected leaf samples, all of them were found to have infected by HSVd
This is reasonable since HSVd is widespread in the world and it may be prevalent in the vineyards GYSVd-1 showed 16% of infection in collected samples The frequency of its infection is much lower as compare to that of HSVd No other grapevine viroids, including GYSVd-2, AGVd, and CEVd were found in the collected samples It is interesting to find that a DNA fragment obtained from RT-PCR analysis when using GYSVd-2 primers and the resulting sequence showed that it was 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 designed the primers for amplified different size of DNA fragment from different viroids The design of the primers were based on conserved regions in the sequence from several plant, in the hope that the same primers could be used in control reactions for all those plants and even for plants for which currently no sequence data are available In order to calculate accurately the melting temperature, the users have to specify which buffer is used for the amplification They can choose between using pre-set polymerase buffers or to specify the ionic content of the buffer and the algorithms they want using the custom-buffer mode The library of sequences to amplify should be provided in the form of a FASTA file The software returns a result page in which all primers are shown and can be downloaded as a FASTA or a
-50- CSV file The validation plot showing the performance of the melting temperature algorithm used on experimental data and the logs of the program are also provided
Most likely some viroids in these extracts is so high that, with the primer concentrations and RT-PCR conditions used, the amplification of the viroid-specific fragment out-competes the amplification of the control fragment This does not pose a real problem in practice, because the purpose of the control is to determine if the extract can support the RT-PCR reaction
The finding that prolonged storage of plant tissue and plant extracts under the proper conditions (-80°C) is possible means that plants can be tested for additional viruses when their sequences become available and specific primers can be designed
This study showed the effectiveness of a multiplex RT-PCR protocol for the contemporary identification of two grapevine infecting viroids The method was validated by testing naturally infected vines from Da-Yeh University of Taiwan and proved to be sensitive and reliable both in the absence and presence of the primer pair designed to amplify a host-derived internal control
The formation of secondary structures by the target sequence, a major determinant affecting its interaction with other molecules, affects availability for hybridization(Murashige 1972) Random amplification approaches, such as the one used in this work, inherently generate non-targeted amplicons which can affect the sensitivity and specificity of hybridization reaction
The genomic sequences of tospoviruses used for comparison were obtained from the National Center for Biotechnology Information (NCBI) databases (http://www ncbi.nlm.nih.gov/) Multiple sequence alignments, comparison of the newly determined sequences to the reference sequences, and translation of the nt sequences to aa residues were performed using the Clustal-W program, the Bl2seq program, and the Six frame program, respectively, of 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
-51- repeated 100 times to generate multiple data sets, and versions of the input data sets were resampled with the Seqboot program of Philip 3.69 A distance matrix for the amino acid sequences was produced using the dnadist.exe program of Phylip 3.69 using the PAM matrixes of the Dayhoff model Phylogenetic branches were set by the Neighbor program of Phylip 3.69 using the neighbor-joining method Finally, phylogenetic trees were produced using the Consense program of Philip 3.69
We compared then HSVd and GYSVd-1 from grapevine in Dacun, Yuanlin, Taiwan samples with previously published sequences in GenBank Results show the mutations on the closest sequence; the similarity was between 99% and 95 %
The entire sequences of HSVd-DY and GYSVd-1-DY have 301 nt and 367 nt, respectively However, it shares the highest nt (95%-98%) identity with those AB742224, AB742225, GQ995466, GQ995464, Y14050 for HSVd and nt (95%-99%) identity with those JF746190, JF746188, GU170805, AY639607, AB028466, Z17225 for GYSVd-1 Phylogenetic analysis of the sequences of all of the viral proteins indicated that HSVd_DY and GYSVd-1-DY are closely related to GQ995464 and JF746188, AB028466, Z17225, respectively
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The rod like secondary structure of Potato Spindle tuber viroid showing the five domains chacrateristic of members of the family Pospiviroidae: the terminal left (TL), pathogenicity (P), central (C), variable (V), and terminal right (TR)