Virus Genes (2009) 38:515–520
DOI 10.1007/s11262-009-0338-1
Molecular characterization of grapevine yellow speckle viroid-2
(GYSVd-2)
Dongmei Jiang Æ Zhixiang Zhang Æ Zujian Wu Æ
Rui Guo Æ Hongqing Wang Æ Peige Fan Æ Shifang Li
Received: 31 December 2008 / Accepted: 12 February 2009 / Published online: 3 March 2009
Ó The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract Grapevine yellow speckle viroid-2 (GYSVd-2)
is a viroid found only in grapevines in China and Australia.
Here, we report the molecular characterization of GYSVd2 isolated from three grapevine varieties in China. A total
of 90 cDNA clones were sequenced including 30 cDNA
clones obtained from each of the Black Olympia, Zaoyu,
and Thomson Seedless isolates. Sequencing analysis
identified 20, 18, and 12 different sequence variants from
the 3 isolates, respectively. Furthermore, each of the isolates included one predominant sequence variant.
Compared to the Australian variant of GYSVd-2 (Accession number: NC_003612), the Black Olympia variant was
identical and the Zaoyu variant contained one substitution.
In contrast, the Thomson Seedless isolate significantly
varied from the Australian variant with three substitutions,
two insertions, and four deletions. In silico structure
D. Jiang Z. Zhang R. Guo S. Li (&)
State Key Laboratory of Biology of Plant Diseases and Insect
Pests, Institute of Plant Protection, Chinese Academy of
Agricultural Sciences, Yuanmingyuan West Road No. 2, Haidian
District, Beijing 100193, People’s Republic of China
e-mail: sfli@ippcaas.cn; lishifang2003@yahoo.com.cn
D. Jiang Z. Wu
Institute of Plant Virology, Fujian Agriculture and Forestry
University, Jinshan, Fuzhou, Fujian 350002, People’s Republic
of China
H. Wang (&)
Department of Fruit Science, College of Agronomy and
Biotechnology, China Agricultural University, Beijing 100193,
People’s Republic of China
e-mail: wanghq@cau.edu.cn
P. Fan
Institute of Botany, Chinese Academy of Sciences, Beijing
100093, People’s Republic of China
analysis predicted that the variations were clustered in the
terminal left, the pathogenicity, and the variable region of
the predicted secondary structure of GYSVd-2.
Keywords Grapevine yellow speckle viroid-2 Genetic
variation Sequencing analysis Secondary structure
Introduction
Viroids are small, circular, single-stranded RNA molecules
that range in size from 246 to 475 nucleotides (nts). Viroids
replicate in host plants and act as phytopathogenic agents;
however, unlike viruses, viroids do not code for proteins.
Viroids are the smallest known plant pathogens and are
responsible for several economically significant crop diseases [1]. Viroids are classified into two families:
Pospiviroidae, which is composed of species with a central
conserved region (CCR) that do not contain a hammerhead
ribozyme, and Avsunviroidae, a family composed of species lacking a CCR but containing a hammerhead ribozyme
that is able to self-cleave both strands of the viroid [2].
Up to now, five viroids have been identified with the
ability to infect grapevines: Hop stunt viroid (HSVd) [3],
Citrus exocortis viroid (CEVd) [4], Australian grapevine
viroid (AGVd) [5], and two Grapevine yellow speckle
viroids (GYSVd-1 and GYSVd-2) [6, 7]. Grapevine viroids
have been divided into three genera based on sequence
homology of their CCRs [8]. GYSVd-1, GYSVd-2, and
AGVd are classified in the Apscaviroid group, whereas
HSVd-g [9] and CEVd-g are classified in the Hostuviroid
and Pospiviroid groups, respectively [10]. Mixed infections
involving these viroids in cultivated grapevines is common,
and in general, grapevine viroids produce very few, if
any, disease symptoms, thereby allowing these viroids to
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Virus Genes (2009) 38:515–520
replicate in a host unnoticed. However, of the five types of
grapevine viroids identified, GYSVd-1 and GYSVd-2 are
associated with disease symptoms such as yellow speckle,
which was originally identified under hot greenhouse
conditions in Australia [6, 7, 11].
GYSVd-2 is composed of 363 nts and was first detected
in Australia when the grapevine cultivar ‘Kyoto’ was
grafted onto the ‘Dogridge’ rootstock and subsequently
became infected with yellow speckle disease [6]. GYSVd-2
is the most closely related viroid to GYSVd-1 with an
overall sequence similarity of 73%, which accounts for the
cross-hybridization that occurs between the two species
[7]. Although GYSVd-2 RNA can be separated from other
grapevine viroid RNA of similar size, including GYSVd-1,
separation occurs only after prolonged electrophoresis
under denaturing conditions [7]. Until now, GYSVd-2 has
only been identified in grapevines from Australia and
China [6, 12].
Due to the low infection rate of cultivated grapevines by
GYSVd-2, little is known about this viroid. The present
work characterizes GYSVd-2 isolates collected from three
different grapevine varieties in China to provide new
information regarding the population diversity and the
genetic variation of the viroid. Differences in viroid
sequences identified among isolates from grapevines in
China as well as in comparison with an Australian GYSVd2 isolate indicate a novel isolate has been identified in
Xinjiang, China, distinct from the other isolates collected
from Beijing, China, and Australia.
Using RT-PCR, cDNAs were generated from viroid RNA.
Briefly, template (1 ll) was mixed with 0.5 ll (20 pmol) of
primer GYSVd-2-P2 (50 -ACTAGTCCGAGGACCTTTTC
TAGCGCTC-30 , complementary to nucleotides 166–187)
and distilled water, then heated at 98°C for 5 min, and
quenched in ice water for more than 2 min. Then 1 ll of
2.5 mM dNTPs, 1 ll (200 U) MMuLV reverse transcriptase
(Promega), 2 ll M-MLV 59 reaction buffer, 0.25 ll (40 U)
Recombinant RNasin ribonuclease inhibitor, and distilled
water were added to the RT mixture for a final volume of
10 ll. The resulting mixture was incubated at 42°C for
60 min, then at 98°C for 5 min. After the RT reaction, 5 ll of
the reverse transcription solution was mixed with 25 ll 29
PCR Ex-Taq Mix, 18 ll distilled water, and 1 ll (20 pmol)
each of primers GYSVd-2-P1 (50 -ACTAGTACTTTCTTCT
ATCTCCGAAGC-30 , homologous to nucleotides 188-208)
and GYSVd-2-P2 (50 -ACTAGTCCGAGGACCTTTTCT
AGCGCTC-30 ) to yield a final reaction volume of 50 ll. The
cycling parameters for PCR amplification consisted of one
cycle of heat denaturation at 94°C for 5 min, 30 amplification cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s,
and a final elongation step at 72°C for 5 min.
Materials and methods
Cloning and sequencing
Viroid sources
After RT-PCR, electrophoresis confirmed the presence of
the expected PCR products before they were purified using
a PCR purification kit (Tiangen). The resulting fragments
were cloned into the pMD18-T vector (Takara) and transformed into E. coli DH5a. Recombinant DNA clones
containing a 363-bp insert were identified by restriction
analysis. Selected clones were sequenced using an automated DNA sequencer (ABI PRISMTM 3730XL DNA
Analyzer) and analyzed by DNAMAN Version 5.2.2.
From 2006 to 2008, young leaves of 89 grapevine samples
from different grape varieties were collected from Xinjiang
autonomous region, Shenyang, Fujian, and Beijing, China.
They were detected by dot-blot or Northern hybridization
using DIG-labeled GYSVd-1 riboprobe followed by
RT-PCR using GYSVd-2 specific primers.
the soluble fraction and collect the low molecular weight
RNAs. The resulting preparation was dissolved in 30 ll of
distilled water.
Reverse transcription-polymerase chain reaction (RTPCR)
Isolation and extraction of GYSVd-2
Low molecular weight RNAs were extracted according to
Li et al. [13]. Briefly, 5 g of tissue were treated with liquid
nitrogen, extracted with 10 ml of 1 M K2HPO4 containing
0.1% b-mercaptoethanol, and homogenized with 10 ml
phenol:chloroform (1:1, v/v). To eliminate polysaccharides
present, extraction with 2-methoxyethanol and Cetyltrimethyl Ammonium Bromide (CTAB) precipitation were
performed, followed by treatment with 2 M LiCl to isolate
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Sequencing analysis and determination of secondary
structures
Sequences were aligned with the Australian GYSVd-2
sequence deposited in the GenBank database (Accession
number: NC_003612) using Clustal W (Ver.1.83). Possible
secondary structures were calculated using the CLC RNA
Workbench package (Version 3.0.1, http://www.clcrna
workbench.com/).
Virus Genes (2009) 38:515–520
Results
Genetic diversity of GYSVd-2 within each isolate
of Black Olympia, Zaoyu, and thomson seedless
GYSVd-2 was positive in only three samples of the 89
grapevine samples examined, i.e., one each from the cultivars Black Olympia, Zaoyu, and Thomson Seedless. The
Black Olympia and Zaoyu samples were collected from the
same grapevine nursery in Beijing, while the Thomson
Seedless sample was collected from a grapevine tree from
the Xinjiang autonomous region estimated to be older than
150 years. Thirty cDNA clones were chosen randomly from
each of the three isolates, and a total of 90 independent
cDNA clones were sequenced. Of them, a total of 50
sequence variants were identified. The variants detected in
the Black Olympia isolate included one predominant
sequence variant, Bv1 (363 nts) and 19 singletons (Fig. 1,
left). In the Zaoyu isolate, one predominant sequence variant, Zv1 (363 nts) was identified along with 17 singletons
(Fig. 1, middle). In the Thomson Seedless isolates, one
predominant sequence variant, Tv1 (361 nts) was detected
along with 11 singletons (Fig. 1, right). Each of the three
isolates consisted of one predominant sequence variant
which occupied 37%, 43%, or 63% of the sequences determined in each isolate (Fig. 1). The overall sequence
homology within each isolate was high, for example, the
homology between the 20 sequence variants of Black
Olympia isolate was 98.90–99.72%. Based on the identification of one predominant sequence variant in each of the
isolates (i.e., Bv1, Zv1, and Tv1), we hypothesize that all the
three GYSVd-2 isolates form a quasispecies with one predominant sequence [11, 12, 14–17].
Genetic diversity among GYSVd-2 isolates from
different grapevine varieties
When the sequences of Bv1, Zv1, and Tv1 were aligned
with the GYSVd-2 sequence derived from an Australian
Fig. 1 GYSVd-2 variants from the three different grape varieties
(Black Olympia, Zaoyu, and Thomson Seedless) in China. Thirty
each of independent cDNA clones, respectively, from each of the
three isolates, in total of 90, were sequenced. One predominant
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grapevine (Accession number: NC_003612), zero, one, and
nine positions showed differences, resulting in an overall
sequence homology of 100%, 99.72%, and 98.34%,
respectively, in each case. The high degree of homology
for the Black Olympia and Zaoyu predominant variants,
except at position 300 (A300 ? G) for Zv1, was consistent
with the origination of the two isolates from the same
nursery yard in Beijing. We hypothesize that Bv1 and Zv1
have the same origin and were geographically separated
rather recently. In contrast, the predominant sequence of
the Thomson Seedless isolate, Tv1, differed from the
Australian isolate with three substitutions at positions 300
(A300 ? G), 361 (C361 ? T), and 362 (T362 ? C), along
with two insertions at positions 134 (-134 ? A) and 147
(-147 ? T), and four deletions at positions 15 (T15 ? -),
349 (C349 ? -), 350 (G350 ? -), and 357 (G357 ? -)
(Fig. 2). The substitution at position 300 (A300 ? G) is the
same as the one identified in the Zaoyu isolate. Given the
greater number of differences identified in Tv1 versus
the Australian sequence variant, Bv1 and Zv1, it appears
that Tv1 is a more isolated viroid variant, consistent with
its collection from a 150-year-old grapevine tree growing
in Xinjiang.
Effects of genetic diversity on predicted secondary
structures
Most of the variations found in the three GYSVd-2 isolates
from China were clustered in the terminal left (TL) region,
the pathogenicity-associated region (P), and the variable
(V) region of the viroid-predicted secondary structure.
Variations were not found in the CCR (from 84–120 nts) or
in the terminal conserved region (TCR) of the TL region
(Fig. 3). In silico analysis of the predicted secondary
structures of the variants suggested that some of the
sequence variants could influence the viroid structure.
Specifically, the two mutations at positions 134 and 147 of
the Thomson Seedless (Tv1) variant were predicted to
sequence was identified for each: Bv1 for Black Olympia, Zv1 for
Zaoyu, and Tv1 for Thomson Seedless. The percentage of variants
associated with singletons (S*) are also indicated in dark gray
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Virus Genes (2009) 38:515–520
Fig. 2 Sequence alignment of
GYSVd-2 variants isolated from
grapevines in China and
Australia. Sequences of viroids
isolated from different
grapevines in China versus a
previously published GYSVd-2
sequence from a grapevine in
Australia (Accession number:
NC_003612). The position
mutations of specific sequence
variants are boxed and the CCR
domain is labeled above the
sequence. (AUS: Australia,
Bv1: China (Black Olympia),
Zv1: China (Zaoyu), Tv1: China
(Thomson Seedless))
affect the predicted secondary structure (Fig. 3). In silico
structure analysis also revealed that the P domain of
GYSVd-2 is purine rich in the upper strand and uridine rich
in the bottom strand.
Discussion
Here, we present, for the first time, the population diversity
and genetic variations of the viroid, GYSVd-2, identified
from three isolates (Black Olympia, Zaoyu, and Thomson
Seedless) collected from grapevines in China. In total of 90
cDNA clones, 30 each of independent cDNA clones from
the 3 isolates were sequenced. Twenty sequence variants
from Black Olympia, 18 sequence variants from Zaoyu,
and 12 sequence variants from Thomson Seedless, in total
of 50, were identified. Although it is possible that the
singletons identified could represent PCR artifacts, we
hypothesize that they are naturally occurring mutations for
the following reasons. First, the frequency of error resulting
from Ex-Taq DNA polymerase ranges from 10-4 to 10-5
[18]. Second, most of the mutations were identified multiple times in different cDNA clones. For example, all the
sequence variants showed an A to G substitution at
nucleotide 300 in both the Thomson Seedless and the
Australian isolates [7]. Additionally, most of the single
mutation sites were located in the TL, P, and V domains of
GYSVd-2, and not in the strictly conserved regions of the
CCR and the TCR of Apscaviroid, supporting the
hypothesis that these mutations are naturally occurring.
Sequence alignments between the variants isolated from
grapevines in Australia and China demonstrate that
GYSVd-2 isolates from different countries do not
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necessarily display regional disparity and variety-specific
sequence variants. For example, Bv1, the predominant
sequence of Black Olympia, was identical to the Australian
sequence and represented the first reported variant of the
Australian isolate. Since Black Olympia and Zaoyu isolates
were collected from the same grapevine nursery in Beijing,
and the homology between the Black Olympia, Zaoyu, and
the Australian isolate were similar, it appears that they
derived from a common ancestor. In contrast, sequence
variations were found at nine different positions in the
Thomson Seedless isolate versus the other grapevine
varieties. Interestingly, six out of the nine single site
mutations were located in the TL region (Fig. 3). This is in
contrast with previous studies that have found the TCR of
Apscaviroid species (CNNGNGGUUCCUGUGG) to be
highly conserved in the TL region [19–21]. This difference
may be explained by the fact that the Thomson Seedless
isolate was collected from a grapevine tree estimated to be
older than 150 years, which is predicted to have survived
by its own root given the absence of any records of grafting
or top-working manipulations. Therefore, the increased
number of mutations in a region otherwise found to be
highly conserved is consistent with the extended period of
isolation experienced by this grapevine tree that grows in a
latitude and climate distinct from the other grapevine
varieties analyzed.
The most stable secondary structures for the three predominant variants (Bv1, Zv1, and Tv1) were derived based
on energy calculations. We also reconfirmed that the P
domain of GYSVd-2 is purine rich in the upper strand and
uridine rich in the bottom strand, resulting in a relatively
high sequence homology between the P domain of
GYSVd-2 and the P domains of other viroids [8]. Like
Virus Genes (2009) 38:515–520
Fig. 3 Predicted secondary structures of GYSVd-2 isolates. Compared to the first reported sequence of GYSVd-2 from a grapevine in
Australia (AUS) (Accession number: NC_003612), of which the
Black Olympia (Bv1) isolate was identical, base changes in the Zaoyu
(Zv1) and Thomson Seedless (Tv1) isolates are boxed. Insertions
other RNAs, the predicted secondary structure of GYSVd-2
contains many loops and bulges flanked by double-stranded
helices, the biological functions of which are mostly
unknown. Recent studies have demonstrated that the loops/
bulges in the rod-shaped secondary structure of the viroid
serve as major functional motifs that likely interact with
cellular factors to accomplish various aspects of replication
and systemic trafficking during infection [22]. Compared to
the Australian variant, most of the variations found in the
Chinese isolates were located in the TL and P domains of
the secondary structure, which may have some influence on
the secondary structure of the GYSVd-2 viroid, particularly
with respect to replication and pathogenicity of the viroid.
However, these possibilities would require further investigation. Among the nucleotide positions that did exhibit
substitutions, the A300 ? G substitution in Zv1 was not
predicted to influence secondary structure, although when
other base changes were present in Tv1, changes in the
secondary structure were predicted (Fig. 4). We hypothesize that this might be due to reciprocity among the bases.
Characterization of the genetic structure and variation of
a viroid population is important to understand the dissemination and evolution of a viroid family. With the
advent of molecular virology, many viroid populations
519
present in the Tv1 isolate are indicated with arrows and the inserted
nucleotides are boxed. Additionally, mutations in Tv1 that affected
secondary structure are indicated with boxed plus signs. (T1, T2: the
terminal regions; P: the pathogenicity region; C: the central conserved
region; V: the variable region)
Fig. 4 Analysis of the terminal conserved region (TCR) of GYSVd-2
isolates in the Apscaviroid group. The sequence proximal to the 30
end of the motif is highly conserved while considerable variability is
found in the nucleotides near the 50 terminus as evidenced in the three
‘‘N’’ nts in the five terminal positions [22]. Sequences from viroids
isolated from different grapevines in China are compared with a
previously published GYSVd-2 sequence from a grapevine in
Australia (AUS*) (Accession number: NC_003612) and a previously
published ASSVd sequence (ASSVd) (Accession number: NC
001340). (Abbreviation of the sample names such as ‘‘Bv1’’ was
described in the text. Samples with asterisk (w) indicate those
obtained from GenBank.)
have been characterized including AFCVd, HSVd, CEVd,
GYSVd-1, and AGVd [15, 16, 19, 23, 24, 25–27, 28, 29].
Given the limited distribution of GYSVd-2 in the world,
very little is known about GYSVd-2 at the molecular level
prior to this study. For example, GYSVd-2 was previously
detected in 11 out of 27 grapevines characterized from
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Australia [30]. However, GYSVd-2 varieties present in
China were not previously reported [12]. Recently, an
intensive survey in Japan of 111 different grapevine cultivars in three different nursery yards and eight cultivars in
several commercial vine yards have been investigated,
however, GYSVd-2 has not been detected (T. Sano, personal communication).
An unusually high GYSVd-2 infection rate in Australian
grapevines, combined with the unique sequence variations
identified in this study of GYSVd-2 Xinjiang isolates,
provide additional information regarding the biology of
GYSVd-2. Insights into the GYSVd-2 viroid can also
provide a possible model for the biology of other grapevine
viroids which have expanded their distribution among the
world viticulture. As a result, more extensive surveys on
grapevine viroids present in Xinjiang, China, are now
underway.
GenBank accession numbers
GenBank accession numbers for the sequence variants
identified for GYSVd-2 isolated from China are FJ490172–
FJ490175 and FJ597915–FJ597947.
Acknowledgments This work was supported by grants from the
National Basic Research and Development Program of China (973
Program) (No. 2009CB119200 and No. 2006 CB100203), the
National Natural Science Foundation of China (No.30771403), the
Beijing Natural Science Foundation of China (No. 6072022),
the National Key Technologies Research and Development Program
(No. 2006 BAD08A14), the National High Technology Research and
Development Program (863 Program) (No. 2006AA10Z432) and
the Opening Project of State Key Laboratory for Biology of Plant
Diseases and Insect Pests, Institute of Plant Protection, Chinese
Academy of Agricultural Sciences. This work was also supported by
NSFC-JSPS Joint Research Project between China and Japan
(30811140157). We specially thank Prof. Teruo Sano at Hirosaki
University of Japan for his valuable comments and the critical reading
of this manuscript.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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