Acta Botanica Sinica
植 物 学 报
2004, 46 (10): 1234-1241
http://www.chineseplantscience.com
Chromosomal Distribution of the 18S-5.8S-26S rDNA Loci and Heterogeneity
of Nuclear ITS Regions in Thinopyrum intermedium (Poaceae: Triticeae)
LI Da-Yong, RU Yan-Yan, ZHANG Xue-Yong*
(Key Laboratory of Crop Germplasm and Biotechnology, Institute of Crop Sciences, Chinese Academy of
Agricultural Sciences, Beijing 100081, China)
Abstract: Fluorescent in situ hybridization (FISH) was used to investigate the chromosomal location of
18S-5.8S-26S rDNA loci in Thinopyrum intermedium (Host) Barkworth et Dewey (2n=6x=42). In all accessions and individuals studied, 3 or 4 pairs of major loci were detected. Subsequent genomic in situ hybridization (GISH) analyses revealed that one pair was located on the ends of the short arms of one pair of
homologous chromosomes of the St genome, while the other 2 or 3 pairs of major loci were located in the
E genomes (including the Ee and Eb). It is suggested that 2 to 3 pairs of major loci were probably lost during
the evolution of this hexaploid species. The variation in rDNA positions and copy numbers between the
diploid donors and Th. intermedium, as well as the diversity among the accessions of Th. intermedium
confirmed that the rDNA gene family conveyed the characters of DNA mobile elements. The internal
transcribed spacer (ITS) regions of the rDNA in Th. intermedium were also investigated. Sequence data of
seven positive clones from one individual suggested high degree of individual heterogeneity exists among
ITS repeats. Phylogenetic analyses showed that there were two distinct types of ITS sequences in Th.
intermedium, one with homology to that of Pseudoroegneria species (St genome) and the other to that of
the E genome diploid species. This showed that the ITS paralogues in Th. intermedium have not been
uniformly homogenized by concerted evolution. The limitation of using the chromosomal location of rDNA
loci for phylogenetic analysis is discussed.
Key words: Thinopyrum intermedium ; 18S-5.8S-26S rDNA; internal transcribed spacer (ITS); concerted evolution; fluorescent in situ hybridization (FISH); genomic in situ hybridization
(GISH)
Ribosomal RNAs (rRNAs) are encoded by two highly
conserved multi-gene families, the 18S-5.8S-26S and 5S
rRNA genes (rDNA) in eukaryotic genomes. The two families are generally arranged in tandem repeats at one or more
chromosomal loci. Each repeating unit of 18S-5.8S-26S rDNA
contains three coding regions (18S, 5.8S, and 26S rDNA)
plus two non-coding internal transcribed spacers (ITSs),
which are located between the 18S and 5.8S coding regions
(ITS1) and between the 5.8S and 26S coding regions (ITS2),
respectively (Wendel et al., 1995). The sequences of the
coding regions are highly conserved while the sequences
of ITS regions are more variable (Booy et al., 2000). In most
cases, the rDNA sequences in one species are often treated
as a single copy sequence because of the force of concerted evolution (Booy et al., 2000).
Owing to the characteristics of its sequences and
organization, the 18S-5.8S-26S rDNA is very easy to locate
physically on chromosomes via fluorescence in situ hybridization (FISH). Therefore, chromosomal localization of
the rDNA loci using FISH has been employed in numerous
plant and animal species for different purposes (e.g. Leitch
and Heslop-Harrison, 1992; Castilho and Heslop-Harrison,
1995; Zhang and Sang, 1999; Li and Zhang, 2002).
Thinopyrum intermedium (syn. Agropyron
intermedium; Elytrigia intermedium) is an important perennial forage grass in North American and Mediterranean regions. It is a wild relative of wheat (Triticum
aestivum) that has long been recognized as a potential
genetic source of many desirable characteristics that
could be used to enhance the sustainability of wheat
disease resistance and production (Larkin et al., 1995;
Wang and Zhang, 1996; Zhang et al., 1996a; 1996b; 2000;
Chen et al., 1998).
Th. intermedium is a hexaploid species (2n=6x=42),
which includes three basic genomes, Ee, Eb and St (Zhang
et al., 1996b; 2000). The Ee and Eb genomes are very closely
related, being subgenome types of the E genome. Its Ee
genome is related to the Ee genome of Th. elongatum, and
Received 7 Jan. 2004 Accepted 10 May 2004
Supported by the National Key Technologies R & D Program in the 10th Five-Year Plan (2001-10) and the Qualified Personal Plan of
Chinese Academy of Agricultural Sciences.
* Author for correspondence. Fax: +86 (0)10 62135294; E-mail: <[email protected]>.
LI Da-Yong et al.: Chromosomal Distribution of the 18S-5.8S-26S rDNA Loci and Heterogeneity of Nuclear ITS Regions in
Thinopyrum intermedium (Poaceae: Triticeae)
the E b genome is related to the Eb (J) genome of Th.
bessarabicum (Savul and Rayss) Á. Löve, respectively.
The St genome of Th. intermedium was donated by a species of the genus Pseudoroegneria Löve (Wang et al., 1996;
Zhang et al., 1996a; 1996b; 1997). Its chromosomes at somatic metaphase are relatively large (about 10-15 µm)
(Zhang et al., 1996a; 1996b; 1997). According to the criteria
of Wendel et al. (1995), Th. intermedium should be an ideal
species for studying concerted evolution of the rDNA following allopolyploid speciation.
In the present study, we employed fluorescent in situ
hybridization (FISH) to determine the number and chromosomal location of the 18S-5.8S-26S rDNA loci in Th.
intermedium. The DNA sequence of the nuclear internal
transcribed spacer (ITS) region was also investigated using PCR amplification and DNA sequencing.
1 Materials and Methods
1.1 Plant materials and DNA extraction
Three accessions of Thinopyrum intermedium (Host)
Barkworth et Dewey (2n = 6x = 42), PI 469214 (Maryland,
USA), PI 578698 (Turkistan, Former USSR) and Z 1141
(Canada), were analyzed in this study. Three of its candidate diploid genome donor species, Th. elongatum
(Z 1371, France), Th. bessarabicum (PI 531712, Ukraine)
and Pseudoroegneria stipifolia (Czern ex Nevski) Á.
Löve (PI 313960; 2n = 2x = 14, Former USSR) were also
used. We follow the standardized genome symbols
given by Wang et al. (1996). Total genomic DNAs of
these plants were extracted from young fresh leaves,
following a modified DNA extraction procedure of Sharp et
al. (1989).
1.2 Chromosome preparation
Seeds were germinated on filter paper wetted by distilled water at room temperature (20-25 ℃). When the roottips were 1-2 cm long, they were excised and pretreated in
ice water for 20 h before fixation in 3:1(V/V) ethanol:acetic
acid fixing solution. Each root-tip was squashed in a drop
of 45% acetic acid. Cover slips were removed via freezing in
liquefied nitrogen, and the slides were air-dried.
1.3 Probe DNA labeling and chromosomal in situ hybridization
The clone pTa71, and total genomic DNA from Ps.
stipifolia (St genome) were used as probes in this study.
The pTa71 contains an 18S-5.8S-26S rDNA repetitive unit,
isolated from Triticum aestivum (Gerlach and Bedbrook,
1979). It was labeled with digoxigenin-11-dUTP using the
DIG-Nick Translation Mix (Boehringer Mannheim, GmbH,
Germany). A 100-ng probe was used for each slide. For
１２３５
GISH analysis, the labeled St genomic DNA was mixed with
a 35 times concentration of non-labeled and sheared blocking DNA, which contained the equivalent genomic DNA
from Th. elongatum and Th. bessarabicum. Details of the
FISH protocol can be found in Li and Zhang (2002). The
hybridization signal was observed under a fluorescence
microscope (OLYMPUS BX60, Japan). The images were
captured by a charge-coupled device system (SPOT TM,
Diagnostic Instruments, Ins., Michigan, USA) and brought
together to make the plate using the software Adobe
Photoshop 6.0.
1.4 PCR amplification, cloning and sequencing
The genomic DNA from one individual of Th.
intermedium (PI 469214) was used in PCR amplification
directly. PCR amplification of ITS regions generally followed
Hsiao et al. (1995) using primers ITS-4 and ITS-L. In this
study, 10 independent reactions were carried out. Amplification products were mixed and purified using the Wizard
PCR Pres DNA purification system (Promega, Madison, WI,
USA). Then, the PCR products were cloned using pGEM-T
Easy Vector Systems (Promega, Madison, Wisconsin,
USA). Seven positive clones designated Thin 1 to Thin 7
were randomly selected and identified by PCR re-amplification using the same primers. Sequencing was done on an
ABI automated 377 DNA Sequencer with a Dye Terminator
Cycle Sequencing Reaction Kit (PE Applied Biosystems,
Foster City, California, USA). The boundaries of the ITS
regions were determined by comparison with the ITS sequence of Th. elongatum (GenBank accession number
L36505) (Hsiao et al., 1995).
1.5 Sequence alignment and phylogenetic analysis
To reconstruct phylogenetic trees of Th. intermedium
and its candidate diploid donor species based on ITS
sequences, the published ITS sequences of Ps. spicata
and Ps. libanotica (2n = 2x = 14, St genome), and of Th.
bessarabicum and Th. elongatum were downloaded from
the website of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and used for the
analysis. Based on the phylogenetic trees of Hsiao et al.
(1995), Hordeum vulgare was selected as outgroup. Their
GenBank accession numbers and lengths of ITS1, 5.8S rDNA
and ITS2 are given in Table 1. All sequences were aligned
using the Clustal Ⅹ program (Thompson et al., 1997). The
sequence divergences were estimated using Kimura twoparameter distances (Kimura, 1980).
Phylogenetic trees were constructed with the maximum
likelihood method (Saitou and Nei, 1987) using the programs of PHYLIP version 3.572c (Felsenstein, 1985). Bootstrap analysis (Felsenstein, 1997) was carried out with
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Ａｃｔａ　Ｂｏｔａｎｉｃａ　Ｓｉｎｉｃａ　植物学报　Ｖｏｌ．４６　Ｎｏ．１０　２００４
Table 1 The length of ITS1, 5.8S rDNA, ITS2 and GenBank accession numbers of the sequences from Thinopyrum intermedium and
its related species
Species/Sequences
Th. intermedium
Thin 1
Thin 2
Thin 3
Thin 4
Thin 5
Thin 6
Thin 7
Pseudoroegneria libanotica
Ps. spicata
Thinopyrum elongatum
Th. bessarabicum
Hordeum vulgare
Genome
ITS1 length
(bp)
5.8S length
(bp)
ITS2 length
(bp)
Total length
(bp)
221
219
221
221
221
222
219
221
221
221
221
217
164
164
164
164
164
164
164
164
164
164
164
164
217
216
217
217
216
217
216
216
216
216
216
217
602
599
602
602
601
603
599
601
601
601
601
598
Sources
EeEbSt
St
St
Ee
Eb
I
1 000 replicates.
2
GenBank
accession number
Results
2.1 Distribution of the 18S-5.8S-26S rDNA loci in Th.
intermedium
At least 10 individuals of each accession of Th.
intermedium were analyzed. In this study, major loci were
defined as those giving large pairs of signals observable in
chromosomes at somatic metaphase under relatively high
stringency washes, while smaller FISH signals or those
found only under lower stringency washes were described
as minor loci. To detect and score the major loci, high stringency washes were effective. In all individuals of the three
accessions of Th. intermedium (PI 469214, PI 578698 and
Z 1141), 6 or 8 major loci were detected on the short arms of
3 or 4 pairs of homologous chromosomes, respectively. A
short arm carried only one major locus. One pair of loci was
located interstitially. The other 2 or 3 pairs were located on
terminal parts (Figs.1, 2, 3b, 4b). Variation of the major locus
number was detected even in different cells within the same
root-tip (Figs.3b, 4b).
Sequential GISH and FISH analyses using St genomic
DNA and pTa 71 as probes, respectively, were employed to
investigate the genomic location of the major loci. Seven
pairs of St-genome chromosomes were hybridized strongly
and visualized yellow (Figs.3a, 4a), which was in agreement
with the presence of one St-genome and two E-genomes.
The FISH results showed that one pair of major rDNA loci
was located terminally on one pair of St-genome
chromosomes, and that the other 2 or 3 pairs were located
on E-genome chromosomes (Figs.3, 4).
Besides the major loci, a very high polymorphism of
minor loci was also observed in Th. intermedium,
AF507802
AF507803
AF507804
AF507805
AF507806
AF507807
AF507808
L36501
L36502
L36505
L36506
Z68921
This study
This study
This study
This study
This study
This study
This study
Hsiao et al.,
Hsiao et al.,
Hsiao et al.,
Hsiao et al.,
Hsiao et al.,
1995
1995
1995
1995
1995
especially at lower washing stringency. The number, size
and distribution pattern of the minor loci varied between
accessions, individuals, even cells. The extent of their signals ranged from very small dots to a dispersion over entire
short arms, even over the whole chromosomes which conveyed the major loci (Figs.1, 2).
Secondary constrictions are usually related to the 18S5.8S-26S rDNA loci. However, in the cells of Z1141 at somatic metaphase, no signals were detected on one pair of
chromosomes conveying a distinctly secondary constriction-like structure (Fig.1).
2.2 Sequence analysis of ITS regions
Seven positive clones (Thin 1 to Thin 7) selected randomly from more than 2 500 positive clones originating
from one individual of PI 469214 were sequenced and
analyzed. The entire ITS regions, including both non-coding spacers (ITS1 and ITS2) and the 5.8S rDNA in Th.
intermedium ranged from 599 bp to 603 bp (Table 1). The
length of ITS1 was 219-222 bp and of ITS2 was 216 or 217
bp (Table 1). Twenty-eight variation sites in the ITS1 regions and 31 in the ITS2 regions were detected. The 5.8S
rDNA sequence was 164 bp long in the seven clones. Only
eight variation sites were found. The G+C content of the
sequences surveyed ranged from 60.96 % to 62.27 %, which
is similar to that of most other Poaceae species analyzed
(Hsiao et al., 1995). The sequence analysis revealed that a
high degree of heterogeneity exists among the nuclear ITS
sequences. The sequences reported in this paper have been
deposited in the GenBank database (accession numbers
AF507802-AF507808; see Table 1).
2.3 Phylogenetic analysis
The phylogenetic tree inferred from ITS sequences of
Th. intermedium (PI 1469214) and its related diploid
LI Da-Yong et al.: Chromosomal Distribution of the 18S-5.8S-26S rDNA Loci and Heterogeneity of Nuclear ITS Regions in
Thinopyrum intermedium (Poaceae: Triticeae)
１２３７
Figs.1-4. FISH and GISH of Thinopyrum intermedium chromosomes at somatic metaphase. 1. Z 1141 after being probed by pTa 71
(yellow). The 18S-5.8S-26S loci occupy whole short arms of one pair of homologous chromosomes. The arrows indicate a pair of
secondary constriction-like chromosomes without observable rDNA loci. 2. PI 578698 probed by pTa 71. The three pairs of homologues
convey a large number of minor loci besides the six major loci. 3, 4. Different cells of the same individual in PI 469214. 3a, a cell was
probed by St genomic DNA and blocked by 35 times of E genomic DNA; 3b, the same cell was re-probed with pTa 71. 4a, a cell was
probed by St genomic DNA and blocked by 35 times of E genomic DNA; 4b, the same cell was re-probed by pTa 71. Subsequent analysis
of GISH and FISH of the same cells clearly showed the genome locations of the major loci.
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Ａｃｔａ　Ｂｏｔａｎｉｃａ　Ｓｉｎｉｃａ　植物学报　Ｖｏｌ．４６　Ｎｏ．１０　２００４
species are shown in Fig.5. Given the controversy concerning the potential distortion induced by allopolyploid
species in cladistic analysis (McDade, 1995), the tree was
compared with the monogenomic species trees of Hsiao et
al. (1995). Figure 5 shows that the topological relationships
among the diploid species were not changed after introducing the polyploid species, Th. intermedium. The two
diploid species conveying the E genome, Th. bessarabicum
(Eb) and Th. elongatum (Ee), formed one clade and the
diploids conveying the St genome, Ps. spicata and Ps.
libanotica, another clade.
The seven sequences were divided into two distinct
types: Thins 2 and 7 formed one branch, and the others
were grouped together. The two types of sequences formed
monophyletic groups with the diploid species of the St and
E genomes, respectively. Therefore, the two types of sequences in Th. intermedium correspond to those of its parental species. This suggests that concerted evolution has
not homogenized the ITS paralogues in the hexaploid
species, Th. intermedium.
Scilla autumnalis, rDNA sites were detected in the A genome only (Vaughan et al., 1993), and in tetraploid Brassica napus, one rDNA site of genome A was lost (Snowdon
et al., 1997).
The number and distribution pattern of the 18S-5.8S26S rDNA in the candidate diploid genome donor species
of Th. intermedium (Th. elongatum, Th. bessarabicum and
Ps. stipifolia) have been described previously (Li and
Zhang, 2002). All three have a similar distribution pattern of
the major 18S-5.8S-26S rDNA: two pairs of 18S-5.8S-26S
rDNA loci in each somatic cell of these species and 2 loci
per haploid genome (Li and Zhang, 2002). One pair was
located at the end of the short arms of one pair of homologous chromosomes, and another pair was located at interstitial regions of the short arms of another chromosome
pair (Li and Zhang, 2002). Th. intermedium is a hexaploid
containing six genomes. It might thus have six pairs of major loci. However, only 3 or 4 pairs were detected in this
study. So the number of the major loci in Th. intermedium
was lower than the expected number based on its
progenitors. This suggests that several major loci (2 or 3
pairs) have been lost during the evolution of this hexaploid
species.
Because the Ee and Eb genomes are very closely related,
the GISH technique could only discriminate the E (including
Ee and Eb) genome from the St genome, but could not discriminate Ee from Eb (Zhang et al., 1996a; 1997; 2000; Chen
et al., 1998). In all individuals observed, there was only one
pair of Th. intermedium St-genome chromosomes carrying
3 Discussion
In newly established polyploid plants, the number of
18S-5.8S-26S rDNA loci may equal the sum of that of their
progenitors (Li and Zhang, 2002; Mishima et al., 2002).
However, loss of loci has been observed in several allopolyploid species. For example, in hexaploid oats (Avena sativa),
there are no rDNA sites on the C genome chromosomes
(Leggett and Markand, 1995). In tetraploid and hexaploid
Fig.5. The phylogenetic tree inferred from ITS sequences from Thinopyrum intermedium and its related diploid species generated by
the maximum likelihood method using the software PHYLIP (version 3.573c). Hordeum vulgare was selected as out-group. The numbers
above the branches represent the bootstrap support in 1 000 replicates.
LI Da-Yong et al.: Chromosomal Distribution of the 18S-5.8S-26S rDNA Loci and Heterogeneity of Nuclear ITS Regions in
Thinopyrum intermedium (Poaceae: Triticeae)
major rDNA loci. These were located at terminal positions
in the short arms. Apparently, a pair of interstitial-type loci
observed in the diploid St species was “lost”(Figs.3, 4). In
the E genomes of Th. intermedium, 2 to 3 pairs of major loci
were observed. One interstitial and one terminal locus were
detected in all individuals. The additive locus was of the
terminal type. This suggests that at least one interstitial
locus existing in the diploid species with an E genome has
been “lost”(Figs.3, 4) and that interstitial type loci were
probably lost more readily than terminally located loci during the evolution of polyploid species (Li and Zhang, 2002).
Studies have shown that there exists a high variability
for the position of rDNAs in eukaryotic genomes and have
revealed that the multi-gene family conveyed the characters of DNA mobile elements (Sánchez-Gea et al., 2000; Li
and Zhang, 2002; Stupar et al, 2002). The rDNA sequences
may have been directly transposed or moved via a certain
transposable element intermediate. There is a possibility
that the rDNA repeat itself or related sequences have the
ability of transposition. The movement of rDNA in genomes
might via some “seeds”sequences intermediate, which may
be mobile elements. Those seed sequences could transpose into novel loci, then duplicate and form tandem arrays.
By certain mechanisms such as unequal crossing over or
gene conversion, the integrated rDNA repeats could be
formed in novel loci. It was noticed that the minor loci are
ˇ k, 1995;
ubiquitous in genomes (Dubcovsky and Dvorá
Sánchez-Gea et al., 2000; Li and Zhang, 2002). Dubcovsky
ˇ k (1995) suggested that the rDNA loci might
and Dvorá
change their positions via dispersion of minor loci. Stupar
et al. (2002) reported that several tandem IGS-related repetitive DNA arrays existed out of the 18S-5.8S-26S rDNA
loci in potato. This supports the hypothesis mentioned
above.
Because of the force of concerted evolution, the rDNA
sequences in one species are often treated as a single copy
sequence. However, the heterogeneity of the ITS has been
reported among individuals of some species throughout all
eukaryotic taxa (Booy et al., 2000). Such heterogeneity may
occur when concerted evolution is not fast enough or even
fails to homogenize rDNA repeated units on some occasions,
e.g. recent hybridization between different species, the development of pseudogenes, a high number of rDNA loci
located on non-homologous chromosomes, and asexual
reproduction (Dover, 1982; Wendel et al., 1995; Zhang and
Sang, 1999; Wendel, 2000; Booy et al., 2000). The frequency
of heterogeneity among rDNA sequences is higher in
alloployploids than that in diploid and autopolyploid
species. Therefore, the ITS sequences may be used either
１２３９
to identify parents or infer the evolution of parental genomes in allopolyploid species (Hodkinson et al., 2002).
The chromosomal location of rDNA loci can offer some
information regarding concerted evolution (Zhang and
Sang, 1999; Li and Zhang, 2002). The terminal or near- terminal location of rDNA may permit unequal crossover without deleterious recombination between non-homologous
chromosomes, which might facilitate the process of sequence homogeneity (Zhang and Sang, 1999; Wendel,
2000). Interstitial location of rDNA has indicated a high
level of sequence polymorphism in some species (Hanson
et al., 1996; Wendel, 2000).
Although our knowledge about Th. intermedium is still
very limited, its allohexaploid origin is an admitted fact.
However, we do not know the detailed evolutionary history of this species, we cannot tell whether it is a novel
polyploid or not. But its characterization of perennial trait,
and possessing the interstitial type rDNA locus localization suggested that the concerted evolution is not fast
enough or even fails to homogenize the rDNA repetitive
units in this species. The sequences analysis supported
the above hypothesis that the ITS paralogues in Th.
intermedium have not been uniformly homogenized by
concerted evolution.
Information from the physical mapping of the rDNA loci,
e.g. locus numbers and locations on chromosomes has been
used for phylogenetic analysis in many studies (Zhang
and Sang, 1999; Mishima et al., 2002; Hodkinson et al.,
2002). However, the difference between the numbers and
positions of rDNA loci in polyploid species and in their
diploid ancestors has revealed the limitation of its phylogenetic significance. Many studies including the present
work have shown that both complex quantitative and qualitative variability exists for 18S-5.8S-26S rDNA loci among
species, subspecies, and populations and even in individuals
ˇ k (1995)
(Sánchez-Gea et al., 2000). Dubcovsky and Dvorá
even described them as “nomads”. In such cases, their
polymorphism may be too high to allow for establishing
phylogenetic relationships on this basis.
Acknowledgements: We appreciate Dr. G. Fedak (Eastern
Cereal and Oilseed Research Centre, Agriculture and AgriFood, Canada) and several anonymous reviewers for their
helpful comments on the manuscript. We also thank Mr.
WANG Chao (Institute of Botany, The Chinese Academy
of Sciences) for phylogenetic analysis and comments.
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(Managing editor: ZHAO Li-Hui)