全 文 ：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: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1234-1241
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 acces-
sions and individuals studied, 3 or 4 pairs of major loci were detected. Subsequent genomic in situ hybridiza-
tion (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); con-
certed 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 fami-
lies 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 con-
certed 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 hy-
bridization (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).
Thinopyru m in t ermedium ( syn. Agropyron
intermedium; Elytrigia intermedium) is an important pe-
rennial forage grass in North American and Mediterra-
nean 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
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) 1235
the Eb 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 spe-
cies of the genus Pseudoroegneria Löve (Wang et al., 1996;
Zhang et al., 1996a; 1996b; 1997). Its chromosomes at so-
matic metaphase are relatively large (about 10-15 mm)
(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 fol-
lowing allopolyploid speciation.
In the present study, we employed fluorescent in situ
hybridization (FISH) to determine the number and chromo-
somal 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 us-
ing 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 can-
didate 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 dis-
tilled water at room temperature (20-25 ℃). When the root-
tips 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 hybrid-
ization
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
GISH analysis, the labeled St genomic DNA was mixed with
a 35 times concentration of non-labeled and sheared block-
ing 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 (SPOTTM,
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. Amplifi-
cation 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-amplifica-
tion 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 se-
quence 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 Infor-
mation (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 two-
parameter distances (Kimura, 1980).
Phylogenetic trees were constructed with the maximum
likelihood method (Saitou and Nei, 1987) using the pro-
grams of PHYLIP version 3.572c (Felsenstein, 1985). Boot-
strap analysis (Felsenstein, 1997) was carried out with
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041236
1 000 replicates.
2 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 strin-
gency 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,
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 sig-
nals ranged from very small dots to a dispersion over entire
short arms, even over the whole chromosomes which con-
veyed the major loci (Figs.1, 2).
Secondary constrictions are usually related to the 18S-
5.8S-26S rDNA loci. However, in the cells of Z1141 at so-
matic metaphase, no signals were detected on one pair of
chromosomes conveying a distinctly secondary constric-
tion-like structure (Fig.1).
2.2 Sequence analysis of ITS regions
Seven positive clones (Thin 1 to Thin 7) selected ran-
domly 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-cod-
ing 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 re-
gions 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
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 Genome
ITS1 length 5.8S length ITS2 length Total length GenBank
Sources
(bp) (bp) (bp) (bp) accession number
Th. intermedium EeEbSt
Thin 1 221 164 217 602 AF507802 This study
Thin 2 219 164 216 599 AF507803 This study
Thin 3 221 164 217 602 AF507804 This study
Thin 4 221 164 217 602 AF507805 This study
Thin 5 221 164 216 601 AF507806 This study
Thin 6 222 164 217 603 AF507807 This study
Thin 7 219 164 216 599 AF507808 This study
Pseudoroegneria libanotica S t 221 164 216 601 L36501 Hsiao et al., 1995
Ps. spicata S t 221 164 216 601 L36502 Hsiao et al., 1995
Thinopyrum elongatum Ee 221 164 216 601 L36505 Hsiao et al., 1995
Th. bessarabicum Eb 221 164 216 601 L36506 Hsiao et al., 1995
Hordeum vulgare I 217 164 217 598 Z68921 Hsiao et al., 1995
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.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041238
species are shown in Fig.5. Given the controversy con-
cerning 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 intro-
ducing 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 se-
quences in Th. intermedium correspond to those of its pa-
rental species. This suggests that concerted evolution has
not homogenized the ITS paralogues in the hexaploid
species, Th. intermedium.
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 allopoly-
ploid 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
Scilla autumnalis, rDNA sites were detected in the A ge-
nome only (Vaughan et al., 1993), and in tetraploid Bras-
sica napus, one rDNA site of genome A was lost (Snowdon
et al., 1997).
The number and distribution pattern of the 18S-5.8S-
26S 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 homolo-
gous chromosomes, and another pair was located at inter-
stitial 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 ma-
jor 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 dis-
criminate 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
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) 1239
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 dur-
ing 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 charac-
ters 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 trans-
pose 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
ubiquitous in genomes (Dubcovsky and Dvorák, 1995;
Sánchez-Gea et al., 2000; Li and Zhang, 2002). Dubcovsky
and Dvorák (1995) suggested that the rDNA loci might
change their positions via dispersion of minor loci. Stupar
et al. (2002) reported that several tandem IGS-related re-
petitive 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 de-
velopment 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
to identify parents or infer the evolution of parental ge-
nomes 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- ter-
minal location of rDNA may permit unequal crossover with-
out deleterious recombination between non-homologous
chromosomes, which might facilitate the process of se-
quence 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 his-
tory 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 localiza-
tion 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 phylo-
genetic significance. Many studies including the present
work have shown that both complex quantitative and quali-
tative variability exists for 18S-5.8S-26S rDNA loci among
species, subspecies, and populations and even in individuals
(Sánchez-Gea et al., 2000). Dubcovsky and Dvorák (1995)
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 Agri-
Food, 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.
References:
Booy G, van der Schoot J, Vosman B. 2000. Heterogeneity of the
internal transcribed spacer 1 (ITS1) in Tulipa (Liliaceae). Plant
ˇ
ˇ
ˇ
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041240
Sys Evol, 225: 29-41.
Castilho A, Heslop-Harrison J S. 1995. Physical mapping of 5S
and 18S-5.8S-26S rDNA and repetitive DNA sequences in
Aegilops umbellulata. Genome, 38: 91-96.
Chen Q, Conner R L, Laroche A, Thomas J B. 1998. Genome
analysis of Thinopyrum intermedium and Thinopyrum
ponticum using genomic in situ hybridization. Genome, 41:
580-586.
Dover G. 1982. Molecular drive: a cohesive mode of species
evolution. Nature, 299: 111-117.
Dubcovsky J, Dvorák J. 1995. Ribosomal RNA multigene loci:
nomads of the Triticeae genomes. Genetics, 140: 1367-1377.
Felsenstein J.1985. Confidence limits on phylogenies: an approach
using the bootstrap. Evolution, 39: 783-791.
Felsenstein J. 1997. PHYLP: Phylogenetic Inference Package.
Version 30572c. Seattle: University of Washington.
Gerlach W L, Bedbrook J R. 1979. Cloning and characterization
of ribosomal RNA genes from wheat and barley. Nucleic Acids
Res, 7: 1869-1885.
Hanson R E, Islam-Faridi M N, Percival E A, Crane C F, Ji Y,
Mcknight T D, Stelly D M, Price H J. 1996. Distribution of
5S and 18S-28S rDNA loci in a tetraploid cotton (Gossypium
hirsutum L.) and its putative diploid ancestors. Chromosoma,
105: 55-61.
Hodkinson T R, Chase M W, Takahashi C, Leitch I J, Bennett M
D, Renvoize S A. 2002. The use of DNA sequencing (ITS and
TrnL-F), AFLP, and fluorescent in situ hybridization to study
allopolyploid Miscanthus (Poaceae). Am J Bot, 89: 279-286.
Hsiao C, Chatterton N J, Asay K H, Jensen K B. 1995. Phyloge-
netic relationships of the monogenomic species of the wheat
tribe, Triticeae (Poaceae), inferred from the nuclear rDNA
(ITS) sequences. Genome, 38: 211-223.
Kimura M. 1980. A simple method for estimating evolutionary
rate of base substitutions through comparative studies of nucle-
otide sequences. J Mol Evol, 16: 111-120.
Larkin P J, Banks P M, Lagudah E S. 1995. Disomic Thinopyrum
intermedium addition lines in wheat with barley yellow dwarf
virus resistance and with rust resistance. Genome, 38: 385-
394.
Leggett J M, Markand G S. 1995. The genomic identification of
some monosomics of Avena sativa L. cv. Sun Ⅱ using ge-
nomic in situ hybridization. Genome, 38: 747-751.
Leitch I J, Heslop-Harrison J S. 1992. Physical mapping of the
18S-5.8S-26S rRNA genes in barley by in situ hybridization.
Genome, 35: 1013-1018.
Li D, Zhang X. 2002. Physical localization of the 18S-5.8S-26S
rDNA and sequence analysis of ITS regions in Thinopyrum
ponticum (Poaceae: Triticeae): implications for concerted
evolution. Ann Bot, 90: 445-452.
McDade L A. 1995. Hybridization and phylogenetics. Hoch P C,
Stephenson A G. Experimental and Molecular Approaches to
Plant Biosystematics. St Louis: Missouri Botanical Garden
Press. 305-331.
Mishima M, Ohmido N, Fukui K, Yahara T. 2002. Trends in site-
number change of rDNA loci during polyploid evolution in
Sanguisorba (Rosaceae). Chromosoma, 110: 550-558.
Rogers S O, Bendich A J. 1987. Ribosomal RNA genes in plants:
variability in copy number and in the intergenic spacer. Plant
Mol Biol, 9: 509-520.
Saitou N, Nei M. 1987. The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol,
4: 406-425.
Sánchez-Gea J F, Serrano J, Galián J. 2000. Variability in rDNA
in Iberian species of the genus Zabrus (Coleoptera: Carabidae)
detected by fluorescence in situ hybridization. Genome, 43:
22-28.
Sharp P J, Chao S, Desai S, Gale M D. 1989. The isolation,
characterization and application in the Triticeae of a set of
wheat RFLP probes identifying each homologous chromo-
some arm. Theor Appl Genet, 78: 342-348.
Snowdon R J, Köhler W, Köhler A. 1997. Chromosomal localiza-
tion and characterization of rDNA loci in the Brassica A and C
genomes. Genome, 40: 582-587.
Stupar R M, Song J, Tek A L, Cheng Z, Dong F, Jiang J. 2002.
Highly condensed potato pericentromeric heterochromatin
contains rDNA-related tandem repeats. Genetics, 162: 1435-
1444.
Thompson J D, Gibson T J, Plewniak F. 1997. The Clustal X
windows interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic Acids Res,
25: 4876-4882.
Vaughan H E, Jamilena M, Ruiz Rejon C, Parker J S, Garrido-
Ramos M A. 1993. Loss of nucleolus-organizer regions during
polyploid evolution in Scilla autumnalis. Heredity, 71: 574-
580.
Wang R R-C, Von Bothemer R, Dvorák J, Fedak G, Linde-Lauren
I, Muramatsu M. 1996. Genome symbols in the Triticeae
(Poaceae). Wang R R-C. Procedings of the 2nd International
Triticeae Symposium, Logan, Utah: Publication Design and
Production Utah State University. 29-34.
Wang R R-C, Zhang X Y. 1996. Characterization of the transloca-
tion chromosomes using FISH and genome-specific RAPD
markers in two wheat translocation lines resistant to wheat
streak mosaic or barley yellow dwarf virus. Chromosome
Res, 4: 583-587.
Wendel J F, Schnabel A, Seelanan T. 1995. Bi-directional interlocus
concerted evolution following alloploid speciation in cotton
(Gossypium). Proc Natl Acad Sci USA, 92: 280-284.
ˇ
ˇ
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) 1241
Wendel J F. 2000. Genome evolution in polyploids. Plant Mol
Biol, 42: 225-249.
Zhang D M, Sang T. 1999. Physical mapping of ribosomal RNA
genes in peonies (Paeonia, Paeoniaceae) by fluorescent in situ
hybridization: implications for phylogeny and concerted
evolution. Am J Bot, 86: 735-740.
Zhang X Y, Banks P M, Larkin P J. 2000. Verification of alien
chromosomes and segments in addition lines derived from
Triticum aestivum -Thinopyrum intermedium partial amphip-
loid Zhong 5. Chin Agr Sci, 3: 53-59.
Zhang X Y, Dong Y S, Wang R R-C. 1996a. Characterization of
genomes and chromosomes in partial amphiploids of the hy-
brids of Triticum aestivum×Thinopyrum ponticum by in situ
hybridization, isozyme analysis, and RAPD. Genome, 39:
1062-1071.
Zhang X Y, Koul A, Petrosk R, Quellet J, Fedak G, Dong Y S,
(Managing editor: ZHAO Li-Hui)
Wang R R-C. 1996b. Molecular verification and characteriza-
tion of BYDV-resistant germ plasms derived from hybrids of
wheat with Thinopyrum ponticum and Th. intermedium. Theor
Appl Genet, 93: 1033-1039.
Zhang X Y, Wang R R-C, Fedak G, Dong Y S. 1997. Determina-
tion of genome and chromosome composition of Thinopyrum
intermedium and partial amphiploids derived from Triticum
aestivum× Th. intermedium by GISH and genome-specific
RAPD markers. Chinese Agricultural Sciences ¡ª for the Com-
pliments to the 40th Anniversary of the Founding of the Chi-
nese Academy of Agricultural Sciences. Beijing: Chinese
Agrtechnology Press. 71-80.
Zhang Z Y, Xin Z Y, Larkin P J. 2001. Molecular characterization
of a Thinopyrum intermedium group 2 chromosome (2Ai-2)
conferring resistance to yellow dwarf virus. Genome, 44: 1129-
1135.