AOBPreview originally published online on September 4, 2002
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Annals of Botany 90: 445-452, 2002
© 2002 Annals of Botany Company
Physical Localization of the 18S-5·8S-26S rDNA and Sequence Analysis of ITS Regions in Thinopyrum ponticum (Poaceae: Triticeae): Implications for Concerted Evolution
1 Key Laboratory of Crop Germplasm and Biotechnology, Institute of Crop Germplasm Resources, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China
* For correspondence. Fax +86 10 62186629, e-mail xueyongz{at}public.bta.net.cn
Received: 5 February 2002; Returned for revision: 30 May 2002; Accepted: 1 July 2002 Published electronically: 4 September 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Fluorescence in situ hybridization was used in Thinopyrum ponticum, a decaploid species, and its related diploid species, to investigate the distribution of the 18S-5·8S-26S rDNA. The distribution of rDNA was similar in all three diploid species (Th. bessarabicum, Th. elongatum and Pseudoroegneria stipifolia). Two pairs of loci were observed in each somatic cell at metaphase and interphase. One pair was located near the terminal end and the other in the interstitial regions of the short arms of one pair of chromosomes. However, all of the major loci in Th. ponticum were located on the terminal end of the short arms of chromosomes, and one chromosome had only one major locus. The maximum number of major loci detected on metaphase spreads was 20, which was the sum of that of its progenitors. The interstitial loci that exist in the possible diploid genome donor species were probably ‘lost’ during the evolutionary process of the decaploid species. A number of minor loci were also detected on whole regions of two pairs of homologous chromosomes. These results suggested that the position of rDNA loci in the Triticeae might be changeable rather than fixed. Positional changes of 18S-5·8S-26S rDNA loci between Th. ponticum and its candidate genome donors indicate that it is almost impossible to find a genome in the polyploid species that is completely identical to that of its diploid donors. The possible evolutionary significance of the distribution of the rDNA is also discussed. Internal transcribed spacer (ITS) regions of nuclear DNA in Th. ponticum were investigated by PCR amplification and sequencing. The sequence data from five positive clones selected at random, together with restriction site analysis, indicated that the ITS repeated units are nearly homogeneous in this autoallodecapolypoid species. Combined with in situ hybridization results, the data led to the conclusion that the ITS region has experienced interlocus as well as intralocus concerted evolution. Phylogenetic analyses showed that the sequences from Th. ponticum have concerted to the E genome repeat type.
Key words: Thinopyrum ponticum, Thinopyrum elongatum, Thinopyrum bessarabicum, Pseudoroegneria, 18S-5·8S-26S rDNA, fluorescence in situ hybridization (FISH), internal transcribed spacer (ITS), concerted evolution.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Thinopyrum ponticum (Podp.) Liu & Wang is a perennial decaploid species (2n = 10x = 70) in the tribe Triticeae. It is known to possess a number of elite genes for wheat improvement, such as those for rust resistance, wheat curl mite resistance, wheat streak mosaic virus (WSMV) resistance, barley yellow dwarf virus (BYDV) resistance, and tolerance to abiotic stresses, such as salinity and drought (Zhang et al., 1996a, b; Chen et al., 1998). All of these characters make this species useful as a source of genes for improving the genetic diversity of cultivated wheat. Because it hybridizes easily with common wheat, a number of useful genes have been transferred from this species to wheat, which has led to the development of many important wheat germplasms and cultivars (Zhang et al., 1996a, b; Fedak et al., 2000).
Th. ponticum is a complex decaploid species that has been used to improve wheat for more than half a century. However, our knowledge of this species is still very limited. The origin and genome composition of Th. ponticum is an interesting and puzzling subject (Dewey, 1984). Genomic relationships among the genomes of Th. ponticum and its related species have been studied several times, and different genome formulae have been proposed. Muramatsu (1990) and Wang et al. (1991) concluded that Th. ponticum was an autodecaploid, and proposed J1J2J3J4J5 and JJJJJ, respectively, as its genome formula based on chromosome pairing. The J genome was from Th. bessarabicum (Savul and Rayss) A Löve (2n = 2x = 14, currently designated as Eb), and was almost identical to the E genome of Th. elongatum (Host) D. R. Dewey (2n = 2x = 14, Ee). Recently, this species was shown to be an autoallodecaploid. Its genome formula was designated by Zhang et al. (1996a, b) as StStEeEbEx, based on genomic in situ hybridization (GISH) and genome-specific markers. The St genome was homologous to the St genome of Pseudoroegneria. However, Chen et al. (1998) proposed JJJJsJs for this decaploid based on the same GISH results. In their paper, the Js genome referred to modified J- or E-genome chromosomes conveying St segments by translocation between the St and E (or J) genomes (Chen et al., 1998). They proposed that Th. ponticum conveyed only segments of the St genome rather than any intact St genome or chromosomes. The major disagreement between Zhang et al. (1996a, b) and Chen et al. (1998) centred on the explanation of GISH results of Th. ponticum probed by St genomic DNA and blocked by E genomic DNA. The St genomic probe, even with a very high rate of E-genome blocking DNA, hybridized all 70 chromosomes, but 28 chromosomes hybridized more strongly at their centromeres and nearby regions. In the reverse GISH analysis, these 28 chromosomes were also hybridized by the E genomic probe except for their centromeric and nearby regions which were completely blocked by the St genomic DNA (Zhang et al., 1996a). Zhang et al. believed that the unexpected signals appearing beyond the probe genome chromosomes were mainly caused by cross-hybridization between St and E genomes because of their too close relationship in Th. ponticum. Therefore, Zhang et al. (1996a) proposed that the centromeres and nearby regions might be critical in the discrimination of St and E genomes. Similar GISH phenomena have also been reported in allotetraploid Triticum dicoccoides (Belyayev et al., 2000) and in the genus Hordeum (Heslop-Harrison, 1996). Inter-genomic translocations may not be appropriate explanations of this phenomenon. Therefore, we accepted the opinion of Zhang et al. (1996a) and used StStEeEbEx as the genome formula for Th. ponticum. Nowadays, the candidate donor species of Th. ponticum have been narrowed down to a few species, including two diploid species of the genus Thinopyrum (Th. bessarabicum and Th. elongatum) and possibly several species of Pseudoroegneria, a unique genome (St) genus (Dewey, 1984).
Fluorescence in situ hybridization (FISH) is a powerful technique used to localize nucleic acid sequences on chromosomes. It provides a tool to construct physical maps, analyse chromosome structure and aberrations, and investigate structure, function and evolution of chromosomes and genomes (Maluszynsky and Heslop-Harrison, 1993; Leitch et al., 1994; Sang and Liang, 2000). The most frequently mapped gene is that for 18S-5·8S-26S rDNA (Zhang and Sang, 1999). In recent years, hybridization of rDNA probes using the FISH technique has been applied to assist karyological and genome analysis in a large number of plant species (Schmidt and Heslop-Harrison, 1998; Snowdon et al., 2000). Physical mapping of rDNAs can also provide information to help understand the relationships and evolution of polyploid species, for example, better understanding of concerted evolution of rDNA (Zhang and Sang, 1999).
That individual members of a gene family evolve as a unit and not independently of other members of the family is frequently referred to as concerted evolution (Elder and Turner, 1994; Waters and Schaal, 1996). Concerted evolution among repeated sequences has been observed in many organisms, from fungi through higher plants and animals (Waters and Schaal, 1996). Generally, the basic unit of repeated sequences homogenizes within individuals and among individuals within a species by the force of concerted evolution, but varies greatly between species.
In the evolution of polyploids, concerted evolution plays an essential role in the maintenance of sequence homogeneity of multigene families (Zhang and Sang, 1999). Studies on concerted evolution have mainly focused on rRNA multigene families (Wang and Zhang, 2000). However, previous studies have shown that the mode and rate of concerted evolution of rDNA differ among various plant groups (Wang et al., 2000). To reveal the evolutionary patterns of rDNA in the formation of allopolyploids, more data are needed. Th. ponticum has been shown to be an autoallodecaploid and has a relatively well-established phylogenetic framework (Zhang et al., 1996a; Chen et al., 1998). According to the criteria of Wendel et al. (1995), it is an ideal plant material for studying the concerted evolution of rDNA following allopolyploid speciation.
In this study, we employed FISH to locate 18S-5·8S-26S rDNA loci on chromosomes of Th. ponticum and its possible diploid progenitors (Th. elongatum, Th. bessarabicum and Ps. stipifolia) and investigated the DNA sequence of internal transcribed spacer (ITS) regions of Th. ponticum by PCR amplification and clone sequencing. Our primary goals were to: (1) determine the number and location of 18S-5·8S-26S rDNA loci in these species; (2) infer evolutionary changes of the rDNA loci; (3) investigate whether ITS sequences have been homogeneous; and (4) determine the direction of concerted evolution of rDNA in Th. ponticum if it has indeed occurred.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plant material
The plants used in this study were Thinopyrum ponticum (Podp.) Liu & Wang (2n = 10x = 70, accessions PI 578683, PI 578684 and PI 578686), Th. elongatum (Host) D. Dewey (2n = 2x = 14, Ee genome, accession Z1371), Th. bessarabicum (Save ex Rayss) A Löve (2n = 2x = 14, Eb genome, accession PI 531712) and Ps. stipifolia (Czern ex Nevski) A Löve (2n = 2x =14, St genome, accession PI 313960). In this paper, we follow the standardized genome symbols designated by Wang et al. (1995).
DNA extraction
Seeds were sown and maintained in a glasshouse. Total genomic DNA was extracted from young fresh leaves collected from ten individuals, following a modified DNA extraction procedure of Sharp et al. (1989).
Chromosome preparation
Seeds were germinated on filter paper wetted by distilled water at room temperature (20–25 °C). When root-tips were 1–2 cm long, they were excised and pre-treated in ice water for 36 h before fixation in 3 : 1 (v/v) ethanol : acetic acid fixing solution. The single root-tip was squashed in a drop of 45 % acetic acid. Cover slips were removed after freezing in liquefied nitrogen, and slides were air-dried.
Probe DNA labelling
The probe, pTa71, a highly tandem repetitive sequence isolated from bread wheat (Triticum aestivum L.) contains a 9 kb EcoRI fragment of the coding sequences for 18S, 5·8S and 26S rDNAs and the non-coding spacer sequences (Gerlach and Bedbrook, 1979). It was labelled with digoxigenin using the DIG-Nick Translation Mix (Boehringer Mannheim, GmbH, Germany).
Fluorescence in situ hybridization
Slides were incubated in 100 µg ml–1 RNase A at 37 °C for 1 h followed by three washings in 2 x SSC for 5 min. They were then dehydrated in a graded ethanol series and air-dried. The hybridization mixture solution containing 100 ng labelled probe (for one slide), 0·125 mg ml–1 salmon sperm DNA, 50 % formamide, 10 % dextran sulfate, 2 x SSC and 0·1 % SDS was denatured at 80 °C for 5 min and chilled on ice for 3–5 min. The solution was added to the slide and covered with a plastic membrane. Slides were put into the hybridization chamber (PTCTM Programmable Thermal Controller; M J Research, Inc., Watertown, MA, USA). The temperature regime was as follows: 75 °C (5 min), 60 °C (2 min), 55 °C (2 min), 50 °C (30 s), 45 °C (1 min), 42 °C (2 min), 40 °C (5 min), 38 °C (5 min), then 37 °C overnight.
Detection of hybridization
Plastic membranes were carefully removed after hybridization. Slides were then washed once at 42 °C in 2 x SSC for 5 min, twice for 5 min each in 0·1 x SSC containing 20 % formamide, three times for 5 min each in 2 x SSC, and then three times for 5 min each in 2 x SSC at 20–25 °C. Slides were washed a final time for 5 min in 4 x SSC containing 0·2 % Tween-20 at 20–25 °C. The slide was then blocked for 5 min at 37 °C by 5 % BSA in 4 x SSC containing 0·2 % Tween-20. Hybridization sites were detected with anti-digoxigenin conjugated by fluorescence isothiocyanate (FITC). The antibody binding reaction was carried out at 37 °C in 5 % BSA in 4 x SSC containing 0·2 % Tween-20 for about 1 h. The slide was washed three times in 5 % BSA in 4 x SSC containing 0·2 % Tween-20 for 8 min each at 37 °C. Chromosomes were counter-stained with propidium iodide (PI), mounted with anti-fade solution (Vector Laboratories Inc., Burlingame, CA, USA). Details of FISH can be found in Leitch et al. (1994).
The hybridization signal was observed using a fluorescence microscope (Olympus BX60, Japan). Images were captured by a charge-coupled device system (SPOTTM; Diagnostic Instruments, Inc., USA) and brought together to make the plate using Adobe Photoshop 6·0 software. During this process, no modification was made to the individual images.
PCR amplification and sequencing
Total genomic DNA from Th. ponticum was used directly in PCR amplifications. PCR amplification of ITS regions generally followed Hsiao et al. (1994, 1995) using primers ITS-4 and ITS-L. PCR was performed in a PTC–100 thermal cycler (MJ-Research, Inc., Watertown, MA, USA) and consisted of 35 cycles (93 °C for 35 s, 49 °C for 35 s, 72 °C for 2 min), followed by a final extension of 7 min at 72 °C. Six independent reactions were carried out in this study. Their amplification products were mixed and purified using the Wizard PCR Pres DNA purification system (Promega, Madison, WI, USA). PCR products were cloned using pGEM-T Easy Vector Systems (Promega). Five positive clones were selected at random and identified by PCR using the same primers (ITS-4 and ITS-L). Sequencing was carried out by the BioAsia Biotechnology Co. Ltd (Shanghai, China) using an ABI 377 DNA Sequencer (Perkin Elmer, Foster City, CA, USA). The boundaries of the ITS regions were determined by comparison with the sequence of Pseudoroegneria spicata (GenBank accession no. L36502) (Hsiao et al., 1995)
Restriction site analysis (PCR-RFLP)
To investigate whether sequences of the ITS region have been homogenous, restriction site analysis was applied following the procedure of Booy et al. (2000). The mixed PCR products of ITS regions were purified and concentrated. Several inner-restriction enzymes (EcoRV, MseI, MluI, MspI, HpaII, Sau3AI) were used to digest the PCR product. Digested fragments were separated on a 4 % agarose gel to determine the polymorphic sites.
The PCR products of ITS regions from the three diploid species (Th. bessarabicum, Th. elongatum and Ps. stipifolia) were also analysed to investigate their homogeneity following the same procedures.
Sequence alignment and phylogenetic analysis
To construct phylogenetic trees and determine the direction of concerted evolution of rDNA, we compared the ITS sequences from Th. ponticum with those of its candidate diploid donor species. Two diploid species containing the St genome, Pseudoroegneria spicata and Pseudoroegneria libantica (Hackel) D. R. Dewey, and two species containing the E genome, Th. bessarabicum (Eb) and Th. elongatum (Ee) were used. The ITS sequences of these species were sequenced by Hsiao et al. (1994). The data were downloaded from the website (http://www.ncbi.nlm.nih.gov), and their GenBank accession numbers were L36501 (Ps. libantica), L36502 (Ps. spicata), L36506 (Th. bessarabicum) and L36495 (Th. elongatum). Based on the phylogenetic trees of Hsiao et al. (1994), Hordeum vulgare L. (GenBank accession no.: Z68921) was selected as outgroup. All of these sequences were aligned using Clustal X program (Thompson et al., 1997).
Phylogenetic trees were constructed using the neighbour-joining method (Saitou and Nei, 1987) using programs of PHYLIP version 3·572c (Felsenstein, 1985). Bootstrap analysis (Felsenstein, 1997) was carried out with 1000 replicates. Phylogenetic trees were constructed with Kimura two-parameter distances (Kimura, 1980).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Fluorescence in situ hybridization
Yellow-green fluorescence signals were indicative of 18S-5·8S-26S rDNA loci (Fig. 1). The pattern of distribution of 18S-5·8S-26S rDNA was similar in all three diploid species. Two pairs of 16S-5·8S-26S rDNA loci were observed on each of the metaphase and interphase spreads of their somatic cell. The number of these loci corresponded to that of satellite chromosomes, and they were located in the region of the secondary constrictions. One pair was located near the terminal end of the short arms, and another pair was located on the interstitial regions of the short arms. Fluorescence signals of all detected loci were strong, indicating that many copies of the 16S-5·8S-26S rDNA repetitive unit existed at these loci (Fig. 1A–C). Kosina and Heslop-Harrison (1996) have described the chromosomal location of rDNA in Th. elongatum (syn. Lophopyrum elongatum). Our result was in accordance with theirs. It was interesting to note that the two loci on homologous chromosomes were unequal in size: one was obviously bigger than the other in all three diploid species.
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For each accession of Th. ponticum, four individuals were evaluated in our study. Hybridization patterns did not differ among accessions or individuals. Eleven integrated and several incomplete metaphase chromosome spreads were observed. All of the major 18S-5·8S-26S rDNA loci were located on the terminal end of the short arms of chromosomes. One chromosome only conveyed one major locus. The maximum number detected on metaphase spreads of root-tips in the present study was 20 (Fig. 1D). The strength of fluorescence signals differed among these loci, indicating that they had different copy numbers of the rDNA repetitive unit. A great number of minor loci were detected on two or more pairs of homologous chromosomes (Fig. 1E).
ITS sequences in Thinopyrum ponticum
The sequences of the five positive clones selected at random among over 1000 positive clones were very similar. Three were sequenced completely. The entire ITS region in Th. ponticum, including both non-coding spacers (ITS1 and ITS2) and the 5·8S rDNA, was 605 base pairs (bp). The ITS1 region was 221 bp, and the ITS2 region was 220 bp. The 5·8S subunit was 164 bp. The other two segments that were successfully sequenced were 530 bp, including the entire ITS1 and 5·8S regions and partial ITS2. These sequences were aligned by the Clustal X program to compare their homogeneity. Results showed that they were almost identical. Only one base ‘g’ was substituted by ‘c’ at position 80 in one of these sequences. The phenomenon could be explained as a point mutation, sequencing or amplification error. These sequences have been nearly homogeneous, suggesting that the ITS repeats have undergone concerted evolution. The sequence reported in this paper has been deposited in the GenBank database (Accession no.: AY090768).
Restriction site analysis
If PCR products contain different sequences, restriction enzymes will cut at heterogeneous sites, and the sum of the sizes of the restriction fragments will be greater than the entire length of the undigested ITS fragment. In this study, restriction site analysis of ITS PCR fragments showed that the sum of the sizes of restriction fragments was equal to the size of the initial amplified fragment itself in each enzyme-cutting reaction. Thus, the ITS repeats in Th. ponticum and its three related diploid species have been homogeneous, at least at the restriction sites detected in this study.
Phylogenetic analysis
The phylogenetic tree inferred from ITS sequences of Th. ponticum and its related diploid species is shown in Fig. 2. Given the controversy concerning the potential distortion induced by species of hybrid-origin allopolyploids in cladistic analyses (McDade, 1995), the tree was compared with the monogenomic species trees of Hsiao et al. (1995). Figure 2 shows that the topological relationships among the diploid species were not changed after the introduction of the polyploid species, Th. ponticum. The two diploid species conveying the E genome, Th. bessarabicum (Eb) and Th. elongatum (Ee), formed a clade. The other two diploid species that conveyed the St genome, Ps. spicata and Ps. libantica, formed another clade. Th. ponticum was grouped with Th. bessarabicum (Eb) and Th. elongatum (Ee), suggesting the ITS region of the rDNA in Th. ponticum has been homogenized or concerted to the E genome type.
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DISCUSSION |
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Most studies have shown that much variability exists for 18S-5·8S-26S rDNA loci among species, subspecies, populations and even individuals (Sánchez-Gea et al., 2000). In most alloployploid plants, the number of 18S-5·8S-26S rDNA loci equals the sum of that of their progenitors (Wang and Zhang, 2000). However, loss of some loci has been observed in several alloployploid species (Vaughan et al., 1993; Leggett and Markand, 1995; Snowdon et al., 1997). Th. ponticum is a decaploid, containing ten genomes in its somatic cells. The related diploid species (Th. bessarabicum, Th. elongatum and Ps. stipifolia) have the same number of 18S-5·8S-26S rDNA loci, with two loci per genome. The expectation is that the decaploid species would have 20 loci if the number of major 18S-5·8S-26S rDNA loci has remained stable during the evolutionary process. The maximum number of major loci detected in Th. ponticum using the present method was 20, which is the sum of that of its progenitors. However, all of the rDNA loci of Th. ponticum were located on the terminal regions of short arms (Fig. 1D and E). The interstitial loci have apparently been ‘lost’. These results suggest that there has been distinct differentiation between Th. ponticum and its diploid relatives during the evolutionary process. It is impossible to determine directly the ancient ancestor(s) of Th. ponticum. The interstitial position is probably an ancestral trait whereas the terminal position is probably derived (Dubcovsky and Dvoøák, 1995). Therefore, the distribution pattern of rDNA loci in the ancient ancestor may be similar to that of the modern diploid species, and it may possess a similar type of interstitial locus. During polyploidization of Th. ponticum, all of the interstitial loci have been deleted or migrated, and ‘novel’ loci have been positioned on terminal regions of the chromosomes. A similar phenomenon has been observed in other species (Gill and Apples, 1988; Dubcovsky and Dvoøák, 1995), though the exact mechanism for this is still unknown. According to studies of comparative linkage maps, Dubcovsky and Dvoøák (1995) suggested that the loci might change position via dispersion of minor loci without structural rearrangements of chromosomes. In addition to the 20 major loci, many minor loci were detected in our study (Fig. 1D), supporting the suggestion of Dubcovsky and Dvoøák (1995). The positional changes of 18S-5·8S-26S rDNA loci between Th. ponticum and its candidate donors indicated that it is almost impossible to find a genome in the polyploid species that is completely identical to that of its diploid donor.
Hybridization between different species with subsequent polyploidization is a prominent process in the evolution of higher plants (Masterson, 1994). In the evolution of polyploid species, concerted evolution plays an essential role in the maintenance of sequence homogeneity in multigene families through inter-chromosomal interactions (Zhang and Sang, 1999). A remarkable example of concerted evolution in plants is cotton (Gossypium). Sequence data from the ITS regions have indicated that rDNA arrays are homogeneous or nearly homogeneous in all five allotetraploids (AD genome) and their diploid progenitors (A genome and D genome). In four tetraploids, the 18S-5·8S-26S rDNA was homogenized to the D genome repeat type, but in the other tetraploid it was concerted to the A genome repeat type, although both A and D conveyed the major rDNA loci in the tetraploid (Wendel et al., 1995; Hanson et al., 1996; Wendel, 2000). Inter-locus homogenization of alternative rDNA repeat types (concerted evolution) has also been reported in other polyploid species such as Microseris, Paeonia and Saxifraga (Wendel, 2000). This phenomenon may be common during polyploidization (Wendel, 2000). Unequal crossing over (unequal exchange) and gene conversion are possible mechanisms for concerted evolution (Zhang and Sang, 1999; Booy et al., 2000; Wendel, 2000).
Physical mapping of rDNA can increase understanding of concerted evolution (Zhang and Sang, 1999). The 5S rDNAs of cotton were located on the interstitial regions of chromosomes. Their sequence homogeneity was very low, indicating a high rate of polymorphism among species and even individuals. Inter-chromosomal exchanges may be facilitated in taxa such as Gossypium and Paeonia by the terminal or near-terminal location of the rDNA loci, which may permit unequal crossing-over without deleterious recombination among non-homologous chromosomes (Zhang and Sang, 1999; Wendel, 2000). The terminal positions of the 18S-5·8S-26S rDNA and the loss of its interstitial loci found in our study suggest that concerted evolution of 18S-5·8S-26S rDNA probably occurred, and the sequences may have become homologous or nearly homogeneous.
Generally, rDNA repeated units are considered to be homogenous as a result of concerted evolution (Dover, 1982, 1989; Dvoøák, 1990; Booy et al., 2000). However, heterogeneity of ITS regions within individuals of some species has been reported (Booy et al., 2000). Such heterogeneity may occur if concerted evolution does not occur quickly enough or if it fails to homogenize rDNA repeated units as a result of recent hybridization, the development of pseudogenes, the large number of rDNA loci, the occurrence of asexual reproduction, and so on (Dover, 1982; Wendel et al., 1995; Zhang and Sang, 1999: Booy et al., 2000; Wendel, 2000).
Although the distribution of major rDNA loci in Th. ponticum implied that concerted evolution may have occurred, the possibility of heterogeneity also exists. The sequence data of the five ITS region clones and the results of restriction site analysis showed high levels of homogeneity, with most of the ITS sequences in Th. ponticum being identical. An alternative explanation for sequence homogeneity is that rDNA loci of one or more genomes have been lost following allopolyploid speciation (Wendel et al., 1995). We can ignore this possibility because the FISH results in our study showed that the number of major rDNA loci in Th. ponticum was the sum of that of its progenitors. Thus, we conclude that the homogeneity of the ITS within Th. ponticum genomes is mainly the result of concerted evolution. The phylogenetic tree suggests that the ITS region of the rDNA in Th. ponticum has been homogenized or concerted to the E genome type. The sequences of the St genome type have possibly been changed into or ‘overwritten’ by those of the E genome type through concerted evolution.
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ACKNOWLEDGEMENTS |
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We thank Dr Douglas Johnson, Dr Steve Larson and Dr Kevin Jensen for their comments on the manuscript. We thank Dr Xiangqi Zhang and Mr Xinming Yang for providing pTa71 and seeds of Th. elongatum (Z1371), respectively; and Mr Chao Wang, Ms Yanyan Ru and Ms Wenhua Yang for help with phylogenetic analysis and technical assistance. This research was supported by the Natural Science Foundation of China (No. 3970494) to X.Y.Z.
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LITERATURE CITED |
|---|
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-
Belyayev A, Raskina O, Korol A, Nevo E. 2000. Coevolution of A and B genomes in allotetraploid Triticum dicoccoides. Genome 43:1021–1026.[Medline]
Booy G, Van der Schoot J, Vosman B. 2000. Heterogeneity of the internal transcribed spacer 1 (ITS1) in Tulipa (Liliaceae). Plant Systematics and Evolution 225: 29–41.[CrossRef]
Chen Q, Conner RL, Laroche A, Thomas JB. 1998. Genome analysis of Thinopyrum intermedium and Thinopyrum ponticum using genomic in situ hybridization. Genome 41: 580–586.[Medline]
Dewey DR. 1984. The genomic system of classification as a guide to intergeneric hybridization with the perennial Triticeae. Stadler Genetic Symposium 16: 209–279.
Dover G. 1982. Molecular drive: a cohesive mode of species evolution. Nature 299: 111–117.[CrossRef][Medline]
Dover G. 1989. Linkage disequilibrium and molecular drive in the rDNA family. Genetics 122: 249–252.
Dubcovsky J, Dvorák J. 1995. Ribosomal RNA multigene loci: nomads of the Triticeae genomes. Genetics 140: 1367–1377.[Abstract]
Dvorák J. 1990. Evolution of multigene families: the ribosomal RNA loci of wheat and related species. In: Brown AHD, Klegg MT, Kahler AL, Weir BS. ed. Plant population genetics, breeding, and genetic resources. Sunderland, MA: Sinauer Associates, 83–97.
Elder JF Jr, Turner BJ. 1994. Concerted evolution at the population level: pupfish Hind III satellite DNA sequences. Proceeding of the National Academy of Sciences of the USA 91: 994–998.
Fedak G, Chen Q, Conner RL, Laroche A, Petroski R, Armstrong KW. 2000. Characterization of wheat-Thinopyrum partial amphi ploids by meiotic analysis and genomic in situ hybridization. Genome 43: 712–719.[Medline]
Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791.[CrossRef][Web of Science]
Felsenstein J. 1997. PHYLP: phylogenetic inference package, version 3.572c. Seattle: University of Washington.
Gerlach WL, Bedbrook JR. 1979. Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acid Research 7: 1869–1885
Gill BS, Apples R. 1988. Relationships between Nor-loci from different Triticeae species. Plant Systematics and Evolution 160: 77–89.
Hanson RE, Islam-Faridi MN, Percival EA, Crane CF, Ji Y, Mcknight TD, Stelly DM, Price HJ. 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.[Web of Science][Medline]
Heslop-Harrison JS, Schwarzacher T. 1996. Genomic southern and in situ hybridization for plant genome analysis. In: Jauhar PP, ed. Methods of genome analysis in plants. Boca Raton, London, Tokyo: CRC Press, 163–179.
Hsiao C, Chatterton NJ, Asay KH, Jensen KB. 1994. Phylogenetic relationships of 10 grass species: an assessment of phylogenetic utility of the internal transcribed spacer region in nuclear ribosomal DNA in monocots. Genome 37: 112–120.
Hsiao C, Chatterton NJ, Asay KH, Jensen KB. 1995. Phylogenetic relationships of the monogenomic species of the wheat Tribe, Triticeae (Poaceae), inferred from the nuclear rDNA (ITS) sequences. Genome 38: 211–223.[Medline]
Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111–120.[CrossRef][Web of Science][Medline]
Kosina R, Heslop-Harrison JS. 1996. Molecular cytogenetics of an amphiploid trigeneric hybrid between Triticum durum, Thinopyrum distichum and Lophopyrum elongatum. Annals of Botany 78: 583–589.
Leggett JM, Markand GS. 1995. The genomic identification of some monosomics of Avena sativa L. cv. Sun II using genomic in situ hybridization. Genome 38: 747–751.
Leitch AR, Schwarzacher T, Jackson D, Leitch IJ. 1994. In situ hybridization: a practical guide. London: BIOS Scientific Publications Ltd.
McDade LA. 1995. Hybridization and phylogenetics. In: Hoch PC, Stephenson AG, ed. Experimental and molecular approaches to plant biosystematics. St Louis, MO: Missouri Botanical Garden, 305–331.
McIntosh RA, Hart GE, Gale MD. 1993. Catalogue of gene symbols for wheat. In: Li ZS, Xin ZY, ed. Proceedings of the 8th International Wheat Genetic Symposium. Beijing: China Agricultural Scientech Press, 1333–1500.
Maluszynsky J, Heslop-Harrison JS. 1993. Physical mapping of rDNA loci in Brassica species. Genome 36: 774–781.
Masterson J. 1994. Stomatal size in fossil plants: evidence for polyploidy in majority of angiosperm. Science 264: 421–424.
Muramatsu M. 1990. Cytogenetics of decaploid Agropyron elongatum (Elytrigia elongatum) (2n=70). I. Frequency of decavalent formation. Genome 33: 811–817.
Petersen G, Seberg O. 1996. ITS regions highly conserved in cultivated barleys. Euphytica 90: 233–234.[CrossRef]
Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406–425.[Abstract]
Sánchez-Gea JF, 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.[Medline]
Sang T, Crawford DJ, Stuessy TF. 1995. Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution. Proceedings of the National Academy of Sciences of the USA 92: 6813–6817.
Sang Y, Liang GH. 2000. Comparative physical mapping of the 18S-5·8S-26S rDNA in three sorghum species. Genome 43: 918–922.[CrossRef]
Schmidt T, Heslop-Harrison JS. 1998. Genomes, genes and junk: the large-scale organization of plant chromosomes. Trends in Plant Science 3: 195–199.[CrossRef][Web of Science]
Sharpe PJ, Chao S, Desai S, Gale MD. 1989. This isolation, characterization and application in the Triticeae of a set of wheat RFLP probes identifying each homologous chromosome arm. Theoretical and Applied Genetics 78: 342–348.
Snowdon RJ, Köhler W, Köhler A. 1997. Chromosomal localization and characterization of rDNA loci in the Brassica A and C genomes. Genome 40: 582–587.
Snowdon RJ, Friedt W, Köhler A, Köhler W. 2000. Molecular cytogenetic localization and characterization of 5S and 25S rDNA loci for chromosome identification in oilseed rape (Brassica napus L.). Annals of Botany 86: 201–204.
Thompson JD, Gibson TJ, Plewniak F. 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Research 25: 4876–4882.
Vaughan HE, Jamilena M, Ruiz Rejon C, Parker JS, Garrido-Ramos MA. 1993. Loss of nucleolus-organizer regions during polyploid evolution in Scilla autumnalis. Heredity 71: 574–580.
Wang JB, Zhang WJ. 2000. Concerted evolution of nuclear rDNA in alloployploid plants. Hereditas (Beijing) 22: 54–56.
Wang JB, Wang C, Shi SH, Zhong Y. 2000. Evolution of parental ITS regions of nuclear rDNA in allopolyploid Aegilops (Poaceae) species. Hereditas 133: 265–270.[CrossRef]
Wang RR-C, Marburger JE, Hu CJ. 1991. Tissue culture-facilitated production of aneuploid haploid Thinopyrum ponticum and amphiploid Hordeum violaceum x H. bogdenii and their use in phylogenetic studies. Theoretical and Applied Genetics 81: 151–156.[CrossRef]
Wang RR-C, Bothemer R, Dvorák J, Fedak G, O’Donoughue, Amstrong KC. 1995. Genome symbols in the Triticeae (Poaceae). In: Wang RR-C, ed. Proceedings of the Second International Triticeae Symposium, Logan, UT, 20–24 June 1994. Logan: Utah State University Publication Design and Production, Utah State University, 29–34.
Waters ER, Schaal BA. 1996. Biased gene conversion is not occurring among rDNA repeats in the Brassica triangle. Genome 39: 150–154.
Wendel JF. 2000. Genome evolution in polyploids. Plant Molecular Biology 42: 225–249.[CrossRef][Web of Science][Medline]
Wendel JF, Schnabel A, Seelanan T. 1995. Bi-directional interlocus concerted evolution following alloploid speciation in cotton (Gossypium). Proceedings of the National Academy of Sciences of the USA 92: 280–284.
Zhang D, Sang T. 1999. Physical mapping of ribosomal RNA genes in peonies (Paeonia, Paeoniaceae) by fluorescent in situ hybridization: implications for phylogeny and concerted evolution. American Journal of Botany 86: 735–740.
Zhang XY, Dong YS, Wang RR-C. 1996a. Characterization of genomes and chromosomes in partial amphiploids of the hybrids of Triticum aestivum x Thinopyrum ponticum by in situ hybridization, isozyme analysis, and RAPD. Genome 39: 1062–1071.
Zhang XY, Koul A, Petrosk R, Quellet J, Fedak G, Dong YS, Wang, RR-C. 1996b. Molecular verification and characterization of BYDV-resistant germplasms derived from hybrids of wheat with Thinopyrum ponticum and Th. intermedium. Theoretical and Applied Genetics 93: 1033–1039.[CrossRef]
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