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Genetics |
2School of Biological and Chemical Sciences, Queen Mary, University of London, E1 4NS, UK; 3Institute of Biophysics, Brno, Czech Republic; 4Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK
Received for publication July 29, 2005. Accepted for publication March 6, 2006.
ABSTRACT
Polyploids have significantly influenced angiosperm evolution. Understanding the genetic consequences of polyploidy is advanced by studies on synthetic allopolyploids that mimic natural species. In Nicotiana, Burk (1973)
and Kostoff (1938)
generated synthetic tobacco (N. tabacum) using the parents 
N. sylvestris x 
N. tomentosiformis. We previously reported rapid genetic changes in the Burk material. Kostoff's material has 24 chromosomes of N. sylvestris origin (S-genome), 24 of N. tomentosiformis origin (T-genome), and a large intergenomic translocation, but not an additive distribution of ribosomal DNA (rDNA) families as expected from the parental contribution. Our new synthetic tobacco lines TR1 and TR2 are chromosomally balanced with no intergenomic translocations and are either sterile or have highly reduced fertility, supporting the nuclear cytoplasmic hypothesis that allopolyploid fertility is enhanced by intergenomic translocations. Two plants of TR1 (TR1-A, TR1-B) have the expected number, structure, and chromosomal distribution of rDNA families, in contrast to Burk's and Kostoff's synthetic tobaccos and to synthetic polyploids of Arabidopsis. Perhaps allopolyploids must pass through meiosis before genetic changes involving rDNA become apparent, or the genetic changes may occur stochastically in different synthetic allopolyploids. The lack of fertility in the first generation of our synthetic tobacco lines may have uses in biopharmacy.
Key Words: chromosomes • evolution • fertility • polyploidy • ribosomal DNA • synthetic tobacco
Polyploidy is a major driving force in flowering plant evolution, with up to 30–80% of species considered polyploid based on chromosome counts (Leitch and Bennett, 1997
). Genome sequencing projects are revealing that even apparently diploid species are in fact cryptic paleopolyploids (Lagercrantz and Lydiate, 1996
; Vision et al., 2000
; Bowers et al., 2003
). The realization of the scale of polyploidy in terms of the number of plant species, their influence on ecosystems (e.g., Tragopogon and Spartina allopolyploids), and their importance for breeding of many important crop plants (e.g., wheat, cotton, soybean, maize, rice, and banana) has elevated interest in polyploid research (Soltis and Soltis, 1995
; Wendel, 2000
; Levy and Feldman, 2002
; Liu and Wendel, 2003
; Osborn et al., 2003
; Soltis et al., 2004
). There is considerable interest in the early events associated with polyploidy, particularly with regard to the stabilization of the de novo polyploid genome and the establishment of populations of polyploid individuals. To address these questions, synthetic polyploids that mimic natural species have been constructed, and the early genetic events recorded. These analyses shed light on why polyploid species are so successful and why polyploidy is a recurrent phenomenon in angiosperm evolution.
Here we present genetic analyses of three artificial allotetraploid lines that closely resemble Nicotiana tabacum (tobacco). The materials studied are a tobacco line (TH7) made by Kostoff (1938)
, which has hitherto not been investigated in depth (Kostoff, 1938
) and another two synthesized by us. The "recreation" of allopolyploid species has also been successfully carried out in other taxa, notably in recent years to synthesize polyploid Triticum (Ozkan et al., 2001
; Shaked et al., 2001
), Gossypium (Liu et al., 2001
), Brassica (Song et al., 1995
; Osborn et al., 2003
) and Arabidopsis (Chen et al., 2004
; Pontes et al., 2004
). We compare our data with those from other synthetic allopolyploids.
Genetic analyses of allopolyploids have yielded some surprising and often contradictory data. In Burk's synthetic tobacco (TH37 line) (Burk, 1973
), novel rDNA loci were associated with the amplification of a novel family of 35S rDNA and conversion of many thousands of rDNA units to this new family (Skalicka et al., 2003
). Likewise, in some synthetic Arabidopsis allopolyploids, changes in the number and distribution of rDNA loci were observed (Pontes et al., 2004
). Rapid, reproducible and non-random elimination of amplified fragment length polymorphisms (AFLPs) are reported in synthetic hybrids and allopolyploids of Triticeae (Ozkan et al., 2001
; Shaked et al., 2001
). This is associated with gene silencing and activation, including elevated retroelement activity (Kashkush et al., 2002
, 2003
). In synthetic Arabidopsis allopolyploids, there is evidence for allopolyploid-induced changes in the transcriptome with different progenitors' genes being expressed in plants both between and within generations (Wang et al., 2004
). Song et al. (1995)
reported both losses and gains of restriction fragment length polymorphisms (RFLPs) in early generations of artificial allotetraploid Brassica napus; the greatest changes occurred in the paternally derived genome. Likewise, Skalicka et al. (2005)
reported in Burk's synthetic tobacco a loss of tandem and dispersed repetitive sequences "targeted" at the paternally derived genome from N. tomentosiformis (Skalicka et al., 2005
). These studies point to rapid genetic change in early allopolyploid generations. However in contrast to all these studies, Liu et al. (2001)
found no changes in AFLP patterns in synthetic allopolyploids of Gossypium, and many natural polyploids appear to be the sum of the parents, when examined at a cytological level (Kovarik et al., 2004
; Lim et al., 2004a
, b
; Melayah et al., 2004
; Pires et al., 2004
). There is now a need to understand whether a reported change is stochastic or directed. Such changes can be studied in similar, but independent synthetic allopolyploids, as done in this report.
In 1973, Burk constructed his artificial tobacco (TH37) using the progenitor species N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). The derived S4 seeds are available and were intensively studied by us (Skalicka et al., 2003
, 2005
; Kovarik et al., 2004
; Lim et al., 2004b
). We have evidence for at least two independent lineages of N. tomentosiformis that differ in the distribution of a tandem repeat (known as NTRS) and in the occurrence of geminivirus-related DNA repeat sequences called GRD3 family sequences (Murad et al., 2002
). Natural tobacco evolved from one of these distinct N. tomentosiformis lineages. Unfortunately Burk (1973)
was unaware of the distinction and almost certainly used a different lineage of N. tomentosiformis. There are no records of the N. tomentosiformis accession used to make the Kostoff line. The hybrids and derived synthetic tobacco lines we report here uses a N. tomentosiformis cultivar that is highly similar to the T-genome found in naturally occurring tobacco. We show an absence of genetic change in the new hybrids and synthetic allopolyploids, in contrast to both Burk's and Kostoff's synthetic tobacco. By comparing the independently synthesized tobacco lines, we attempt to discriminate between those events associated with de novo polyploidy that appear directed and those events that are stochastic.
MATERIALS AND METHODS
Plant material
F1 hybrid 4A-7 is from cross N. sylvestris ‘Salzburg' (Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany) x N. tomentosiformis NIC 479/84 (Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany) and chromosomally doubled with oryzalin via tissue culture to produce the new synthetic Nicotiana tabacum TR2. F1 hybrid 1A-4 is from a cross N. sylvestris ‘Seita' (Institut du Tabac, Bergerac, France) x N. tomentosiformis NIC 479/84 (Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany) that was chromosomally doubled with oryzalin via tissue culture to produce synthetic Nicotiana tabacum TR1-A and TR1-B. Nicotiana tabacum cv. Kostoff (TH7) is from the USDA (North Carolina State University, USA) seed bank.
Making F1 hybrids and synthetic tobacco
N. sylvestris x N. tomentosiformis F1 hybrids were made by pollination with a paintbrush, and the seeds produced were sterilized with 10% (v/v) bleach in water for 10 min and plated onto agar. The number of seeds per capsule was variable and in most cases less than normal. The fertility of seeds was close to 95%. Young seedlings were transferred to pots and grown. From N. sylvestris ‘Salzburg' x N. tomentosiformis cross, 64 F1 plants were obtained; from N. sylvestris ‘Seita' x N. tomentosiformis 58 plants were produced.
Nodal segments of 4A-7 and 1A-4 were collected from glasshouse-grown plants and used for in vitro culture. They were sterilized with 0.5% (w/v) sodium dichloroisocyanurate and a drop of Tween-20 for 30 min, washed three times with sterile water, and cultured on semi-solid Murashige–Skoog (MS) salts and vitamins (Murashige and Skoog, 1962
). Leaves were collected from the developed shoot cultures after 4 weeks, trimmed to 2 cm x 1 cm discs, and treated with oryzalin by culturing on basal MS medium containing 3% sucrose and 5 µM oryzalin in a 90 mm petri dish for 1, 2, 3, and 7 d. Treated leaf discs were transferred to fresh MS medium containing 3% sucrose and 0.5 µM thidiazuron (TDZ). Cultures were kept at 23 ± 2°C on a 12/12 h photoperiod at 1500 lux. Adventitious buds developed on the leaves were removed once they grew to a length of about 0.5 cm long and subcultured on basal MS medium. Shoots were left in this medium until they grew to a length of at least 3 cm with three or more nodes. Nodal segments were collected from these plants and grown on half strength MS medium with 1 µM naphthalene acetic acid (NAA) for rooting. Fully developed plants were transferred to sterilized vermiculite in jars. Leaves from these plants were harvested, and the ploidy determined using propidium iodide stained nuclei and flow cytometry as described in Bennett et al. (2003)
.
Fluorescent in situ hybridization (FISH)
Root tips from pot-grown synthetic tobacco plants were pretreated with a saturated aqueous solution of Gammexane (hexachlorocyclohexane, Sigma, UK) for 4 h and fixed in 3 : 1 absolute ethanol to glacial acetic acid for 24 h. Chromosome squashes were prepared on glass slides following enzyme softening of material as described by Leitch et al. (2001)
. Fluorescent in situ hybridization (FISH) with cloned pTa71 against 35S rDNA (Gerlach and Bedbrook, 1979
) and genomic in situ hybridization (GISH) using total genomic DNA as probes was carried out as described in Lim et al. (1998)
. Briefly, chromosomes on slides were denatured in 70% formamide in 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate) at 70°C for 2 min. For GISH, we used 8 µg · mL–1 digoxigenin-11-dUTP labelled N. sylvestris total genomic DNA and 8 µg · mL–1 biotin-16-dUTP labelled N. tomentosiformis total genomic DNA. For FISH with cloned probes, we used 2 µg · ml–1 digoxigenin-11-dUTP labelled NTRS (Matyasek et al., 1997
) and 2 µg · ml–1 biotin-16-dUTP pTa71. The hybridization mix included 50% (v/v) formamide, 10% (w/v) dextran sulphate, and 0.1% (w/v) sodium dodecyl sulphate in 2x SSC. After overnight hybridization at 37°C, slides were washed in 20% (v/v) formamide in 0.1x SSC at 42°C at an estimated hybridization stringency of 80–85%. Sites of probe hybridization were detected using 20 µg · ml–1 fluorescein conjugated anti-digoxigenin IgG (Roche Biochemicals, UK) and 5 µg · ml–1 Cy3 conjugated avidin (Amersham Pharmacia Biotech, UK). Chromosomes were counterstained with 2 µg · mL–1 DAPI (4',6-diamidino-2-phenylindole) in 4x SSC, mounted in Vectashield (Vector Laboratories, California, USA) medium, examined using a Leica DMRA2 epifluorescent microscope, photographed with a Orca ER camera, and analyzed using Improvision Openlab software (UK). Images were processed for color balance, contrast, and brightness uniformity.
Restriction endonuclease digestion and Southern hybridization
Genomic DNA was extracted from young leaves using the cetylammonium method described previously (Saghai-Maroof et al., 1984
; Kovarik et al., 1994
). DNAs were digested to completion with excess restriction endonuclease and fractionated in 1% agarose by gel electrophoresis. DNA was transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech). Southern hybridization was carried out under high stringency conditions using a heat-denatured 32P-labelled rDNA probe that was a 220-bp fragment of the 26S rRNA gene subunit from N. tabacum obtained by PCR amplification of the 3' end region (details in Lim et al. [2000a
]). Labelling of DNA probes was carried out by a random primed method using 32P-dCTP (Dekaprime kit, Ambion, UK.
RESULTS
Making the synthetic hybrids and derived allopolyploids
We established 122 hybrid N. sylvestris (using ac. ‘Salzburg' or ‘Seita') x N. tomentosiformis plants. From these two, hybrids were selected that represented a ‘Salzburg' (cross 4A-7) and ‘Seita' (cross 1A-4) line, and successfully doubled using tissue-culture oryzalin treatment. Three allotetraploid plants were generated (plants TR1-A, TR1-B from cross 1A-4; TR2 from cross 4A-7).
Oryzalin-treated leaf discs of hybrid material produced 35–60 shoot buds after 2 weeks on a medium containing 0.5 µM TDZ. Only leaf sections treated with oryzalin for 2 days produced chromosomally doubled shoots. Leaves treated with oryzalin for 1 day yielded no doubled plants, whereas the leaf section treated with oryzalin for 3 and 7 days produced stunted shoots. The stunted shoots were slow growing and failed to produce any quality shoot cultures for ploidy analyses. There were morphological differences between the hybrids and the oryzalin-treated, genome-doubled plants while in tissue culture. The leaves of the doubled plants had a length to breadth ratio smaller than the hybrids, and the leaf tips were more rounded. In addition, the leaves were greener and appeared healthier.
Morphology of F1 hybrids and derived allopolyploids
All F1 hybrids were vigorous and had morphological characters of both parental species. The flowers resembled Burk's (1973) material and some wild collected tobacco with their soft-pink coloration. All synthetic materials were perennial rather than annual as observed in most tobacco cultivars, and all had collapsed anthers without pollen. In doubled, synthetic allotetraploids TR1-A (Fig. 1), TR1-B, and TR2, plants were more robust, and, although their anthers were full of pollen, the grains were collapsed. However, DAPI-stained fixed pollen grains looked normal, and germination was low (<5%) on sucrose media (data not shown). We attempted 50–100 pollinations, both selfings and crosses between plants, but failed to produce viable seeds.
|
) in the long arm of some chromosomes, and these label strongly by GISH (red) in some preparations (depending on amount of DNA loss during DNA denaturation). When this signal is overlaid with DAPI fluorescence (blue), the fluorescence is pink, although the less strongly labelled regions without the satellite repeats stain less brightly and appear purple.
|
; Skalicka et al., 2003
; Kovarik et al., 2004
). The major band of N. tabacum had a different mobility from the parental bands, consistent with gene conversion of parental rDNA units in tobacco (Volkov et al., 1999
; Lim et al., 2000b
). In N. sylvestris ‘Seita,' all three BstNI bands were of similar intensity, whereas N. sylvestris ‘Salzburg' had a marked reduction of the 3.1- and 2.7-kb bands and a concomitant increase in the 2.4-band (Fig. 3A). These changes keep the copy number of rDNA approximately the same in ‘Seita' and ‘Salzburg' (Fig. 3A). Thus, there are quantitative differences in the proportion of rRNA gene families among different N. sylvestris accessions. In the F1 diploid and derived polyploids, parental bands were faithfully inherited, and no qualitative and quantitative changes were observed.
|
, and the chromosomes were named according to similar translocations observed in other tobacco accessions/cultivars.
We analyzed 10 TH7 plants by Southern blot hybridization (Fig. 3B). Genomic DNAs were digested with BstNI and hybridized as described previously. The TH7 plants had rDNA units similar to those from N. sylvestris, N. tomentosiformis, and natural N. tabacum. No novel bands were found to indicate an absence of major changes at the unit sequence level. However, there were qualitative changes. The bands inherited from both diploid species were relatively faint; of these the 2.4-kb band was most prominent and the 2.7-kb band of N. sylvestris origin may have been lost. In all TH7 plants, the 4.0-kb band was predominant, accounting for more than 60% of total rDNA signal, suggesting that most rDNA units of TH7 are of a tobacco type. Direct DNA sequencing of PCR amplified internally transcribed spacer (ITS) regions of the rDNA unit gave two products that differed at 24 positions that correspond to differences between ITS sequences of N. sylvestris and N. tomentosifomis (Chase et al., 2003
). There were consistent major and minor peaks; the major ones were identical to N. tomentosiformis, and the alternative sequence using the minor peaks was identical to that of N. sylvestris. The reduced number of copies of N. sylvestris type of sequence is consistent with the loss of two thirds of the expected number of N. sylvestris type of rDNA units, as seen in Southern analyses (Fig. 3B).
FISH with pTa71 for 35S rDNA to metaphases of TH7 revealed four pairs of rDNA-carrying chromosomes (Fig. 2C–E). These sites occurred on chromosomes T3 of N. tomentosiformis origin and chromosomes S10, S11, and S12 of N. sylvestris origin, as expected (Fig. 2E), but one of the S-genome chromosomes (S11) is unusual in nature. The 35S rDNA locus is split in two by the insertion of a chromatin segment that does not bear rDNA. The rDNA locus is partially decondensed on each side of the chromatin insertion, indicating either transcriptional activity or the potential for transcriptional activity (Fig. 2F, G).
GISH reveals the genetic nature of the rDNA units at each locus. The discrimination presumably arises by differential labelling of the genomic probes to the variable regions of the rDNA locus, especially the IGS. All rDNA loci label orange (indicating N. tomentosiformis origin) except for two sites distal to the chromatin insertion on each chromosome S11 that labels yellow/green (indicating N. sylvestris origin) (Fig. 2G).
DISCUSSION
The data from our synthetic allopolyploids, as well as from the Burk (1973)
and Kostoff (1938)
allopolyploids, did not reveal any common trends in the genetic changes. These polyploids differ in the accessions used as parents, and this may be important in controlling or influencing genetic outcome. Only in synthetic allopolyploids of Triticum are there reports of reproducible and nonrandom genetic change associated with allopolyploidy. These changes involve coding and noncoding sequences that are both chromosome- and genome-specific (Ozkan et al., 2001;
Shaked et al., 2001
). However, comparing de novo allopolyploids of different genera reveals no common patterns that might be used to predict outcome. Perhaps the genetic events that do occur are simply stochastic, or perhaps outcomes occur over widely different time frames so that many generations are needed before common trends become apparent. Whichever argument is true, some allopolyploids can show dramatic genetic changes within a few generations, e.g., in synthetic allopolyploids of Triticum (Ozkan et al., 2001
; Shaked et al., 2001
; Kashkush et al., 2002
), Arabidopsis (Pontes et al., 2003
), and Nicotiana (Skalicka et al., 2003
, 2005
). One of the reasons the allopolyploids are thought to be so successful in nature is that they can benefit from allelic diversity in the gene pools of both parents if there are multiple, recurrent allopolyploid origins (Soltis and Soltis, 2000
). Clearly, if allopolyploidy also stimulates genetic change, then this might also be a major source of variation upon which selection can act.
Generation of synthetic tobacco and intergenomic translocations
The three allotetraploid plants generated in this study (TR1-A, TR1-B from cross 1A-4; TR2 from cross 4A-7) appeared sterile, although there was some pollen germination on sucrose media and the generative and vegetative nuclei looked normal with DAPI staining (data not shown). Because the synthetic material is perennial, repeated attempts will be made to obtain an F2 generation using the methods of Burk (1973)
.
GISH using N. sylvestris- and N. tomentosiformis-genomic DNA labels chromosomes according to their genomic origin. The F1 hybrids analyzed (4A-7, 1A-4) had 12 chromosomes from each parent as expected. The derived synthetic allopolyploids (TR1-A, TR1-B, TR2) all had the expected 24 chromosomes from each parent. It may be significant that this synthetic material has no intergenomic translocations and is sterile. It has been proposed that the generation of fertile diploid hybrids requires recombination between homologous chromosome sets (Rieseberg et al., 1996
). Gill (1991)
proposed in his nucleocytoplasmic interaction (NCI) hypothesis that there were adverse interactions between the paternal genome and maternal cytoplasm in newly formed allopolyploid. He suggested that a "bottleneck of sterility" (p48) is overcome by species-specific translocations. Potentially, these translocations can influence absolute and relative gene expression through altered gene activities. Certainly in wheat x rye hybrids, nucleolar activity at the 1R locus is influenced by the presence of particular chromosomes and chromosome arms, e.g., the presence of the long arm of chromosome 1R (Vieira et al., 1990
) and of chromosome 2R from rye (Neves et al., 1997
) inhibits activity of rDNA, on the short arm of chromosome 1R. Jiang and Gill (1994)
identified "species-specific" translocations in two tetraploid wheat species, and Leitch and Bennett (1997)
noted there were translocations common to two cultivars of tobacco (cvs. 095–55 and 35466). To our surprise, similar translocations were also observed in some of Burk's (1973) synthetic tobacco plants (Lim et al., 2004a
). Here we showed a pair of chromosomes with a large intergenomic translocation in the Kostoff tobacco (Fig. 2C–G), although in this case they may have been inherited from natural tobacco (see section "TH7 Kostoff allopolyploid).
TH7 (Kostoff allopolyploid)
The allopolyploid TH7 had 12 chromosomes of N. tomentosiformis origin and 12 of N. sylvestris origin as expected. It also carries three intergenomic translocations (Fig. 2E). One of these, S2t on chromosome S2, is similar to translocations found in all cultivated tobaccos and in two of three plants of Burk's synthetic material. The other translocations are on chromosome T7 (T7ss) and are found in two cultivars of tobacco that are not recorded elsewhere. It is possible that TH7 is not a synthetic allopolyploid N. sylvestris x N. tomentosiformis as reported (Kostoff, 1938
) but instead may have been created by crossing a synthetic allotetraploid or hybrid with normal tobacco. Further evidence for this hypothesis is from Southern hybridization data that use 35S rDNA as a probe and indicate additivity of signal assuming this cross (Fig. 3). Indeed Burk (1973)
had previously speculated that TH7 may be a hybrid with introgressed tobacco genomic DNA. However, an alternative and intriguing possibility is that a new family of rDNA unit has evolved and amplified in TH7. If so, this new family is the same size as in natural tobacco. Support for this hypothesis comes from F4 descendents of Burk's synthetic allotetraploid, which in many cases have new rDNA families (Skalicka et al., 2003
). In addition, a new family of rDNA units was amplified in N. plumbaginifolia addition lines that carry individual N. sylvestris chromosomes (Chen et al., 2002
).
Ribosomal DNA
All our allopolyploid material had the expected number of rDNA loci, with one site on T3 homologues and three sites on S10, S11, and S12 homologues. This pattern is additive with respect to parental contributions. Similar results were found in natural tobacco (Lim et al., 2000a
). There was no indication of variable numbers of rDNA loci in contrast to synthetic Arabidopsis suecica (Pontes et al., 2004
) or in Burk's synthetic tobacco line TH37 (Skalicka et al., 2003
). However, these studies involved allotetraploid plants of the second and fourth generations, respectively. It is possible that rearrangements of rDNA loci occur after the first meiosis and in subsequent generations. Pontes et al. (2004)
speculated that interchromosomal translocations, leading to loss or gain of rDNA loci, might be mediated by increased transposon activity, but in our previous study using the F1 hybrid material, we found no genetic change in two classes of pararetroviral sequences (Skalicka et al., 2005
), and preliminary results suggest no genetic activity in several retrotransposons of the Tnt family (M.-A. Grandbastien, l'Institut National de la Recherche Agronomique, Versailles, France, personal communication). However, these too could be activated by meiosis, and certainly we do find evidence for deletion of pararetroviral sequences in the fourth generation TH37 synthetic tobacco (Skalicka et al., 2005
). In contrast, there are reproducible genomic changes in F1s used to make synthetic wheat (Ozkan et al., 2001
).
The TH7 material did show deviations from additivity of rDNA. At the sequence level, units inherited from both parents have been largely eliminated and perhaps overwritten by those of a tobacco type. Whether the dominant tobacco-like rDNA units in TH7 amplified de novo and represent convergent evolution of rDNA modifications or were inherited from an unintentional cross to tobacco is unknown. Assuming that N. sylvestris has about three times the amount of rDNA compared to tobacco and N. tomentosiformis (Lim et al., 2000a) (Fig. 3B), it is clear that more than two thirds of N. sylvestris units are now lost in TH7. In contrast, Burk's synthetic tobacco does not carry IGS characteristic of tobacco, showed no changes to the IGS derived from N. sylvestris and had no evidence of intergenomic homogenization (between S and T genomes), although it did have intragenomic homogenization of rDNA families associated with T-genome loci (Skalicka et al., 2003
). Therefore in natural tobacco and synthetic allopolyploids (TR1A, TR1B, TR2, TH7, and TH37), there are different responses with regard to homogenization of IGS. Perhaps the presence of intergenomic homogenization of rDNA relates to the ages of the material (TR1A, TR1B, TR2—1 year without passage through meiosis; TH7-1938 and an unknown number of generations; TH37-1973 analyzed in generation 4). Certainly after c. 40 generations, there is evidence for concerted evolution of rDNA between parental genomes in natural Tragopogon allopolyploids (Kovarik et al., 2005).
The TH7 material at the cytogenetic level revealed an unusual rDNA distribution on putative chromosome S11. The rDNA units were split over two closely linked loci by a chromatin segment (Fig. 2F). Both linked loci were partially decondensed, indicating transcriptional activity or at least the potential for activity. GISH paints the rDNA loci in a characteristic color, indicative of an origin from either N. sylvestris or N. tomentosiformis (Fig. 2G). Thus the rDNA units in the distal chromosome region likely originated from, or were converted to, the N. sylvestris type. In contrast, the rDNA units in the proximal region likely originated from N. tabacum or N. tomentosiformis (Fig. 2E, G). Southern hybridization of BstNI-restricted DNA probed for 26S rDNA revealed that there is only one prominent (of three expected) band (2.4 kb) of N. sylvestris in TH7 (Fig. 3B). Therefore, the yellow/green locus on chromosome S11 is probably comprised of N. sylvestris units generating the 2.4-kb band. Previously, Chen et al. (2002)
, using N. sylvestris addition lines to N. plumbaginifolia, mapped this gene family to chromosome 11 (or 10) (chromosome 6 by their nomenclature).
The observation of either N.-tabacum-specific units or N.-tomentosiformis-origin units occurring on chromosome S11 in additional to N. sylvestris-type units in TH7 (Fig. 2G) can be explained by (1) insertion and/or amplification of these units at a new site on the N.-sylvestris-derived chromosome 11 or; (2) translocation of the rDNA locus from N. sylvestris chromosome 11 to tobacco chromosome S11 (which carries tobacco-type units; Lim et al. 2000b
). If the latter argument is correct, then the situation resembles N. tabacum and N. otophora hybrids in which N. otophora heterochromatin segments have been translocated to N. tabacum chromosomes (Gersel and Burns, 1967
; reviewed in Comai, 2000
). In some chromosomes, translocated repeats became unstable and amplified leading to abnormally large chromosomes. The S11 chromosome of TH7 was larger than chromosome 11 of N. sylvestris or S11 of N. tabacum, although its size does not reach that of the megachromosomes observed by Gersel and Burns (1967)
. Previously we speculated that intergenomic translocations might accelerate homogenization of rDNA units in Nicotiana genomes (Kovarik et al., 2004).
Uses and future directions
Attempts will be made to establish future generations from our synthetic allotetraploids. Nevertheless, the sterile material, its perennial habit, and ability to propagate clonally may have considerable uses. Tobacco is a particularly useful plant for biopharmacy (Powledge, 2001
; Ma et al., 2003
), including the manufacture of pharmaceutical drugs and industrial chemicals. One problem with growing genetically modified plants in field conditions is the potential of the spread of transgenes to the wild. First generation synthetic tobacco, with sterility or greatly reduced fertility, presents an opportunity to grow genetically modified plants without the potential of cross hybridization to wild or cultivated species.
FOOTNOTES
1 The authors thank Prof. V. Sisson for the supply of TH7 seeds, L. Hansen for assistance, and Dr I. Leitch for helpful comments on the manuscript. The work was supported by NERC and the Grant agency of the Czech Republic (grant no. 521/01/0775). 
6 Author for correspondence (a.r.leitch{at}qmw.ac.uk
)
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