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Genetics and Molecular Biology |
School of Biological Sciences, Queen Mary, University of London, London, E1 4NS, UK; Institute of Biophysics, Academy of Sciences of the Czech Republic, CZ-61265 Brno, Czech Republic
Received for publication August 28, 2001. Accepted for publication January 10, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: ancestors • evolution • geminivirus-related DNA • GRD • integration • Nicotiana • Solanaceae • tobacco
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Gerstel, 1963
; Goldberg, 1985; Bland, Matzinger, and Levings, 1985
; Shinshi et al., 1988; Sperisen, Ryals, and Meins, 1991
). The paternal ancestor, which donated the T genome to tobacco, is more controversial, with ancestors of N. tomentosiformis, N. otophora (both section Tomentosae, 2n = 2x = 24), or an introgression hybrid implicated (Kenton et al., 1993
; Parokonny and Kenton, 1995
).
From interspecific crossing experiments and analysis of flower morphology, Goodspeed (1954)
concluded that hybrids of N. sylvestris and N. tomentosiformis were the most similar to tobacco. However, Goodspeed (1954)
also noted that the hybrid N. sylvestris x N. tomentosiformis was female sterile, while N. sylvestris x N. otophora was not, and that the present-day distribution of N. sylvestris overlaps only with N. otophora. Gerstel (1960)
made crosses of tobacco with N. otophora and N. tomentosiformis and revealed more chromosome pairing between tobacco chromosomes and N. tomentosiformis, which suggests a closer homology of tobacco with N. tomentosiformis.
Riechers and Timko (1999)
examined the putrescine N-methyltransferase gene family in tobacco, N. tomentosiformis, and N. otophora and found that two of the gene members in tobacco have separate origins in the diploid species. Similar observations were obtained in amplified fragment length polymorphism (AFLP) studies of these species (Ren and Timko, 2001
). These data can be explained either by introgression of N. tomentosiformis and N. otophora in the formation of the T genome, or by the independent loss of sequences in the diploids after the formation of tobacco. Most isozyme (Sheen, 1972
; Gray et al., 1974) and sequence data suggest that N. tomentosiformis is the closest relative of the T genome. The randomly amplified polymorphic DNA (RAPD) sequence phylogeny of Bogani et al. (1997)
places N. tomentosiformis closer to tobacco than N. otophora. Furthermore, sequence data of 18S–5.8S–26S ribosomal DNA (rDNA) suggest that most units in tobacco are of the N. tomentosiformis type (Borisjuk et al., 1997
; Volkov et al., 1999
; Lim et al., 2000a
).
Using genomic in situ hybridization (GISH), the S and T genomes can be distinguished in metaphase spreads of tobacco. Kenton et al. (1993)
and Papp et al. (1996)
used total genomic DNA from N. otophora and N. tomentosiformis as a probe and concluded that genomic DNA from N. otophora gave the highest signal strength to the T-genome chromosomes of tobacco. However, no such distinction was observed by Lim et al. (2000b)
in their GISH experiments that also used nine cloned repetitive sequences as probes in fluorescent in situ hybridization (FISH) experiments to determine the ancestry of tobacco and phylogenetic relationships within section Tomentosae. The probes included variants of GRD (geminiviral-related DNA) and NTRS, both tandemly repeated sequences. The GRD itself is a family of repeats that arose by integration of a geminivirus rep gene into an ancestor within section Tomentosae (Ashby et al., 1997
). Lim et al. (2000b)
showed that the arrangement of repeats in N. tomentosiformis was the most similar to the T genome of tobacco while N. otophora had many differences and lacked both GRD and NTRS, which are both found in N. tomentosiformis and N. tabacum. However, there remained small but significant differences in the occurrence and distribution of two GRD classes (GRD3 and GRD53) in tobacco and in the N. tomentosiformis accession examined by Lim et al. (2000b)
.
In order to resolve the apparently contradictory data concerning the origin of the T genome of tobacco, we embarked on a molecular and cytological study of several N. tomentosiformis accessions. The most parsimonious explanation of the results is that tobacco formed after Tomentosae speciation and after divergence within N. tomentosiformis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DNA probes
(1) The following GRD probes isolated from N. tabacam (Bejarano et al., 1996
) were used for FISH and Southern hybridization experiments: GRD5 (1.45-kb monomeric units), GRD53 (1.33-kb monomeric units), and GRD3 (890-bp monomeric units). (2) NTRS is a tandem array of a 212–219 bp monomeric unit isolated from N. tabacum (Matyá
ek et al., 1997
) and used for FISH. Five different NTRS units cloned in pBluescript vector were randomly ligated and labelled for FISH. (3) GRS is a 180–182 bp tandem repeat (Gazdova et al., 1995
) in N. tomentosiformis, N. kawakamii, N. tomentosa, N. otophora, and N. setchellii (Parokonny and Kenton, 1995
; Lim et al., 2000b
) and was used for FISH. (4) The T-genome intergenic spacer of the 5S rDNA unit of N. tabacum was amplified from genomic DNA using primers (5SLF 5-CCT GGG AAT TCC TCG TGT T-3; 5SLR 5-TGC GTT AAA GCT T-3; Fulnecek et al., 2002
) and used in Southern hybridization experiments to determine DNA loading and GRD class lengths.
DNA extraction, restriction enzyme digestion, and Southern hybridization
DNA was extracted from young leaves or whole germinating plants using standard procedures (Saghai-Maroof et al., 1984
) with some modifications (Kova
ík et al., 1997
). The quantity and quality of DNA preparations were checked by absorbance at 260/280 nm. Purified DNA was digested with an excess of restriction endonucleases (5 U/µg DNA) overnight, and then size fractionated by standard agarose gel electrophoresis. Ethidium bromide-stained gels were photographed and blotted onto membranes (Hybond N+, Amersham Pharmacia Biotech., Buckinghamshire, UK) as described by Sambrook, Fritsch, and Maniatis (1989)
. Labelling of the DNA probes, hybridization, and detection of signal were performed as described in Kovaík et al. (1997)
. The Southern membrane used (Fig. 3) was stripped by the denaturation step (twice for 10 min each in 0.2 mol/L sodium hydroxide and 1% [mass/volume] sodium dodecyl sulphate) and reprobed three times.
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; database accession number U81301). The F1, R1, and R2 primers are sequenced as: F1: 5-ACT TAT CCT CAG TGT TCT C-3;
R1: 5-CAC CAA GTA AGT TGA GTG G-3;
R2: 5-GTG TTT CAA TTT TAA ATA GGC-3.
In situ hybridization
The cloned probes GRD5, GRD3, and GRD53 were labelled by nick translation with digoxigenin-11-dUTP (Roche Biochemicals, Sussex, UK) or biotin-16-dUTP (Sigma-Aldrich, Dorset, UK). Cell spreads were prepared according to the modifications in Lim et al. (1998) of standard procedures (Schwarzacher and Leitch, 1994). The in situ hybridization reactions were as described in Leitch et al. (1994) with a stringency of probe hybridization to allow sequences with 80–85% sequence identity to remain hybridized. Sites of hybridization of the digoxigenin labelled probes were detected using fluoresceinated anti-digoxigenin (Roche Biochemicals) and of biotin-labelled probes with Cy3 conjugated avidin (Amersham Pharmacia Biotech.). Chromosomes were counterstained with DAPI (4',6-diamidino-2-phenylindole), mounted in Vectashield (Vector Laboratories, Peterborough, UK) medium, examined using a Leitz Aristoplan epifluorescent microscope, and photographed using Fujicolor 400 color film. Photographs were scanned and adjusted using Adobe (Adobe Systems, Edinburgh, UK) photoshop. All images were treated for color contrast and brightness uniformly.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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showed that the distribution and occurrence of repeats in N. tomentosiformis ac. TW142 karyotype was similar, but not identical, to the T genome of N. tabacum cv. 095-55 (Fig. 1). The repeat NTRS labelled N. tomentosiformis ac. TW142 at two locations, one locus interstitial to the long arm of chromosome 3, and a second at the distal end of chromosome 6 (Lim et al., 2000b
). In N. tomentosiformis ac. NIC 479/84, described for the first time here, there is only one NTRS locus as in N. tabacum, occurring on chromosome 3 (Figs. 1, 2A, B).
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). This led to the suggestion that GRD3 arose with the formation of N. tabacum. However, we show here that N. tomentosiformis ac. NIC 479/84 also has a separate GRD3 site on chromosome 2. This locus is found interstitially on the long arm and proximal to a medium-sized GRS band. This is characteristic of chromosome T2 of tobacco (Figs. 1, 2F–H, I–K).
A third variant of GRD, called GRD53, was isolated from a tobacco lambda clone and is linked to GRD3 in tandem (Bejarano et al., 1996
). It has high sequence similarity to GRD3 and less to GRD5. In N. tabacum cv. 095-55, the GRD53 probe hybridizes in situ to the GRD3 locus on chromosome T2, the GRD5 locus on chromosome T4, and to dispersed sequences on chromosome T3 (Lim et al., 2000b
). Only this dispersed GRD53 on chromosome T3, which does not co-localize with any other GRD probes, is shown on the ideogram (Fig. 1). When GRD53 is used in situ to N. tomentosiformis ac. NIC 479/84, it labels strongly the GRD3 locus on chromosome 2 and weakly the GRD5 locus on chromosome 4 (Fig. 2C–E) as it does on chromosomes T2 and T4 of N. tabacum, respectively. The distribution of the probe GRS to N. tomentosiformis ac. NIC 479/84, ac. TW142, and tobacco is similar for chromosomes 3, 4, and 5 in N. tomentosiformis and for T3, T4, and T5 in tobacco (Figs. 1, 2I–K). Chromosome 2, which carries GRD3 in N. tomentosiformis ac. NIC 479/84, is linked to a GRS site that occurs on chromosome T2 of tobacco. A GRS site on chromosome 1 in both accessions of N. tomentosiformis is missing in N. tabacum cv. 095–55. Nicotiana tomentosiformis ac. NIC 479/84 also has an extra GRS site on chromosome 6 or 7 (based on size). Thus, molecular cytogenetics reveals that N. tomentosiformis ac. NIC 479/84 has the closest similaritiy to the T genome of tobacco with only minor differences in the distribution of GRS and GRD53 (Fig. 1).
Distribution and occurrence of GRD
The differences in the distribution and occurrence of GRD types in two accessions of N. tomentosiformis were analyzed by Southern hybridization and compared with two cultivars of tobacco. Southern hybridization of BamHI-restricted DNA against the GRD3 probe (Fig. 3A) revealed bands corresponding to monomers and dimers of GRD3 (0.89-kb monomer size) and GRD3
(truncated variants of GRD3 [Ashby et al. 1997
], 0.74-kb monomer size) in two cultivars of N. tabacum. In N. tomentosiformis ac. NIC 479/84, GRD3 and GRD3 monomers and multimers were also found as in N. tabacum (Fig. 3A). However, there was no signal in the lane loaded with DNA from N. tomentosiformis ac. TW142 and N. sylvestris (Fig. 3A) as previously published (Ashby et al., 1997
). There is sequence similarity between GRD53 and units of GRD3 and GRD5. When GRD53 is used as a probe in Southern hybridization experiments, the GRD3 but not GRD3 bands are labelled, with additional weak labelling of the GRD5 bands (Fig. 3C). There is not a unique GRD53 monomeric band, although there is a putative GRD53 dimeric band; however, this size category cannot be distingushed from a GRD3 trimer (both approximately 2.6 kb Fig. 3C). The GRD53 probe does not label DNA from N. tomentosiformis ac. TW142 nor N. sylvestris, as expected from previous work (Ashby et al., 1997
). All cultivars of N. tabacum and all accessions of N. tomentosiformis analyzed carry GRD5, and the restriction profiles were similar, generating monomers and dimers of GRD5 and the truncated variant GRD5 (Fig. 3B).
Because Southern hybridization does not generate an unequivocal GRD53 band, PCR primers were designed to specifically amplify the sequence from genomic DNA. One forward and two reverse primers were used to generate predicted GRD53 fragment sizes of 840 and 415 bp, respectively. Four cultivars of tobacco, the tobacco cell culture TBY-2 and N. tomentosiformis ac. NIC 479/84, ac.W0062-001, ac.W0062-002 and ac.TW142, and N. sylvestris were analyzed by PCR. Only the N. tabacum cultivars and TBY-2, and N. tomentosiformis ac. NIC 479/84 generated PCR bands of the expected lengths (Figs. 4, 5). The absence of a GRD53 and a GRD3 product in the other accessions Nicotiana tomentosiformis is consistent with past work (Ashby et al., 1997
; Lim et al., 2000b
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Gerstel, 1960
; Sheen, 1972
; Gray et al., 1974; Kenton et al., 1993
; Parokonny and Kenton, 1995
; Reichers and Timko, 1999
; Lim et al., 2000b
; Ren and Timko, 2001
). Lim et al. (2000b)
showed that the occurrence and distribution of nine tandem repeats in the karyotype of N. otophora was widely different from the T genome of tobacco, while N. tomentosiformis was very similar. The even closer similarity with N. tomentosiformis ac. NIC 479/84 reported here makes it even less likely that an ancestor of N. otophora was the parent as suggested from GISH analysis (Kenton et al., 1993
; Parokonny and Kenton, 1995
) and some crossing experiments (Gerstel, 1960
). The possiblity that N. otophora has partly contributed to the T genome of N. tabacum, perhaps via hybrids with tobacco or with the ancient N. tomentosiformis parent before tobacco allopolyploidy, cannot be ruled out entirely. However, if this did occur, no sign of such an event is detected in the distribution and occurrence of known repetitive sequences. What is clear, however, is that paternity studies in tobacco should use N. tomentosiformis cv. NIC 479/84 rather than any other accession.
Understanding the genetic consequences of plant polyploidy is important, since it is likely that polyploidy is a major force in plant evolution (cf. Leitch and Bennett, 1997
). Recent data is contradictory; newly synthesized Triticeae hybrids show rapid genome change (Ozkan, Levy, and Feldman, 2001
) while the reverse is observed for synthesised Gossypium hybrids (Liu et al., 2001
). Much work is required to resolve the differences. Lim et al. (2000b)
attributed the small genomic differences observed between N. tomenotosifomis ac. TW142 and the T genome of tobacco to have occurred in N. tabacum during or after tobacco's formation. For example, the sequences GRD3 and GRD53 were thought to be unique to tobacco, but we show here that this is not the case since they are present in one accession of N. tomentosiformis and absent in others. Therefore GRD3 and GRD53 probably appeared de novo in certain populations of N. tomentosiformis plants, either as a second independent viral integration event or perhaps through sequence translocation and amplification associated with sequence modification. Whichever happened, N. tomentosiformis plants from this population probably gave rise to tobacco.
It will be interesting to know if N. tabacum has a single or multiple allopolyploid origin. Multiple origins of Tragopogon allopolyploids are known to have occurred since the introduction of diploid Tragopogon species into America about 80 yr ago (Soltis and Soltis, 2000
). Tobacco is an ancient allopolyploid, perhaps up to 6 million years old (Okamuro and Goldberg, 1985
), and by using GRD3, GRD53, and NTRS in assays we can search for evidence for multiple origins in tobacco as well.
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FOOTNOTES |
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4 Author for reprints requests (A.R.Leitch{at}qmul.ac.uk
; Tel: +44 (0)20 7882 5294; Fax: +44 (0)20 8983 0973)
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bejarano E. R. A. Khashoggi M. Witty C. P. Lichtenstein 1996 Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution. Proceedings of National Academy of Sciences, USA 93: 759-764
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Fulnecek J. K. Y. Lim A. R. Leitch A. Kovaík R. Matyáek 2002 Characterization of two families of 5S rDNA in allotetraploid Nicotiana tabacum show a lack of genetic interaction between parental loci. Heredity 88: 19-25[CrossRef][ISI][Medline]
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Liu B. C. L. Brubaker G. Mergeai R. C. Cronn J. F. Wendel 2001 Polyploid formation in cotton is not accompanied by rapid genomic changes. Genome 44: 321-330[Medline]
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