Nicotine in floral nectar pharmacologically influences bumblebee learning of floral features.

Many plants defend themselves against herbivores by chemical deterrents in their tissues and the presence of such substances in floral nectar means that pollinators often encounter them when foraging. The effect of such substances on the foraging behaviour of pollinators is poorly understood. Using artificial flowers in tightly-controlled laboratory settings, we examined the effects of the alkaloid nicotine on bumblebee foraging performance. We found that bumblebees confronted simultaneously with two equally rewarded nicotine-containing and nicotine-free flower types are deterred only by unnaturally high nicotine concentrations. This deterrence disappears or even turns into attraction at lower nectar-relevant concentrations. The alkaloid has profound effects on learning in a dose-dependent manner. At a high natural dose, bees learn the colour of a nicotine-containing flower type more swiftly than a flower type with the same caloric value but without nicotine. Furthermore, after experiencing flowers containing nicotine in any tested concentration, increasing numbers of bumblebees stay more faithful to these flowers, even if they become a suboptimal choice in terms of reward. These results demonstrate that alkaloids enhance pollinator flower constancy, opening new perspectives in co-evolutionary process between plants and pollinators.

Silenced rRNA genes are activated and substitute for partially eliminated active homeologs in the recently formed allotetraploid, Tragopogon mirus (Asteraceae).

To study the relationship between uniparental rDNA (encoding 18S, 5.8S and 26S ribosomal RNA) silencing (nucleolar dominance) and rRNA gene dosage, we studied a recently emerged (within the last 80 years) allotetraploid Tragopogon mirus (2n=24), formed from the diploid progenitors T. dubius (2n=12, D-genome donor) and T. porrifolius (2n=12, P-genome donor). Here, we used molecular, cytogenetic and genomic approaches to analyse rRNA gene activity in two sibling T. mirus plants (33A and 33B) with widely different rRNA gene dosages. Plant 33B had ~400 rRNA genes at the D-genome locus, which is typical for T. mirus, accounting for ~25% of total rDNA. We observed characteristic expression dominance of T. dubius-origin genes in all organs. Its sister plant 33A harboured a homozygous macrodeletion that reduced the number of T. dubius-origin genes to about 70 copies (~4% of total rDNA). It showed biparental rDNA expression in root, flower and callus, but not in leaf where D-genome rDNA dominance was maintained. There was upregulation of minor rDNA variants in some tissues. The RNA polymerase I promoters of reactivated T. porrifolius-origin rRNA genes showed reduced DNA methylation, mainly at symmetrical CG and CHG nucleotide motifs. We hypothesise that active, decondensed rDNA units are most likely to be deleted via recombination. The silenced homeologs could be used as a 'first reserve' to ameliorate mutational damage and contribute to evolutionary success of polyploids. Deletion and reactivation cycles may lead to bidirectional homogenisation of rRNA arrays in the long term.

Ecological and genetic factors linked to contrasting genome dynamics in seed plants.

The large-scale replacement of gymnosperms by angiosperms in many ecological niches over time and the huge disparity in species numbers have led scientists to explore factors (e.g. polyploidy, developmental systems, floral evolution) that may have contributed to the astonishing rise of angiosperm diversity. Here, we explore genomic and ecological factors influencing seed plant genomes. This is timely given the recent surge in genomic data. We compare and contrast the genomic structure and evolution of angiosperms and gymnosperms and find that angiosperm genomes are more dynamic and diverse, particularly amongst the herbaceous species. Gymnosperms typically have reduced frequencies of a number of processes (e.g. polyploidy) that have shaped the genomes of other vascular plants and have alternative mechanisms to suppress genome dynamism (e.g. epigenetics and activity of transposable elements). Furthermore, the presence of several characters in angiosperms (e.g. herbaceous habit, short minimum generation time) has enabled them to exploit new niches and to be viable with small population sizes, where the power of genetic drift can outweigh that of selection. Together these processes have led to increased rates of genetic divergence and faster fixation times of variation in many angiosperms compared with gymnosperms.

Mobilization of retrotransposons in synthetic allotetraploid tobacco.

Allopolyploidy is a major driving force in plant evolution and can induce rapid structural changes in the hybrid genome. As major components of plant genomes, transposable elements are involved in these changes. In a previous work, we observed turnover of retrotransposon insertions in natural allotretraploid tobacco (Nicotiana tabacum). Here, we studied the early stages of allopolyploid formation by monitoring changes at retrotransposon insertion sites in the Th37 synthetic tobacco. We used sequence-specific amplification polymorphism (SSAP) to study insertion patterns of two populations of the Tnt1 retrotransposon in Th37 S4 generation plants, and characterized the nature of polymorphic insertion sites. We observed significant amplification of young Tnt1 populations. Newly transposed copies were amplified from maternal elements and were highly similar to Tnt1A tobacco copies amplified in response to microbial factors. A high proportion of paternal SSAP bands were not transmitted to the hybrid, corresponding to various rearrangements at paternal insertion sites, including indels or the complete loss of the Tnt1/flanking junction. These data indicate that major changes, such as retrotransposon amplification and molecular restructuring in or around insertion sites, occur rapidly in response to allopolyploidy.

Making a functional diploid: from polysomic to disomic inheritance.

One little understood feature of polyploid speciation is the transition from polysomic to disomic inheritance, and much recent attention has focused on the role of pairing genes in this process. Using computer simulations we studied the effects of mutations, chromosomal inversions, chiasma, neofunctionalization, subfunctionalization and selection on the evolution of disomic inheritance in a polyploid over 10 000 generations. We show that: the evolution of pairing genes is not essential for the establishment of disomic inheritance, as genetic drift, coupled with a threshold for homologue pairing fidelity, is sufficient to explain the transition from polysomic to disomic inheritance; high rates of recombination increase the number of generations required for disomic inheritance to become established; both neofunctionalization and subfunctionalization speed up the transition to disomic inheritance. The data suggest that during polyploid species establishment, selection will favour reduced chiasma number and/or more focused distribution. The data also suggest a new role for subfunctionalization in that it can drive disomic inheritance. The evolution of subfunctionalization in genes across the genome will then act to maintain genes in syntenic blocks and may explain why such regions are so highly conserved.

The asymmetric meiosis in pentaploid dogroses (Rosa sect. Caninae) is associated with a skewed distribution of rRNA gene families in the gametes.

In pentaploid dogroses, Rosa section Caninae (2n=5x=35), the pollen transmits one basic genome (x=7) derived from the seven segregating bivalents, whereas the egg transmits four basic genomes (4x=28) one set derived from the segregation of seven bivalents and three sets of univalent-forming chromosomes. Chromosomes from all five genomes carry 18-5.8-26S nuclear ribosomal DNA (rDNA) sites. This mode of sexual reproduction, known as permanent odd polyploidy, can potentially lead to the independent evolution of rDNA on bivalent- and univalent-forming chromosomes. To test this hypothesis, we analyzed rRNA gene families in pollen and somatic leaf tissue of R. canina, R. rubiginosa and R. dumalis. Six major rRNA gene families (alpha, beta, beta' gamma, delta and epsilon) were identified based on several highly polymorphic sites in the internal transcribed spacers (ITSs). At least two of the major rRNA gene families were found in each species indicating that rDNAs have not been homogenized across subgenomes. A comparison of ITS1 sequences from leaf and pollen showed differences: the shared beta rRNA gene family was more abundant among pollen clones compared to leaf clones and must constitute a major part of the rDNA loci on bivalent-forming chromosomes. The gamma and delta families were underrepresented in pollen genomes and are probably located predominantly (or solely) on the univalents. The results support the hypothesis that pentaploid dogroses inherited a bivalent-forming genome from a common proto-canina ancestor, a likely donor of the beta rDNA family. Allopolyploidy with distantly related species is likely to have driven evolution of Rosa section Caninae.

Genomic plasticity and the diversity of polyploid plants.

Polyploidy, a change whereby the entire chromosome set is multiplied, arises through mitotic or meiotic misdivisions and frequently involves unreduced gametes and interspecific hybridization. The success of newly formed angiosperm polyploids is partly attributable to their highly plastic genome structure, as manifested by tolerance to changing chromosome numbers (aneuploidy and polyploidy), genome size, (retro)transposable element mobility, insertions, deletions, and epigenome restructuring. The ability to withstand large-scale changes, frequently within one or a few generations, is associated with a restructuring of the transcriptome, metabolome, and proteome and can result in an altered phenotype and ecology. Thus, polyploid-induced changes can generate individuals that are able to exploit new niches or to outcompete progenitor species. This process has been a major driving force behind the divergence of the angiosperms and their biodiversity.

The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae).

BACKGROUND: In studies looking at individual polyploid species, the most common patterns of genomic change are that either genome size in the polyploid is additive (i.e. the sum of parental genome donors) or there is evidence of genome downsizing. Reports showing an increase in genome size are rare. In a large-scale analysis of 3008 species, genome downsizing was shown to be a widespread biological response to polyploidy. Polyploidy in the genus Nicotiana (Solanaceae) is common with approx. 40 % of the approx. 75 species being allotetraploid. Recent advances in understanding phylogenetic relationships of Nicotiana species and dating polyploid formation enable a temporal dimension to be added to the analysis of genome size evolution in these polyploids. METHODS: Genome sizes were measured in 18 species of Nicotiana (nine diploids and nine polyploids) ranging in age from <200,000 years to approx. 4.5 Myr old, to determine the direction and extent of genome size change following polyploidy. These data were combined with data from genomic in situ hybridization and increasing amounts of information on sequence composition in Nicotiana to provide insights into the molecular basis of genome size changes. KEY RESULTS AND CONCLUSIONS: By comparing the expected genome size of the polyploid (based on summing the genome size of species identified as either a parent or most closely related to the diploid progenitors) with the observed genome size, four polyploids showed genome downsizing and five showed increases. There was no discernable pattern in the direction of genome size change with age of polyploids, although with increasing age the amount of genome size change increased. In older polyploids (approx. 4.5 million years old) the increase in genome size was associated with loss of detectable genomic in situ hybridization signal, whereas some hybridization signal was still detected in species exhibiting genome downsizing. The possible significance of these results is discussed.

Evolutionary implications of permanent odd polyploidy in the stable sexual, pentaploid of Rosa canina L.

In Rosa canina (2n = 5x = 35), the pollen and ovular parents contribute, respectively, seven and 28 chromosomes to the zygote. At meiosis I, 14 chromosomes form seven bivalents and 21 chromosomes remain as univalents. Fluorescent in situ hybridization to mitotic and pollen mother cells (PMC) of R. canina showed that 10 chromosomes (two per genome) carry ribosomal DNA (rDNA) loci. Five chromosomes carry terminal 18S-5.8S-26S rDNA loci; three of these also carry paracentric 5S rDNA loci and were designated as marker chromosomes 1. Five chromosomes carry only 5S rDNA loci and three of these were designated as marker chromosomes 2. The remaining four of the 10 chromosomes with rDNA loci were individually identifiable by the type and relative sizes of their rDNA loci and were numbered separately. At PMC meiosis, two marker chromosomes 1 and two marker chromosomes 2 formed bivalents, whereas the others were unpaired. In a gynogenetic haploid of R. canina (n = 4x = 28), obtained after pollination with gamma-irradiated pollen, chromosomes at meiosis I in PMC remained predominantly unpaired. The data indicate only one pair of truly homologous genomes in R. canina. The 21 unpaired chromosomes probably remain as univalents through multiple generations and do not recombine. The long-term evolutionary consequence for the univalents is likely to be genetic degradation through accumulated mutational change as in the mammalian Y chromosome and chromosomes of asexual species. But there is no indication that univalents carry degenerate 5S rDNA families. This may point to a recent evolution of the R. canina meiotic system.

Preferential elimination of repeated DNA sequences from the paternal, Nicotiana tomentosiformis genome donor of a synthetic, allotetraploid tobacco.

Nicotiana tabacum (tobacco, 2n = 4x = 48) is a natural allotetraploid combining two ancestral genomes closely related to modern Nicotiana sylvestris and Nicotiana tomentosiformis. Here we examine the immediate consequences of allopolyploidy on genome evolution using 20 S4-generation plants derived from a single synthetic, S0 plant made by Burk in 1973 (Th37). Using molecular and cytogenetic methods we analysed 14 middle and highly repetitive sequences that together total approximately 4% of the genome. Two repeats related to endogenous geminiviruses (GRD5) and pararetroviruses (NtoEPRV), and two classes of satellite repeats (NTRS, A1/A2) were partially or completely eliminated at variable frequency (25-60%). These sequences are all from the N. tomentosiformis parent. Genomic in situ hybridization revealed additivity in chromosome numbers in two plants (2n = 48), while a third was aneuploid for an N. tomentosiformis-origin chromosome (2n = 49). Two plants had homozygous translocations between chromosomes of the S- and T-genomes. * The data demonstrate that genetic changes in synthetic tobacco were fast, targeted to the paternal N. tomentosiformis-donated genome, and some of the changes showed concordance with changes that presumably occurred during evolution of natural tobacco.

Molecular cytogenetics and tandem repeat sequence evolution in the allopolyploid Nicotiana rustica compared with diploid progenitors N. paniculata and N. undulata.

Nicotiana rustica (2n = 4x = 48) is a natural allotetraploid composed of P and U genomes which are closely related to genomes of diploid species N. paniculata and N. undulata. Genomic in situ hybridization (GISH) also confirms that the diploid parents, or close relatives, are the ancestors of N. rustica. In order to study genetic interactions between ancestral genomes in the allotetraploid, we isolated three families of repetitive sequences, two from N. paniculata (NPAMBE and NPAMBO) and one from N. undulata (NUNSSP). Southern blot hybridization revealed that the sequences are digested with a range of restriction enzymes into regular ladder patterns indicating a tandem arrangement of high copy repeats possessing monomeric units of about 180 bp. The three-tandem sequences belong to a larger Nicotiana tandem repeat family called here the HRS-60 family. Members of this family are found in all Nicotiana species studied. Fluorescence in situ hybridization (FISH) analysis localized the satellite repeats to subtelomeric regions of most chromosomes of N. paniculata and N. undulata. The pattern of sequence distribution on the P- and U-genomes of N. rustica was similar to the putative parents N. paniculata and N. undulata respectively. However, NPAMBO repeats appear to be reduced and rearranged in N. rustica that may suggest evolution within the P genome. GISH and FISH with the tandem repeat probes failed to reveal intergenomic translocations as might be predicted from the nucleocytoplasmic interaction hypothesis.

Rapid concerted evolution of nuclear ribosomal DNA in two Tragopogon allopolyploids of recent and recurrent origin.

We investigated concerted evolution of rRNA genes in multiple populations of Tragopogon mirus and T. miscellus, two allotetraploids that formed recurrently within the last 80 years following the introduction of three diploids (T. dubius, T. pratensis, and T. porrifolius) from Europe to North America. Using the earliest herbarium specimens of the allotetraploids (1949 and 1953) to represent the genomic condition near the time of polyploidization, we found that the parental rDNA repeats were inherited in roughly equal numbers. In contrast, in most present-day populations of both tetraploids, the rDNA of T. dubius origin is reduced and may occupy as little as 5% of total rDNA in some individuals. However, in two populations of T. mirus the repeats of T. dubius origin outnumber the repeats of the second diploid parent (T. porrifolius), indicating bidirectional concerted evolution within a single species. In plants of T. miscellus having a low rDNA contribution from T. dubius, the rDNA of T. dubius was nonetheless expressed. We have apparently caught homogenization of rDNA repeats (concerted evolution) in the act, although it has not proceeded to completion in any allopolyploid population yet examined.

Dynamic changes in the distribution of a satellite homologous to intergenic 26-18S rDNA spacer in the evolution of Nicotiana.

An approximately 135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentosiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as an independent satellite (termed an A1/A2 satellite) outside of rDNA loci in species of Nicotiana section Tomentosae. The chromosomal location, patterns of genomic dispersion, and copy numbers of its tandemly arranged units varied between the species. In more distantly related Nicotiana species the A1/A2 repeats were found only at the nucleolar organizer regions (NOR). There was a trend toward the elimination of the A1/A2 satellite in N. tabacum (tobacco), an allotetraploid with parents closely related to the diploids N. sylvestris and N. tomentosiformis. This process may have already commenced in an S(3) generation of synthetic tobacco. Cytosine residues in the IGS were significantly hypomethylated compared with the A1/A2 satellite. There was no clear separation between the IGS and satellite fractions in sequence analysis of individual clones and we found no evidence for CG suppression. Taken together the data indicate a dynamic nature of the A1/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions.

The origin and evolution of geminivirus-related DNA sequences in Nicotiana.

A horizontal transmission of a geminiviral DNA sequence, into the germ line of an ancestral Nicotiana, gave rise to multiple repeats of geminivirus-related DNA, GRD, in the genome. We follow GRD evolution in Nicotiana tabacum (tobacco), an allotetraploid, and its diploid relatives, and show GRDs are derived from begomoviruses. GRDs occur in two families: the GRD5 family's ancestor integrated into the common ancestor of three diploid species, Nicotiana kawakamii, Nicotiana tomentosa and Nicotiana tomentosiformis, on homeologous group 4 chromosomes. The GRD3 family was acquired more recently on chromosome 2 in a lineage of N. tomentosiformis, the paternal ancestor of tobacco. Both GRD families include individual members that are methylated and diverged. Using relative rates of synonymous and nonsynonymous nucleotide substitutions, we tested for evidence of selection on GRD units and found none within the GRD3 and GRD5 families. However, the substitutions between GRD3 and GRD5 do show a significant excess of synonymous changes, suggesting purifying selection and hence a period of autonomous evolution between GRD3 and GRD5 integration. We observe in the GRD3 family, features of Helitrons, a major new class of putative rolling-circle replicating eukaryotic transposon, not found in the GRD5 family or geminiviruses. We speculate that the second integration event, resulting in the GRD3 family, involved a free-living geminivirus, a Helitron and perhaps also GRD5. Thus our data point towards recurrent dynamic interplay between geminivirus and plant DNA in evolution.

Ribosomal DNA evolution and gene conversion in Nicotiana rustica.

Genomic in situ hybridisation was used to confirm that Nicotiana rustica (2n=4x=48) is an allotetraploid between N. paniculata (2n=2x=24, maternal P-genome donor) and N. undulata (2n=2x=24, paternal U-genome donor), their progenitors or species closely related to them. Fluorescent in situ hybridisation showed that N. paniculata has one 5S and two 18-5.8-26S rDNA loci whereas N. undulata has an additional 18-5.8-26S rDNA locus. N. rustica has the sum of the loci found in these putative parents. The sizes of the 18-5.8-26S rDNA loci indicate that the number of rDNA units on the U-genome chromosomes has amplified; perhaps this is associated with a concomitant reduction in the number of units on P-genome chromosomes. Restriction fragment length polymorphism analysis of the intergenic spacer (IGS) of the 18-5.8-26S rDNA units in N. rustica and the two progenitor diploids revealed that about 80% of IGS sequences in N. rustica are of an N. undulata type and 20% of N. paniculata type. These data indicate that interlocus sequence homogenisation has caused the replacement of many N. paniculata-type IGSs in N. rustica with an N. undulata-type of sequence. It is probable that subsequent to this replacement there has been sequence divergence at the 5' end of the IGS. As in tobacco, an allotetraploid between N. sylvestris and N. tomentosiformis, the direction of the IGS interlocus conversion is towards the paternal genome donor.

Evolution of 5S rDNA unit arrays in the plant genus Nicotiana (Solanaceae).

Nicotiana tabacum (tobacco, Solanaceae) has two 5S ribosomal DNA (rDNA) families, one of unit length approximately 646 bp and the other -430 bp, that differ in the length of the 5S rDNA non-transcribed spacer (NTS). The long 5S rDNA family, found on the T genome of tobacco and in Nicotiana tomentosiformis, contains a GC-rich subregion that is absent in the short family. We designed primers for this subregion and generated a probe that we used against a range of Nicotiana and related Solanaceous species. We demonstrated the presence of the GC-rich subregion in a range of Nicotiana species, but it was absent in Nicotiana sylvestris, Nicotiana longiflora, and two closely related genera, Petunia and Solanum. These data suggest that this subregion of the NTS is likely to have evolved with the genus Nicotiana. The absence of the subregion in N. sylvestris and N. longiflora is likely to have arisen by a deletion event in the evolution of section alatae. We demonstrate patterns of evolution in the 5S rDNA unit cluster in relation to a phylogenetic reconstruction of species relationships in section tomentosae. Nicotiana glutinosa diverged early from the section and contains a 5S rDNA family based on a 550-bp unit. After this divergence, 430- and 650-bp rDNA unit families evolved. The 650-bp family is found in all species of tomentosae (except N. glutinosa) and in tobacco. The 430-bp family within tomentosae includes the GC-rich subregion and is thus unrelated to the 430-bp family in N. sylvestris. Nicotiana setchellii is unusual in that it has three 5S rDNA loci, including one locus that is exceptionally large. This species, unique to tomentosae, has a very abundant 900-bp unit family. It is possible that this 900-bp family occurs on this one large locus. In N. tomentosa and N. kawakamii, the 650-bp family is predominant, whereas N. tomentosiformis and N. otophora have only the 650-bp family. There is no clear relationship between the number of 5S families and the number of 5S rDNA loci. Certainly the replacement of 5S rDNA units, perhaps by gene conversion, has occurred repeatedly in the evolution of genus Nicotiana.

Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species.

Nicotiana tabacum (tobacco) is an allotetraploid derived from ancestors of the modern diploids, N. sylvestris and N. tomentosiformis. We identified and characterized two distinct families of 5S ribosomal DNA (rDNA) in N. tabacum; one family had an average 431 bp unit length and the other a 646 bp unit length. In the diploid species, N. sylvestris and N. tomentosiformis, the 5S rDNA unit lengths are 431 bp and 644 bp respectively. The non-coding spacer sequence of the short unit in tobacco had high sequence homology to the spacer of N. sylvestris5S rDNA, while the longer spacer of tobacco had high homology with the 5S spacer of N. tomentosiformis. This suggests that the two 5S families in tobacco have their origin in the diploid ancestors. The longer spacer sequence had a GC rich sub-region (called the T-genome sub-region) that was absent in the short spacer. Pulsed field gel analysis and fluorescent in situ hybridization to tobacco metaphase chromosomes showed that the two families of 5S rDNA units are spatially separate at two chromosomal loci, on chromosomes S8 (short family) and T8 (long family). The repeat copy number at each chromosomal locus showed heterogeneity between different tobacco cultivars, with a tendency for a decrease in the copy number of one family to be compensated by an increase in the copy number of the second family. Sequence analysis reveals there is as much diversity in 5S family units within the diploid species as there is within the T and S-genome 5S family units respectively, suggesting 5S diversification within each family had occurred before tobacco speciation. There is no evidence of interlocus homogenization of the two 5S families in tobacco. This is therefore substantially different to 18-26S rDNA where interlocus gene conversion has substantially influenced most sequences of S and T genome origin; possible reasons are discussed.

Loss and recovery of Arabidopsis-type telomere repeat sequences 5'-(TTTAGGG)(n)-3' in the evolution of a major radiation of flowering plants.

Fluorescent in situ hybridization and Southern blotting were used for showing the predominant absence of the Arabidopsis-type telomere repeat sequence (TRS) 5'-(TTTAGGG)(n)-3' (the 'typical' telomere) in a monocot clade which comprises up to 6300 species within Asparagales. Initially, two apparently disparate genera that lacked the typical telomere were identified. Here, we used the new angiosperm phylogenetic classification for predicting in which other related families such telomeres might have been lost. Our data revealed that 16 species in 12 families of Asparagales lacked typical telomeres. Phylogenetically, these were clustered in a derived clade, thereby enabling us to predict that the typical telomere was lost, probably as a single evolutionary event, following the divergence of Doryanthaceae ca. 80--90 million years ago. This result illustrates the predictive value of the new phylogeny, as the pattern of species lacking the typical telomere would be considered randomly placed against many previous angiosperm taxonomies. Possible mechanisms by which chromosome end maintenance could have evolved in this group of plants are discussed. Surprisingly, one genus, Ornithogalum (Hyacinthaceae), which is central to the group of plants that have lost the typical telomere, appears to have regained the sequences. The mechanism(s) by which such recovery may have occurred is unknown, but possibilities include horizontal gene transfer and sequence reamplification.

Comparative analysis of DNA methylation in tobacco heterochromatic sequences.

Cytosine methylation levels and susceptibility to drug-induced hypomethylation have been studied in several Nicotiana tabacum (tobacco) DNA repetitive sequences. It has been shown using HapII, MspI, BamHI and Sau3AI methylation-sensitive restriction enzymes that the degree of 5'-mCmCG-3' methylation varied significantly between different repeats. There were almost saturation levels of 5-methylcytosine at the inner (3') cytosine position and variable degrees of methylation at the outer (5') cytosine at the enzyme recognition sites. The non-transcribed high copy satellite sequences (HRS60, GRS) displayed significant heterogeneity in methylation of their basic units while middle repetitive sequences (R8.1, GRD5, 5S rDNA) were more uniformly modified at both cytosine residues. Dihydroxypropyladenine (DHPA) treatment, which is thought to reduce DNA methyltransferase activity by increasing S-adenosylhomocysteine levels, resulted in extensive demethylation of the outer cytosine in all repeats, and the partial hypomethylation of cytosines at the inner positions in less densely methylated repeats such as HRS60 and GRS. The results suggest that hypomethylation of 5'-mCmCG-3' sites with DHPA is a gradual non-random process proceeding in the direction mCmCG-->CmCG-->CCG. The 18S-5.8S-25S rDNA was remarkably hypomethylated relative to the 5S rDNA at all restriction sites studied. Fluorescence in-situ hybridization showed that DNA decondensation within and between the 18S-5.8S-25S and 5S rDNA loci was variable in different nuclei. All nuclei had condensed and decondensed sequence. The chromatin of 18S-5.8S-25S rDNA was more readily digested with micrococcal nuclease than the 5S rDNA suggesting that the overall levels of decondensation were higher for 18S-5.8S-25S rDNA. Variable decondensation patterns within and between loci were also observed for GRS and HRS60. Cytosine methylation of the tobacco repeats is discussed with respect to transcription, overall levels of condensation and overall structure.

Molecular cytogenetic analyses and phylogenetic studies in the Nicotiana section Tomentosae.

Phylogenetic schemes based on changing DNA sequence have made a major impact on our understanding of evolutionary relationships and significantly built on knowledge gained by morphological and anatomical studies. Here we present another approach to phylogeny, using fluorescent in situ hybridisation. The phylogenetic scheme presented is likely to be robust since it is derived from the chromosomal distribution of ten repetitive sequences with different functions and evolutionary constraints [GRS, HRS60, NTRS, the Arabidopsis-type telomere repeat (TTTAGGG)n, 18S-5.8S-26S ribosomal DNA (rDNA), 5S rDNA, and four classes of geminiviral-related DNA (GRD)]. The basic karyotypes of all the plant species investigated Nicotiana tomentosiformis, N. kawakamii, N. tomentosa, N. otophora, N. setchellii, N. glutinosa (all section Tomentosae), and N. tabacum (tobacco, section Genuinae) are similar (x=12) but the distribution of genic and non-genic repeats is quite variable, making the karyotypes distinct. We found sequence dispersal, and locus gain, amplification and loss, all within the regular framework of the basic genomic structure. We predict that the GRD classes of sequence integrated into an ancestral genome only once in the evolution of section Tomentosae and thereafter spread by vertical transmission and speciation into four species. Since GRD is similar to a transgenic construct that was inserted into the N. tabacum genome, its fate over evolutionary time is interesting in the context of the debate on genetically modified organisms and the escape of genes into the wild. Nicotiana tabacum is thought to be an allotetraploid between presumed progenitors of N. sylvestris (maternal, S-genome donor) and a member of section Tomentosae (T-genome donor). Of section Tomentosae, N. tomentosiformis has the most similar genome to the T genome of tobacco and is therefore the most likely paternal genome donor. It is known for N. tabacum that gene conversion has converted most 18S-5.8S-26S rDNA units of N. sylvestris origin into units of an N. tomentosiformis type. Clearly if such a phenomenon were widespread across the genome, genomic in situ hybridisation (GISH) to distinguish the S and T genomes would probably not work since conversion would tend to homogenise the genomes. The fact that GISH does work suggests a limited role for gene conversion in the evolution of N. tabacum.