Species-specific evolution of telomeric and rDNA repeats in the tobacco composite genome

In order to investigate possible interactions between parental genomes in the composite genome of Nicotiana tabacum we have analyzed the organization of telomeric (TTTAGGG)_n and ribosomal gene (rDNA) repeats in the progenitor genomes Nicotiana sylvestris and Nicotiana tomentosiformis or Nicotiana otophora. Telomeric arrays in the Nicotiana species tested are heterogeneous in length ranging from 20 to 200 kb in N. sylvestris, from 20 to 50 kb in N. tomentosiformis, from 15 to 100kb in N. otophora, and from 40 to 160kb in N. tabacum. The patterns of rDNA repeats (18S, 5.8S, 25S RNA) appeared to be highly homogeneous and speciesspecific; no parental rDNA units corresponding to N. sylvestris, N. tomentosiformis or N. otophora were found in the genome of N. tabacum by Southern hybridization. The results provide evidence for a species-specific evolution of telomeric and ribosomal repeats in the tobacco composite genome.     

Comparison of the mitochondrial genome of Nicotiana tabacum with its progenitor species

Summary Mitochondrial DNAs from Nicotiana tabacum, an amphiploid, and its putative progenitor species, N. sylvestris and N. tomentosiformis were compared in structure and organization. By using DNA transfer techniques and cloned fragments of known genes from maize and N. sylvestris as labeled probes, the positions of homologous sequences in restriction digests of the Nicotiana species were analyzed. Results indicate that the mitochondrial DNA of N. tabacum was inherited from N. sylvestris. Conservation in organization and sequence homology between mtDNAs of N. tabacum and the maternal progenitor, N. sylvestris, provide evidence that the mitochondrial genome in these species is evolutionarily stable. Approximately one-third of the probed restriction fragments of N. tomentosiformis mtDNA showed conservation of position with the other two species. Pattern variations indicate that extensive rearrangement of mtDNA has occurred in the evolution of these Nicotiana species.     

Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units

We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nico-tiana sylvestris (2n=2x=24) and N. tomentosiformis (2n=2x=24) and compared these with patterns in N. tabacum (tobacco, 2n=4x=48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are likely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotiana sylvestris has approximately three times as much rDNA as the other two species, resulting in much condensed rDNA at interphase. This species also has three classes of IGS, indicating gene conversion has not homogenised repeat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at inactive genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, restriction enzymes showed hybridisation patterns that were similar to, but different from, those of N. tabacum. In addition, TBY-2 has elevated rDNA copy number and variable numbers of rDNA loci, all indicating rDNA evolution in culture. Received: 17 November 1999; in revised form: 3 February 2000 / Accepted: 3 February 2000     

The chloroplast genome of Nicotiana sylvestris and Nicotiana tomentosiformis: complete sequencing confirms that the Nicotiana sylvestris progenitor is the maternal genome donor of Nicotiana tabacum

The tobacco cultivar Nicotiana tabacum is a natural amphidiploid that is thought to be derived from ancestors of Nicotiana sylvestris and Nicotiana tomentosiformis. To compare these chloroplast genomes, DNA was prepared from isolated chloroplasts from green leaves of N. sylvestris and N. tomentosiformis, and subjected to whole-genome shotgun sequencing. The N. sylvestris chloroplast genome comprises of 155,941 bp and shows identical gene organization with that of N. tabacum, except one ORF. Detailed comparison revealed only seven different sites between N. tabacum and N. sylvestris; three in introns, two in spacer regions and two in coding regions. The chloroplast DNA of N. tomentosiformis is 155,745 bp long and possesses also identical gene organization with that of N. tabacum, except four ORFs and one pseudogene. However, 1,194 sites differ between these two species. Compared with N. tabacum, the nucleotide substitution in the inverted repeat was much lower than that in the single-copy region. The present work confirms that the chloroplast genome from N. tabacum was derived from an ancestor of N. sylvestris, and suggests that the rate of nucleotide substitution of the chloroplast genomes from N. tabacum and N. sylvestris is very low. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Tobacco single-copy DNA is highly homologous to sequences present in the genomes of its diploid progenitors

Summary We compared the single-copy DNA sequences of the tetraploid tobacco plant, Nicotiana tabacum, with those of its diploid progenitors N. sylvestris and N. tomentosiformis. We observed that 65% of N. sylvestris and N. tomentosiformis single-copy DNA fragments reacted with each other using moderately stringent hybridization conditions (60° C, 0.18 M Na⁺). An additional 10% sequence homology was detected when the hybridization temperature was reduced by 10° C. The thermal stability of interspecific single-copy DNA duplexes indicated that they were approximately 6% more mispaired than homologous single-copy DNA duplexes. In contrast, we observed almost no single-copy DNA divergence between N. tabacum and its diploid progenitors. Greater than 99% of N. sylvestris and N. tomentosiformis single-copy DNAs reacted with N. tabacum DNA using moderately stringent hybridization conditions. The thermal stability of these duplexes indicated that they contained no more sequence mismatch than homologous single-copy duplexes. Together, our results show that significant single-copy DNA sequence divergence has occurred between the diploid N. sylvestris and N. tomentosiformis genomes. However, by applying our experimental criteria these single-copy DNAs are indistinguishable from their counterparts in the hybrid N. tabacum nucleus.     

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. Received: 8 November 1999; in revised form: 23 February 2000 / Accepted: 1 March 2000     

A plant culture (BY‐2) widely used in molecular and cell studies is genetically unstable and highly heterogeneous

Nicotiana tabacum (tobacco, 2n = 4x = 48) is an allotetraploid with 24 S‐genome chromosomes (from a diploid related to N. sylvestris) and 24 T‐genome chromosomes (from a diploid related to N. tomentosiformis). The BY‐2 suspension cell culture, derived from N. tabacum cultivar Bright Yellow 2, has been used extensively for research in molecular and cell biology for almost 40 years; a Web of Knowledge search reveals that it has been used over 150 times since 2008 alone, largely for cell cycle and plant physiology studies. However, we show that this culture is unstable and, as with other long‐term cultures, exists as a community of cells with different karyotypes reflected in different chromosome numbers, morphologies and distributions of satellite repeats, At least one rearranged chromosome type was found in all cells investigated in detail. In comparison with N. tabacum, one satellite repeat, NTRS, has become dispersed across several chromosomes and there is complete homogenization of 35S rRNA genes towards T‐genome type rDNA units. Karyotype divergence should be considered when using BY‐2 cells for plant physiology or cell cycle/development studies in the future. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 459–471.     

Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T‐DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana

Nicotiana species carry cellular T‐DNA sequences (cT‐DNAs), acquired by Agrobacterium‐mediated transformation. We characterized the cT‐DNA sequences of the ancestral Nicotiana tabacum species Nicotiana tomentosiformis by deep sequencing. N. tomentosiformis contains four cT‐DNA inserts derived from different Agrobacterium strains. Each has an incomplete inverted‐repeat structure. TA is similar to part of the Agrobacterium rhizogenes 1724 mikimopine‐type T‐DNA, but has unusual orf14 and mis genes. TB carries a 1724 mikimopine‐type orf14‐mis fragment and a mannopine‐agropine synthesis region (mas2‐mas1‐ags). The mas2′ gene codes for an active enzyme. TC is similar to the left part of the A. rhizogenes A4 T‐DNA, but also carries octopine synthase‐like (ocl) and c‐like genes normally found in A. tumefaciens. TD shows a complex rearrangement of T‐DNA fragments similar to the right end of the A4 TL‐DNA, and including an orf14‐like gene and a gene with unknown function, orf511. The TA, TB, TC and TD insertion sites were identified by alignment with N. tabacum and Nicotiana sylvestris sequences. The divergence values for the TA, TB, TC and TD repeats provide an estimate for their relative introduction times. A large deletion has occurred in the central part of the N. tabacum cv. Basma/Xanthi TA region, and another deletion removed the complete TC region in N. tabacum. Nicotiana otophora lacks TA, TB and TD, but contains TC and another cT‐DNA, TE. This analysis, together with that of Nicotiana glauca and other Nicotiana species, indicates multiple sequential insertions of cT‐DNAs during the evolution of the genus Nicotiana.     

Heterosis,transmission genetics,and selection for increased growth rate in a <Emphasis Type="Italic">N. tabacum</Emphasis> × synthetic tobacco cross

Cultivated tobacco (Nicotiana tabacum L.) is a classic amphidiploid, and hybrids between this cultivated species and closely related diploid Nicotiana relatives often exhibit heterotic effects for growth rate and yield. Crosses between N. tabacum and synthetic tobaccos, 4x(Nicotiana sylvestris × Nicotiana otophora) or 4x(N. sylvestris × Nicotiana tomentosiformis), may provide superior routes for genome-wide introgression from diploid relatives and allow increased potential to capitalize on heterotic effects in tobacco. Significant levels of mid-parent heterosis were observed for yield and growth rate in F₁ hybrids between synthetic tobaccos and a standard tobacco cultivar. Microsatellite marker genotyping of an F₂ population derived from a K326 × [4x(N. sylvestris × N. otophora)] cross was carried out to preliminarily investigate the relative importance of different types of gene action on observed heterosis in the original interspecific cross. Results suggested a role for both partial dominance and overdominance. Marker genotyping also indicated an overall reduced level of recombination in the N. tabacum × synthetic tobacco cross relative to a N. tabacum × N. tabacum cross but no evidence of genomic regions with severely restricted levels of recombination. Results suggest that populations derived from N. tabacum × synthetic tobacco crosses may be more efficient for introgressing germplasm from diploid relatives, as compared to populations derived from crosses between N. tabacum and diploid forms where preferential pairing between N. tabacum homologues can reduce the potential for introgression of alien chromatin. Such materials may be useful as sources of favorable alleles influencing quantitative characters in tobacco.     

A protein in the oxygen-evolving complex in the chloroplast is associated with symptom expression on tobacco leaves infected with cucumber mosaic virus strain Y

To elucidate the molecular basis of symptom expression in virus-infected plants, the changes in proteins between tobacco, Nicotiana tabacum cv. Ky57, leaves inoculated with cucumber mosaic virus strain Y [CMV(Y)] and strain O [CMV(O)], were compared by 2-dimensional (2-D) gel electrophoresis. The appearance of chlorotic spots in CMV(Y)-inoculated tobacco leaves accompanied an increase of 3 polypeptides and a decrease in 6 polypeptides, as compared with those in the CMV(O)-inoculated tobacco which showed no clear symptoms. The decrease in the amounts of two polypeptides of 22 and 23 kDa was particularly significant: these two polypeptides were compared with a 24 kDa polypeptide, which co-migrated with them in 2-D gel electrophoresis but did not clearly decrease at an early stage of infection, as well as major other proteins of CMV(Y)-inoculated tobacco leaves. However, the 22, 23 and 24 kDa polypeptides showed the same peptide mapping pattern. Furthermore, the 12 amino acid residues at N-termini of the three polypeptides match those of the extrinsic 23 kDa polypeptide of an oxygen-evolving complex from spinach. A comparative analysis of the 22, 23 and 24 kDa polypeptides in N. tabacum and its ancestral parents, N. sylvestris and N. tomentosiformis, revealed that the 22 kDa polypeptide derives from N. sylvestris and the 23 kDa polypeptide from N. tomentosiformis; the 24 kDa polypeptide derives from both ancestral Nicotiana species. The results indicate that the polypeptides whose amounts differentially decrease with the progress of symptom expression in N. tabacum inoculated with CMV(Y) are one component of the oxygen-evolving complex in photosystem II.     

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots

Summary Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.     

Genomic factors controlling the lethality exhibited in the hybrid between Nicotiana suaveolens Lehm. and N. tabacum L.

Interspecific hybrid plants between Nicotiana suaveolens and N. tabacum exhibit lethal symptoms at the seedling stage and cannot grow to maturity. In this investigation, an attempt was made to clarify the genomic factors responsible for this lethality. N. suaveolens was crossed to N. sylvestris (genomic constitution: SS) and N. tomentosiformis (TT), these latter two species being the progenitors of N. tabacum (SSTT). From the cross N. suaveolens x N. tomentosiformis, many seedlings were obtained through ovule culture, and these subsequently grew to maturity without exhibiting any lethality. In the reciprocal crossing between N. sauvelons and N. sylvestris, only a few hybrid seedlings were obtained through ovlue culture and all died after unfolding their cotyledons when cultured at 28 °C. This lethality could be avoided by culturing the ovules at 36 °C. These features of hybrid lethality resembled those observed in the interspecific hybrid between N. suaveolens and N. tabacum. These findings suggest that the S genome in N. tabacum is responsible for the lethality exhibited in the hybrid between N. suaveolens and N. tabacum.     

Possible involvement of genes on the Q chromosome of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> in expression of hybrid lethality and programmed cell death during interspecific hybridization to <Emphasis Type="Italic">Nicotiana debneyi</Emphasis>

Hybrid seedlings from the cross between Nicotiana tabacum, an allotetraploid composed of S and T subgenomes, and N. debneyi die at the cotyledonary stage. This lethality involves programmed cell death (PCD). We carried out reciprocal crosses between the two progenitors of N. tabacum, N. sylvestris and N. tomentosiformis, and N. debneyi to reveal whether only the S subgenome in N. tabacum is related to hybrid lethality. Hybrid seedlings from reciprocal crosses between N. sylvestris and N. debneyi showed lethal characteristics identical to those from the cross between N. tabacum and N. debneyi. Conversely, hybrid seedlings from reciprocal crosses between N. tomentosiformis and N. debneyi were viable. Furthermore, hallmarks of PCD were observed in hybrid seedlings from the cross N. debneyi × N. sylvestris, but not in hybrid seedlings from the cross N. debneyi × N. tomentosiformis. We also carried out crosses between monosomic lines of N. tabacum lacking the Q chromosome and N. debneyi. Using Q-chromosome-specific DNA markers, hybrid seedlings were divided into two groups, hybrids possessing the Q chromosome and hybrids lacking the Q chromosome. Hybrids possessing the Q chromosome died with characteristics of PCD. However, hybrids lacking the Q chromosome were viable and PCD did not occur. From these results, we concluded that the Q chromosome belonging to the S subgenome of N. tabacum encodes gene(s) leading to hybrid lethality in the cross N. tabacum × N. debneyi.     

Genome-wide identification,evolution and expression analysis of cyclic nucleotide-gated channels in tobacco (Nicotiana tabacum L.)

Tobacco (Nicotiana tabacum) serve as the top leading commercial, non-food, and model crop worldwide. Cyclic nucleotide-gated channels (CNGCs) are ligand-gated, calcium-permeable, divalent, cation-selective channels, involved in important biological functions. Here, we systematically characterized thirty-five CNGC genes in the genome of Nicotiana tabacum, and classified into four phylogenetic groups. Evolutionary analysis showed that NtabCNGC family of N. tabacum originated from the parental genome of N. sylvestris and N. tomentosiformis, and further expanded via tandem and segmental duplication events. Tissue-specific expression analysis showed that twenty-three NtabCNGC genes are involved in the development of various tobacco tissues. Subsequent RT-qPCR analyses indicated that these genes are sensitive towards external abiotic and biotic stresses. Notable performances were exhibited by group-I and IV CNGC genes against black shank, Cucumber mosaic virus, Potato virus Y, cold, drought, and cadmium stresses. Our analyses also suggested that NtabCNGCs can be regulated by phosphorylation and miRNAs, and multiple light, temperature, and pathogen-responsive cis-acting regulatory elements present in promotors. These results will be useful for elaborating the biological roles of NtabCNGCs in tobacco growth and development.     

Genetic diversity and heterosis in nicotiana

Summary Two flue-cured varieties of N. tabacum were crossed to putative progenitor species and to distantly related species. Heterosis for yield, plant height, and number of leaves was largest for crosses to progenitor species, particularly to N. otophora and N. tomentosiformis. The magnitude of this heterosis appeared to be greater than estimates presented in the literature for crosses among varieties of N. tabacum. An additional study presented some evidence for the genomic basis of heterosis in crosses of N. tabacum with N. tomentosiformis and N. sylvestris.

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Zusammenfassung Zwei für R?hrentrocknung geeignete Sorten von N. tabacum wurden mit vermutlichen Ausgangs- und mit entfernt verwandten Arten gekreuzt. Die Heterosis für Ertrag, Pflanzenh?he und Blattanzahl war am st?rksten bei Kreuzungen mit den Ausgangsarten, besonders mit N. otophora und N. tomentosiformis. Das Ausma? dieser Heterosis schien die in der Literatur berichteten Sch?tzungen für Kreuzungen zwischen Sorten von N. tabacum zu übertreffen. Eine weitere Untersuchung erbrachte Hinweise für die genomatische Grundlage der Heterosis bei Kreuzungen von N. tabacum mit N. tomentosiformis und N. sylvestris.

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Dedicated to Dr. George F. Sprague on the occasion of his 65^th birthday.

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Paper Number 2318 of the Journal Series of the North Carolina Agricultural Experiment Station. This investigation was supported in part by Public Health Service Research Grant GM 11546 from the Division of General Medical Sciences.     

Differential impact of retrotransposon populations on the genome of allotetraploid tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

LTR-retrotransposons contribute substantially to the structural diversity of plant genomes. Recent models of genome evolution suggest that retrotransposon amplification is offset by removal of retrotransposon sequences, leading to a turnover of retrotransposon populations. While bursts of amplification have been documented, it is not known whether removal of retrotransposon sequences occurs continuously, or is triggered by specific stimuli over short evolutionary periods. In this work, we have characterized the evolutionary dynamics of four populations of copia-type retrotransposons in allotetraploid tobacco (Nicotiana tabacum) and its two diploid progenitors Nicotiana sylvestris and Nicotiana tomentosiformis. We have used SSAP (Sequence-Specific Amplification Polymorphism) to evaluate the contribution retrotransposons have made to the diversity of tobacco and its diploid progenitor species, to quantify the contribution each diploid progenitor has made to tobacco's retrotransposon populations, and to estimate losses or amplifications of retrotransposon sequences subsequent to tobacco's formation. Our results show that the tobacco genome derives from a turnover of retrotransposon sequences with removals concomitant with new insertions. We have detected unique behaviour specific to each retrotransposon population, with differences likely reflecting distinct evolutionary histories and activities of particular elements. Our results indicate that the retrotransposon content of a given plant species is strongly influenced by the host evolutionary history, with periods of rapid turnover of retrotransposon sequences stimulated by allopolyploidy.     

Root‐specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium‐derived T‐DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2′ gene (TB‐mas2′). This gene is similar to the Agrobacterium rhizogenes A4‐mas2′ gene that encodes the synthesis of the Amadori compound deoxyfructosyl‐glutamine (DFG or santhopine). In this study we show that TB‐mas2′ is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB‐mas2′ expression levels. The TB‐mas2′ promoter sequences of low‐ and high‐expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low‐ and a high‐expression cultivar. pTB‐mas2′‐GUS and pA4‐mas2′‐GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB‐mas2′ expression contain detectable levels of DFG.     

Polymorphism of a photosystem I subunit caused by alloploidy in Nicotiana

The photosystem I complex from Nicotiana tabacum, which has an alloploid genome, contains subunits of 17.5 and 18.5 kilodaltons whose N-terminal amino acid sequences are highly homologous. Comparative analysis of photosystem I subunits among N. tabacum and its ancestral plants, N. tomentosiformis and N. sylvestris, revealed that the 17.5 kilodalton subunit of N. tabacum derives from N. sylvestris, and the 18.5 kilodalton subunit from N. tomentosiformis.     

Faithful inheritance of cytosine methylation patterns in repeated sequences of the allotetraploid tobacco correlates with the expression of DNA methyltransferase gene families from both parental genomes

The widespread occurrence of epigenetic alterations in allopolyploid species deserves scrutiny that DNA methylation systems may be perturbed by interspecies hybridization and polyploidization. Here we studied the genes involved in DNA methylation in Nicotiana tabacum (tobacco) allotetraploid containing S and T genomes inherited from Nicotiana sylvestris and Nicotiana tomentosiformis progenitors. To determine the inheritance of DNA methyltransferase genes and their expression patterns we examined three major DNA methyltransferase families (MET1, CMT3 and DRM) from tobacco and the progenitor species. Using Southern blot hybridization and PCR-based methods (genomic CAPS), we found that the parental loci of these gene families are retained in tobacco. Homoeologous expression was found in all tissues examined (leaf, root, flower) suggesting that DNA methyltransferase genes were probably not themselves targets of uniparental epigenetic silencing for over thousands of generations of allotetraploid evolution. The level of CG and CHG methylation of selected high-copy repeated sequences was similar and high in tobacco and its diploid progenitors. We speculate that natural selection might favor additive expression of parental DNA methyltransferase genes maintaining high levels of DNA methylation in tobacco, which has a repeat-rich heterochromatic genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AM946602–AM946620 and FM872474–FM872476.