ABam HI family of tobacco highly repeated DNA:

ABamHI family of highly repeated DNA sequences of theNicotiana tabacum nuclear genome, denoted as a HRS60-family, was recently isolated. It comprises about 2% of the tobacco nuclear genome. Monomeric units are 182–184 bp long. Members of the HRS60-family isolated till now are closely related. DNA-DNA hybridization experiments with DNA of the two tobacco progenitors,N. tomentosiformis andN. sylvestris, revealed that the HRS60-family was present in many copies inN. sylvestris, the amount being about 1.7 times that inN. tabacum. InN. tomentosiformis as well as in some other species of the genusNicotiana, the HRS60-family is present in a small amount. Sequences related to the HRS60-family were revealed using DNA-DNA hybridization at low stringency. With respect to quantity, the HRS60-family could be considered as a species-specific DNA repeat which may be a useful genetic marker in genetic manipulations withN. tabacum.     

Nuclear DNA,heterochromatin and phylogeny of Nicotiana amphidiploids

There are significant differences in nuclear DNA amount between both diploid and amphidiploid species of Nicotiana. Owing to the higher DNA density in the interphase nuclei of the amphidiploids DNA amounts tend to be underestimated by microdensitometry. After applying necessary corrections to amphidiploid readings it was found that: (1) The nuclear DNA amount in the tetraploid N. rustica is not significantly different from the sum of nuclear DNA amounts in reputed diploid parents, N. undulata and N. paniculata. (2) It is well established that N. sylvestris is one of the diploid progenitors of N. tabacum. The sum of the nuclear DNA amounts in N. sylvestris and N. tomentosiformis is not significantly different from that of the amphidiploid N. tabacum. In contrast the sum of the DNA amounts in N. sylvestris and N. otophora is significantly higher than that in N. tabacum. Observations and measurements of the amount and distribution of heterochromatin in interphase nuclei of the diploid and tetraploid species give further support to the conclusion that N. tomentosiformis rather than N. otophora is the second diploid progenitor of N. tabacum.     

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.     

Serological relationships of Fraction I proteins from species in the genusNicotiana

The serological relationships of Fraction I proteins from 62 species of the genusNicotiana have been determined by qualitative double diffusion in agar gels, using antisera raised against crystalline Fraction I proteins from four species,N. glauca, N. glutinosa, N. gossei, andN. tabacum. The relationships obtained are, in most cases, similar to the relationships of the species based on morphological and cytogenetic criteria. In general, Fraction I proteins from species within the same section are serologically identical. However, serological examination of Fraction I proteins draws attention to certain species, for example,N. glauca, N. sylvestris, andN. acaulis, whose classification by traditional methods has been difficult.     

CENTRORADIALIS/TERMINAL FLOWER 1 gene homolog is conserved inN. tabacum, a determinate inflorescence plant

A mutation in theCENTRORADIALIS (CEN) gene ofAntirrhinum and in theTERMINAL FLOWER 1 (TFL1) gene ofArabidopsis causes their indeterminate inflorescence to determinate. We clonedCEN/TFL1 homologs fromNicotiana tabacum, the wild-type of which has a determinate inflorescence. TheCEN gene was expressed in the inflorescnece meristem and kept its inflorescence meristem identity, whereas the tobacco homolog (NCH) was expressed at a low level throughout the plant’s development. AlthoughCEN andNCH are highly homologous genes, they may have been recruited to different developmental functions during their evolution. TwoNCH genes are derived from amphidiploidN. tabacum, but both of them hybridized with its diploid parents,N. sylvestris andN. tomentosiformis. Southern blotting, and the genomic organization ofTFL1 inArabidopsis revealed that anotherCEN homolog exists in the genome ofArabidopsis. These results suggest that there are two copies of theCEN homolog per diploid plant. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology” These two authors contributed to this work equally.     

COSII genetic maps of two diploid Nicotiana species provide a detailed picture of synteny with tomato and insights into chromosome evolution in tetraploid N. tabacum

Using single-copy conserved ortholog set (COSII) and simple sequence repeat (SSR) markers, we have constructed two genetic maps for diploid Nicotiana species, N. tomentosiformis and N. acuminata, respectively. N. acuminata is phylogenetically closer to N. sylvestris than to N. tomentosiformis, the latter two of which are thought to contribute the S-genome and T-genome, respectively, to the allotetraploid tobacco (N. tabacum L., 2n = 48). A comparison of the two maps revealed a minimum of seven inversions and one translocation subsequent to the divergence of these two diploid species. Further, comparing the diploid maps with a dense tobacco map revealed that the tobacco genome experienced chromosomal rearrangements more frequently than its diploid relatives, supporting the notion of accelerated genome evolution in allotetraploids. Mapped COSII markers permitted the investigation of Nicotiana–tomato syntenic relationships. A minimum of 3 (and up to 10) inversions and 11 reciprocal translocations differentiate the tomato genome from that of the last common ancestor of N. tomentosiformis and N. acuminata. Nevertheless, the marker/gene order is well preserved in 25 conserved syntenic segments. Molecular dating based on COSII sequences suggested that tobacco was formed 1.0MYA or later. In conclusion, these COSII and SSR markers link the cultivated tobacco map to those of wild diploid Nicotiana species and tomato, thus providing a platform for cross-reference of genetic and genomic information among them as well as other solanaceous species including potato, eggplant, pepper and the closely allied coffee (Rubiaceae). Therefore they will facilitate genetic research in the genus Nicotiana.     

The PELPIII glycoproteins in Solanaceae: stylar expression and transfer into pollen tube walls

The class III pistil-specific extensin-like proteins (PELPIII) of Nicotiana tabacum accumulate in the intercellular matrix (IM) of the style transmitting tissue (TT). After pollination, the 110–140 kDa PELPIII is translocated from the IM into the pollen tube walls. PELPIII-like sequences have been found in several solanaceous species. These sequences are expressed in mature non-pollinated styles at both RNA and protein level. Of the genus Nicotiana, the species N. alata, N. x sanderae and N. sylvestris (section Alatae), and N. tomentosiformis and N. otophora (section Tomentosae) showed an expression level of PELPIII homologues similar to that in mature styles of N. tabacum. PELPIII genes were absent in the most ancient species studied, namely N. trigonophylla (section Trigonophyllae). To study the species dependence of the translocation of PELPIII into the pollen tube wall in tobacco, interspecific pollinations on N. tabacum pistils were carried out with pollen from the incongruous species N. rustica, N. trigonophylla and Petunia hybrida, where PELPIII homologues are absent in the style. Immunocytological tests showed that the N. tabacum PELPIII is translocated into the pollen tube walls of all three species. Thus, the pollen tube walls of these species do not form a barrier for IM compounds such as the 110–140 kDa PELPIII and the absence of any possible effect of PELPIII on pollen tube growth cannot be due to failure of PELPIII transport through the wall. The importance of these findings is discussed with respect to the evolutionary origin of PELPIII, the pollen pistil interaction, the function of style TT-specific proteins and the physical properties of pollen tube walls.     

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.     

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.     

ITS sequences from nuclear rDNA suggest unexpected phylogenetic relationships between Euro-Mediterranean, East Asiatic and North American taxa ofQuercus (Fagaceae)

Nucleotide sequences from the internal transcribed spacer (ITS) regions of the 18S–26S nuclear ribosomal DNA have been studied from ten species ofQuercus (representing four subgenera),Castanea sativa andFagus sylvatica, as a preliminary molecular contribution to the still poorly understood systematics and evolution ofFagaceae. The resulting matrix has been used to calculate pair-wise sequence divergence indices and to construct a maximum parsimony tree forQuercus coding indels as a fifth state. Divergence is greater forQuercus vs.Fagus than forQuercus vs.Castanea. The tree for theQuercus taxa studied reveals two clearly divergent clades. In clade I the evergreen W MediterraneanQ. suber appears in a basal position as sister to more distal deciduous taxa, i.e. the E MediterraneanQ. macrolepis and the E AsiaticQ. acutissima (all formerly united as different sections under the apparently polyphyletic subg.Cerris), andQ. rubra (a representative of the N American subg.Erythrobalanus), forming a pair withQ. acutissima. In clade II the evergreen southeastern N AmericanQ. virginiana is basal and sister to the remaining three branches, i.e. a pair of evergreen Mediterranean taxa withQ. ilex andQ. coccifera (subg.Sclerophyllodrys), the deciduous but otherwise plesiomorphic SE European/SW AsiaticQ. cerris (type species of subg.Cerris), and the related but more apomorphic European pairQ. petraea andQ. robur (subg.Quercus). These results partly conflict with current taxonomic classification but are supported by some anatomical and morphological characters. They document polyphyletic lines from evergreen to deciduous taxa and suggest Tertiary transcontinental connections within the genus.     

Construction of phylogenetic tree forNicotiana species based on RAPD markers

To apply random amplified polymorphic DNA for analysis of phylogenetic relationships, we used 34 synthetic oligonucleotides as primers to examine interspecific and intraspecific variations among 18 genotypes, nine species ofNicotiana. The nine species used in this study belong to sectionsTomentosae andAlatae. In addition, we attempted to clarify the taxonomic position ofN. sylvestris. A total of 354 distinct DNA fragments were obtained by polymerase chain reaction. Pair-wise comparisons of unique and shared amplification products were used to generate Jaccard's similarity coefficients and Nei and Li's similarity coefficients with the computer software of numerical taxonomy and multivariate analysis system. On the basis of the dendrogram constructed with the similarity coefficients, the 18Nicotiana genotypes were divided into two clusters. The classification analyzed by RAPD markers is in accordance with the classification of Goodspeed thatN. sylvestris is a member of sectionAlatae.     

Cytological studies inCyclamen subg.Cyclamen (Primulaceae)

Although theCyclamen subg.Cyclamen spp. are morphologically variable, previous studies suggest a superficial cytological uniformity. New chromosome numbers and an indication of karyotypic instability are reported inC. hederifolium andC. africanum that reveal that the cytology of the subgenus is more complicated than previous accounts suggest. The possible significance of these phenomena is discussed. The diploid status ofC. purpurascens has been confirmed and a distinction between the three diploid karyotypes has been described. The cytological variation may help to explain the well documented morphological variation exhibited in these species.     

Biochemical and serological characterization of b-proteins fromNicotiana species

Summary Proteins associated with the hypersensitive response (b-proteins) were purified from variousNicotiana species and compared biochemically and serologically. The method developed to purify proteins b₁, b₂ and b₃ ofN. tabacum cv. Xanthi-nc was used to purify b-proteins present inN. sylvestris (b₀, b₁ and b₃) andN. tomentosiformis (b₂), the parental species ofN. tabacum, and b₁″ from bothN. glutinosa andN. debneyi. Ultracentrifugation and amino acid analysis of some of these proteins has shown that they are very similar and that they are all monomers in their native form (mol wt = 15 700 for b₀, b₁, b₂ and b₃; mol wt = 13 800 for b₁″). Based on their reactions to an antiserum produced against protein b₁ ofN. tabacum cv. Xanthi-nc, 3 serological groups can be recognized which are independent of the source species (I) b₀ and b₁, (II) b₁″ and b₂, (III) b₃. Thus, proteins in the same serological group but from different species are more closely related than the b-proteins in different serological groups but present in the same species. The implication of this site on the possible phylogeny of b-proteins is discussed. Serological tests confirmed the b-protein present as a constitutive component in the virus resistant interspecific hybrids ofN. glutinosa ×N. debneyi as protein b₁″.     

Revision of the genus Clibadium (Asteraceae, Heliantheae)

Clibadium L. (Asteraceae, Heliantheae) is a genus of 29 species distributed throughout latin America, from Mexico to Peru, and in the West Indies, with high numbers of species in Costa Rica, Colombia, and Ecuador.Clibadium includes shrubs and small trees; usually with loosely aggregated capitula; herbaceous phyllaries arranged in 1–5 series; receptacles usually paleaceous throughout; corollas of pistillate florets 2–4-lobed; corollas of the staminate florets 4–5-lobed; purple to black anthers; and chromosome numbers alln=16. Two sections of species previously recognized are here considered as subgenera (subg.Paleata and subg.Clibadium) containing two and four sections, respectively.Clibadium subg.Paleata contains five species distributed in sects.Eggersia (3 spp.) andTrixidium (2 spp.), and subg.Clibadium has 24 species distributed among sects.Clibadium (6 spp.),Glomerata (9 spp.),Grandifolia (5 spp.), andOswalda (4 spp.).     

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     

Physical maps of Nicotiana chloroplast DNA constructed by an efficient procedure

Summary The restriction profiles of chloroplast DNA (cpDNA) from Nicotiana tabacum, N. sylvestris, N. plumbaginifolia, and N. otophora were obtained with respect to AvaI, BamHI, BglI, HindIII, PstI, PvuII, SalI, and XhoI. An efficient mapping method for the construction of cpDNA physical maps in Nicotiana was established via a computer-aided analysis of the complete cpDNA sequence of N. tabacum for probe selection. The efficiency of this approach is demonstrated by the determination of cpDNA maps from N. sylvestris, N. plumbaginifolia, and N. otophora with respect to all of the above restriction endonucleases. The size and basic structure of the cpDNA from the three species are almost identical, with an addition of approximately 80 bp in N. plumbaginifolia. The restriction patterns and hence the physical maps between N. tabacum and N. sylvestris cpDNA are identical and there is no difference in the Pvull digests of cpDNA from all four species. Restriction site variations in cpDNA from different species probably result from point mutations, which create or eliminate a particular cutting site, and they were observed spanning the whole chloroplast molecule but highly concentrated in both ends of the large, single-copy region. The results presented here will be used for the forthcoming characterization of chloroplast genomes in the interspecies somatic hybrids of Nicotiana, and will be of great value in completing the exploration of the phylogenetic relationships within this already extensively studied genus.     

Polyphenol content,polyphenoloxidase and peroxidase activity in certain Nicotiana species,varieties and interspecific hybrids

Summary Eight Nicotiana species including the putative progenitors of N. tabacum, Kostoff's amphidiploid (N. sylvestris × N. tomentosiformis), and 19 cultivars have been compared for total polyphenols, polyphenoloxidase and peroxidase activity in the leaf and/or root by a small plant technique. Greater variations for these chemical constituents occurred in the species than in the cultivars. N. tomentosiformis was highest in polyphenol content. Root extracts contained more polyphenoloxidase than the leaf, but its peroxidase content may not exceed the concentration in the leaf. The Kostoff's amphidiploid tended to resemble more the low oxidase and polyphenol parent. An additional study based on mature green leaves of Burley 21, the progenitor species, and their F ₁ hybrids confirmed the quantitative differences of these chemical constituents in the species. The magnitude of the heterosis appeared to be greater in the hybrids of N. tomentosiformis or N. otophora crossed to N. sylvestris than those between the Tomentosae members or involving Burley 21 as the parent. An exception was the hybrid Burley 21 × N. tomentosiformis which showed heterosis for oxidase activities.

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Zusammenfassung Acht Nicotiana-Spezies einschl. der vermutlichen Eltern von N. tabacum, Kostoffs Amphidiploid (N. sylvestris × N. tomentosiformis) und 19 Sorten wurden auf ihren Gehalt an Polyphenolen und auf die Polyphenoloxidase- und Peroxidaseaktivität in den Blättern und/oder Wurzeln in einem Pflanzen-Kleinversuch verglichen. Bei den Spezies ergaben sich größere Abweichungen für diese chemischen Substanzen als bei den Sorten. N. tomentosiformis hatte den höchsten Polyphenolgehalt. Wurzelextrakte enthielten mehr Polyphenoloxidase als Blattextrakte, der Peroxidasegehalt dürfte aber die Konzentration in den Blättern nicht übersteigen. Kostoffs Amphidiploid schien mehr dem Elter mit niedriger Oxidase-aktivität und niedrigem Polyphenolgehalt zu ähneln. Eine weitere Untersuchung anhand von ausgewachsenen grünen Blättern von Burley 21 als Elter-Spezies und ihren F₁-Hybriden bestätigte die quantitativen Unterschiede in diesen chemischen Bestandteilen der Spezies. Das Ausmaß der Heterosis schien stärker in den Hybriden von N. tomentosiformis oder N. otophora bei Kreuzung mit N. sylvestris als innerhalb der Tomentosae oder bei Einbeziehung von Burley 21 als Elter. Eine Ausnahme bildete die Hybride aus Burley 21 × N. tomentosiformis, die bezüglich der Oxidaseaktivität Heterosis zeigte.

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Contribution from the Tobacco and Health Research Institute, and the Department of Agronomy, University of Kentucky, Lexington, Kentucky 40 506. This investigation was supported by a contract with the Agricultural Research Service, U.S. Department of Agriculture, administered by the Crops Research Division, Plant Industry Station, Beltsville, Maryland.     

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.