Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.     

Nicotiana chloroplast genome

Summary A physical map containing six restriction sites of the Nicotiana tabacum chloroplast genome, together with the BamHI maps of N. tabacum, N. otophora and N. knightiana, and the SmaI maps of N. acuminata, N. plumbaginifolia, N. langsdorffii, N. otophora, N. tabacum, N. tomentosiformis and N. knightiana was constructed. In Nicotiana chloroplast genomes, the most frequently observed variations are point mutations. Deletions are also detected. Most of the observed changes are confined to one area of the large single copy region, which is designated as the hot spot. Based on the evidence obtained from Nicotiana chloroplast genomes, an origin of the inverted repeats in this genus is proposed. We suggest that the inverted repeats represent a vestige of what were once two identical, complete chloroplast genomes joined together in a head-to-head and tail-to-tail fashion, and that deletions generated the current chloroplast genome organization.     

Quantitative chromosome maps and rDNA localization in the T subgenome of Nicotiana tabacum L. and its putative progenitors

Using DAPI-stained prometaphase chromosomes, quantitative idiograms were constructed for the T subgenome of Nicotiana tabacum (2n = 4x = 48, SSTT) and two putative candidates for its T subgenome progenitor, Nicotiana otophora and Nicotiana tomentosiformis (both have 2n = 24, TT). The large chromosomes of the three karyotypes could be identified from the distributional pattern of the DAPI signal. Fluorescence in situ hybridization (FISH) with 5S rDNA gave not only good cytogenetical landmarks for identification of small chromosomes of the karyotypes but also phylogenetical information. In all three idiograms, 5S rDNA was localized in the proximal region of the long arm of a small submetacentric pair, but an additional 5S rDNA locus was detected terminally on the short arm of a small metacentric pair in N. otophora. The 18S rDNA locus detected here corresponded to satellite regions in all three karyotypes. Two satellited pairs in N. otophora and one satellited pair in N. tomentosiformis had single large subterminal DAPI blocks and two interstitial DAPI bands on their long arms, respectively. For the T subgenome component of N. tabacum, the single intense DAPI band was depicted on the center of the long arm of a satellited pair in the idiogram, although two interstitial bands were often detected on the long arm of the satellited pair in some spreads. Therefore, it was suggested that the T component of N. tabacum was more similar to that of N. tomentosiformis than N. otophora, especially in respect of the number and location of rDNA and the distributional patterns of DAPI signals. Received: 25 October 1999 / Accepted: 24 March 2000<@head-com-p1a.lf>Communicated by Y. Gleba     

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.     

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.     

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.     

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.     

Effect of Radiation Spectral Composition on Nicotiana spp. Seedlings Grown in vitro

The aim of this work was to assess the responses of seedlings of five species of Nicotiana genus to red and far red radiation. N. acuminata exhibits positive photoblastic behaviour and germination was completely inhibited under far red and darkness. In N. glauca germination was reduced under far red and darkness, but the other species showed neutral behaviour. The hypocotyl elongation was inhibited in N. glauca and N. tabacum under white and far red radiation. In N. langsdorffii and N. debneyi hypocotyl was elongated under far red radiation. Only in N. acuminata red radiation promote greater hypocotyl elongation than dark condition. On the phylogenetic tree obtained from restriction analysis N. glauca and N. acuminata are grouped in one branch, while the other species, N. langsdorffii, N. debneyi and N. tabacum, are grouped in the other branch cluster. Moreover, the N. debneyi behaviours under different radiation treatments were similar to those of N. tabacum. These two species are allopolyploid members of the genus Nicotiana, as also was confirmed by this study. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cybrids of Nicotiana tabacum and Petunia hybrida have an intergeneric mixture of chloroplasts from P. hybrida and mitochondria identical or similar to N. tabacum

Summary The mitochondrial genomes of cybrids of Nicotiana tabacum containing chloroplasts of Petunia hybrida were characterized by restriction endonuclease digestion and agarose gel electrophoresis. Cybrids that displayed normal growth and development contained mitochondrial DNA indistinguishable from N. tabacum mitochondrial DNA. Cybrids that displayed abnormal growth and development contained mitochondrial DNA that differed from N. tabacum either by possessing a few additional fragments, by lacking a few fragments, or both. In spite of these differences, the mitochondrial DNA of cybrids showing abnormal growth and development was much more similar to N. tabacum than to P. hybrida mitochondrial DNA. In those cybrids that contained P. hybrida chloroplasts and N. tabacum mitochondria, cotransfer of cytoplasmic organelles did not occur. Although P. hybrida chloroplasts can interact compatibly with the N. tabacum nucleus, no cybrids were found in which P. hybrida mitochondria coexisted with the N. tabacum nucleus.     

Purification of chloroplast elongation factor Tu and cDNA analysis in tobacco: the existence of two chloroplast elongation factor Tu species

We have purified a chloroplast elongation factor Tu (EF-Tu) from tobacco (Nicotiana tabacum) and determined its N-terminal amino acid sequence. Two distinct cDNAs encoding EF-Tu were isolated from a leaf cDNA library of N. sylvestris (the female progenitor of N. tabacum) using an oligonucleotide probe based on the EF-Tu protein sequence. The cDNA sequence and genomic Southern analyses revealed that tobacco chloroplast EF-Tu is encoded by two distinct genes in the nuclear genome of N. sylvestris. We designated the corresponding gene products EF-Tu A and B. The mature polypeptides of EF-Tu A and B are 408 amino acids long and share 95.3% amino acid identity. They show 75–78% amino acid identity with cyanobacterial and chloroplast-encoded EF-Tu species.     

Inheritance behavior of information coding for small subunit polypeptides of fraction 1 protein

In various genera of plants, the small subunit of fraction 1 protein is often composed of more than one kind of polypeptide; these differ in isoelectric points and amino acid composition. Previous analysis of numerous individual progeny of Nicotiana tabacum (two kinds of polypeptides), N. glauca + N. langsdorffii parasexual hybrids (three kinds), and other examples showed no change in F-1 protein composition as a consequence of alternation of generations. Experiments reported here show that absence of one member of each of the 24 different pairs of chromosomes in an N. tabacum monosomic series and also absence of the “S” pair in a nullisome did not affect F-1 protein composition. Absence of the “E” pair caused reduction in the amount of the least acidic of the two kinds of N. tabacum small subunit polypeptides. The question of how many individual progeny of self-fertile hybrids would have to be analyzed to detect segregation of genes coding for F-1 protein small subunit polypeptides, if segregation occurs, was answered by analysis of F₁ hybrids between N. otophora and N. tomentosiformis, and two subspecies of N. suaveolens, together with their F₂ progeny. In both cases, analysis of 16 progeny was sufficient to demonstrate a segregation pattern of two F₁ hybrid type to one each of the two parental types. Therefore, in the absence of segregation, it is likely that coding information for different kinds of F-1 protein small subunit polypeptides is sequestered on heterologous chromosomes, as postulated in previous reports.     

Evaluation of Nicotiana otophora as a Source of Resistance to Meloidogyne incognita Race 4 for Tobacco

No currently available tobacco cultivar possesses resistance to Meloidogyne incognita race 4, nor has any source of resistance been reported within Nicotiana tabacum. The purpose of this study was to evaluate N. otophora acc. La Quinta as a source of resistance to this pathogen. Plants of tobacco cvs. NC 95 and NC 2326, N. otophora La Quinta and N. repanda were inoculated with second-stage juveniles of M. incognita race 4. Gall indices and egg-mass ratings were assessed at 4 and 8 weeks after inoculation. The two N. tabacum cultivars were heavily galled and had numerous egg masses at both rating periods. Nicotiana repanda was only weakly resistant. The galls on this species were very small and present at a low to moderate level; however, egg-mass ratings approaching those of the tobacco cultivars were observed 8 weeks after inoculation. In contrast, low gall indices and egg-mass ratings were found for N. otophora La Quinta at both the 4- and 8-week rating periods. In addition, little variability was observed within this species for either disease rating. Therefore, it appears that the La Quinta accession of N. otophora is a very promising source of M. incognita race 4 resistance for transfer to N. tabacum.     

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.     

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.     

Genomic factors responsible for abnormal morphology of pollen tubes in the interspecific cross Nicotiana tabacum ×N. rustica

Nicotiana tabacum shows unilateral pollen-pistil incongruity with N. rustica. If N. tabacum is pollinated with N. rustica, growth of the pollen tube is arrested in the middle of the style, and abundant callose deposition, tube swelling and tube winding are observed. An attempt was made to clarify the genomic factors responsible for this pollen-pistil incongruity. N. tabacum was pollinated with N. paniculata or N. undulata, progenitors of amphidiploid N. rustica. When pollinated with N. undulata, growth of the pollen tube was arrested in the middle of the style and showed abnormal morphology similar to that with N. rustica, but when pollinated with N. paniculata the pollen tube reached near the base of the style and was almost normal in appearance. These observations suggest that the factors responsible for the pollen tube abnormality of N. rustica are derived from the N. undulata genome.We also used N. sylvestris, N. glutinosa and N. otophora as pistilar parents and N. rustica or its progenitors as pollen parents to examine the genomic factors of the pistilate parents. The pollen tube features of these three species in the pistils of N. sylvestris were similar to those in the pistil of N. tabacum. Received: 25 October 1999 / Revision accepted: 2 February 2000     

NTRS, a new family of highly repetitive DNAs specific for the T1 chromosome of tobacco

Species-specific repeated DNAs are important for identifying genomic components of hybrid organisms in plant breeding and in taxonomic studies, and we have previously described the HRS60 and GRS families of highly repetitive DNA sequences in tobacco. Here we describe a new family of highly repetitive DNA sequences termed NTRS (SspI family) that we have isolated from Nicotiana tomentosiformis (Goodspeed) and characterized and that is specific for the genomes of several species of the subgenus Tabacum. In situ hybridization showed that NTRS sequences are present in three pairs of chromosomes of N. tomentosiformis, six pairs of chromosomes of N. kawakamii, and only one pair of chromosomes of N. tabacum at an intercalary site. The NTRS family is not present in the N. otophora genome. The majority of NTRS sequences appeared to be organized in tandem arrays in which local DNA structures sensitive to single strand-specific chemical probes, potassium permanganate, and osmium tetroxide complexed with pyridine revealed a periodicity of 220 bp, equal to the length of the repeat unit. The inner cytosine in CCGG and CC(A/T)GG sequences of the NTRS family is frequently methylated. Cloned and sequenced NTRS monomeric units are 212–219 bp in length and show 83.5%–95% mutual homology. They exhibit properties characteristic for molecules that possess stable intrinsic curvature, but there are differences among individual monomers in the degree of curvature. NTRS sequences like HRS60 and GRS sequences, were found to specify nucleosome positions. Received: 12 November 1996 / in revised form: 12 May 1997 / Accepted: 12 May 1997     

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.     

Possible tobacco progenitors share long-tongued hawkmoths as pollen vectors

The putative ancestors of the allopolyploid hybrid Nicotiana tabacum have distinct flower features, apparently suited either for hawkmoth or bat pollination. This suggests that progenitors were reproductively isolated by mechanical and ethological barriers. However, the present data show that in natural populations pollen vectors could be shared by two of the possible progenitors. Pollen vectors of one of the possible male progenitors (N. otophora) were short- and long-tongued hawkmoths and a nectar-feeding bat, while those of the female ancestor (N. sylvestris) were only long-tongued hawkmoths. The latter are then the most likely vectors responsible for the presumed spontaneous hybridization. These data also suggest that interspecific pollen transfer occurred more likely in one direction.     

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.